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J. Biol. Chem., Vol. 277, Issue 21, 18313-18321, May 24, 2002
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From the
Received for publication, October 17, 2001, and in revised form, February 20, 2002
Using a transgenic approach, we studied the role
of GATA-3 in T cells. As previously shown, enforced GATA-3 expression
in transgenic mice inhibits Th1 differentiation of CD4 T cells, but unexpectedly, both type 1 (interferon Upon antigenic activation, naive CD4 T cells differentiate into at
least two functionally distinct subsets of effector cells that differ
in their cytokine expression pattern and in their effect in the
regulation of the immune response (1). T helper 1 (Th1)1 CD4 cells produce
interleukin-2 (IL-2), interferon How the Th1/Th2 differentiation of CD4 T cells is regulated at the
transcriptional level has been the subject of intensive research these
last years. Among transcription factors, an essential role has been
assigned to the signal transducer and activator of transcription 4 (STAT4) and 6 (STAT6) in the IL-12- and IL-4-mediated differentiation
of CD4 T cells toward Th1 or Th2. In addition, Th1 commitment requires
the expression of T-bet, a T box transcription factor that induced
IFN In contrast to CD4 T cells, CD8 T cells have been described as a
homogeneous class of cells producing IFN To study the consequences of GATA-3 overexpression in CD4 and CD8 T
cells, we generated transgenic mice where GATA-3 expression is under
the transcriptional control of the human CD2 promoter and locus control
region, a cassette that allows an expression restricted to T cells in
adult (23). The expression pattern of the cytokines genes in transgenic
mice CD8 T cells indicated that the role of GATA-3 might be different
in CD8 and in CD4 T cells and showed a function of GATA-3 in the
transcriptional regulation of the IL-13 gene through a complex network
of motifs characterized in this study.
DNA Constructs and Transgenic Mice--
The
CD2-hGATA-3 construct was obtained by inserting the human
GATA-3 (hGATA-3) cDNA at the EcoRI
cloning site of the CD2 minigene cassette (23). After a
SalI and XbaI digestion of the recombinant plasmid, the 12-kbp DNA fragment containing the transgene was purified
by agarose gel electrophoresis and microinjected into the pronuclei of
fertilized oocytes from a cross of B6D2F1 animals (C57BL/6 × DBA/2). Southern blot analysis of tail DNA was used to identify
transgenic animals, to determine the copy number, and to assess
integration patterns.
Northern Analysis--
Total RNA of thymus was prepared using
TRIZOL (Invitrogen). 20 µg of each sample were electrophoresed in a
1% agarose gel containing 2 M formaldehyde in 10 mM phosphate buffer, pH 7, and then transferred to a Nylon+
membrane (Hybond-N+) by capillary blotting. The blot was probed with a
BglII-BamHI DNA fragment complementary to the 3'
end of the transgenic RNA.
Western Analysis--
Cellular extracts of thymus were
obtained and Western blotting was performed as previously
described (24) using the mouse anti-hGATA-3 antibody (HG3-35, Santa
Cruz Biotechnology) and a rabbit anti-mouse horseradish
peroxidase-linked antibody (Promega). Immunoblots were then developed
using electrochemiluminescence (ECL, Amersham Biosciences).
Purification and Activation of Splenic T
Lymphocytes--
Spleens were removed from mice and homogenized in
RPMI 1640 medium containing 1% fetal calf serum, 50 µM
Reverse Transcription (RT) PCR--
Total RNA from thymus,
spleen, or purified and activated CD4 or CD8 T cells was purified with
TRIZOL (Invitrogen) and concentrated by ethanol precipitation. RNAs
were then treated with DNase I (Roche Molecular Biochemicals). cDNA
was prepared from 1 µg of total RNA using a oligo-dT primer and the
Superscript II reverse transcriptase (Invitrogen) in 50 µl. All
RT-PCR reactions were standardized by level of murine hypoxanthine
phosphoribosyltransferase (mhprt) expression. Conditions of
PCR were chosen according to the primers used. Amplification of
cDNA was performed using the following primers: mhprt
5', GTAATGATCAGTCAACGGGGGAC, and 3', CCAGCAAGCTTGCAACCTTAACCA;
hGATA-3 5', GCTCATCTTAGGGGTTGGTTTC, and 3',
GTGCATGACTCACTGGAGGAC; mIFN Intracellular Staining of IFN RNase Protection Assay--
The mCK-1 multi-probe template set
(BD PharMingen) was used for multiple cytokine gene RNase protection
assay. Total RNA was used, and RNase protection was performed following
the manufacturer's recommendations (BD PharMingen). Protected RNA were
resolved on a 6% denaturing polyacrylamide gel. Quantification was
using ImageQuant (Molecular Dynamics, Sunnyvale, CA). Normalization of
RNA loading was done by measuring the intensities of the protected
fragments encoding L32.
Cloning and Expression of the Chicken GATA-2 DNA Binding
Domain--
DNA sequences coding for amino acids 277-396 of chicken
GATA-2 were modified during PCR amplification to contain an
NcoI site at the 5' end and a BamHI site at the
3' end. The PCR product was inserted into pET 11D (Novagen) between
these sites. Cysteine 334, which is not involved in metal
co-ordination, was changed to serine. Direct expression from this
vector resulted in the addition of a methionine and an alanine to the
NH2 terminus of the peptide. The bacteria (BL21(DE3) strain
of Escherichia coli) were grown overnight and induced with
isopropyl-1-thio- Nuclear Extracts and DNA Binding Assays--
Nuclear extracts
were prepared from 107 Jurkat T cells. Briefly, cell lysis
was performed in Hepes 20 mM, NaCl 10 mM,
MgCl2 1.5 mM, EDTA 0.2 mM, glycerol
20%, Triton X-100 0.1%, dithiothreitol 1 mM, and
phenylmethylsulfonyl fluoride 1 mM, pH 7.7. After
centrifugation, nuclear proteins were extracted from the pellet by a
high salt buffer (the same buffer containing 400 mM NaCl).
DNA binding assays were performed essentially as described (25).
Poly(dI-dC) was used as a nonspecific competitor. The competitor
oligonucleotide was added at a 100-fold molar excess, and 200 ng of
mouse anti-hGATA-3 antibody (HG3-35, Santa Cruz Biotechnology) was
used for the supershift experiment.
