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and IFN-
Posttranscriptionally Down-Regulate the IL-4-Induced IL-4 Receptor Gene Expression1
Department of Biological Science and Institute for Basic Science, SungKyunKwan University, Suwon, Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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/
and IL-4 counterregulate
diverse immune functions. In particular, IFN- and IFN- have been
reported to markedly suppress the IL-4-induced IgE production and type
II IgE receptor (Fc
RII/CD23) expression. Because modulation of IL-4R
may be an important mechanism in the regulation of IL-4 response, we
have investigated the effect of IFN-/ on IL-4R expression and
signal transduction mechanisms involved in this process. In human
mononuclear cells and B cells isolated from tonsil or peripheral blood,
IL-4 up-regulates IL-4R() expression at surface protein and mRNA
levels, and the IL-4-induced IL-4R() is significantly down-regulated
by both IFN- and IFN- to a similar extent. The inhibitory effects
of IFN-/ on the IL-4R mRNA expression require a lag period of
about 8 h, and are sensitive to cycloheximide treatment, which
suggests that the suppressive effect of IFNs on IL-4R gene expression
is a secondary response requiring de novo synthesis of IFN-induced
factors. Under such conditions that the inhibitory effects of IFNs are
observed, IFNs do not affect the IL-4-induced STAT6 activation and
IL-4R transcription, as analyzed by EMSA and nuclear run-on assays,
respectively. Subsequently, mRNA stability studies have indicated that
the action of IFN-/ is primarily mediated by an accelerated decay
of IL-4-induced IL-4R mRNA. Thus, it appears that, as already shown in
the case of the IL-4-induced FcRII regulation, posttranscriptional
inhibition of IL-4-inducible genes by mRNA destabilization is a common
mechanism by which type I and II IFNs antagonize the IL-4 response in
human immune cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-4 are prototypic Th1 and Th2
cytokines, respectively, whose antagonistic actions against each other
have been well known. These include Th1/Th2 cell differentiation, IgG
subclass switching, IgE production, and modulated expression of class
II MHC, IL-1R, FcRI, and FcRII (2, 3, 4, 5, 6, 7, 8, 9).
IL-4 is a pluripotent cytokine whose regulatory effects on cell
growth and differentiation are exerted in diverse cell types, such as B
cells, T cells, and monocytes, as well as cells of nonhemopoietic
origin (10). The signal transmission of IL-4 is mediated
by the high affinity receptor, of which two types are known: type I
IL-4R composed of IL-4R -chain (p140) plus common -chain, and
type II receptor composed of IL-4R plus IL-13R (11).
Although IFN- and IFN- are primarily produced by different cell
types and act on target cells through distinct receptors, IFN- has
been also recognized as a cytokine promoting Th1 differentiation
(12, 13). Thus, its regulatory effects on the IL-4 action
have recently become a subject of interest. In fact, IFN- and
IFN- both effectively suppress the IL-4-induced IgE production
(4, 14) and the low affinity IgE receptor (FcRII/CD23)
expression (9, 15). It has been reported that while the
inhibitory action of IFN- on the IgE production is exerted by a
direct suppression of the IL-4-induced IgE C region transcript
transcription (16), the down-regulation of the
IL-4-induced CD23 by IFN- mainly involves posttranscriptional
inhibition of CD23 mRNA (17). The mechanism of IFN-
inhibition on the IL-4-evoked responses, however, remains largely
unknown.
