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*
Lymphocyte Cell Biology Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal Diseases, National Institutes of Health, and
Division of Cytokine Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible genes. We
identified several genes up-regulated by IL-12, namely, MIP-1,
MIP-1ß, IL-1RA, and IFN regulatory factor-1 (IRF-1). IRF-1 is a
transcription factor regulated by IFNs that is also essential for Th1
responses. We demonstrated that IL-12 directly up-regulates IRF-1 to
the same extent as IFN- in normal human T cells and in NK cells. We
showed that IL-12 had a direct effect on IRF-1, an effect not mediated
indirectly by the induction of IFN-
production. Furthermore, IL-2
and IL-12 synergistically induced IRF-1, whereas IFN- and IL-12 did
not. The participation of STAT4 in the regulation of IRF-1 was
demonstrated in two ways. First, STAT4 was required for the
IL-12-dependent transactivation of an IRF-1 reporter construct, and
second, STAT4 binding to the IRF-1 promoter was shown using EMSA. In
contrast to IL-12, no up-regulation of IRF-1 was found in
IL-4-stimulated cells, and IL-4 did not block IL-12-dependent
up-regulation of IRF-1. Therefore, IRF-1 may be an important
contributor to IL-12 signaling, and we speculate that the defective
IL-12 responses seen in IRF-1-/- mice might be
attributable, in part, to the absence of this transcription
factor. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(2). IL-12 induces tyrosine
phosphorylation of JAK2 and TYK2 (3), which, in turn, phosphorylate
other substrates, including the transcription factors STAT4, STAT1 (4),
and STAT3 (5, 6); STAT4-/- mice, like
IL-12-/- mice, have defective Th1 differentiation and
impaired cell-mediated immunity. Despite the importance of IL-12, there
is relatively little information pertaining to IL-12-inducible genes
and genes known to be regulated by STAT4 (7). In contrast to IL-12,
however, many IFN-inducible genes have been characterized (8, 9). While
it is clear that STAT1 is essential for many IFN- responses,
IFN-, like IL-12, activates STAT4. In addition, both IFN- and
IL-12 activate TYK2 (10, 11). This suggests that IFN- and IL-12 may
share a subset of target genes. The similarity in IFN- and IL-12
signaling prompted us to define commonalities and differences in genes
regulated by IFN- and IL-12.
One well-characterized IFN-inducible gene is IFN regulatory factor-1
(IRF-1)2 (12). Interestingly,
IRF-1 knockout mice have a broad range of defects. For instance,
IRF-1-deficient mice have impaired ability to undergo DNA
damage-induced cell cycle arrest (13, 14). These mice also have defects
in inflammatory and innate immune responses (15) and disruption of NK
and T cell development (16, 17). Importantly, IRF-1 knockout mice
demonstrate the involvement of IRF-1 in multiple stages of the Th1
differentiation (18, 19). IRF-1-/- mice have defective
IL-12 and IFN- production and exaggerated IL-4 secretion. The lack
of NK cells in IRF-1-/- mice contributes, in part, to the
impairment in IFN- production. In addition, there is defective IL-12
production, which has been attributed to the presence of a potential
IRF-1-responsive element in the IL-12p40 gene (20). Most importantly,
however, in addition to impaired IL-12 production, CD4+ T
cells from IRF-1-/- mice have been shown to have impaired
responsiveness to IL-12, as measured by IFN- production after IL-12
stimulation (18, 19).
Thus, although IRF-1 was initially described as an IFN-inducible
transcription factor, the phenotype of IRF-1-deficient mice is
dramatically different from that of IFN- or IFN-ß
receptor-deficient mice, which argues that IRF-1 serves functions other
than simply transmitting IFN-mediated signals (reviewed in 21).
This was the second issue that led us to investigate the potential
regulation of IRF-1 by IL-12.
In the present study, we report that IFN- and IL-12 up-regulate a
number of genes in common, including macrophage-inflammatory protein
(MIP)-1, MIP-1ß, and IL-1RA. Importantly, in addition to IFN-,
IL-12 and IL-2, but not IL-4, directly up-regulate IRF-1 expression.
Thus, although IRF-1 was initially characterized as an IFN-inducible
gene, IRF-1 can be viewed as a transcription factor that is also
regulated by cytokines that participate in Th1 responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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rhIL-12 and a human IFN- immunoassay kit were purchased from
R&D Systems (Minneapolis, MN). Human IL-2 was provided by Dr. C.
