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*
Department of Immunology and Infectious Diseases, Harvard School of Public Health, and
Department of Medicine, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies suggest that IL-13 might play a more important role than IL-4 or IL-5 as an effector molecule in the pathogenesis of allergic asthma. Adoptive transfer of Ag-specific Th2 cells derived from IL-4-deficient mice still induced airway hyperresponsiveness (AHR)3 and overproduction of mucus (6, 7). Blockade of IL-13, even after the development of allergic asthma in an animal model, ablated AHR and mucus overproduction (8, 9). In contrast, IL-4 blockade prevented the development of allergic asthma only when applied during the phase of sensitization (10). Furthermore, administration of rIL-13 to airways induced AHR, influx of eosinophils, mucus production, and an increase in serum total IgE (8, 9).
Although the roles of IL-13 in the pathogenesis of allergic asthma have been well studied, very little is known about the transcriptional regulation of the IL-13 gene. The expression of most of the Th2 cytokine genes, such as IL-4 and IL-5, is regulated at the level of transcription and is induced by stimulation with anti-CD3 or PMA/ionomycin (iono). Sequence analysis revealed that the sequences immediately upstream of the first exon of the IL-13 gene are highly conserved through evolution and contain potential binding sites for NF-AT, GATA, and AP-1 (11); however, the functional significance of these binding sites has not been investigated. The IL-13 gene maps to the same chromosomal locus as the IL-4 and IL-5 genes, and the expression of these Th2 cytokine genes is directly related to the chromosomal accessibility of this locus. Indeed, several Th2 cell-specific DNaseI hypersensitivity sites (DHSs) have been identified along the IL-13/IL-4/IL-5 locus (12). Three of the DHSs are located in the IL-13 gene; however, their roles in regulating the expression of the IL-13 gene remain elusive. Recently, it was shown that the intergenic region between IL-4 and IL-13 functions as a coordinate regulator of IL-4, IL-5, and IL-13 (13). Despite close chromosomal linkage of the Th2 cytokine genes, several pieces of evidence suggest that there is significant heterogeneity of cytokine gene expression by individual Th cells or clones (14, 15, 16). However, the IL-13 gene was not examined in these studies, and it remains unclear whether IL-13 is always coexpressed with IL-4 or IL-5 in normal Th2 cells on a single cell basis.
In agreement with the heterogeneity in cytokine expression, previous
studies have suggested that each of the Th2 cytokine genes might be
regulated by independent mechanisms. For example, it was shown that the
sequence-specific binding of GATA-3, a Th2 cell-specific transcription
factor, is critical for the expression of the IL-5 gene
(17, 18), whereas a high level of IL-4 gene
expression requires the presence of c-maf, another
functionally critical Th2 cell-specific transcription factor (19, 20). In addition, Th2 cells derived from
c-maf-deficient mice continue to produce IL-5 and IL-13 at
levels comparable to those of wild-type (WT) Th2 cells
(21). Developing Th1 cells derived from GATA-3 transgenic
mice expressed several Th2 cytokine genes, but not IL-13. In contrast,
overexpression of an anti-sense GATA-3 in a Th2 clone, D10,
inhibited the expression of almost all Th2 cytokine genes, including
IL-13 (22). In a transgenic mouse model, the
expression of IL-13 and other Th2 cytokines was significantly reduced
in Th2 cells derived from mice carrying a "dominant negative"
GATA-3 transgene. However, the mechanism that leads to this effect
remains unclear because the mutant GATA-3 also inhibited the expression
of IFN-
and was as potent as WT GATA-3 in inducing the expression of
Th2 cytokine genes in vitro (23, 24). Although forced
expression of WT GATA-3 in developing Th1 cells resulted in the
expression of Th2 cytokine genes, including IL-13
(23), it is unclear whether GATA-3 directly transactivates
the IL-13 promoter or indirectly induces IL-13 expression by
redirecting the differentiation program into the Th2 pathway.