Because receptor modulation is an important event during cytokine
signal transduction, we have been studying the molecular mechanism of
the IL-4R regulation as a part of our ongoing investigation on the
mechanism of IgE production and responses. Previously, we have reported
that IL-4 and anti-CD40 up-regulate IL-4R via tyrosine
kinase-dependent pathways, and that the costimulatory effect of
anti-CD40 on the IL-4-induced response in B cells is partly due to
the increase in IL-4R expression by CD40-mediated signal
(18). In the present study, we have examined regulation
mechanisms of IFN- and IFN- on the IL-4R() expression, and
present strong evidence that in human primary immune cells IFN- and
IFN- both down-regulate the IL-4-induced IL-4R expression as a
delayed response requiring an IFN-induced protein factor, and the
inhibition occurs not at the transcriptional, but at the
posttranscriptional level by decreasing stability of IL-4R mRNA. The
accelerated decay of IL-4R mRNA may serve as a means to suppress the
IL-4-induced response and thus constitutes one of the mechanisms
underlying the counteraction between IL-4 and IFNs. Furthermore, when
taken together with our previous report on the mechanism of IFN-
inhibition of FcRII/CD23 expression (17), mRNA
destabilization of the IL-4-stimulated genes may be a common mechanism
by which type I and II IFNs counterregulate the IL-4 response in
various immune functions such as allergy, inflammation, and
infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mononuclear cells were isolated from freshly excised human
tonsils or peripheral blood using Ficoll-Hypaque (Pharmacia Biotech,
Uppsala, Sweden) density-gradient centrifugation. Typically, the
mononuclear preparation from tonsils contained about 70% B cells,
25
30% T cells, and less than 5% non-B/T cells, including
monocytes. B cells were further purified from mononuclear cells by
negative selection after rosetting twice with
2-aminoethylisothiouronium bromide-treated SRBC and subsequently
removing adherent cells. The purity of B cells was determined by
staining with anti-CD20, anti-CD3, and anti-CD14 mAbs, and
confirmed to be 
98%. Cells were cultured in RPMI media containing
10% FBS (Life Technologies, Grand Island, NY), 10 mM HEPES, 2 mM
L-glutamine, 5 x 10-5 M 2-ME,
50 µg/ml gentamicin, and 50 µg/ml amphotericin B (Sigma, St. Louis,
MO). Recombinant human IL-4 (R&D Systems, Minneapolis, MN, and KRIBB,
Tajeon, Korea), IFN- (R&D Systems, and LG Biotech, Tajeon, Korea),
IFN- (Schering-Plough, Madison, NJ), cycloheximide
(CHX),3 and
actinomycin D (Sigma, St. Louis, MO) were added to cells at indicated
times and the cells were cultured in humidified 5%
CO2 at 37°C.
Flow cytometric analysis of IL-4R
Mononuclear or purified B cells (1 x
106) were cultured in the presence of IL-4,
IFN-, or IFN- for 48 h. The cells were washed with PBS and
recultured for 2 h in the fresh media according to Zuber et al.
(19). The levels of IL-4R expression were then analyzed by
staining cells with mouse anti-IL-4R() mAbs (M56, kindly
provided by Dr. S. Gillis, Immunex, Seattle, WA) as a primary Ab and
goat anti-mouse IgG FITC (Immunotech, Marseille, France) as a
secondary Ab in HBSS containing 3% FBS and 1%
NaN3 for 30 min and 4°C using
fluorescence-activated cell scanner (FACSCalibur; Becton Dickinson,
Mountain View, CA). An aliquot of each treated cell sample was stained
with the secondary Ab alone as control. The surface IL-4R levels were
expressed as the mean fluorescence intensity (MFI) (7).
MFI was calculated as MFI of cells stained with anti-IL-4R and
anti-mouse IgG FITC - MFI of cells stained with
anti-mouse IgG FITC alone. Each experiment was repeated several
times and the values represent a mean of two independent
determinations.
Northern analysis of IL-4R mRNA
Total cytoplasmic RNAs were isolated from mononuclear or
purified B cells (1 x 108) after treatment
with IL-4, IFN-, or IFN- with and without actinomycin D or CHX
for various durations, as indicated, using guanidium isothiocyanate and
cesium chloride through ultracentrifugation. For Northern blots, 10
µg of total RNA from each preparation was separated on a 1%
agarose-formaldehyde gel, and transferred to nylon membranes
(Genescreen Plus; New England Nuclear, Boston, MA). A cDNA probe of
IL-4R() (provided by Dr. S. Gillis, Immunex) or STAT6 (provided by
Dr. B. Groner, Goethe University, Frankfurt, Germany) was labeled with
[-32P]dCTP (3000 Ci/mmol; Amersham,
Arlington Heights, IL) at sp. act. of 5 x
108 cpm/µg and used for hybridization. RNA
concentration was determined by OD measurement, and the amount of
loaded RNA on the gel was confirmed by ethidium bromide (EtBr)
(8) staining. Blots were reprobed using an adenosyl
phosphoribosyl transferase (APRT) (9) probe as internal
control (20). Northern analyses were conducted several
times for each experiment, and a representative blot is shown.