The sequences of the electrophoretic mobility shift assay (EMSA) probes
(sense strands) were as follows.
Determination of Dissociation Constants--
Titrations with the
GATA-2 DNA binding domain and various oligonucleotides were performed
in 10 µl of 50 mM Tris, pH 7, 0.0125% Triton, 3.2%
Ficoll, 2 mM EDTA, and 4 µg/ml poly(dI-dC). Samples were
electrophoresed on 8% acrylamide gels in 10 mM HEPES, 10 mM Tris, and 1 mM EDTA. The amount of bound (B)
versus free (F) DNA was determined with a Molecular Dynamics
PhosphorImager, and the B/F ratio was plotted against the concentration
of bound DNA in mol/liter. The dissociation constants
(Kd) were determined by Scatchard analysis from the
reciprocal of the slope of the best linear fit as described previously.
The sequences of the binding sites (sense strands) were as follows.
IL-13 Promoter Reporter Constructs--
A 176-bp DNA fragment of
the murine IL-13 promoter spanning nucleotides -110 to +66 relative to
the transcription initiation start site was amplified using the
primers 5'-CGGGGTACCAATTCAAGATGAGTAAAGATGTGG-3' and
5'-GAAGATCTAGCCCAGAGCCAGTGAGAGA-3'. These primers add the KpnI and BglII restriction sites, respectively,
at the 5' and 3' ends of the amplified fragment. The fragment was then
inserted upstream from the firefly luciferase-coding sequence in the
pGL3 vector plasmid. Mutated promoters where one or several of the GATA
binding sites were disrupted ( the mutations are as described under
"Nuclear Extracts and DNA Binding Assays") were generated by the
use of different 5' primers during PCR amplification. All constructs
were checked by DNA sequencing.
Transient Transfections and Luciferase
Assays--
107 Jurkat or HeLa cells were electroporated
as described (25). Ten µg of reporter gene plasmid, 2 µg of pRL-TK
plasmid (Promega) for normalization of transfection efficiency, and for
HeLa cells, various amounts of pECE-hGATA-3 expression plasmid or empty
pECE vector were used. Two hours after electroporation, Jurkat cells were activated by 50 ng/ml PMA and 1 µM ionomycin. Cells
were harvested 24 h after transfection, and luciferase activities
were determined using the dual-luciferase reporter assay system as indicated by the manufacturer (Promega).
CD2-GATA-3 Transgenic Mice--
Transgenic constructs were made by
inserting the human full-length GATA-3 cDNA into the
EcoRI site of an hCD2 minigene (23) containing 4.5 kb of
5'-flanking sequence, the first exon and intron as well as the last
exon of the human CD2 gene, and 5.5 kb of 3' CD2 locus control
region (Fig. 1A). The
CD2-GATA-3 DNA fragment was purified and microinjected into the
pronuclei of fertilized (C57BL/6 × DBA/2) F1 oocytes. Two
transgenic lines were generated, line 1 (L1) and line 3 (L3). Southern
analysis of tail DNA showed that L1 contained 8 copies of the
transgene, whereas L3 contained 3 copies (data not shown).
Northern blot analysis showed the presence of transgenic hGATA-3
mRNA in the thymus of L1 heterozygous and homozygous mice (Fig.
1B), whereas a very faint signal was obtained with mRNA from L3 thymus (data not shown). To precisely define the difference in
the expression of the transgene in the two transgenic lines, we used
RT-PCR analysis and found that the hGATA-3 mRNA level was about
eight times lower in the thymus of line 3 (Fig. 1C). Transgenic hGATA-3 protein expression was studied by Western blot analysis using protein extracts from the thymus of L1 mice. As shown in
Fig. 1D, a higher level of GATA-3 was found in both
heterozygous and homozygous L1 mice, and similar results were obtained
using EMSA (data not shown). The GATA-3 protein found in the thymus of
wild type (WT) mouse is accounted for by the cross-reactivity of the
antibody against human GATA-3 with mouse GATA-3. In purified CD4 or CD8
T cells isolated from the spleen of homozygous L1 mice, hGATA-3
expression was studied by RT-PCR, and a higher level of expression was
found in CD8 T cells (Fig. 1E).
Transgenic mice were healthy and of normal fertility in the two lines.
Thymus and spleen of transgenic mice were of similar size compared with
wild type mice. The percentage of immature double negative
CD4 Cytokines Expression after Activation of CD4 Lymphocytes from
Transgenic Mice--
Numerous studies have shown that overexpression
of GATA-3 in CD4 T cells leads to an elevation of type 2 cytokines and
a reduction of type 1 cytokines. It has also been demonstrated that
GATA-3 overexpression mediated by retroviral transduction induced an increased expression of the endogenous GATA-3 gene in murine T cells
(11, 26). To validate the transgenic CD2-GATA-3 model we have
generated, we compared the expression level of mGATA-3 mRNA and of
two known GATA-3 targets, IL-4 and IFN Cytokine Expression after Activation of CD8 Lymphocytes from
Transgenic Mice--
We therefore used these mice to determine the
consequence of GATA-3 overexpression in CD8 lymphocytes. Cell-sorted
purified CD8 T cells from the spleen of L1 or L3 mice or from
non-transgenic age- and sex-matched control mice were activated for 6 days by PMA and ionomycin and further stimulated for 24 h with
immobilized anti-CD3 in the presence of IL-2. The mRNA levels of
mGATA-3, IL-4, IL-13, and IFN
To quantify the respective expression of the different cytokine
mRNAs after CD8 lymphocyte activation, RNase protection assay was
performed. IFN
Because it has been demonstrated that in CD4 T cells GATA-3 expression
induced type 2 cytokines expression (7) and reduced IFN The Mouse or Human IL-13 Gene Promoter Contains Three Clustered
GATA Binding Sites--
Because the IL-13 mRNA level was markedly
increased by the overexpression of hGATA-3 in CD8 T cells, we wondered
if IL-13 gene transcription was directly regulated by GATA-3. We first looked at the sequences of the mouse and human IL-13 gene promoters (27) and found a region of high homology between human and mouse, respectively, located between
We first studied the binding of hGATA-3 to the consensus GATA binding
site using a T cell nuclear extract and a short oligonucleotide that
does not contain either of the two GATG motifs and found very weak
hGATA-3 binding (data not shown). This result prompted us to study
hGATA-3 binding to the mouse Affinities of the Different IL-13 Promoter GATA Binding Sites for
the GATA Zinc Fingers--
To precisely evaluate the relative binding
affinities of the three GATA sites found in the IL-13 gene promoter, we
determined by Scatchard analysis their affinity for a GATA-2
recombinant peptide that contained the DNA binding domain of GATA-2.