Reynolds (National Cancer Institute, Frederick, MD). Polyclonal rabbit
anti-STAT4 Ab was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Rabbit polyclonal anti-STAT1 Ab was provided by Dr. A.
Larner (Cleveland Clinic Foundation, Cleveland, OH). An RNA extraction
kit (RNAgents Total RNA Isolation System) was purchased from Promega
(Madison, WI), and RiboQuant Multiprobe RNase Protection System was
purchased from PharMingen (San Diego, CA). Cell culture supernatants of
cell were collected at 4 h, and IFN- levels were analyzed using
a commercial ELISA kit (R&D Systems) according to the manufacturer’s
instructions. The plasmid p3xGAS-luc, containing the STAT binding site
of the IRF-1 gene (22), was provided by Dr. R. Pine (The Public Health
Research Institute, New York, NY), and pcDNA3-STAT4 was provided
by Dr. J. Ihle (St. Jude Children’s Hospital, Memphis, TN). Plasmids
containing the cDNA for IL-12Rß1 and IL-12Rß2 in expression vector
pEF-BOS were provided by Dr. U. Gubler (Hoffmann-La Roche,
Nutley, NJ). LipofectAMINE was purchased from Life Technologies
(Gaithersburg, MD).
Cells
NK3.3 cells were provided by Dr. J. Kornbluth (Arkansas Cancer Research Center, Little Rock, AR). PBMC from healthy donors were isolated by Ficoll-Paque (Pharmacia, Piscataway, NJ) gradient centrifugation, activated with PHA (2 µg/ml) for 72 h, and cultured for an additional day in the presence of IL-2 (40 IU/ml), as described previously (3, 5).
RNase protection assay and Northern analysis
Total RNA was extracted from cytokine-activated cells using
RNAgents (Promega). The RNase protection assay was conducted as
follows: 32P-labeled RNA probe was synthesized using SP6
RNA polymerase for GAPDH, ISG15, ISG54, and DNA templates or with T7
RNA polymerase for granylate-binding protein (GBP), IFN--inducible
protein-10, IRF-1, and the multiprobe template set (PharMingen). DNA
was digested with DNase I (Boehringer Mannheim, Indianapolis, IN), and
RNA probes were extracted with phenol and chloroform and precipitated
with ethanol. Labeled RNA probes were hybridized overnight at 56°C to
target RNA (5 µg) and were digested with T1 RNase (Life
Technologies). The protected mRNA fragment was extracted with phenol
and chloroform, precipitated with ethanol, resolved on a 6%
denaturating polyacrylamide gel, and subjected to autoradiography. Gene
transcripts were identified by the length of the protected fragments.
Equal loading of RNA was estimated from the amounts of protected
fragments of two housekeeping genes, namely, L32 and GAPDH.
For Northern blots, RNA samples (20 µg) were electrophoresed on 1% agarose gels containing formaldehyde (Ambion, Austin, TX), transferred to BrightStar Plus membranes (Ambion), and cross-linked to the membrane by UV light (UV Stratalinker, Spectronics, Rochester, NY). A 32P-labeled RNA IRF-1 probe was synthesized, and 106 cpm/ml of radiolabeled probe was hybridized to membranes for 16 h. The membranes were washed at 62°C and exposed to film.
Luciferase assay
The NIH 3T3 cells stably transfected with the , ß, and
c chains of the IL-2R and JAK3 (NIH 3T3ßJAK3)
(23) and NIH 3T3 cells (3 x 105 cells per point) were
transfected with 0.3 µg of 3xGAS-luciferase reporter plasmid, the
IRF-1-promoter luciferase construct, pcDNA3-STAT4, pEF-BOS-IL-12Rß1,
and pEF-BOS-IL-12Rß2 using LipofectAMINE (Life Technologies). The
luciferase reporter construct alone (pGL2-B) and that containing the
IRF-1 promoter (1.3-kb 5' flanking region of the human IRF-1 gene) or
the mutant STAT binding element (SBE) site at position -123 of the
IRF-1 promoter (mutant sequence 5'-GATTTCCCCtccAT-3') were provided by
Dr. Thomas A. Hamilton (Cleveland Clinic Foundation, Cleveland, OH)
(24). Transfected cells were stimulated with cytokines, lysed, and
assayed for luciferase activity (Promega).