Here we report that the murine IL-13 gene is not always
coexpressed with the IL-4 and IL-5 genes in
normal Th2 cells on a single cell basis. In addition, we identified a
2-kb genomic fragment, containing the murine IL-13 promoter, which is
sufficient to confer cell type specificity and response to PMA/iono
stimulation in vitro, and mapped a minimal IL-13 promoter that retains
both properties. The cell type specificity of the minimal IL-13
promoter is mediated by a functionally critical cis-acting
element that binds endogenous GATA-3 proteins that is located in the
proximal region of the IL-13 promoter, whereas the response to PMA/iono
is mediated by distinct cis-acting elements that are
conserved in sequence between human and mouse. Ectopic expression of
GATA-3, but not c-maf, is sufficient to transactivate the
endogenous IL-13 promoter in Th1 cells, which concomitantly express
high levels of IFN-. Furthermore, ectopic expression of both
c-maf and GATA-3 in differentiated Th1 cells
synergistically transactivates the endogenous IL-4 promoter, but does
not have any synergistic effect on the endogenous IL-13 promoter. Taken
together, our data suggest that IL-13 is transcriptionally regulated by
a mechanism distinct from that regulating the expression of the
IL-4 gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine B cell lines, M12 and 18.81, and EL4 (a murine thymoma cell line) cells were maintained in RPMI 1640 supplemented with 10% FCS. NIH3T3 cells were maintained in DMEM supplemented with 10% FCS. D10 (a Th2 clone) cells were maintained in RPMI 1640 supplemented with 10% FCS and 10% conditioned medium of Con A-stimulated rat splenocytes. D1.1 (a Th1 clone) cells were maintained in RPMI 1640 supplemented with 10% FCS, 5% conditioned medium of Con A-stimulated rat splenocytes, 1% T-STIM (Collaborative Biomedical Products, Bedford, MA), and recombinant human IL-2 at 50 U/ml. Transfection of tumor cell lines was performed by electroporation as described previously (19). For transfection experiments, 10 µg of each murine IL-13 promoter reporter construct, in the backbone of pGL2Basic (Promega, Madison, WI), alone or in combination with 10 µg of a GATA-3 expression vector, pcDNA-GATA3 (a gift from J. Leiden, Abbott Laboratories, Abbott Park, IL) or 10 µg of empty pcDNA-3 vector (Invitrogen, Carlsbad, CA), was used. In addition, 10 ng of a Renilla luciferase reporter vector, pRL-TK (Promega), was added into each transfection as an internal control for transfection efficiency. The transfected cells were left untreated, treated with PMA (10 ng/ml) and iono (1 µg/ml) 1 h posttransfection, or stimulated with plate-bound anti-CD3 (1 µg/ml, for D10 cells). Twenty-four hours after transfection, cell extract was harvested and subjected to luciferase assay by using the Dual Luciferase Reporter System (Promega) according to the manufacturer’s instructions. The firefly luciferase activity thus obtained was normalized against the respective Renilla luciferase activity.
In vitro differentiation of Th cells
CD4+Mel14high
naive Th cells were purified from splenocytes and lymph node
cells of BALB/c or C57B/6 mice by a Vantage cell sorter (BD
Biosciences, Mountain View, CA) and stimulated in vitro with
plate-bound anti-CD3 mAb (2C11) and anti-CD28 at 1 µg/ml each
in the presence of human IL-2 at 100 U/ml, along with anti-IFN-
mAb (XMG1.2) at 10 µg/ml and IL-4 (10 ng/ml) (Th2 skewing conditions)
or with anti-IL-4 (11B11) at 10 µg/ml and IL-12 (1 ng/ml) (Th1
skewing conditions). Afterward, the stimulated cells were cultured for
another 6 days before use. All Abs and cytokines were purchased from BD
PharMingen (San Diego, CA).
Retroviral transduction of Th cells
The bicistronic retroviral vector (RV) and the GATA-3-expressing retroviral vector, RV-GATA3, were provided by K. Murphy (Washington University School of Medicine, St. Louis, MO). Retroviral transduction of Th cells was described previously (23). Briefly, the packaging Phoenix cells were cotransfected with RVs along with CMV-Env and RSV-Gag-Pol-Lyt-2 vectors (gifts from G. Nolan, Stanford University, Stanford, CA) by using Effectene Transfection Reagent (Qiagen, Valencia, CA). Supernatant from transfected Phoenix cells was used as the source of retrovirus. Naive Th cells were purified from WT C57B/6 or c-maf transgenic (mafTG) mice (20) and differentiated in vitro under Th1 skewing conditions. On the seventh day of in vitro differentiation, the differentiated Th1 cells were restimulated with anti-CD3 under Th1 skewing conditions. Twenty-four hours later, the restimulated cells were incubated with the virus-containing supernatant at 37°C for 24 h. The infected Th1 cells were washed once, replated in fresh complete medium containing the necessary cytokines, and cultivated for another 6 days before use. We routinely achieve 60% transduction efficiency by using this protocol.
Intracellular cytokine staining
The protocol for intracellular cytokine staining was described
previously (25). Briefly, Th cells were stimulated with
PMA (10 ng/ml) and iono (1 µg/ml) for 2 h, then 2 µM monensin
(Sigma, St. Louis, MO) was added for an additional 2 h. Cells were
collected, washed, fixed in 4% paraformaldehyde, and permeabilized
with saponin buffer (0.1% saponin in PBS). The fixed and permeabilized
cells were incubated with PE-, FITC-, or biotin-conjugated
anti-cytokine Abs or control Abs for 30 min at room temperature.
