Interleukin-12 Induces Expression of Interferon Regulatory
Factor-1 via Signal Transducer and Activator of Transcription-4 in
Human T Helper Type 1 Cells*
Eliana M.
Coccia
,
Nadia
Passini§,
Angela
Battistini¶,
Carlo
Pini
,
Francesco
Sinigaglia§, and
Lars
Rogge§
From the Laboratories of
Immunology and
¶ Virology, Istituto Superiore di Sanità, I-00161 Rome
and § Roche Milano Ricerche, Via Olgettina 58,
I-20132 Milan, Italy
 |
ABSTRACT |
IRF-1-deficient mice show a striking defect in
the development of T helper 1 (Th1) cells. In the present report, we
investigate the expression of IRF-1 during differentiation of human T
helper cells. No significant differences of IRF-1 mRNA expression
were found in established Th1 and Th2 cells; however, interleukin 12 (IL-12) induced a strong up-regulation of IRF-1 transcripts in Th1 but
not in Th2 cells. We demonstrate that IL-12-induced up-regulation of
IRF-1 is mediated by signal transducer and activator of
transcription-4, which binds to the interferon (IFN)-
-activated
sequence present in the promoter of the IRF-1 gene. Strong
IL-12-dependent activation of a reporter gene construct
containing the IRF-1 IFN-
-activated sequence element provides
further evidence for the key role of signal transducer and activator of
transcription-4 in the IL-12-induced up-regulation of IRF-1 transcripts
in T cells. IRF-1 expression was strongly induced after stimulation of
naive CD4+ T cells via the T cell receptor,
irrespective of the cytokines present at priming, indicating that this
transcription factor does not play a major role in initiating a
Th1-specific transcriptional cascade in differentiating helper T cells.
However, our finding that IRF-1 is a target gene of IL-12 suggests that
some of the IL-12-induced effector functions of Th1 cells may be
mediated by IRF-1.
 |
INTRODUCTION |
The discovery of functionally distinct subsets of T helper cells
has provided a better understanding for the heterogeneity of immune
responses in normal and pathological situations (1-4). However, the
transcriptional programs that control the differentiation of naive
CD4+ T cells into polarized
Th11 and Th2 cells are just
beginning to be elucidated (5).
The differentiation process is initiated by stimulation of the TCR and
directed by cytokines present during the initiation of a T cell
response (6). IL-4 promotes Th2 development (7, 8), whereas IL-12
produced by antigen presenting cells is a potent inducer of Th1 cells
(9-13). Signaling of these two cytokines is mediated through the
activation of specific signal transducer and activator of transcription
(STAT) proteins. STAT-6 is activated by IL-4 in Th2 cells (14-16), and
IL-12 activates STAT-4 in Th1 cells (17-21). Studies on knockout mice
for these transcription factors clearly indicate the pivotal role of
STAT-6 and STAT-4 in T helper cell differentiation. STAT-6-deficient T
lymphocytes fail to differentiate into Th2 cells in response to IL-4
(22-24), and the analysis of STAT-4
/
T cells has
revealed an impaired production of IFN-
upon antigen receptor
triggering, indicative of a defect in Th1 differentiation (25, 26).
More recently, additional transcription factors have been studied and
characterized for their role in the Th2-specific expression of the IL-4
gene. The nuclear factor of activated T cells (NF-AT) has been shown to
regulate IL-4 expression in Th2 cells through a cooperative binding
with AP-1 family members (27). NF-AT functions are potentiated by the
transcription factor NIP45 (for NF-ATp interacting protein) (28). The
proto-oncogene c-maf is expressed in Th2 clones but not in
Th1 clones and has also been identified as a potent transactivator of
the IL-4 gene (29). More recently, it has been shown that the
transcription factor GATA-3 is expressed at a high level in naive
CD4+ T cells and differentiating and effector Th2 cells,
whereas its expression is suppressed in Th1 cells (30). Less is known
about the regulation of the IFN-
gene in Th1 cells that appears to involve the interaction of numerous transcription factors, including NF-kB and NF-AT (31), or the cooperative binding of STAT-4 dimers (32).