EMSA (10)
Cells were pretreated with IFN- or IFN- for 3–24 h and
stimulated with IL-4 for indicated durations, after which nuclear
extract preparations and EMSA were performed essentially as described
(21). IL-4-responsive element (IL-4RE) (11)
sequence (FcRIIb IFN--activated site (GAS) (12):
5'-GGGTGAATTTCTAAGAAAGGG-3') was labeled using
[-32P]dCTP and Klenow, and the binding
reaction with the nuclear extract was performed in the buffer
containing 10 mM Tris-Cl (pH 7.5), 50 mM NaCl, 10 mM
MgCl2, 1 mM DTT, 1 mM EDTA, 10% glycerol, 1 mM
NaF, and 2 µg poly(dI)·(dC) for 20 min at room temperature.
Mobility shift of the oligomer was then analyzed by a 5% PAGE in 0.5x
TBE buffer.
Immunoprecipitation and immunoblots
Cells were pretreated with IFNs for 3–24 h and stimulated with IL-4 for indicated periods, after which total cell extracts were prepared using a lysis buffer containing 1% Nonidet P-40, as described (22). The extracts (1–2 mg) were immunoprecipitated by incubating with rabbit polyclonal anti-STAT6 Abs (Upstate Biotechnology, Lake Placid, NY), and then with anti-rabbit IgG agarose, after which precipitated samples were subjected to 10% denaturing SDS-PAGE. Gels were transferred to nitrocellulose membrane, which were then blotted with anti-phosphotyrosine mAbs (4G10; Upstate Biotechnology), and reprobed with anti-STAT6 Abs after stripping. The blots were developed using an enhanced chemiluminescence detection kit (Amersham).
Nuclear run-on transcription
Nuclei were prepared by incubating cells (1 x
108) for 5 min on ice in lysis buffer containing
20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2,
0.5 mM DTT, 0.3 M sucrose, and 0.2% Nonidet P-40. In vitro
transcription reactions were then performed in 200 µl reaction buffer
(10 mM Tris-HCl, pH 7.5, 35% glycerol, 5 mM
MgCl2, 80 mM KCl, 0.1 mM EDTA, 0.5 mM DTT, and 4
mM each of ATP, CTP, GTP, and 200 µCi
[-32P]UTP (3000 Ci/mmol; Amersham)). The
nuclei were digested with RNase-free DNase I and proteinase K. Nuclear
RNA was then purified as described (23) and used to
hybridize linearized plasmids containing 2 µg each of IL-4R()
cDNA, GAPDH (13), cDNA, and pBluescript DNA, which were
previously blotted on nylon membranes. Conditions for hybridization and
washing were as described by Celano et al. (24).
Densitometric analysis
Densitometric analysis was performed to quantify the relative intensity of radioactive bands using Image QuaNT phosphor imager system (Bio-Rad, Hercules, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN- both suppress the IL-4-induced IL-4R expression
in human primary immune cells
Although it has been widely reported that IFN-/ often
antagonize the ability of IL-4 to induce specific gene expression in
various cell types, relatively little is known about the mechanism by
which IFNs down-regulate such IL-4-induced responses. In fact, results
from several studies have indicated that the detailed mechanism used by
IFNs to inhibit the IL-4-induced response may differ depending on
biological effects and cell types analyzed (9, 16, 25, 26, 27, 28, 29). Since we have been studying the mechanism of
counterregulation between IL-4 and IFN- in human B cells, we have
first analyzed the regulatory effects of IFN-/ on the
IL-4-induced IL-4R() expression using tonsillar mononuclear cells
that represent an enriched human B cell source. As shown in Fig. 1
, IL-4 induced a noticeable increase (2-
to 3-fold) in surface IL-4R() expression on these cells as analyzed
by flow cytometry, and IFN- (type II IFN) significantly inhibited
the IL-4-induced IL-4R() expression in a dose-dependent manner.