Abs against murine IL-4, IL-5, and IFN- were purchased from BD
PharMingen; biotinylated polyclonal Ab against murine IL-13 was
purchased from R&D Systems (Minneapolis, MN). The cells incubated with
biotin-conjugated Abs were washed twice and stained with
Tri-color-conjugated streptavidin (Caltag Laboratories, Burlingame, CA)
for an additional 20 min at room temperature. The stained cells were
washed with saponin buffer twice and resuspended in PBS. The
resuspended cells were subjected to flow cytometric analysis on FACS
(BD Biosciences) and analyzed with CellQuest software.
EMSA
T4 polynucleotide kinase was used to end label 100 ng of
double-stranded (ds) oligonucleotides with
[32P]ATP (DuPont-NEN Research Products,
Wilmington, DE). The labeled ds-oligonucleotides were isolated and
purified on 12% polyacrylamide gels, eluted overnight at 37°C in 1x
TE (10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0)), and precipitated
in ethanol. Binding assays were performed at room temperature for 20
min using 5 µg of nuclear extract prepared from
anti-CD3-stimulated D10 cells, 500 ng of poly(dI-dC), 20,000 cpm of
probe in a 15-µl volume of 20 mM HEPES (pH 7.9), 100 mM KCl, 5%
glycerol, 1 mM EDTA, and 5 mM DTT. For supershift assay, 1 µl of
monoclonal anti-GATA-3 or control Ab (Santa Cruz Biotechnology,
Santa Cruz, CA) was added. For cold competition, unlabeled WT or
mutant ds-oligonucleotides in 50-fold molar excess were added. The
samples were then fractionated in 4% nondenaturing polyacrylamide gels
containing 0.5x TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA (pH
8.0)) at room temperature. The sequences of the WT
oligonucleotide correspond to -110 to -30 (see Fig. 3
B) of
the murine IL-13 gene.
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Cultured cells at 0.5 x 106
million/ml were stimulated with PMA (10 ng/ml) and iono (1 µM) for
24 h. The supernatants were collected, and ELISA was performed as
described previously (19). The capturing and detecting Abs
against murine IL-13 were purchased from R&D Systems. For RT-PCR,
cultured cells were stimulated as described. Six hours later, total RNA
was prepared from stimulated cells. Total RNA (0.5 µg) was used for
each RT-PCR by using the ACCESS RT-PCR SYSTEM (Promega) according to
the manufacturer’s instruction. The sequences of primers used are
IL-13 sense 5'-GACTGCAGTCCTGGCTCTTGC-3', IL-13 antisense
5'-TGAGTCCACAGCTGAGATGCC-3', 
-actin sense
5'-GTGGGCCGCTCTAGGCACCA-3', and -actin antisense
5'-CGGTTGGCCTTAGGGTTCAGGGGGG-3'.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-13 is produced mainly by T, in particular Th2, cells. However,
it is unclear whether the IL-13 gene is always coexpressed
with other Th2 cytokine genes on a single cell basis. To address this
question, we purified naive CD4+ Th cells from WT mice and
stimulated them in vitro under Th2 skewing conditions. On day 7, the
differentiated Th2 cells were restimulated with PMA/iono and analyzed
by intracellular cytokine staining. As shown in Fig. 1, whereas IL-13 was coexpressed with
IL-4 in a majority of Th2 cells (
50%), a significant number of
cells produced either IL-13 or IL-4, but not both. A similar scenario
was observed for IL-4 and IL-5 staining. In contrast, nearly all
IL-5-producing cells also expressed IL-13, but not vice versa. These
results demonstrate that the IL-13 gene is not always
coexpressed with other Th2 cytokine genes.
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The murine IL-13/IL-4 locus contains several Th2
cell-specific DHSs (12). Three of the DHSs (A, B, and C)
fall within the IL-13 gene (Fig. 2A). To examine the function
of these DHSs in dictating the expression of the IL-13 gene,
we first isolated a 2-kb genomic fragment, corresponding to -1990
to +48 of the murine IL-13 gene and containing DHS-A and
DHS-B, by PCR amplification. The 2-kb IL-13 promoter was cloned into
the pGL2Basic vector in a 5' to 3' orientation. The resulting reporter
construct was used to transfect a variety of cell lines and Th clones.
As shown in Fig. 2B, the activity of the 2-kb IL-13 promoter
was induced in EL4 (a murine thymoma line) and D10 (a Th2 clone) by
stimulation with PMA/iono, but not in M12 (a murine B lymphoma line),
18.81 (a human B cell line), D1.1 (a murine Th1 clone), and NIH3T3
cells. More importantly, the PMA/iono-induced activity of the 2-kb
IL-13 promoter correlated well with the expression of the
IL-13 gene as examined by RT-PCR and ELISA (Fig. 2C). Somewhat unexpectedly, addition of the second intron,
containing DHS-C, of the IL-13 gene into the 2-kb IL-13
promoter reporter construct, downstream to the transcriptional unit,
did not further enhance the promoter activity (data not shown). Taken
together, these results demonstrate that the 2-kb IL-13 promoter,
containing only DHS-A and DHS-B, is sufficient to confer the expression
pattern and kinetics of the murine IL-13 gene in vitro.