Recently, it has been suggested that transcription factors belonging to
the interferon regulatory factor (IRF) family are necessary for Th1
development (33, 34). The IRF family includes at least 10 members:
IRF-1, IRF-2, IRF-3, ISGF3
/p48, ICSBP, Pip/ICSAT/IRF-4, IRF-5,
IRF-6, IRF-7, and viral IRF, recently identified in the genome of HHV-8
(35). IRF expression is either constitutive and/or induced upon
treatment with interferons (IFNs) or other cytokines or in response to
viral infection. Moreover, some IRFs are specific for hematopoietic
cells (ICSBP and IRF-4), whereas others are expressed in multiple
tissues and cell lines. However, all members of this family share
significant homology in the amino-terminal 115 amino acids, which make
up the DNA binding domain that mediates the interaction with a specific
consensus IRF binding sequence motif, termed IRF-E (36). Proteins of
the IRF family have been shown to be involved in the regulation of the
pleiotropic activities elicited by IFNs and other cytokines, including
modulation of the immune response, inflammation, hematopoiesis, cell
proliferation, and differentiation (37, 38).
Studies of IRF-1-deficient mice have revealed that this factor is
implicated in the regulation of several immune processes, such as
T-cell selection and maturation (39, 40), development of NK cells
(41-43), and development of Th1 cells (33, 34). The compromised Th1
differentiation was associated with defects in the expression of p40
subunit of IL-12 by cells of myeloid origin (33, 34). Indeed, a
potential IRF-1-responsive element was found in the promoter region of
the IL-12 p40 gene (44, 45). Together, these results suggest that IRF-1
may be a master gene, directly or indirectly controlling Th1 responses
at multiple stages.
In the present report, we have analyzed the expression of IRF-1 in
developing human Th1 and Th2 cells and its regulation in response to
cytokines in polarized Th1 and Th2 cells. IRF-1 transcripts were
strongly induced after stimulation of naive CD4+ T cells
via the T cell receptor, irrespective of the cytokines present at
priming, and no differences in IRF-1 mRNA expression were
detectable in established Th1 and Th2 cells. Interestingly, IL-12
treatment up-regulated IRF-1 transcripts only in established Th1 cells.
This up-regulation does not depend on de novo protein synthesis. IL-12 induced binding of STAT-4 to the IFN-
-activated sequence (GAS) of the IRF-1 gene promoter in Th1 cells but not in Th2
cells. Strong IL-12-dependent activation of a reporter gene
construct containing the IRF-1 GAS element further emphasizes the key
role of STAT-4 for IL-12-induced up-regulation of IRF-1 transcripts in
T cells.
 |
EXPERIMENTAL PROCEDURES |
Generation of Th1 and Th2 Lines from Cord Blood
Leukocytes--
Human neonatal leukocytes were isolated from freshly
collected, heparinized, neonatal blood by Ficoll-Paque (Amersham
Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation.
CD8+ T cells were removed by positive selection with
anti-CD8 microbeads and magnetic-activated cell sorting according to a
protocol supplied by the manufacturer (Miltenyi Biotec, Bergisch
Gladbach, Germany). Cells were stimulated with 2 µg/ml
phytohemagglutinin (Wellcome, Beckenham, United Kingdom) in the
presence of 2.5 ng/ml IL-12 (Hoffmann-La Roche Inc., Nutley, NJ) and
200 ng/ml neutralizing anti-IL-4 antibodies (18500D, Pharmingen, San
Diego, CA) for Th1 cultures or 1 ng/ml IL-4 (Pharmingen) and 2 µg/ml
neutralizing anti-IL-12 antibodies 17F7 and 20C2 (kindly provided by M. Gately, Hoffmann-La Roche Inc.) for Th2 cultures. Cells were washed on day 3 and expanded in complete RPMI 1640 medium (Life Technologies, Inc.) supplemented with 5% FetalClone I (HyClone, Logan, UT), 2 mM L-glutamine, 1 mM sodium
pyruvate, 100 units/ml penicillin-streptomycin) containing 100 units/ml
IL-2 (Hoffmann-La Roche Inc.). The cells were treated with IL-12 (2.5 ng/ml) or IFN-
(1000 units/ml) followed by the extraction of total
RNA. To determine whether de novo protein synthesis is
required for IL-12-induced up-regulation of IRF-1 transcripts, cells
were treated with the protein synthesis inhibitor cycloheximide (Sigma,
10 µg/ml) 45 min prior to the addition of IL-12 (2.5 ng/ml). RNA was
extracted 16 h after the addition of IL-12.
Purification and Stimulation of Naive CD4+ T
Cells--
CD4+/CD45RO
T cells were purified
from cord blood leukocytes by negative selection using a
CD4+ T cell isolation kit and CD45RO microbeads according
to a protocol supplied by the manufacturer (Miltenyi Biotec). The
purity of the CD4+/CD45RO
T cells using this
procedure was typically >98% as determined by flow cytometry.