EMSA
Cell extracts were prepared from cytokine-stimulated NK3.3 cells, and EMSAs were performed as described (25) using a 32P-labeled double-stranded oligonucleotide corresponding to the human IRF-1 promoter (5'-AGCTTCAGCCTGATTTCCCCGAAATGACGGA-3') and a control oligonucleotide (5'-CTGCAGTAACGCCATTTTGCAAGGCATGAA-3'. In supershift assays, complexes were incubated with 1 µl of STAT1 Ab, STAT4 Ab, or preimmune rabbit serum at 4°C for 30 min. The complexes were electrophoresed through a 5% nondenaturing acrylamide gel and subjected to autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and IL-12-induced genes
Since there appear to be some similarities in IFN- and IL-12
signaling, we surveyed IFN-inducible genes to determine whether any of
these were IL-12 inducible. IFN--inducible genes are relatively well
characterized and include GBP, ISG15, and ISG54, as well as MIP-1,
MIP-1ß, and IL-1RA (26, 27, 28); we confirmed this in the experiment
shown in Fig. 1
A. As is
evident in this experiment, IL-12 up-regulated a subset of these genes.
Specifically, as with IFN-, we demonstrate that IL-12 up-regulated
IL-1RA, MIP-1, and MIP-1ß. In contrast, IL-12 did not up-regulate
GBP, ISG15, and ISG54. This is not unexpected, as the latter genes
contain IFN-stimulation response element in their promoters, which bind
STAT1/STAT2/p48 complexes, and IL-12 has not been shown to activate
STAT2. IL-12 has previously been shown to up-regulate granzyme B (29).
Interestingly, we found that IFN- also up-regulated this gene.
Notably, we found that another gene implicated in the apoptotic
pathway, RVP1, was up-regulated by both IFN- and IL-12. As control,
the expression of DAD1 is shown; neither IFN- nor IL-12 affected
expression of this gene or GAPDH.
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Because of the number of genes we found that were commonly
regulated by IFN- and IL-12, we next analyzed another gene typically
induced by IFNs, namely IRF-1, a well-characterized transcription
factor that is important in regulating a variety of genes. We therefore
sought to determine whether IRF-1 was uniquely regulated by IFN- or
whether IL-12 also up-regulated its expression. Because NK3.3 cells are
a useful model system to study IL-12 signaling, we first tested
inducibility in these cells. As shown in Fig. 1
B, IFN-
induced expression of IRF-1 as expected. Interestingly, IL-12 also
induced IRF-1 mRNA expression to the same extent as IFN- (Fig. 1B, lane 3 vs lane 2); increased IRF-1
mRNA accumulation in IL-12-stimulated NK3.3 cells was reproducibly
detected in 10 different experiments. Because IL-12 and IL-2 have
overlapping effects on some genes (e.g., IFN-; data not shown), we
next sought to determine whether IL-2 had similar or distinct effects
relative to IL-12 and IFN-. We found that IL-2 stimulation also
induced IRF-1 mRNA (Fig. 1B, lane 4).
To confirm that IL-12 and IL-2 up-regulated IRF-1 in normal human T
cells, we stimulated activated T cells with IFN-, IL-12, and IL-2.
As shown in Fig. 1C, stimulation with IFN-, IL-12, and
IL-2 induced IRF-1 mRNA up-regulation (lanes 2–4);
increased IRF-1 mRNA accumulation in human T cells was reproducibly
detected in five different experiments, using cells from five different
donors. The basal level of IRF-1 was higher in T cells than in NK3.3
cells, presumably because it was necessary to preactivate T cells (see
Materials and Methods) to induce IL-12R and IL-2R -chain
expression. Therefore, we used NK3.3 cells in subsequent experiments.
Since IRF-1 is regulated by IFNs, and since IL-12 and IL-2 are potent
inducers of IFN-, we next considered the possibility that the IL-12
and IL-2 induction of IRF-1 was indirectly mediated by the production
of IFN-. We used several approaches to address this issue. First, we
directly examined the effect of IFN- on IRF-1, and under conditions
tested, stimulation of the NK3.3 cells with IFN- did not induce
IRF-1. The lack of effect of IFN- was due to an absence of the
IFN--R ß chain in this cell line, as determined by RNase
protection assay. In addition, we attempted to inhibit the IRF-1
induction with Abs against IFN-. Anti-IFN- antibodies have no
effect on IL-12-up-regulation, whereas anti-IL-12 Abs completely
inhibited its induction of IRF-1. Finally, we also measured the amount
of IFN- in the culture supernatants after 4 h of stimulation
and found that no IFN- was produced at this time point (data not
shown).