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To map functionally important cis-acting elements
within the 2-kb IL-13 promoter, we compared the sequences of the 2-kb
promoter between murine and human IL-13 genes and identified
two regions, henceforth called consensus sequence (CS)1 and 2 (Fig. 3
). Each of the CSs is at least 80 bp in
length and is >85% similar in sequence between mouse and human.
Interestingly, even the locations, relative to the transcriptional
start site, of the CSs are also maintained through evolution, and
correspond well to those of DHS-A and DHS-B, respectively. To determine
the function of each CS, we generated a series of deletion mutants of
the 2-kb IL-13 promoter reporter, which were used to transfect EL4 and
M12 cells. EL4 and M12 cells were chosen because they are easily
transfectable and each represents an IL-13 producer and an IL-13
nonproducer, respectively. Although deletion of CS1 (-1535 and -579
promoter constructs in Fig. 4) resulted
in an 50% decrease in the PMA/iono-induced activity of the IL-13
promoter, the -579 deletion mutant could still be induced by PMA/iono
and retained the cell type specificity. Deletion of the intervening
sequences between CS1 and CS2 did not result in any further loss of
promoter activity. Interestingly, a smaller fragment, containing only
the 3' half of CS2 (-109 promoter construct), did not respond to
PMA/iono but was still sufficient to retain cell type-specific
activity. Similar results were obtained from D10 cells that were
stimulated with anti-CD3. In contrast, all the deletion mutants of
the IL-13 promoter remained inactive in M12 cells. Taken together,
these results demonstrated that the -254 to +48 fragment contained the
minimal murine IL-13 promoter and that the cell type specificity and
induction by PMA/iono of the murine IL-13 promoter are mediated by
distinct cis-acting elements.
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To identify and clone trans-acting factors that
regulate the cell type-specific expression of the murine
IL-13 gene, we performed a yeast one-hybrid screen by using
CS2 region as bait. One of the cDNA clones thus obtained encoded the
zinc finger domain of GATA-3 (data not shown). In agreement with this
data, overexpression of GATA-3 in M12 cells resulted in an 25-fold
increase of the activity of the 2-kb IL-13 promoter (Fig. 5). Deletion analysis revealed that
GATA-3-induced transactivation required only the 3' half of CS2 (Fig. 5, -109 promoter construct). The 3' half of CS2 contains at least four
potential GATA-3 binding sites, located at -105, -95, -82, and -44,
respectively (Figs. 3 and 6A).
To identify the functionally critical GATA site within the minimal
IL-13 promoter, we performed EMSAs by using the 3' half of CS2 as a
probe. As shown in Fig. 6B, the 3' half of CS2 bound at
least two protein complexes (a and b) present in D10 cell extract. The
mobility of both complexes was further retarded by anti-GATA-3 Abs,
but not by control Abs. In addition, the formation of the complexes was
inhibited by an unlabeled WT probe or by an unlabeled probe bearing a
mutation in the -105 (Mut1), -82 (Mut3), or -44 (Mut4) GATA sites.
In contrast, the formation of the complexes was only negligibly, or not
at all, inhibited by an unlabeled probe bearing a mutation in the -95
(Mut2) GATA site, all GATA sites (Mut1234), or by an unrelated probe
containing a consensus NF-AT binding site (Fig. 6, A and
B). The EMSA results demonstrate that the -95 GATA site is
capable of binding endogenous GATA-3 proteins. To further examine the
function of the -95 GATA site, we introduced Mut1 and/or Mut2
mutations into the -109 IL-13 promoter reporter construct. The
resulting constructs and a GATA-3 expression vector, pcDNA-GATA3, were
used to transfect M12 cells. As shown in Fig. 6C, left
panel, the Mut2 mutation dramatically attenuated GATA-3-induced
transactivation. The same result was obtained with the Mut1 and Mut2
double mutant (Mut1/Mut2). In contrast, no such attenuation was
observed with the Mut1 mutant. Furthermore, Mut2 or Mut1/Mut2, but not
Mut1, nearly completely abrogated the cell type-specific activity of
the -109 IL-13 promoter in EL4 cells (Fig. 6C, right
panel). Taken together, these results demonstrate that the -95
GATA site can bind endogenous GATA-3 proteins and is functionally
critical for GATA-3-induced transactivation and cell type-specific
activity of the minimal IL-13 promoter.