Purified naive T cells were stimulated with plate-bound anti-CD3 mAb
(TR66 (46)) in the absence of exogenously added cytokines, in the
presence of IL-12 (2.5 ng/ml) and neutralizing anti-IL-4 mAb (200 ng/ml), or in the presence of IL-4 (1 ng/ml).
Single Cell Analysis of Intracellular IFN-
and IL-4
Production--
Single cell analysis of IFN-
and IL-4 production
was performed as described previously (47). Briefly, T cell lines were collected 7 days after priming and washed, and 106 cells
were restimulated with phorbol 12-myristate 13-acetate (50 ng/ml)
(Sigma) and ionomycin (1 µg/ml) (Sigma) for 2 h at 37 °C in
complete medium. Brefeldin A (10 µg/ml) (Sigma) was added to the
cultures and the cultures were incubated for an additional 2 h.
Then, the cells were fixed with 4% paraformaldehyde and permeabilized with saponin. Fixed cells were stained with anti-human IFN-
-FITC (Pharmingen) and anti-human IL-4-PE (Pharmingen) following a protocol provided by the manufacturer and analyzed with a FACScan flow cytometer
(Becton Dickinson, Mountain View, CA).
Ribonuclease Protection Assays--
To obtain the pBS IRF-1
construct, the plasmid pUC IRF-1 (a generous gift of T. Taniguchi) was
digested with SmaI, and the 400-base pair-long fragment was
cloned into the same sites of pBluescript/KS (Stratagene, La Jolla,
CA). To generate the 32P-labeled 280-base pair-long
antisense IRF-1 RNA probe, the plasmid pBS IRF-1 was linearized with
EcoRI and transcribed by T7 polymerase. A 327-base pair DNA
fragment encompassing the cytoplasmic region of the human IL-12R
2
subunit was subcloned in pGEM 3Z (21). This construct was linearized
with EcoRI, and radiolabeled antisense transcripts were
synthesized with SP6 polymerase and a commercial kit according to the
manufacturer's protocol (Promega, Madison, WI). RNA was extracted from
T cell lines using Ultraspec total RNA extraction reagent (Biotecx
Laboratories Inc., Houston, TX). The antisense RNA probes were
hybridized to 5 µg of total RNA, and ribonuclease protection assays
were performed with a commercial kit (Ambion Inc., Austin, TX)
according to the company's protocol. Products were resolved on 6%
denaturing polyacrylamide gels, and the protected fragments were
visualized by autoradiography. The radioactivity present in the
protected fragments was also quantitated using a MolecularImager
(Bio-Rad). An 18 S RNA probe was used as a control for equal RNA loading.
Reporter Gene Assays--
Jurkat cells were co-transfected with
the following constructs: (i) pBOS-IL-12R
1, pBOS-IL-12R
2 (48)
(kindly provided by Dr. U. Gubler, Hoffmann-La Roche Inc.) and
pBOS-Stat4 (kindly provided by Dr. R. Chizzonite, Hoffmann-La Roche
Inc.), (ii) pIRF-luc, a luciferase reporter plasmid containing three
copies of an oligonucleotide representing the GAS of the IRF-1 promoter
(5'-CTGATTTCCCCGAAATGAC-3') inserted upstream of the SV40 promoter in
pGL3-promoter (Promega), and (iii) pCMV-
-gal, a mammalian expression
vector containing the
-galactosidase gene driven by the
cytomegalovirus promoter. 30 µg of plasmid DNA (7 µg each of
pIRF-luc, pBOS-IL-12R
1, pBOS-IL-12R
2, and pBOS-Stat4 and 2 µg
of pCMV-
-gal) were used to electroporate 8 × 106
cells in 400 µl of RPMI medium with a 0.4-cm gap electroporation cuvette at 960 microfarads and 300 V using a Gene Pulser (Bio-Rad). Four ml of complete medium were added immediately after transfection, and the cells were seeded in 2 wells of a 24-well plate. Four h after
transfection, 4 ng/ml IL-12 was added to half of the cultures, and the
cells were incubated for additional 20 h. Luciferase activity was
measured using a luciferase reporter gene assay (Boehringer Mannheim),
and
-galactosidase activity was determined with the
-galactosidase reporter gene assay (Boehringer Mannheim). Both activities were measured in a LB 9507 Lumat luminometer (EG & G
Berthold, Milano, Italy). Luciferase activities were normalized using
-galactosidase activity.