Among the GATA factors, the DNA binding domains of GATA-2 and -3 are very similar to one another. Unlike most GATA factors, the
N-fingers of both proteins can bind to DNA independently
(28), and the specificity of GATA-2 and -3 was shown to be virtually
identical in binding site selection experiments (29). Each of the three sites displayed a low affinity, the dissociation constant being, respectively, 10, 67, and 73 nM for sites 1, 2, or 3 (Fig.
7A). As expected from the EMSA
experiment, the association of the three sites or of the consensus GATA
binding site with the site 2 enhanced affinity between 5- and 10-fold
(Fig. 7B). These experiments demonstrated that the GATA
binding motif present in the IL-13 promoter is a high affinity motif
resulting from the association of low affinity binding sites.
Using higher peptide concentrations, a second more slowly migrating
complex (2:1 complex) can be seen with the wild type IL-13 probe or
with sites 1 and 3 (Fig. 7B). This complex is due to the
association of two molecules of peptide with the DNA and occurs with
the probes containing the first and the last GATA sites. No 3:1
complexes were observed.
It can be seen in Fig. 7A that the migration of complexes
with probes containing only site 1 or 2 is slower than that of the wild
type or the site 3 probe. However, combining site 1 and 2 on a single
probe results predominantly in a complex that migrates in the position
of the wild type probe and faster than either single site alone (Fig.
7B). This suggests that the presence of both binding sites
on a single DNA allows the GATA-2 peptide to bind in a different
conformation than when only one site is available. In contrast, the
combination of site 1 and site 3 appears additive since both the slow
migrating (site 1) and fast migrating (site 3) complexes are visible
with the 1,3 probe in the 1:1 complex. However, adding site 2 to the
probe (wild type) causes a reduction of the slow migrating form (Fig.
7B), again suggesting a change in complex conformation. As
previously shown, the N-finger of GATA proteins may
participate in binding to double sites, causing conformational changes
in the protein-DNA complexes. In conclusion, sites 1 and 3 can each
independently bind a molecule of protein, whereas site 2 increases the
affinity, probably through binding of the N-finger.
Expression Driven by the IL-13 Promoter in a T Cell Line--
To
study the function of the GATA binding sites of the IL-13 gene
promoter, a 176-bp DNA fragment of the murine IL-13 promoter spanning
nucleotides Transactivation by hGATA-3 of the IL-13 Gene Promoter in a
Heterologous Cell Line--
To determine directly the ability of
hGATA-3 to activate the IL-13 gene promoter and to define the relative
role of the different GATA-3 binding sites in this activation, we
studied the function of the previously described promoters (WT or
mutated) in a heterologous cell line (HeLa) that does not express
hGATA-3 (Fig. 9). In the absence of
hGATA-3, all the constructs displayed a similar transcriptional activity, which was less than twice the transcriptional activity of the
empty vector (data not shown). When hGATA-3 was co-transfected with the
WT promoter, its transcriptional activity was 8-fold increased.
Disruption of site 2 or site 3, respectively, reduced transactivation
to about 6- or 4.5-fold, whereas disruption of sites 2 and 3 resulted
in a 2.5-fold transactivation. Thus, the consensus GATA binding site
only accounted for 30% of the hGATA-3 transactivation, suggesting that
cooperativity between this site and non-consensus GATA binding sites is
required for proper regulation of the IL-13 gene promoter.
Quantitative Requirement of hGATA-3 in the IL-13 Gene Promoter
Activity--
During T lymphocyte activation, the level of the hGATA-3
protein is regulated as a consequence of the different stimuli leading to differentiation toward the Th1 or the Th2 pathway. Thus, we thought
that it was of interest to evaluate the quantitative requirement of the
hGATA-3 protein for the function of the different elements of the IL-13
gene promoter. Precisely, we wanted to test whether site 1 alone or
sites 1, 2, and 3 behaved similarly in response to the amount of
hGATA-3 protein expressed. These two constructs were co-transfected
with increasing amounts of hGATA-3 expression vector, and after
normalization, the reporter gene activity was determined. As shown in
Fig. 10, the two promoter constructs
displayed a very different dependence on hGATA-3 protein level. The WT
promoter reached 50% of its full activity when less than 200 ng of the hGATA-3 expression vector was co-transfected and was sensitive to
variations of hGATA-3 concentration under 50 ng, whereas the mutated
IL-13 gene promoter, which only contained site 1, required 400 ng of
hGATA-3 expression vector for 50% transcriptional activity and started
to be sensitive to hGATA-3 expression only when more than 200 ng of the
expression plasmid was used. Altogether, these results demonstrated
that the association of several sites of low affinity for hGATA-3 could
lead to a fine tuning of the transcriptional activity of the IL-13 gene
promoter, which becomes highly sensitive to minute variations of GATA-3
protein level.
In this paper we used transgenic mice that expressed hGATA-3 under
the CD2 locus regulatory region to study the effects of enforced
hGATA-3 expression on the transcription of the cytokine genes in CD4
and CD8 T cells. We found no difference between transgenic and wild
type control mice in the T lymphoid populations of thymus or spleen,
and none of the transgenic mice obtained developed a thymic lymphoma.
These results are different from the ones recently published using the
same CD2 locus region but containing a mouse GATA-3 cDNA tagged
with three hemagglutinin epitopes (30). Using this construct, Nawjin
et al. have shown a significant reduction of CD8 T
cells in the spleen and lymph nodes and the induction of thymic
lymphoma in 50% of the transgenic mice (30). The differences between
these results and the ones shown in this paper could be accounted for
1) by the presence of the hemagglutinin tag that seems to affect
post-transcriptional regulation of the mouse GATA-3 protein and 2) by
the genetic background, which is C57BL/6 for the transgenic mice we
studied and FVB for the mice previously described. Indeed
genetic backgrounds can lead to completely different phenotypes, as
shown by the studies done on different strains of mice carrying the
same gene inactivation (31).