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To determine whether GATA-3 is sufficient to induce the expression
of endogenous IL-13 in normal Th cells, we infected Th1 cells,
generated by in vitro differentiation under Th1 skewing conditions for
7 days, with bicistronic retroviruses expressing both green fluorescent
protein (GFP) and GATA-3 (RV-GATA3), or GFP only (RV). It was shown
that expression of retroviral GATA-3 in developing, but not fully
developed, Th1 cells could reprogram Th cell differentiation into the
Th2 pathway and subsequently enhance the production of all Th2
cytokines. To avoid this effect of GATA-3, we chose to use fully
developed Th1 cells in this experiment to isolate the effect of GATA-3
on the endogenous IL-13 promoter. The infected Th1 cells were then
restimulated with PMA/iono, and the production of IL-13 was examined by
intracellular cytokine staining. Previously, it was shown that
transduction with RV-GATA3 was insufficient to effectively
transactivate the endogenous IL-4 promoter in developed Th1 cells
(23). In agreement with the previous observation,
retroviral GATA-3 induced only a small number (7%) of the developed
Th1 cells (GFP+ of WTTh1/RV-GATA3) to express IL-4 (Fig. 7A). As controls, no
more than 1% of the uninfected (GFP-) or RV-infected (GFP+ of
WTTh1/RV) Th1 cells and 68% of WT Th2 cells (WTTh2/RV) produced
IL-4. In contrast, a significant number (40%) of the RV-GATA3-infected
Th1 cells, as compared with 72% of WT Th2 cells and 2–2.5% of the
uninfected or RV-infected Th1 cells, stained positive for IL-13 (Fig. 7B). These data demonstrate that forced expression of GATA-3
alone efficiently transactivates the endogenous IL-13, but not IL-4,
promoter in Th1 cells.
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It was demonstrated that overexpression of c-maf or, to
a lesser degree, GATA-3 in tumor cell lines transactivated an IL-4
promoter reporter (17, 19). To determine whether GATA-3
and c-maf have synergistic effects on the endogenous IL-13
and IL-4 promoters in Th1 cells, we also infected mafTG Th1 cells
(20), generated by in vitro differentiation under Th1
skewing conditions for 7 days, with RV or RV-GATA3 viruses. This
protocol allows us to generate Th1 cells ectopically expressing
c-maf alone or c-maf with GATA-3. In agreement
with our previous report (20), ectopic expression of
c-maf alone resulted in only a small number (7%) of Th1
cells (mafTGTh1/RV) expressing IL-4 (Fig. 7A); however,
ectopic expression of c-maf and GATA-3 synergistically
induced endogenous IL-4 production (21% of GFP+mafTGTh1/RV-GATA3). In
contrast to its role in enhancing the production of IL-4, ectopic
expression of c-maf alone did not transactivate the
endogenous IL-13 promoter (5% of mafTGTh1/RV vs 2% of WTTh1/RV) (Fig. 7B). Furthermore, no synergistic effect between
c-maf and GATA-3 was observed in the production of IL-13
(43% of GFP+mafTGTh1/RV-GATA3 vs 40% of GFP+WTTh1/RV-GATA3).
Interestingly, while a substantial number of the
RV-GATA3-infected WT or mafTG Th1 cells expressed IL-13, they continued
to produce IFN- at levels comparable to those of WT Th1 (Fig. 8). A similar result was obtained when we
gated on the RV-GATA3-infected mafTG Th1 cells that stained positive
for IL-4 (data not shown). This observation suggests that forced
expression of GATA-3 and c-maf cannot reverse the Th1
phenotype and that the expression of IFN- and Th2 cytokines is not
mutually exclusive.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Retroviral transduction of GATA-3 into WT or mafTG Th cells is an ideal system for examining the effect of ectopic expression of GATA-3 and/or c-maf on the endogenous promoters of Th2 cytokine genes in differentiated Th1 cells. Recently, it was shown that forced expression of GATA-3 in Th1 cells and a Th1 clone resulted in the remodeling of the IL-13/IL-4 locus as examined by DNaseI hypersensitivity assays (26, 27). This would imply that GATA-3 could induce the production of both cytokines. However, we found that only IL-13, but not IL-4, was produced at a significant level by the GATA-3-expressing Th1 cells. This discrepancy indicates that the expression of the IL-13 and IL-4 genes, while only 10 kb away from each other in the murine genome, is regulated by distinct transcriptional mechanisms. For the IL-13 gene, GATA-3 may function as both a chromatin remodeling factor and a critical cell type-specific transcription factor. In contrast, high level expression of IL-4 appears to require both GATA-3 and c-maf. Ectopic expression of GATA-3 or c-maf alone in Th1 cells has been shown to be insufficient to induce significant levels of IL-4 production (20, 23). Here, we directly demonstrate, for the first time, that GATA-3 and c-maf synergistically transactivate the endogenous IL-4 promoter, even in Th1 cells. Although c-maf is a potent transactivator of the IL-4 gene, it did not play any critical role in transactivating the endogenous IL-13 gene. This is in agreement with a previous report showing the production of IL-13 by c-maf-deficient Th2 cells was not impaired (21).