DNA Electrophoretic Mobility Shift Assay (EMSA)--
For gel
shift assays, polarized human Th1 and Th2 cells were harvested 6 days
after priming, washed, and resuspended in complete medium.
107 cells were incubated 30 min at 37 °C in 1 ml of
complete medium with or without IL-12 (2.5 ng/ml) for 1 h or
IFN-
(1000 units/ml), IFN-
(1000 units/ml), or IL-4 (1 ng/ml) for
15 min. The cells were washed once with ice-cold PBS and lysed in 20 µl of ice cold whole cell extraction buffer (20 mM Hepes,
pH 7.9; 300 mM NaCl; 10% glycerol; 0.5% Nonidet P-40; 1 mM dithiothreitol; 0.1 mM EDTA and EGTA; 10 µg/ml aprotinin, leupeptin, and NaF; 1 mM sodium orthovanadate; and Pefabloc SC (Boehringer Mannheim)). The lysate was
incubated 30 min on a shaker at 4 °C, and insoluble debris was
removed by centrifugation (13,000 rpm at 4 °C for 20 min). After
addition of 1 volume of a solution containing 20 mM
Hepes/NaOH, pH 7.9, 0.2 mM EDTA, pH 8.0, 0.25% Nonidet
P-40, and 20% glycerol, the lysate was stored at
80 °C. For gel
shift analysis, a double-stranded oligonucleotide with the GAS from the
human IRF-1 gene promoter (5'-CCCCTGATTTCCCCGAAATGACCCC-3') (49) was
end-labeled with [
-32P]ATP using T4 polynucleotide
kinase according to standard protocols. Five µg of total cell extract
were incubated with labeled probes (2-3 × 104 cpm)
for 20 min at room temperature in 20 µl of buffer containing 10 mM Tris, pH 7.5, 2 mM MgCl2, 25 mM NaCl, 1 mM dithiothreitol, 1 mM
EDTA, 5% glycerol, 0.3 mg/ml bovine serum albumin, and 2 µg of
poly(dI-dC). The reactions were analyzed by electrophoresis in a
nondenaturing 5% polyacrylamide gel in 0.5× Tris-borate-EDTA buffer.
The gels were then dried and exposed at
80 °C for autoradiography. For supershift analysis, total cell extracts (5 µg) were incubated with 3 µg of anti-STAT-1, anti-STAT-3, anti-STAT-4, and an unrelated control antibody (SantaCruz Biotechnology, Inc., Santa Cruz, CA) prior
to the addition of the radiolabeled IRF-1 GAS oligonucleotide.
 |
RESULTS |
IRF-1 Expression in Human Th1 and Th2 Cells--
Recent reports
have demonstrated that IRF-1-deficient mice lack the capacity to
develop a Th1 response (33, 34). To study whether the transcription
factor IRF-1 may also play a role in human T helper cell development,
we have analyzed expression of IRF-1 transcripts in human Th1 and Th2
cells generated from cord blood (21, 50). The T helper phenotype has
been determined by the analysis of intracellular IFN-
and IL-4
production after restimulation of T cells with phorbol 12-myristate
13-acetate and ionomycin (Fig. 1).

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Fig. 1.
Cytokine profiles of human Th1 and Th2 cell
lines generated from cord blood leukocytes. Human Th1 and Th2 cell
lines were generated by stimulating cord blood leukocytes that had been
depleted of CD8+ cells with mitogen in the presence of
IL-12 and anti-IL-4 mAbs and in the presence of IL-4 and anti-IL-12
mAbs, respectively. Cells were harvested 7 days after stimulation, and
the intracellular production of IL-4 and IFN- was analyzed by flow
cytometry as described under "Experimental Procedures."
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We examined the expression of IRF-1 transcripts in Th1 and Th2 cells
treated with or without IL-12 or IFN-
by RNase protection assays. We
could not detect significant differences of the IRF-1 mRNA levels
in human Th1 and Th2 cells 2 weeks after priming (Fig. 2A, lanes 1 and 7).
Interestingly, we found that IRF-1 expression is clearly induced by
IL-12 in Th1 but not in Th2 cells (Fig. 2A, lanes 2, 3, 8, and 9). The IL-12-induced up-regulation of IRF-1 transcripts
is not blocked by cycloheximide indicating that de novo
protein synthesis is not required for IL-12 action (Fig. 2A, lane
4). Treatment of Th1 cells with cycloheximide in the absence of
IL-12 does not result in an increased IRF-1 mRNA expression (data
not shown). IFN-
induced IRF-1 transcripts in both T helper subsets
(Fig. 2A, lanes 5, 6, 10, and 11). A quantitative
analysis of the results is shown in Fig. 2B. Treating Th1
cells for 4 h with IL-12 resulted in a 5-fold up-regulation of
IRF-1 transcripts; a 9-fold up-regulation was detected after 16 h.