We first validated the mouse model generated by studying the CD4 T
cells. We showed that after activation by PMA-ionomycin and
reactivation by anti-CD3, i.e. under nonpolarizing
conditions allowing commitment to both Th1 or Th2 phenotype, the CD4 T
cells of transgenic mice displayed a shift in the Th1/Th2 balance, as demonstrated by an increased expression of IL-4 mRNA associated with a decreased expression of IFN A major finding of this study is that GATA-3 could directly enhance
IL-13 gene expression in CD8 T cells under neutral or Tc1 conditions.
This result is different from the transgenic data of Zheng and Flavell
(7) in CD4 T cells showing a major effect of GATA-3 on IL-4 gene
expression but minimal effects on IL-5 and IL-13 genes. However, our
result is in perfect accordance with the up-regulation of IL-13 in T
cells that overexpressed GATA-3 and the inhibition of IL-13 genes seen
in transgenic mice expressing a dominant-negative mutant of GATA-3 (8,
37). Many reports have described the regulation of the IL-4 and IL-5 genes. As expected, proximal and distal cis-acting elements regulate their expressions and GATA-3, c-Maf, and nuclear factor AT are among the major trans-acting factors that control these two genes (5).
The IL-5 promoter is directly transactivated by GATA-3, and two distal
regulatory elements are necessary for the transactivating effect of
GATA-3 on IL-4 gene (7, 37-39). Conversely, and despite the importance
of IL-13 in diseases such as asthma and allergy, there is very limited
information on the regulation of the IL-13 gene. During the immune
responses, the co-expression of IL-4, IL-5, and IL-13 in conditions
associated with the appearance of Th2 cells indicated a co-regulation
of these cytokines genes consistent with their clustered position over
150 kb of genomic DNA in both human and mouse (40). Recently, an
IL-4/IL-13 intergenic regulatory region that might be involved in the
co-regulation of the IL-4 and IL-13 genes has been characterized, and
GATA-3 together with STAT6 seems to induce chromatin remodeling of this
regulatory region (41). However, no direct role of this regulatory
region on IL-13 gene expression has been shown. Because the IL-13
mRNA level was increased in the hGATA-3 transgenic mice we studied, we first analyzed the IL-13 gene promoter and found an
evolutionary-conserved consensus GATA binding site possibly involved in
the GATA-3-mediated activation of the IL-13 gene. Using EMSA, we showed
that this GATA binding site alone bound GATA-3 with poor affinity,
whereas the combination of this motif and two GATG sequences located
immediately upstream constituted a high affinity sequence for GATA-3. A
similar result was obtained in the study of the IL-5 promoter, where a consensus GATA binding site is of weak affinity, whereas an overlapping and inverted TGATT motif dramatically increased the GATA-3 affinity and
trans-activation mediated by the consensus GATA binding site (37).
Using peptides that represent the two GATA-2 zinc fingers, we were able
to determine that the presence of multiple GATA sites increases the
binding affinity and alters the conformation of the complexes formed on
the IL-13 gene promoter. Two molecules of protein can bind to the IL-13
gene GATA sequences through sites 1 and 3 but not to other pairs of
sites. The addition of site 2 increases the affinity but does not
result in the binding of a third protein molecule, suggesting the
participation of the N-finger. The role of site 2 could be
to ensure that sites 1 and 3 are both occupied by increasing the local
concentration of GATA-3 or by stabilizing the binding of two molecules
of GATA-3 through formation of a more favorable protein/DNA
conformation. At high GATA-3 concentrations site 2 may be unimportant,
but it could be critical at stages of development when GATA-3 is
limiting. Although the sites 1 and 2 association lead to a higher
affinity for GATA proteins than the sites 1 and 3 association, we
showed that sites 1 and 3 association is transcriptionally more
efficient. This result together with our Scatchard analysis indicated
that the number of GATA-3 molecules bound on this region is more
important than their DNA binding affinities in the transcriptional
activity of the IL-13 promoter and is in line with a recent study that showed that high affinity binding sites for GATA-1 did not always lead
to high transactivation (42).
The association of consensus and non-consensus GATA binding sites found
in the IL-13 or the IL-5 genes promoters might be important for the
fine regulation of the expression of cytokine genes during the immune
response. This hypothesis was tested by transfections using increasing
amounts of the hGATA-3 expression vector and IL-13 promoters containing
the consensus GATA binding site alone or the association of motifs
found in the IL-13 gene promoter. Indeed, we found that the wild type
promoter was highly sensitive to minute amounts of hGATA-3, whereas the
promoter containing only the consensus GATA binding site required a
higher amount of hGATA-3 for trans-activation. Such results have
already been found for other trans-acting factors but not for members
of the GATA family (43). Because most of the studies performed on the functions of the GATA family members have been done on constitutively expressed target genes, the results presented in this study might reveal an arrangement of cis-acting motifs that mediate inducible expression by GATA-3.
Several studies indicate that GATA-3 might be a potential therapeutic
target for the treatment of pathologies such as asthma and allergy (44,
45). These studies are based on inhibition of GATA-3 activity in T
cells by a dominant negative mutant of GATA-3 or by a blockade of
GATA-3 expression using antisense RNA. In this paper we show that the
production of cytokines by CD8 T cells is also modulated by the
expression level of GATA-3 but, at least for IFN We thank Dr. Helene Jouault, Isabelle
Bouchaert, and Nicolas Lebrun for flow cytometry, Frank Letourneur and
Nicolas Lebrun for sequencing, Christophe Dez for maintenance of mice
colonies, and Drs. Sophie Ezine, Flora Zavala, and Yves Levy for
assistance and helpful discussions.
*
This work was supported in part by grants from the INSERM
and the Ligue Nationale Contre Le Cancer (Equipe Labellisée).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by the Association Pour La Recherche Sur Le Cancer.