IFN- (type I IFN) also exerted a similar inhibitory effect. At 100
ng/ml, IFN- and IFN- produced about 80% and 70% inhibition of
IL-4-induced IL-4R(), respectively. With B cells further purified
from the tonsillar mononuclear cells, basically the same response was
observed (Fig. 2A). In
addition, very similar results were obtained with other sources of
human primary immune cells, such as purified B cells isolated from
peripheral blood of normal donor (Fig. 2B). Through multiple
experiments, we have confirmed that these results were
reproducible with tonsil or blood samples obtained from different
donors. The data strongly indicate that antagonistic regulation of
IL-4R by IL-4 and IFN-/ occurs in human primary immune cells.
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and IFN- inhibit IL-4R mRNA expression as a delayed
response requiring de novo protein synthesis
To further examine the mechanism of IFN inhibition of the
IL-4-induced surface IL-4R, we have analyzed the kinetics of inhibition
at IL-4R mRNA level. Cells were first treated with IL-4 for at least
6 h to initiate induction of IL-4R and then cultured in the
absence or presence of IFN- or IFN- for various periods. Fig. 3A shows that IL-4-induced
IL-4R mRNA gradually accumulates with time. In fact, we have previously
reported that IL-4R mRNA starts to increase after 24 h of IL-4
stimulation in tonsillar B cells, and becomes saturated at about
12 h posttreatment (18). Fig. 3 also demonstrates
that the counterregulatory effect of IFNs on the IL-4-induced gene
expression appears as a rather delayed response. The suppressive effect
of IFN- was not apparent at 2 h, and the inhibition became
prominent by 8 h after IFN- treatment (A). In case
of IFN-, the inhibition was not significant up to 4–6 h and was
gradually evident by 8–12 h after IFN- treatment (B).
These data indicate that the inhibitory action of IFNs requires a lag
period of 6–8 h. Thus, we wanted to examine the effect of IFN
pretreatment before IL-4 stimulation. As expected, pretreatment of B
cells with IFN- or IFN- for 12 h followed by IL-4 treatment
for 12 h yielded an effective inhibition of the IL-4 response
(Fig. 4). Importantly, CHX
(14), a translational inhibitor, abolished the suppressive
effect of IFN- or IFN-; i.e., in the presence of CHX, IFN- had
no inhibitory effect on the IL-4-inducible level of IL-4R mRNA
(A, lanes 5–7). The same effect was observed for
the case of IFN- (B, lanes 4 and
5). CHX itself caused only slight change (0.9–1.1-fold) in
the control or the IL-4-inducible level of IL-4R mRNA. The control
STAT6 blot (C) demonstrates that IL-4 or IFNs do not
apparently affect the STAT6 mRNA level under our experimental
conditions, ruling out a possibility that counterregulation of IL-4R
mRNA by IL-4 and IFN- occurs via modulation of STAT6 gene
expression.
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/ on the IL-4-stimulated
IL-4R gene expression is delayed, requiring a lag period and ongoing
protein synthesis. The observed lag period for IFN inhibition seems to
be necessary for the production of an IFN-induced protein factor, i.e.,
a liable and/or newly synthesized protein factor playing a role in
mediating the inhibitory action of IFNs on the IL-4R gene
expression.
Pretreatment of IFN- or IFN- only transiently attenuates
STAT6 activation without affecting IL-4R transcription
Although STAT6 has been strongly implicated in the activation of
several IL-4-inducible genes, such as IgE C region transcript,
FcRII, IL-4, and IL-1R and murine IL-4R (30, 31),
direct role of STAT6 in human IL-4R gene regulation has not been
reported. Therefore, we wanted to examine the possibility that the
inhibitory effect of IFN-/ on the IL-4R mRNA is exerted through
the attenuation of STAT6 activity by IFNs, thereby regulating
IL-4-induced IL-4R gene activation. We conducted STAT-DNA-binding
assays using an IL-4RE GAS probe as a STAT6-binding sequence. As shown
in Fig. 5A, IL-4 induced
specific activation of a STAT factor, which binds to the IL-4RE
sequence. The immunoreactivity of the complex to anti-STAT6 Ab
confirmed that the IL-4-activated factor is STAT6 (18).