The differences in transcriptional regulation that we observed between the IL-13 and IL-4 genes provide an attractive explanation for the observations that many Th2 cells express either IL-4 or IL-13, but not both. Heterogeneity in the expression of other Th2 cytokine genes has been previously reported (14, 15, 16), but we clearly demonstrate that IL-13 is not always coexpressed with other Th2 cytokines in normal Th2 cells on a single cell basis. Recently, a report by Kelly et al. (28) showed that approximately half of 23 IL-4-producing Th2 clones also expressed the IL-13 gene as examined by RT-PCR. Our results concur with this observation and further demonstrate that the IL-13 gene can be expressed by non-IL-4-producing Th2 cells. It remains unclear how a Th2 cell chooses to express IL-13, IL-4, and/or IL-5, and whether a Th2 cell can alternate the expression of these cytokine genes. In a Th2 cell, because the IL-13/IL-4/IL-5 locus is easily accessible, the expression of IL-13, IL-4, and/or IL-5 might therefore be determined by the relative levels of c-maf, GATA-3, and, possibly, other Th2 cell-specific transcription factors. This hypothesis requires further examination.
Several groups have reported that GATA-3, in addition to promoting Th2
cell differentiation, can repress the production of the opposing Th1
cytokine, IFN-, by various degrees (23, 27). Here, we
show that ectopic expression of GATA-3 alone or in combination with
c-maf was sufficient to force developed Th1 cells to produce
high levels of IL-13 or IL-4 but did not significantly suppress the
expression of IFN-. This was true even when we examined only the
bright GFP-positive population of the RV-GATA3-infected mafTG Th1
cells. The discrepancy still cannot be satisfactorily explained and
might be due to differences in the source of Th cells (WT vs TCR
transgenic) or the modality of stimulation (anti-CD3 vs Ag/APCs).
Our observation that expression of Th2 cell-specific transcription
factors did not fully suppress the expression of IFN- then led us to
question whether Th1 and Th2 cytokines could be coexpressed on a single
cell basis. Our data demonstrates that retroviral GATA-3 enabled Th1
cells to express both Th1 and Th2 cytokine genes, and further suggests
that the expression of Th1 and Th2 cytokine genes in effector Th
cells is not mutually exclusive. This result is somewhat contradictory
to a recent report by Farrar et al. (29), in which nearly
all Th clones derived from Th1 cells transduced with GATA-3 produced
IL-4 but not IFN-. The discrepancy might arise from timing of
retroviral transduction. In the experiments in Farrar et al., Th1 cells
were infected at 36 h after initial stimulation, a stage when
developing Th cells are not fully committed to either Th1 or Th2
pathway and are more responsive to the Th2-polarizing effect of
retroviral GATA-3. It was recently demonstrated that, as early as
1 h after initial stimulation of naive Th cells under either Th1
or Th2 skewing conditions, transcripts of both IL-4 and IFN- were
detected by real-time PCR, and that both IL-4 and
IFN- genes were positioned apart from centromeric
chromatin domains. Repositioning of the IL-4 or
IFN- allele in apposition to centromeric chromatin then
took place in effector Th1 or Th2 cells, respectively, as a way to
stabilize the cytokine profile (30). Thus, it will be
interesting to examine whether the repositioning process is reversed in
retroviral GATA-3-transduced Th1 cells, which produce high levels of
both Th1 and Th2 cytokines. Finally, the generation and demonstration
of effector Th cells capable of producing high levels of both Th1 and
Th2 cytokines might have therapeutic implications in treating
autoimmune and allergic diseases in which the balance between Th1 and
Th2 responses is critical in determining clinical outcomes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. I-Cheng Ho, Department of Immunology and Infectious Diseases, Harvard School of Public Health, FXB, Room 205, 652 Huntington Avenue, Boston, MA 02115.
3 Abbreviations used in this paper: AHR, airway hyperresponsiveness; CS, consensus sequence; DHS, DNaseI hypersensitivity site; iono, ionomycin; RV, retroviral vector; ds, double-stranded; GFP, green fluorescent protein; mafTG, c-maf transgenic; WT, wild type.