Published, JBC Papers in Press, March 13, 2002, DOI 10.1074/jbc.M110013200
The abbreviations used are:
Th, T helper;
IL, interleukin;
IFN, interferon;
STAT, signal transducer and activator of
transcription;
Tc, T cytotoxic;
PMA, phorbol 12-myristate 13-acetate;
RT, reverse transcription;
mhprt, murine hypoxanthine
phosphoribosyltransferase;
EMSA, electrophoretic mobility shift assay;
WT, wild type;
h, human;
m, mouse.
Interleukin-13 Gene Expression Is Regulated by GATA-3 in T
Cells
ROLE OF A CRITICAL ASSOCIATION OF A GATA AND TWO GATG
MOTIFS*
§,,, and
Institut Cochen (INSERM, CNRS,
Université Paris V), Département d'Hematologie, Maternite
Port-Royal, 123 Bd de Port-Royal, 75014 Paris, France and
¶ Laboratory of Molecular Biology, NIDDK, National Institutes of
Health, Bethesda, Maryland 20892
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) and type 2 (interleukin (IL)-4 and IL-13) cytokine genes were activated in the transgenic CD8 T
cells. Because IL-13 gene expression was highly enhanced in
vivo by GATA-3 expression, we studied the human and the mouse IL-13 gene promoters and found an evolutionary-conserved association of
a consensus GATA binding site and two GATG motifs. We showed that
efficient GATA-3 binding to this regulatory sequence required these
three motifs and that the affinity of the GATA zinc fingers for this
association was five times higher than for the consensus GATA binding
site alone. Transfections in a T cell line or transactivation by GATA-3
showed that the combination of the three sites was required for full
transcriptional activity of the IL-13 gene promoter. Finally we showed
that this association of binding sites causes a very high sensitivity
of the IL-13 gene promoter to small variations in the level of GATA-3
protein. Altogether, these results indicate an important role of GATA-3
in CD8 cytokine gene expression and demonstrate that a critical network
of GATA binding sites highly modulates GATA-3 activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(IFN), and tumor necrosis
factor 
and are implicated in cell-mediated immune response, whereas
Th2 CD4 cells produce IL-4, IL-5, IL-10, and IL-13 and are mostly
involved in humoral immunity. In several pathological conditions like
allergy or autoimmune diseases, the balance between Th1 and Th2
responses seems to be impaired (2, 3). In asthma, Th2 CD4 T cells are
increased in the airways of patients after antigen challenge, and Th2
cytokines seem to be required for the development of airway
eosinophilia and elevation of IgE serum level (4).
and IL-12 receptor 2 gene expression, whereas nuclear
factor IL-6 and c-Maf are expressed in Th2 cells and regulate IL-4 gene
expression (5, 6). Finally, numerous reports demonstrate that GATA-3 is
a major regulator of Th1/Th2 polarization (7-11). GATA-3 is a member
of the GATA family of transcription factors whose common structure is a
conserved double zinc finger motif that binds to the consensus DNA
sequence 5'-(A/T)GATA(A/G)-3'. GATA-3 is widely expressed during mouse
development, and its deficiency is lethal on mouse embryonic day 12 (12-14). In adults, its expression is essentially restricted to T and
natural killer cells. GATA-3 is absolutely required for the development
of the T cell lineage and for the Th1/Th2 differentiation of naive CD4
T cells (15, 16). The increase of GATA-3 mRNA level during Th2
development is induced by the IL-4/STAT6 pathway, whereas its decrease
during Th1 development is under the control of the IL-12/STAT4 pathway (8). In CD4 T cells, GATA-3 induces the expression of type 2 cytokines
like IL-4 or IL-5 and represses type 1-specific genes like the IFN
and the IL-12 2 receptor subunit genes (7-9).
and tumor necrosis factor
and are implicated in cytotoxicity (17). However, some studies
indicate that CD8 T cells can also differentiate toward two different
phenotypes, leading to the concept of Tc1 and Tc2 CD8 T cells (18, 19).
Tc2 cells have been detected in AIDS patients and in lepromatous
leprosy, and the relative in vivo roles of the Tc1 and Tc2
subsets in immune regulation are currently being investigated in
pathological conditions such as autoimmunity, viral infections, or bone
marrow graft (20-22).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, and 1% penicillin and streptomycin. Red blood
cells were lysed by osmotic shock in lysis buffer (NH4Cl
155 mM, KHCO3 10 mM, EDTA 0.1 mM). Non-dissociated cells and tissue debris were filtered on a cell strainer 70-µm nylon (Falcon, 35-2350). Splenic B cells were depleted using sheep anti-mouse IgG microbeads (Dynal 11201), and
CD4 and CD8 T cells were further purified by positive selection after
staining, respectively, with fluorescein isothiocyanate-conjugated anti-mouse CD3 (clone 145-2C11) and R-phycoerythrin-conjugated anti-mouse CD4 (clone GK1.5) or fluorescein isothiocyanate-conjugated anti-mouse CD3 and R-PE-conjugated anti-mouse CD8 (clone 53-6.7) by
sorting on a flow cytometer (Epics Coulter). Both T cells subsets were
more than 95% pure. These T cells were cultured in RPMI 1640 medium
complemented with Glutamax, penicillin, and streptomycin and
containing 10% fetal calf serum, 50 µM
2-mercaptoethanol, 10 ng/ml phorbol 12-myristate 13-acetate (PMA), 500 ng/ml ionomycin, and 50 units/ml human lymphocyte IL-2 (Roche Molecular
Biochemicals). Tc1 conditions included 16 ng/ml recombinant murine
IL-12 (BD PharMingen) and 5 µg/ml neutralizing antibody against mouse
IL-4. Tc2 conditions included 10 ng/ml recombinant murine IL-4 (BD
PharMingen) and 50 µg/ml neutralizing antibody against mouse IFN.
After 6 days, cells were harvested and restimulated (2 × 105 cells/200 µl) with coated anti-mouse CD3 (clone
145-2C11) in the presence of IL-2 (50 units/ml) for 24 h. All
antibodies used were purchased from BD PharMingen.