The pretreatment of IFN- or IFN- for various periods caused a
gradual inhibition of the IL-4-mediated STAT6 binding to the IL-4RE at
30 min post-IL-4 stimulation (Fig. 5A). The
immunoprecipitation and Western blot analysis of total cellular
extracts revealed that IL-4-stimulated tyrosine phosphorylation of
STAT6, and IFN- or IFN-, upon pretreatment, suppressed the
IL-4-induced tyrosine phosphorylation of STAT6 (Fig. 5B). To
examine whether the ability of IFNs to modulate STAT6 activity
correlates with IFN regulation of IL-4-induced IL-4R mRNA, as observed
in Fig. 4
, cells were pretreated with IFN- or IFN- for 16 h,
a duration sufficient to exert inhibitory effects, and further
incubated with IL-4 for 4–10 h to allow substantial induction of IL-4R
mRNA by IL-4. Under this condition, however, no inhibitory effects of
IFNs on STAT6 activity were observed (Fig. 5C). There was
also no apparent inhibitory effects of IFNs on the tyrosine
phosphorylation of STAT6 (data not shown). To find out whether even
transient attenuation of STAT6 activity by IFNs underlies the molecular
mechanism of IFN-mediated down-regulation of IL-4R mRNA level via
suppression of IL-4-induced IL-4R gene transcription, we have performed
nuclear run-on assays. In Fig. 6, we have
observed an enhancement of IL-4R gene transcription by IL-4.
Densitometric analysis revealed that IL-4 induced a significant
increase (2- to 3.9-fold) in nuclear IL-4R transcription over untreated
samples, while the levels of control GAPDH transcripts were basically
not affected by IL-4. The treatment of IFN- or IFN- at least for
12 h, a duration sufficient for the manifestation of IFN-induced
down-regulation of IL-4R mRNA, however, did not affect the
IL-4-activated IL-4R gene transcription. These results indicate that
while IFN- and IFN- can cause the transient inhibition of the
IL-4-induced STAT6 activation, they do not adversely affect the overall
transcriptional activity of IL-4 gene, suggesting that modulation of
STAT6 activity is not responsible for the down-regulation of
IL-4-induced IL-4R gene expression by IFNs in these cells.
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and IFN- posttranscriptionally
down-regulate IL-4R through mRNA destabilization
Having observed that IFNs down-regulate the IL-4-induced steady
state IL-4R mRNA level without affecting transcription of IL-4R gene,
we conducted mRNA stability studies to examine a possible
posttranscriptional control by IFNs. Mononuclear cells were first
treated with IL-4 for 12–15 h to induce the IL-4 activation of IL-4R
gene expression in the presence or absence of IFNs. Actinomycin D
(15) was then added to block further synthesis of IL-4R
mRNA, and time-dependent changes in mRNA levels by spontaneous
degradation were analyzed. As shown in Fig. 7, A and B, an
accelerated decay of IL-4R mRNA in cells treated with IL-4 plus IFN-
or IFN- was noted compared with cells treated with IL-4 alone. The
t1/2 of IL-4R mRNA was found to be
significantly reduced in both IFN- (80 vs 180 min) and IFN- (50
vs 95 min)-treated cells. With purified B cells, very similar results
were obtained (C), in that the stability of IL-4R mRNA was
drastically decreased by IFN- or IFN- treatment. These data
strongly suggest that IFN-/ posttranscriptionally rather than
transcriptionally modulate IL-4R through accelerating mRNA decay.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) (32, 33, 34). Among the known two types of IL-4R
complex, IL-4R() is a common and essential subunit (35, 36). In fact, modulation of IL-4R() has been recognized as an
important event during the IL-4-mediated signal transduction, which
leads to the regulation of IL-4-induced responses. It has been reported
that in human lymphoblastoid cell line Jiyoye, immediately after IL-4
treatment, the surface IL-4R undergoes a transient down-regulation via
internalization and degradation. Then, most likely through IL-4-induced
de novo synthesis, the surface IL-4R is up-regulated by 24 h,
which is thought be required for the IL-4 induction of FcRII/CD23
expression (33). According to the well-established Th1/Th2
modality, diverse antagonistic actions have been recognized between
IL-4 and IFN-. Therefore, as a part of our study on the regulation
of IgE response, we investigated the molecular mechanism of IL-4R()
modulation by these cytokines.