Received for publication May 29, 2001. Accepted for publication August 8, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 Y. Matsuno, Y. Ishii, K. Yoh, Y. Morishima, N. Haraguchi, N. Kikuchi, T. Iizuka, T. Kiwamoto, S. Homma, A. Nomura, et al. Overexpression of GATA-3 Protects against the Development of Hypersensitivity Pneumonitis Am. J. Respir. Crit. Care Med., November 15, 2007; 176(10): 1015 - 1025. [Abstract] [Full Text] [PDF]
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T. Yoshimoto, T. Yoshimoto, K. Yasuda, J. Mizuguchi, and K. Nakanishi IL-27 Suppresses Th2 Cell Development and Th2 Cytokines Production from Polarized Th2 Cells: A Novel Therapeutic Way for Th2-Mediated Allergic Inflammation J. Immunol., October 1, 2007; 179(7): 4415 - 4423. [Abstract] [Full Text] [PDF]
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S. Rose, M. Lichtenheld, M. R. Foote, and B. Adkins Murine Neonatal CD4+ Cells Are Poised for Rapid Th2 Effector-Like Function J. Immunol., March 1, 2007; 178(5): 2667 - 2678. [Abstract] [Full Text] [PDF]
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 R. B. Webster, Y. Rodriguez, W. T. Klimecki, and D. Vercelli The Human IL-13 Locus in Neonatal CD4+ T Cells Is Refractory to the Acquisition of a Repressive Chromatin Architecture J. Biol. Chem., January 5, 2007; 282(1): 700 - 709. [Abstract] [Full Text] [PDF]
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P. J. Kim, S.-Y. Pai, M. Brigl, G. S. Besra, J. Gumperz, and I-C. Ho GATA-3 Regulates the Development and Function of Invariant NKT Cells J. Immunol., November 15, 2006; 177(10): 6650 - 6659. [Abstract] [Full Text] [PDF]
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R. Shinnakasu, M. Yamashita, K. Shinoda, Y. Endo, H. Hosokawa, A. Hasegawa, S. Ikemizu, and T. Nakayama Critical YxKxHxxxRP Motif in the C-Terminal Region of GATA3 for Its DNA Binding and Function J. Immunol., November 1, 2006; 177(9): 5801 - 5810. [Abstract] [Full Text] [PDF]
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J. Shoemaker, M. Saraiva, and A. O'Garra GATA-3 Directly Remodels the IL-10 Locus Independently of IL-4 in CD4+ T Cells J. Immunol., March 15, 2006; 176(6): 3470 - 3479. [Abstract] [Full Text] [PDF]
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 J. Rubenfeld, J. Guo, N. Sookrung, R. Chen, W. Chaicumpa, V. Casolaro, Y. Zhao, V. Natarajan, and S. Georas Lysophosphatidic acid enhances interleukin-13 gene expression and promoter activity in T cells Am J Physiol Lung Cell Mol Physiol, January 1, 2006; 290(1): L66 - L74. [Abstract] [Full Text] [PDF]
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 S. Monticelli, D. U. Lee, J. Nardone, D. L. Bolton, and A. Rao Chromatin-based regulation of cytokine transcription in Th2 cells and mast cells Int. Immunol., November 1, 2005; 17(11): 1513 - 1524. [Abstract] [Full Text] [PDF]
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 Y. Tanaka, T. So, S. Lebedeva, M. Croft, and A. Altman Impaired IL-4 and c-Maf expression and enhanced Th1-cell development in Vav1-deficient mice Blood, August 15, 2005; 106(4): 1286 - 1295. [Abstract] [Full Text] [PDF]
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L.-O. Tykocinski, P. Hajkova, H.-D. Chang, T. Stamm, O. SOzeri, M. LOhning, J. Hu-Li, U. Niesner, S. Kreher, B. Friedrich, et al. A Critical Control Element for Interleukin-4 Memory Expression in T Helper Lymphocytes J. Biol. Chem., August 5, 2005; 280(31): 28177 - 28185. [Abstract] [Full Text] [PDF]
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Y.-K. Jee, J. Gilmour, A. Kelly, H. Bowen, D. Richards, C. Soh, P. Smith, C. Hawrylowicz, D. Cousins, T. Lee, et al. Repression of Interleukin-5 Transcription by the Glucocorticoid Receptor Targets GATA3 Signaling and Involves Histone Deacetylase Recruitment J. Biol. Chem., June 17, 2005; 280(24): 23243 - 23250. [Abstract] [Full Text] [PDF]
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 R. Grenningloh, B. Y. Kang, and I-C. Ho Ets-1, a functional cofactor of T-bet, is essential for Th1 inflammatory responses J. Exp. Med., February 22, 2005; 201(4): 615 - 626. [Abstract] [Full Text] [PDF]
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A. Masuda, Y. Yoshikai, H. Kume, and T. Matsuguchi The Interaction between GATA Proteins and Activator Protein-1 Promotes the Transcription of IL-13 in Mast Cells J. Immunol., November 1, 2004; 173(9): 5564 - 5573. [Abstract] [Full Text] [PDF]