5', GGTT GAC ATG AAA ATC CTG
CAG AGC, and 3', CGC TGG ACC TGT GGG TTG TTG ACC; mIL-4 5', AGG AGA AGG GAC GCC ATG CAC GGA, and 3', ATC GAA AAG CCC GAA AGA GTC
TCT G; mIL-13 5', TCT TGC TTG CCT TGG TGG TCT CGC, and 3', GAT GGC ATT GCA ATT GGA GAT GTT G. All these primers span an
intron except hGATA-3 transgene primers. The sizes of the
PCR products were 250 bp for the hGATA-3 transgene, 370 bp
for mhprt, 340 bp for mIFN, 200 bp for
mIL-4, and 220 bp for mIL-13. The number of
amplification cycles was always within the exponential amplification phase. All PCR products were run on 2% agarose gels with ethidium bromide and visualized by UV transillumination.
--
Expression of IFN by
activated CD8 T cells was examined by intracellular staining. Cells
were treated with Golgistop (BD PharMingen) for 6 h as indicated
by the manufacturer, collected, and stained with R-phycoerythrin
anti-mouse CD8, fixed, permeabilized-stained with fluorescein
isothiocyanate-anti-mouse IFN (clone XMG1.2), and analyzed by flow cytometry.
-D-galactopyranoside for 4 h at
37 °C. The peptide purification was essentially as previously
described. The amino acid sequence of the GATA-2 double finger peptide
is
MAEGRECVNCGATATPLWRRDGTGHYLCNACG}LYHKMNGQNRPLIKPKRRLSAARRAGTSCANCQTTTTTLWRRNANGDPVCNACGLYYKLHNV- NRPLTMKKEGIQTRNRKMSNKSKKSKKG.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
CD2/hGATA-3 vector and transgene expression.
A, transgenic construct. The hGATA-3 cDNA is inserted in
a CD2 minigene cassette containing 4.5 kb of the CD2 5'-flanking
sequence (hatched), the first exon and intron
(black), the fifth exon (gray) and 5.5 kb of the
3'CD2 locus control region (dotted). B,
Northern blot analysis of hGATA-3 expression in transgenic L1 mice. 20 µg of total RNA isolated from the thymus of WT or L1 heterozygous
(HET) and homozygous (HO) transgenic mice was
hybridized with a probe that specifically recognized the 3' end of the
transgenic mRNA. The length of the transgenic mRNA is 2.6 kb.
The amount of ribosomal RNA was used as a control for the amount of
total RNA loaded per lane. C and E, RT-PCR
analysis of hGATA-3 expression in transgenic mice. RT-PCR experiments
were performed on 1 µg of total RNA. The RT-PCR reactions were
normalized by mhprt PCR. C, hGATA-3 expression in
the thymus of L1 heterozygous, L3 heterozygous, and homozygous
transgenic mice. E, hGATA-3 expression in purified CD4 and
CD8 splenocytes from WT or homozygous L1 mice. D, expression
of the hGATA-3 protein in transgenic L1 mice. Whole cell lysates were
prepared from thymus of heterozygous (HET), homozygous
(HO), or WT mice, and an equal amount of total protein was
loaded on a 12% SDS-PAGE, transferred to Hybond-P polyvinylidene
difluoride membrane (Amersham Biosciences), and probed with the HG3-35
anti-hGATA-3 antibody that recognized the human and the murine GATA-3
protein. The 50-kDa GATA-3 protein is indicated by an
arrow.
CD8, double positive CD4+
CD8+, or single positive CD4+ or
CD8+ was similar in the thymus of transgenic and WT mice as
was the percentage of CD4+ or CD8+ T cells in
the spleen (data not shown).
, in CD4 lymphocytes isolated
from the spleen of homozygous L1 mice or of non-transgenic age- and
sex-matched control mice. Cell-sorted purified CD4 T cells were
activated for 6 days with PMA and ionomycin and further stimulated for
24 h with immobilized anti-CD3 in the presence of IL-2. The
mRNA expression level of mGATA-3, IFN, and IL-4 was studied by
RT-PCR. As shown in Fig. 2, the mGATA-3
level was not modified by the presence of the transgenic hGATA-3
protein, but the IFN mRNA level was lower in CD4 lymphocytes of
homozygous mice compared with WT CD4 T cells, whereas IL-4 mRNA
level was higher in the CD4 lymphocytes of these transgenic mice. These results agreed with previous reports and indicate that the CD2-GATA-3 mice produced are suitable to study the consequences of GATA-3 overexpression in T cells.
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Fig. 2.
Effect of hGATA-3 on mGATA-3,
IFN , and IL-4 genes expression in CD4 T
cells. RT-PCR experiments were performed using total RNA isolated
from activated cell-sorted CD4 T cells of L1 homozygous mice
(HO) and wild type littermate (WT). These cells
were collected after 6 days of PMA-ionomycin activation followed by
anti-CD3 reactivation in the presence of IL-2. The amount of cDNA
used for PCR was normalized by hprt PCR.
were first studied by RT-PCR. As shown
in Fig. 3A, although the
mRNA level of mGATA-3 was identical in WT and transgenic CD8 T
cells, the expression level of type 2 cytokines (IL-4 and IL-13)
mRNAs was higher in the transgenic CD8 lymphocytes. Surprisingly,
the level of IFN mRNA was also higher, albeit to a lesser
extent. The same results were obtained using CD8 lymphocytes from an L1
heterozygote mouse (Fig. 3B), and an elevation of IL-13 mRNA was also demonstrated in CD8 lymphocytes from a L3 homozygous mouse (data not shown).
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Fig. 3.
Effects of hGATA-3 expression on the mGATA-3
and cytokines genes expression in CD8 T cells activated under neutral
conditions. RT-PCR experiments were performed on total RNA
isolated from activated cell-sorted splenic CD8 T cells of sex- and
age-matched control (WT) and L1 homozygous mice
(HO) (A) or non-transgenic littermate
(WT) and heterozygous (HET) mice (B).
These cells were collected after 6 days of PMA-ionomycin activation
followed by anti-CD3 reactivation in the presence of IL-2. The amount
of cDNA used for PCR was normalized by hprt PCR.
mRNA was highly expressed and could be easily detected, whereas among Th2 cytokines, IL-13 mRNA was the only mRNA whose level was high enough to be detected by RNase protection assay (Fig. 4A). After
quantification of the different signals, the IFN mRNA level in
the CD8 T cells from the homozygous L1 mice was found to be twice the
level of control mice, whereas the IL-13 mRNA was 13-fold higher.