Although IFN- has been primarily recognized for antiviral effects
rather than immunoregulatory effects, recent studies suggest a
potential role of IFN- on diverse immunomodulatory functions, such
as activation of macrophages and NK cells (37, 38),
differentiation of Th1 cells (12), as well as regulation
of cytokine secretion and Ig production. Many of these biological
effects of IFN- are shared by IFN- in the antagonistic regulation
of IL-4 action, and IFN- and IFN- both use partially overlapping
signaling mechanisms (30, 31). Hence, we explored the
possibility that IFN- and IFN- both employ a common mechanism to
down-regulate the IL-4-evoked response by modulating the IL-4R
expression. Indeed, IFN- and IFN- both significantly reduced the
IL-4-induced IL-4R expression at the surface protein as well as steady
state mRNA level in tonsillar mononuclear cells. Basically, the same
pattern of induction and antagonistic regulation of IL-4R gene
expression by IL-4 and IFNs was also observed when purified B cells
were used (Figs. 1 and 2). With purified T cells from tonsils or
peripheral blood, a low magnitude of IL-4R induction by IL-4 and
down-regulation by IFNs were observed (data not shown). It should be
noted that our tonsillar mononuclear population contained about
70% B cells, 2530% T cells, and less than 5% of non-B/T cells,
and we have been obtaining basically the same effects of IL-4/IFN-
on the IL-4R expression using mononuclear cells or purified B cells
from tonsil throughout the experiments conducted in this study.
Distinct from the IL-4-induced IL-4R up-regulation that is thought to
be manifested as a primary response involving tyrosine kinase-dependent
STAT6 action in a CHX-independent manner (18) (Fig. 4),
IFN-/-induced inhibitory effects represent a secondary response.
The effects of IFN- and IFN- were both sensitive to CHX, and
required a lag period of 6–8 h, a duration probably necessary for the
de novo synthesis of IFN-/-induced factors (Figs. 3 and 4). Yet,
the signal transmission of IFN-/ to leading to such secondary
response occurred within minutes, in that pretreatment of IFN-/
for less than 1 h, subsequent washing, and continued incubation of
cells in the presence of IL-4 for 16 h produced the same
inhibitory effects (data not shown). It is likely that the
IFN-/-induced factors are products of IFN-inducible genes whose
transcription is mediated by STAT1 activation rapidly occurring upon
IFN treatment. Although it has been suggested that STAT1 and STAT6 can
recognize the same GAS site, and competitive DNA binding by the two
STATs may influence the transcriptional activities of target genes
counterregulated by IFN and IL-4 (39), no direct roles of
STAT1 and STAT6 in the repression of IL-4-inducible or IFN-inducible
genes, respectively, have yet been demonstrated. Aside from STAT1
activation, pretreatment of IFN- or IFN- caused a substantial
attenuation of the IL-4-induced STAT6 activity, as demonstrated by
IL-4RE binding and tyrosine phosphorylation of STAT6 (Fig. 5, A and B). However, such negative modulation of
STAT6 by IFNs was found to be only transient, and was not reflected in
the regulation of IL-4R gene transcription, in that under the condition
in which IFN-inhibitory action on IL-4R mRNA level is exerted, IFNs do
not affect the IL-4-induced STAT6 activity (Figs. 5C and 6).
This suggests that down-regulation of STAT6 activity by IFNs does not
constitute the mechanism of IFN inhibition of the IL-4-induced IL-4R
gene expression observed in this study. In fact, although
IL-4-stimulated STAT6 activation is correlated with the increased rate
of IL-4R transcription by IL-4, the essential role of STAT6 in human
IL-4R gene activation has not been demonstrated, possibly due to the
unavailability of the exact promoter structure of human IL-4R gene. In
case of murine IL-4R gene, a recent article reported that STAT6 binds
the GAS site of murine IL-4R promoter and activates the transcription
(34). Whatever the authentic role of STAT6 in IL-4R gene
regulation in human cells, our data still imply that the inhibitory
action of IFNs on the IL-4-induced IL-4R gene expression involves not
direct transcriptional repression of IL-4R, but posttranscriptional
down-regulation via decreasing IL-4R mRNA stability (Figs. 6 and 7).