Consistent with previously published data (51), we observed that
IFN-
up-regulates IRF-1 in human Th2 but not Th1 cells (data not
shown). These results demonstrate that IRF-1 is a target gene of IL-12
in human Th1 cells.

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Fig. 2.
IL-12 induces IRF-1 transcripts in human Th1
cells. A, Th1 and Th2 lines generated from cord blood
were harvested 2 weeks after stimulation. The cells were treated as
indicated with IL-12 (2.5 ng/ml) or IFN- (1000 units/ml) followed by
the extraction of total RNA. To determine whether de novo
protein synthesis is required for IL-12-induced up-regulation of IRF-1
transcripts, cells were treated with the protein synthesis inhibitor
cycloheximide (CHX) (10 µg/ml) 45 min prior to the
addition of IL-12 (2.5 ng/ml). RNA was extracted 16 h after the
addition of IL-12. Transcripts encoding IRF-1 (top panel)
and 18 S RNA as loading control (bottom panel) were analyzed
by ribonuclease protection assays as described under "Experimental
Procedures." B, the radioactivity present in the protected
fragments was quantitated using a MolecularImager. The black
bars correspond to the relative expression levels of IRF-1
mRNA after normalization with an 18 S RNA probe.
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|
STAT-4 Binds to a GAS Element within the IRF-1 Promoter--
To
analyze whether the IL-12-induced expression of IRF-1 is mediated by a
specific STAT protein, we performed gel-shift experiments using a
specific oligonucleotide corresponding to the GAS within the IRF-1
promoter (49, 52). Total cell extracts were prepared from untreated Th1
and Th2 cells or from cells incubated with IL-12, IFN-
, IFN-
, and
IL-4 and were used in gel-shift experiments shown in Fig.
3. Consistent with our previous findings
(21) and those of other laboratories (19, 20, 53), IL-12 activated a
STAT complex only in Th1 cells but not in Th2 cells. IFN-
strongly induced two STAT complexes with different electrophoretic mobility both
in Th1 and Th2 cells, whereas only Th2 cells were sensitive to IFN-
.
The activation of a STAT molecule by IFN-
in Th2 but not in Th1
cells correlates with the selective expression of the IFN-
receptor
chain on Th2 cells (51, 54, 55).

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Fig. 3.
Cytokines induce binding of transcription
factors to the GAS element in the IRF-1 promoter. Th1 and Th2
cells were harvested 6 days after priming, washed, and resuspended in
complete medium. 107 cells were incubated at 37 °C in 1 ml of complete medium with or without IL-12 (2.5 ng/ml) for 1 h or
IFN- (1000 units/ml), IFN- (1000 units/ml), or IL-4 (1 ng/ml) for
15 min, followed by the preparation of whole cell extracts. Gel shift
assays were performed with a 32P end-labeled double
stranded oligonucleotide corresponding to the GAS element present
within the promoter of the IRF-1 gene, as described under
"Experimental Procedures."
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In order to define which STAT molecules bind to the IRF-1 GAS element
following IL-12 treatment, we performed supershift assays using
specific antibodies against STAT proteins. As shown in Fig. 4, anti-STAT-1 antibodies supershifted
the fast migrating complex, whereas the anti-STAT-4 antibodies
abrogated the slow migrating complex induced by IFN-
both in Th1 and
Th2 cells. These findings are consistent with the previously described
observation that IFN-
activates STAT-4 in human mitogen-activated
lymphoblasts and in human Th1 and Th2 cells (50, 56). The complex
induced in IL-12-treated Th1 cells is abrogated with STAT-4 specific
antibodies, confirming previously published results (17, 20, 21).
Addition of anti-STAT-3 antibodies did not significantly change the
DNA-binding complexes, suggesting that STAT-3 does not play a major
role in activating the IRF-1 promoter in response to the cytokines
tested in this study. These results suggest that STAT-4 can induce
IRF-1 expression in Th1 cells in response to IL-12.

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Fig. 4.
IL-12 induces binding of STAT-4 to the IRF-1
GAS element in human Th1 cells. Supershift assays were performed
as described in Fig. 3; however, extracts were preincubated with
antibodies specific for STAT molecules prior to the addition of the
32P end-labeled double stranded oligonucleotide
corresponding to the IRF-1 GAS element. Somewhat reduced band
intensities were also observed in extracts preincubated with unrelated
control antibodies (data not shown). The weaker signal in the samples
pretreated with anti-STAT-1 and anti-STAT-3 antibodies therefore does
not appear to be a specific effect of these antibodies.