IFN was also evaluated at the protein level by intracellular
staining with a monoclonal antibody to IFN and flow cytometry. The
percentage of CD8 T cells expressing IFN was 22% for control mouse
and 34% for transgenic mouse (Fig. 4B), indicating that
hGATA-3 expression did not impair IFN expression. Altogether, these
results indicated that the consequences of GATA-3 overexpression are
different in CD4 and in CD8 T cells and that the IL-13 gene is
regulated by GATA-3 in vivo.
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Fig. 4.
Quantitative effects of hGATA-3 on IL-13 and
IFN genes expression in CD8 T cells activated
under neutral conditions. Cell-sorted CD8 T cells were activated
as described under "Experimental Procedures." A, total
RNA was prepared from these cells and IL-13 and IFN mRNAs
expression was detected by RNase protection assay using the mouse
cytokine set 1 probe (BD PharMingen). After quantification, IFN and
IL-13 mRNA levels were found to be, respectively, 2- and 13-fold
higher in transgenic (HO) than in control (WT)
CD8 T cells. B, CD8 T cells were fixed, permeabilized,
stained with anti-CD8 and anti-IFN monoclonal antibodies, and
analyzed by flow cytometry. The percentage of cells expressing IFN
was determined in gated CD8 cells. Both experiments were conducted
three times, and the result of a representative experiment is
shown.
expression
under Th1-skewing conditions (9), we studied, under Tc1- or
Tc2-polarizing conditions, the mRNA levels of IFN and IL-13,
which is the only highly expressed type 2 cytokine in CD8 T
cells (Fig. 4A). The mRNA levels of the endogenous
mGATA-3 and of the transgenic hGATA-3 were also studied (Fig.
5). Under Tc1 conditions mGATA-3 mRNA
level was nearly undetectable, whereas the same level of mGATA-3
mRNA was observed in WT or transgenic CD8 T cells under Tc2
conditions. Under Tc1 conditions, no IL-13 mRNA was seen in WT,
whereas a high expression of this mRNA was detected in transgenic
CD8 T cells. Under Tc2 conditions, we found no obvious difference of
the IL-13 mRNA level between transgenic and non-transgenic CD8 T
cells. Finally, no clear difference of the IFN gene expression could
be detected between transgenic and non-transgenic cells under
Tc1 or Tc2 conditions, the IFN mRNA level being higher in Tc1
conditions as expected. Thus, ectopic expression of hGATA-3 in CD8 T
cells induced expression of IL-13 under Tc1-polarizing or neutral
activation conditions. Under Tc2-polarizing conditions, the mGATA-3
level is high, and ectopic expression of hGATA-3 has no obvious
effect.
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Fig. 5.
Effects of hGATA-3 expression on mGATA-3,
IL-13, and IFN genes expression in CD8 T cells
activated under Tc1 or Tc2 conditions. Sorted splenic CD8 T cells
of sex- and age-matched control (WT) and L1 homozygous mice
(HO) were activated as described under "Experimental
Procedures" in the presence of IL-12 and anti-IL-4 for Tc1 conditions
and in the presence of IL-4 and anti-IFN for Tc2 conditions. RT-PCR
experiments were performed on total RNA isolated from these activated
CD8 T cells as described under "Experimental Procedures."
125 to 77 (human) and 107 to 59
(mouse) upstream from the site of transcription initiation. This region
contains a consensus GATA binding site (site 1) and two GATG motifs
(sites 2 and 3) highly conserved between human and mouse (Fig.
6A).
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Fig. 6.
The human and mouse Il-13 genes promoters
contain three clustered GATA binding sites that can bind hGATA-3.
A, comparison of the 5' regions of the human and mouse IL-13
genes. The 125/90 (human) and 107/72 (mouse) regions of the IL-13
gene shared a consensus GATA binding site (site 1) and two GATG motifs
(site 2 and 3). B, binding of hGATA-3 to the mIL-13
110/70 5' region. EMSAs with Jurkat T cell nuclear extracts were
performed using oligonucleotide probes that contain the three GATA
binding sites (lanes 1-4), site 1 only (lane 5),
sites 2 and 3 (lane 6), sites 1 and 3 (lane 7),
or sites 1 and 2 (lane 8). This binding was performed in the
presence of a 100-fold molar excess of the unlabeled "three GATA
sites probe" (lane 1) or in the presence of anti-hGATA-3
antibody (lane 3). The black arrow indicates the
hGATA-3 complex, the hatched arrow indicates the hGATA-3
supershifted complex, and the empty arrow indicates the free
probe.
110 to 70 DNA fragment that contained
the three putative GATA binding sites. This fragment specifically bound
hGATA-3, as shown by competition (Fig. 6B, lane
1) and supershift (Fig. 6B, lane 3)
experiments, and displayed a much higher affinity for hGATA-3 than the
consensus GATA binding site alone (Fig. 6B, compare
lanes 4 and 5). Disruption of the consensus GATA
binding site led to weak hGATA-3 binding (lane 6) and,
although disruption of site 3 (lane 8) had a modest affect on hGATA-3 binding affinity, disruption of site 2 (lane 7)
decreased binding. Thus, the presence of the GATG sites seems to
enhance the affinity of a consensus GATA binding site, which is a weak binding site on its own.
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Fig. 7.
Affinity of IL-13 promoter GATA binding sites
for GATA zinc finger. Approximately 6 pmol of GATA-2 double finger
peptide was titrated with 32P-labeled oligonucleotides
(0.25, 0.5, 1, 1.5, and 2 pmol) containing the three IL-13 gene GATA
binding sites in various combinations as shown. The numbers
above the gels indicate which GATA sites are present on each
individual probe. The dissociation constants (Kd),
as determined by Scatchard analysis, are shown below the gels:
N is the number of experiments, SD is the
standard deviation, and ND is not determined. A,
titrations are with probes containing a single GATA site. B,
titrations are with probes containing the wild type site or two of the
three sites. For this gel, 20 pmol of peptide was used, but the
Kd determinations were from experiments with less
peptide.