There is a recent article describing that IFNs inhibit the IL-4-induced
STAT6 activation in monocytes that may be associated with the
repression of IL-4-induced IL-1R by IFNs (40). In this
study, the authors obtained the inhibitory effect of IFN- upon
pretreatment of monocytes with IFN- for 1 h and subsequent
stimulation of cells with IL-4 for 30 min. Considering that their
observation of the suppressive effect of IFN- on the IL-4-induced
IL-1R gene expression in monocytes was made at 6-h incubation of cells
cotreated with IL-4 and IFN-, it remains to be further explored
whether the attenuation of STAT6 activity observed at 30 min of IL-4
treatment is the major mode of IFN action to down-regulate the IL-1R
gene, or other delayed responses involving IFN-induced factors also
play a role in this process. It is quite possible that IL-4 or IFN-
can transiently down-regulate STAT1 or STAT6 activation, respectively,
in the early phase of cytokine signal transduction, especially when one
cytokine is treated to cells before the other. Because IL-4 and IFN-
both require Janus kinase activity 1 for STAT6 and STAT1 activation
(30, 31), either prior receptor occupation or depletion of
Janus kinase 1 that is caused by one cytokine may cause a transient
suppression of the other’s activity. However, as much as observed
biological effect of both IFN- and IFN- on IL-4R was manifested
as a delayed secondary response in the present study (Figs. 3 and 4),
there seems to be dissociation between the ability of IFNs to modulate
STAT6 activity and to down-regulate the IL-4-induced IL-4R gene
expression.
There still is a possibility that IFNs may influence the IL-4R
transcription machinery via other constitutive or IFN-inducible
transcriptional factors, including proteins of SOCS/SSI/CIS (suppressor
of cytokine signaling/STAT-induced STAT inhibitors/cytokine-inducible
inhibitors of signaling) (16, 17, 18) family, which have been
suggested to play a role in the feedback inhibition of cytokine
response (41, 42, 43). Such possibility, however, is clearly
ruled out by nuclear run-on transcription assays, the results of which
demonstrate that IFN- or IFN- does not directly or indirectly
suppress the IL-4-induced IL-4R transcription rate (Fig. 6). A
conclusive evidence that IFN- and IFN- both down-regulate the
IL-4-induced IL-4R by accelerating the mRNA decay was then provided by
the mRNA stability studies conducted using both mononuclear and
purified B cells (Fig. 7). Although the absolute
t1/2 of IL-4R mRNA turned out to be
somewhat different in experiments using primary cells derived from
tonsils provided by different donors, 50% of reduction in the
t1/2 of IL-4R mRNA was obtained for
IFN- or IFN-. Such a rapid turnover rate is characteristic for
mRNAs of primary response genes induced by mitogens or other growth
stimuli, which include mRNAs of a number of protooncogenes, cytokines,
and cytokine receptors (44, 45, 46). These mRNAs usually
possess AU-rich sequences in their 3' untranslated region (UTR), and
the decay mechanism of the mRNAs has been suggested to involve the
action of mRNA-destabilizing factor binding to the consensus UUAUUUA
motifs. Considering that IL-4R is induced by IL-4 as a primary response
gene and that there is an AU-rich motif in the 3'UTR of human IL-4R
(47), it is likely that IL-4R mRNA is a target of rapid
turnover upon specific extracellular stimuli. The observation that the
effects of IFNs for the down-regulation of IL-4-induced IL-4R mRNA
required a lag time and ongoing protein synthesis strongly argues for
the role of a liable and/or IFN-induced protein factor mediating
destabilization of IL-4R mRNA. Although potential candidates of such
IFN-/-inducible factor include a (2'-5') oligoadenylate-dependent
endonuclease (RNase L) that is shown to preferentially recognize U-rich
sequence of 3'UTR of many mRNAs via (2'-5') adenylate oligomer, an
allosteric activator of RNase L (48, 49), more specific
IFN-induced factors responsible for mediating the decay of IL-4R as
well as other IL-4-inducible genes are to be identified in future
investigations. A recent paper by Mozo et al. (50)
reported that while the modulation of IL-4R mRNA stability is an
important mechanism for IL-4R regulation by PMA and glucocorticoids,
IL-4-induced down-regulation of IL-4R by glucocorticoids may involve
translational or posttranslational mechanisms in human PBMCs.