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T. Katsumoto, M. Kimura, M. Yamashita, H. Hosokawa, K. Hashimoto, A. Hasegawa, M. Omori, T. Miyamoto, M. Taniguchi, and T. Nakayama STAT6-Dependent Differentiation and Production of IL-5 and IL-13 in Murine NK2 Cells J. Immunol., October 15, 2004; 173(8): 4967 - 4975. [Abstract] [Full Text] [PDF]
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G. Matsumoto, E. Kubota, Y. Omi, U. Lee, and J. M. Penninger Essential Role of LFA-1 in Activating Th2-Like Responses by {alpha}-Galactosylceramide-Activated NKT Cells J. Immunol., October 15, 2004; 173(8): 4976 - 4984. [Abstract] [Full Text] [PDF]
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M. Yamashita, M. Ukai-Tadenuma, T. Miyamoto, K. Sugaya, H. Hosokawa, A. Hasegawa, M. Kimura, M. Taniguchi, J. DeGregori, and T. Nakayama Essential Role of GATA3 for the Maintenance of Type 2 Helper T (Th2) Cytokine Production and Chromatin Remodeling at the Th2 Cytokine Gene Loci J. Biol. Chem., June 25, 2004; 279(26): 26983 - 26990. [Abstract] [Full Text] [PDF]
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M. Inami, M. Yamashita, Y. Tenda, A. Hasegawa, M. Kimura, K. Hashimoto, N. Seki, M. Taniguchi, and T. Nakayama CD28 Costimulation Controls Histone Hyperacetylation of the Interleukin 5 Gene Locus in Developing Th2 Cells J. Biol. Chem., May 28, 2004; 279(22): 23123 - 23133. [Abstract] [Full Text] [PDF]
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 S.-Y. Pai, M. L. Truitt, and I-C. Ho GATA-3 deficiency abrogates the development and maintenance of T helper type 2 cells PNAS, February 17, 2004; 101(7): 1993 - 1998. [Abstract] [Full Text] [PDF]
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A. Valanciute, S. le Gouvello, B. Solhonne, A. Pawlak, P. Grimbert, L. Lyonnet, S. Hue, P. Lang, P. Remy, R. Salomon, et al. NF-{kappa}B p65 Antagonizes IL-4 Induction by c-maf in Minimal Change Nephrotic Syndrome J. Immunol., January 1, 2004; 172(1): 688 - 698. [Abstract] [Full Text] [PDF]
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M. S. Sundrud, S. M. Grill, D. Ni, K. Nagata, S. S. Alkan, A. Subramaniam, and D. Unutmaz Genetic Reprogramming of Primary Human T Cells Reveals Functional Plasticity in Th Cell Differentiation J. Immunol., October 1, 2003; 171(7): 3542 - 3549. [Abstract] [Full Text] [PDF]
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 T. S. Absi, T. M. Sundt III, W. S. Tung, M. Moon, J. K. Lee, R. R. Damiano Jr, and R. W. Thompson Altered patterns of gene expression distinguishing ascending aortic aneurysms from abdominal aortic aneurysms: complementary DNA expression profiling in the molecular characterization of aortic disease J. Thorac. Cardiovasc. Surg., August 1, 2003; 126(2): 344 - 357. [Abstract] [Full Text] [PDF]
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S. Kusam, L. M. Toney, H. Sato, and A. L. Dent Inhibition of Th2 Differentiation and GATA-3 Expression by BCL-6 J. Immunol., March 1, 2003; 170(5): 2435 - 2441. [Abstract] [Full Text] [PDF]
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N. Takemoto, K.-i. Arai, and S. Miyatake Cutting Edge: The Differential Involvement of the N-Finger of GATA-3 in Chromatin Remodeling and Transactivation During Th2 Development J. Immunol., October 15, 2002; 169(8): 4103 - 4107. [Abstract] [Full Text] [PDF]
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A. Masuda, Y. Yoshikai, K. Aiba, and T. Matsuguchi Th2 Cytokine Production from Mast Cells Is Directly Induced by Lipopolysaccharide and Distinctly Regulated by c-Jun N-Terminal Kinase and p38 Pathways J. Immunol., October 1, 2002; 169(7): 3801 - 3810. [Abstract] [Full Text] [PDF]
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M. J. Loza and B. Perussia Peripheral Immature CD2-/low T Cell Development from Type 2 to Type 1 Cytokine Production J. Immunol., September 15, 2002; 169(6): 3061 - 3068. [Abstract] [Full Text] [PDF]
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S. Santangelo, D. J. Cousins, N. E. E. Winkelmann, and D. Z. Staynov DNA Methylation Changes at Human Th2 Cytokine Genes Coincide with DNase I Hypersensitive Site Formation During CD4+ T Cell Differentiation J. Immunol., August 15, 2002; 169(4): 1893 - 1903. [Abstract] [Full Text] [PDF]
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