110 to +66 relative to the transcription initiation start
site was amplified and cloned upstream from the firefly luciferase
coding sequence in the pGL3 basic vector. We also constructed different
mutated promoters in which one or several of the GATA binding sites
were disrupted. The resulting plasmids were transiently transfected in
Jurkat T cell line stimulated by PMA and ionomycin, and firefly
luciferase activity was determined after 24 h. The transfection
efficiency of each assay was evaluated by cotransfection of a plasmid
expressing Renilla luciferase under the control of the
herpes simplex virus thymidine kinase promoter (pRL-TK). When
the WT promoter was transfected, transcriptional activity was 4.2-fold
higher than when the three GATA sites were disrupted (Fig.
8). Disruption of site 2 did not affect
the transcriptional activity of the IL-13 gene promoter, whereas
disruption of site 3 or sites 2 and 3 resulted in a 40% decreased
transcriptional activity. These results demonstrated that full activity
of the IL-13 gene promoter in T cells is dependent on the presence of at least two GATA binding sites. However, the role of sites 2 and 3 in
their association with site 1 does not seem to correlate with their
affinity for GATA proteins since site 2, which increases GATA-3 binding
affinity, seems dispensable for the promoter activity in Jurkat
cells.
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Fig. 8.
Role of the three GATA binding sites in the
IL-13 gene promoter activity. The various pGL3 promoter constructs
together with pRL-TK were transiently cotransfected into Jurkat T cell
line activated by PMA and ionomycin as described under "Experimental
Procedures." The firefly luciferase (LUC) activities were
determined, and normalization for transfection efficiency was obtained
by determination of Renilla luciferase activity. Data
represent the fold increase in luciferase activity over that obtained
for cells transfected with the IL-13 gene promoter construct containing
none of the GATA binding sites (mean results of three transfections).
The hatched box represents the consensus GATA binding site,
whereas the dotted box and gray box represent the
two GATG sites.
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Fig. 9.
Transactivation of the human IL-13 gene
promoter by hGATA-3. HeLa cells were cotransfected with the
different IL-13 promoter constructs, pRL-TK and 1 µg of pECE-hGATA-3
or empty pECE vector. Luciferase (LUC) activities were
determined as described under "Experimental Procedures" and Fig. 8.
The fold increase by GATA-3 transactivation was calculated as the ratio
of luciferase activity in the presence of pECE hGATA-3 or in the
presence of the empty pECE vector (mean results of three
transfections). The hatched box represents the consensus
GATA binding site, whereas the dotted box and gray
box represent the two GATG sites.
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Fig. 10.
Effect of hGATA-3 protein level on the
transcriptional activity of the human IL-13 gene promoter. Two
constructs containing the three GATA binding sites (WT) or
only the consensus GATA binding site (Site 1 alone) were
co-transfected into HeLa cells with pRL-TK and increasing amounts of
the pECE-hGATA-3 expression vector. The transcriptional activities of
the two promoters were evaluated as described under "Experimental
Procedures" and Fig. 8. The fold increase of the transcriptional
activity of the two promoters was calculated as the ratio of luciferase
activity in the presence of the different amounts of pECE hGATA-3 or in
the presence of the same amount of the empty pECE vector. The two
constructs reached 50% of their full activity with 200 ng for the WT
construct and 400 ng for the construct containing the consensus GATA
binding site only.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mRNA in the transgenic mice. These results are in a good agreement with the ones obtained by retroviral tagging of splenic lymphocytes with GATA-3 or by enforced GATA-3 expression in CD4 T cells using the CD4 gene promoter or the CD2
locus control region and further demonstrated that GATA-3 is a major
regulator of the Th1/Th2 differentiation of CD4 T cells (7, 8, 32). No
activation of the endogenous mGATA-3 gene could be detected, and this
result is different from previous studies (11, 26). One explanation for
this discrepancy might be the origin of ectopic GATA-3 expression,
i.e. retroviral-mediated expression or transgenic mice.
Because the CD2 cassette used also drove expression of the transgene in
CD8 T cells, we studied the expression of the cytokine genes in these
cells. After activation, the CD8 T cells of wild type mice displayed a
type1 phenotype (expressing essentially IFN mRNA), as
classically described, whereas the CD8 T cells of the transgenic mice
displayed over an intermediate phenotype with simultaneous
expression of both IL-13 and IFN mRNAs, whose levels were,
respectively, more than 10- and 2-fold higher than in control-activated
CD8 T cells. This pattern of expression was maintained under Tc1
conditions. The IFN gene regulation is different in CD4 and CD8 T
cells since CD4 T cells required STAT4 activation to express IFN,
whereas CD8 T cells displayed a STAT4-independent activation of this
gene (33). At the molecular level, two regulatory elements located in
the IFN gene promoter have been implicated in the induction of
IFN gene transcriptional activity during T cell activation, and the
function of these two elements is different in CD4 and CD8 T cells
(34). Both elements are active in primed CD4 T cells, whereas only the
distal element, which contained a consensus GATA binding site, is
active in primed CD8 T cells (35). The results presented in this paper
corroborated the differential regulation of the IFN gene in CD4 and
CD8 T cells and showed that the GATA-3 expression level also
participates in this differential regulation. However, these results
are different from the results recently published (32) where CD8 T
cells from GATA-3-expressing transgenic mice displayed a lower level of
positive cells for intracellular expression of IFN than control
mice. The differences between the transgenic mice used (genetic
background, hemagglutinin tag) and the differences in the
protocols of activation might again account for the discrepancy. WT and
transgenic CD8 T cells have a similar cytokine expression pattern under
Tc2 conditions. This result could be explained by the high level of
expression of mGATA-3 in these conditions and is in accordance with
recent results demonstrating the inhibitory effect of IFN
transduction pathway on GATA-3 expression and IL-4 production (36).
, in a different way
than in CD4 T cells. Thus, depending on the target T cell, the blockade
of GATA-3 might have different consequences on specific cytokine
expression and immune response. As for the IL-13 gene, our results
demonstrate that it is a true GATA-3 target gene that is highly
sensitive to GATA-3 expression level. Recently, it has been shown that
inhibition of IL-13 production results in a severe inhibition of mucus
production and airway hyperresponsiveness in a murine model of asthma
(46, 47). Our results indicate that modulation of GATA-3 expression might be a promising therapy in the treatment of this disease.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.:
33-153104384; Fax: 033-143251167; E-mail:
maxaudit@cochin.inserm.fr.
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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