In this regard, any possible regulations of IL-4-induced IL-4R by IFNs
at translational or posttranslational levels are also worthy of
investigation.
The results of the present study strongly support our previous report
on IFN- regulation mechanism for the IL-4-induced FcRII/CD23 gene
expression, in which we observed the same pattern of
posttranscriptional regulation by IFN- (17). In fact,
we have found that IL-4R and FcRII, two major allergy-associated
immune cell receptors, are regulated by cytokines and other
costimulatory signals in a highly coordinated manner (18, 51, 52), which may be important for a fine-tuned control of IgE
response in normal and disease conditions. In conclusion, our study
collectively suggests that mRNA destabilization of IL-4-stimulated
genes by IFN-/-induced factors may provide a common mechanism by
which allergy-associated immune cell receptors are regulated via
posttranscriptional modulation by type I and II IFNs, and constitute in
part, a molecular basis of counterregulation by Th1 and Th2
cytokines.
|
Acknowledgments |
|---|
cDNA and
anti-IL-4R mAb (M56) by Dr. S. Gillis at Immunex, and
STAT6 cDNA by Dr. B. Groner at Institute for Biomedical
Research, Goethe University. We are also grateful to Drs. B.
S. Cho and J. S. Cho at Kyung Hee University for the
valuable help provided in our experiments with tonsils, and to Mr.
J. H. Kim for excellent technical assistance. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Choong-Eun Lee, Laboratory of Immunology, Department of Biological Science, SungKyunKwan University, 300 Cheon-Cheon Dong, Jang-An Ku, Suwon, 440-746, Korea.
3 Abbreviations used in this paper: CHX, cycloheximide; APRT, adenosyl phosphoribosyl transferase; EtBr, ethidium bromide; GAS, IFN--activated site; IL-4RE, IL-4-responsive element; MFI, mean fluorescence intensity; UTR, untranslated region.
Received for publication July 6, 2000. Accepted for publication August 9, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and B cell stimulatory factor reciprocally regulate Ig isotype production. Science 236:944. and and prostaglandin E2. Proc. Natl. Acad. Sci. USA 85:880. and
IL-4. J. Immunol. :3524.
R membrane expression and FcR-mediated cytotoxic activity of human monocytes. J. Immunol. 144:3046.[Abstract]
RII receptor (CD23) on normal human B lymphocytes. J. Exp. Med. 169:149. and IFN- on the production of human IgE binding factor (soluble CD23). J. Immunol. 142:134.[Abstract]
increases the frequency of interferon -producing human CD4+ T cells. J. Exp. Med. 178:1655., IFN-, interleukin (IL)-2, and arachidonic acid metabolites modulate IL-4-induced IgE synthesis similarly in healthy persons and in atopic dermatitis patients. Allergy 48:189.[Medline]
and regulate Fc R2/CD23 mRNA expression on normal human B cells. Mol. Immunol. 27:129.[Medline]
represses germline transcription and subsequently down-regulates switch recombination to . Int. Immunol. 4:515.
down-regulation of the interleukin-4-induced CD23/FcRII expression in human B cells: post-transcriptional modulation by interferon-. Mol. Immunol. 30:301.[Medline]
-induced factor binding to the interleukin-4-responsive element of CD23b promoter in human tonsillar mononuclear cells: role in transient up-regulation of interleukin-4-induced CD23b mRNA. Mol. Immunol. 35:239.[Medline]
R II/CD23 gene expression by cytokines and specific ligands (IgE and anti-FcR II monoclonal antibody): variable regulation depending on the cell type. J. Immunol. 141:1376.[Abstract]
and IFN-. Cytokine 6:407.[Medline]
and interferon- via an interferon--activated site and its flanking regions. Eur. J. Biochem. 240:667.[Medline]
. Cytokine 10:75.[Medline]
chain: a functional component of the interleukin-4 receptor. Science 262:1880.1 chain and reconstitution with the IL-4R of a functional IL-4/IL-13 receptor complex. FEBS Lett. 401:163.[Medline]
. Cell. Immunol. 75:390.[Medline]
and IL-4 response element present in the germ-line 1 Ig promoter. J. Immunol. 154:4513.[Abstract]
chain expression by different posttranscriptional mechanisms. J. Allergy Clin. Immunol. 162:968.
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