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|
Mechanisms Underlying the IRF-1 Expression by IL-12--
We next
analyzed the mechanism by which IL-12 induces IRF-1 expression. Three
copies of the GAS element present in the IRF-1 promoter were inserted
upstream of a luciferase reporter gene. The resulting construct, termed
IRF-1-luc, was transiently transfected in Jurkat cells. Because Jurkat
cells do not respond to human IL-12, we cotransfected expression
vectors encoding human IL-12R
1, IL-12R
2, and STAT-4. The cells
were treated for 20 h with or without IL-12 prior to the
preparation of cell extracts. Strong IL-12-dependent
activation of the IRF-1-luc construct was detected when the components
of the IL-12 signaling machinery, i.e. IL-12R
1, IL-12R
2, and STAT-4, were simultaneously expressed in Jurkat cells
(Fig. 5). In contrast, no
IL-12-dependent induction of luciferase activity was
observed when the cDNAs encoding IL-12R
2 or STAT-4 were omitted.
Treatment of transfected Jurkat cells for 6 h with IL-12 also
resulted in increased luciferase activity (data not shown). To exclude
the possibility that the up-regulation of IRF-1 is secondary to IFN-
production induced in response to IL-12 we determined IFN-
in the
supernatant of the transfected cells by enzyme-linked immunosorbent
assays. There was no detectable IFN-
-production by the transfected
cells, indicating that IL-12-dependent induction of
luciferase activity is not mediated by IFN-
(data not shown). These
results provide strong evidence that IL-12-induced activation of the
IRF-1 gene is mediated by STAT-4 and depends on the presence of
functional IL-12 receptors on the cell surface. Moreover, our data
suggest that IRF-1 expression in human Th1 cells is regulated by IL-12
through STAT-4 activation.

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Fig. 5.
The IL-12-induced activation of an IRF-1 GAS
reporter gene construct is mediated by STAT-4. Jurkat T cells were
transiently transfected with a reporter gene construct containing three
copies of the GAS element present in the IRF-1 gene promoter. Where
indicated, cells were co-transfected with expression vectors encoding
IL-12R 1, IL-12R 2, and/or STAT-4. Cells were left untreated
(unstim.) or were treated for 20 h with 2.5 ng/ml IL-12
(IL-12) prior to the preparation of lysates. Luciferase
assays and normalization of results were performed as described under
"Experimental Procedures."
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Regulation of IRF-1 Expression in Differentiating T Helper
Cells--
Two recent studies have demonstrated that IRF-1-deficient
mice show a striking defect in the development of Th1 cells (33, 34).
Interestingly, these two reports differ in their conclusions regarding
the relevance of IRF-1 in CD4+ T cells. To determine the
role of IRF-1 in the development of human T helper cells, we analyzed
the expression of IRF-1 along the differentiation of naive T cells
under neutral, Th1-polarizing, or Th2-polarizing conditions. Purified
CD4+, CD45RO
T cells isolated from cord blood
were stimulated with plate-bound anti-CD3 mAb in the absence of
exogenously added cytokines, in the presence of IL-12 and neutralizing
anti-IL-4 mAb to induce Th1 development, or in the presence of IL-4 to
promote Th2 development. The expression of transcripts encoding
IL-12R
2 was used to monitor T helper cell differentiation (Fig.
6A, middle panel). Consistent with our previous findings, IL-12R
2 transcripts were not detectable in purified naive T cells but were induced by T cell receptor triggering. Transcripts encoding the IL-12R
2 subunit were expressed at significantly higher levels in T cells stimulated in the presence of
IL-12 than in T cells stimulated in the presence of IL-4 or without
exogenously added cytokines (21). Next, we analyzed IRF-1 expression in
differentiating Th1 and Th2 cells (Fig. 6A, top panel).
IRF-1 transcripts were detectable in naive CD4+ T cells
(Fig. 6A, lane 1) but were strongly up-regulated by TCR triggering (lane 2). IRF-1 mRNA was slightly more
abundant in cells stimulated in the presence of Th1-inducing conditions
than in cells stimulated in the presence of IL-4 or with anti-CD3 mAb alone (Fig. 6A, lanes 2-10). IL-4 does not suppress
expression of IRF-1 transcripts to the same extent as it does with
IL-12R
2 transcripts. Only a slight down-regulation of IRF-1 mRNA
is detectable when comparing cultures stimulated in the presence of
IL-4 or with anti-CD3 mAb alone (Fig. 6A, compare lane
2 with 4 and lane 5 with 7). A
quantitation of the data is shown in Fig. 6B. These results
show that T cell receptor triggering strongly induces expression of
IRF-1 irrespective of whether the stimulation was performed in the
presence of Th1- or Th2-inducing cytokines.

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Fig. 6.
Antigen receptor triggering induces
expression of IRF-1 transcripts in naive CD4+ T cells.
CD4+/CD45RO T cells were purified by negative
selection from cord blood as described under "Experimental
Procedures." Purified CD4+/CD45RO T cells
(3 × 106) were stimulated with plate-bound anti-CD3
mAb with or without the addition of IL-12 (2.5 ng/ml) or IL-4 (1 ng/ml). RNA was extracted from unstimulated
CD4+/CD45RO T cells (lane 1) or at
the indicated time after CD3-stimulation. Transcripts encoding IRF-1
(top panel), IL-12R 2 (middle
panel), and 18 S RNA as loading control (bottom
panel) were quantitated in RNase protection assays. B,
the radioactivity present in the protected fragments was quantitated
using a MolecularImager. The open and filled bars
correspond to the relative expression levels of IL-12R 2 and IRF-1
mRNAs, respectively. Results were normalized using an 18 S RNA
fragment.
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 |
DISCUSSION |
In this study, we analyzed whether the transcription factor IRF-1
plays a role in the differentiation of human naive CD4+ T
cells into polarized T helper cell subsets. Previous results obtained
with IRF-1-deficient mice revealed a striking deficiency of Th1 cell
development. Compromised Th1 development was shown to be associated
with an impaired production of IL-12 by macrophages (33, 34) and
defective development of natural killer cells (41-43). It remained
controversial whether compromised Th1 development also resulted from a
defect of CD4+ T cells from IRF-1
/
mice to
develop into Th1 effector cells. Cell transfer experiments indicated
that IRF-1
/
CD4+ T cells could develop into
Th1 cells when transferred to recombination activating gene-1-deficient
(IRF-1+/+) mice, lacking T and B cells (34). However, the
analysis of CD4+ T cells purified from
IRF-1
/
TCR transgenic mice showed a deficiency in Th1
development when stimulated in vitro with peptide presented
by wild-type antigen presenting cells (33). Here, we have examined
IRF-1 expression and regulation along human T helper cell differentiation.
Analysis of IRF-1 transcripts in human Th1 and Th2 cells generated from
cord blood leukocytes 2 weeks after stimulation revealed no significant
differences in the two populations. However, after treatment with
IL-12, IRF-1 transcripts were strongly induced in Th1 but not in Th2
cells. This finding demonstrates that IRF-1 is a target gene of IL-12.
The IL-12-induced up-regulation of IRF-1 transcripts could already be
detected after 4 h and did not depend on de novo
protein synthesis, arguing against a potential secondary effect
mediated by IFN-
induced in response to IL-12. In addition, previous
results have shown that Th1 cells do not respond to IFN-
, because
they do not express the IFN-
R
subunit (Fig. 3 and Refs. 51, 54,
and 55).
Previous studies have demonstrated that induction of the IRF-1 gene in
response to interferons and IL-6 is mediated by the binding of
activated STAT-1 and STAT-3 to the palindromic GAS element present in
the promoter of the IRF-1 gene (49, 52, 57-59). In this report, we
demonstrate that IL-12 induces IRF-1 gene transcription via activated
STAT-4. We observed strong binding of an IL-12-induced factor to the
GAS element of the IRF-1 promoter in extracts prepared from Th1 cells;
conversely, IFN-
induced a gel shift only in Th2 cells. These
results are consistent with the previously described selective
expression of the IL-12 receptor
2 (IL-12R
2) subunit and of the
IFN-
receptor
(IFN-
R
)-chain on Th1 and Th2 cells,
respectively (21, 51, 53-55). The IL-12- and IFN-
-induced
protein-DNA complexes migrated with a different mobility, indicating
the binding of different STAT molecules to the IRF-1 GAS element. To
determine which STAT molecules activated in response to IL-12 and
IFN-
bound to the IRF-1 GAS element, we used specific antisera
against STAT-1, STAT-3, and STAT-4 to supershift the protein-DNA
complexes. These experiments revealed that the faster migrating complex
formed in response to IFN-
contains mainly STAT-1, whereas the
slower migrating band induced by IL-12 contains STAT-4. Binding of
STAT-4 to the IRF-1 GAS element is also induced by IFN-
, confirming
the recently demonstrated IFN-
-induced activation of STAT-4 in human
mitogen-stimulated lymphoblasts (56) and in human Th1 and Th2 cells
(50). Taken together, the gel shift experiments demonstrated that
activated STAT-4 binds to the GAS element in the IRF-1 promoter. It is
also of interest to note that the optimal recognition sequences for STAT-4, TTCCGGGAA (32) and (T/A)TTCC(C/G)GGAA(T/A) (60), as determined
by binding site selection, are very similar to the DNA sequence of the
GAS element in the human IRF-1 promoter, TTTCCCCGAAA (49, 52).
Transient transfection assays with a reporter gene construct containing
three copies of the IRF-1 GAS element in Jurkat cells provided strong
evidence that the transcriptional activation of the IRF-1 gene in
response to IL-12 is mediated by STAT-4. Because Jurkat cells do not
express detectable levels of functional IL-12 receptors and STAT-4
(data not shown), we co-transfected expression constructs encoding
IL-12R
1, IL-12R
2, and STAT-4. A strong
IL-12-dependent activation of the IRF-1 GAS reporter gene
construct was observed when the components of the IL-12 signaling
machinery are co-expressed in T cells.
To determine the role of IRF-1 in the differentiation of T helper
subsets, we analyzed IRF-1 expression at different time intervals early
after the stimulation of naive T cells under neutral, Th1-inducing, or
Th2-inducing conditions. We compared IRF-1 expression to the expression
of the IL-12R
2 subunit, which is induced during differentiation of
human naive cells along the Th1 but not the Th2 pathway (21, 53). In
contrast to IL-12R
2, IRF-1 transcripts are detectable in naive
CD4+ T cells. Cross-linking of the TCR strongly induces
IRF-1 mRNA even in the absence of exogenously added cytokines. At
later time intervals after T cell stimulation, IRF-1 transcripts were
slightly more abundant in cultures that were stimulated in the presence of IL-12. Consistent with the lack of functional IL-12 receptors, we
did not observe any IL-12-induced up-regulation of IRF-1 transcripts in
naive T cells (data not shown). Taken together, these data show that
activated STAT-4 is able to transactivate IRF-1 expression in
differentiating Th1 cells accounting for a sustained expression of
IRF-1.
In conclusion, our findings point to a potential hierarchy of
transcriptional events during the differentiation of naive T cells into
polarized Th1 cells; TCR ligation is an essential prerequisite for the
initial expression of functional IL-12 receptors on developing Th1
cells, although the mechanisms and factors regulating this phase have
not yet been uncovered. In the second phase, IL-12 induces further
up-regulation of the IL-12R
2, IRF-1, and other, yet to be
identified, IL-12-regulated genes. The rapid tyrosine phosphorylation
and activation of STAT-4 by IL-12 and the phenotype of the
STAT-4
/
mice predict that this phase is regulated by
STAT-4. In a third phase, transcription factors, such as IRF-1, and
other, so far unknown, regulatory proteins induced by STAT-4 may
themselves regulate target genes that are important for the effector
functions of Th1 cells. IRF-1 appears to be the first member of a
probably large family of regulatory proteins induced by IL-12. It will be of great interest to identify additional members of this family and
to analyze their functions with respect to the differentiation and
effector functions of T helper cells.
 |
ACKNOWLEDGEMENTS |
We thank T. Taniguchi for pUC IRF-1, M. Gately for the IL-12 and anti-IL-12 antibodies, U. Gubler and D. Presky
for pBOS-IL-12R
1 and pBOS-IL-12R
2 expression constructs, and R. Chizzonite for pBOS-Stat4.
 |
FOOTNOTES |
*
This work was supported in part by the Project on
Tuberculosis of the Istituto Superiore di Sanità.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.
To whom correspondence should be addressed. Tel.:
39-02-2884-804; Fax: 39-02-2153-203; E-mail:
lars.rogge{at}roche.com.
 |
ABBREVIATIONS |
The abbreviations used are:
Th1, T helper type
1;
IL, interleukin;
IRF-1, interferon regulatory factor-1;
STAT, signal
transducer and activator of transcription;
IFN, interferon;
GAS, IFN-
-activated sequence;
TCR, T cell antigen receptor;
NF-AT, nuclear factor of activated T cell;
mAb, monoclonal antibody.
 |
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