To confirm that the enhancement of expression of IRF-1 by IL-12 was a
direct effect, we next added cycloheximide (CHX), a potent protein
synthesis inhibitor, to our cultures. As shown in Fig. 2B, CHX did not inhibit the
IL-12 induction of IRF-1 mRNA; in fact, CHX augmented the induction
(lane 3 vs lane 2). This was not due to
CHX alone, since CHX, in the absence of stimulation, did not alter
IRF-1 mRNA levels as compared with the control (data not shown). This
up-regulation was reproducibly detected in six different experiments
and suggests the existence of a labile inhibitor.
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. As shown in Fig. 3A, IL-12, IL-2, and IFN-
induced IRF-1 mRNA (lanes 2–4). The combination of
IFN- and IL-12 induced IRF-1 mRNA to the same extent as IL-12 alone
(Fig. 3A, lane 6), whereas IL-12 and IL-2 had a
synergistic effect (Fig. 3A, lane 5). As a
control, the induction of ISG15 was also assessed; IL-2 and IL-12
either separately or in combination had no effect on this gene (Fig. 3A, lanes 2, 3, and 5). To
confirm that the effect of the combination of IL-2 and IL-12 was direct
and not mediated by the induction of another cytokine, the experiment
was repeated using pretreatment with CHX. As shown in Fig. 3B, IL-12, IL-2, and IFN- induced IRF-1 expression
(lanes 2–4). When tested together, IL-12 and IL-2
again had a synergistic effect (lane 5) in the
presence of CHX, whereas IL-12 and IFN- did not (lane
6). These results determined by RNase protection assay were also
confirmed by Northern analysis (Fig. 3C). Densitometry
showed an 18-fold induction of IRF-1 with IL-12, a 15-fold induction
with IL-2, and a 43-fold induction with the combination of both
cytokines.
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treatment, quantitating the fold induction of IRF-1 by densitometry. As
shown in Fig. 4, CHX alone had no effect
as compared with the untreated cells (Ctl lanes). Both with or without
CHX, IL-12 had little effect on IRF-1 expression after 30 min or 1
h, but the effect was more pronounced after 4 h (14- and 33-fold
induction, respectively). In contrast, IRF-1 induction by IFN- is
maximal after 1-h stimulation (23-fold induction) and declined by
4 h. Since both IL-12 and IFN- exert their effect by similar
mechanisms, there is no obvious explanation for this difference. One
possible explanation would be that IL-12 activates IRF-1 through STAT4,
whereas the major STAT induced by IFN- is STAT1. Perhaps
differential levels of expression of these STATs can explain the
different kinetics of IRF-1 induction.
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STAT4 serves an essential role in transmitting some but not all
IL-12-dependent signals (30, 31). To determine whether STAT4 was
involved in IL-12-induced expression of IRF-1, we assayed
transactivation of a luciferase reporter gene driven by the STAT
binding site from the IRF-1 gene (22). Since T cells and NK cells
already express STAT4, confounding any conclusions, we performed these
experiments in fibroblasts (NIH 3T3) specifically because they lack
STAT4. NIH 3T3 cells were transiently transfected with this reporter
gene construct along with cDNAs encoding STAT4 and IL-12R subunits. As
seen in Fig. 5A, IL-12 induced
transactivation of the reporter construct in cells transfected with
receptor subunits and STAT4, but not in cells transfected with
receptor subunits alone, suggesting that STAT4 is required for
IL-12-induced activation of the IRF-1 gene. IL-2 also induced
transactivation of the luciferase reporter gene in NIH 3T3ßJAK3
cells, but this did not require STAT4. This was expected, as IL-2 has
been shown to activate STAT1, STAT3, STAT5a, and STAT5b. To
confirm that IRF-1 transactivation was mediated by STAT4, we
transfected NIH 3T3 cells with a plasmid construct in which a reporter
gene was linked to a fragment of the IRF-1 gene containing the
transcription promoter-enhancer region (24). As shown in Fig. 5C, this promoter fragment was able to respond to IL-12, and
this response was dependent on an intact SBE at position -123 to
-113, as indicated by the loss of IL-12 sensitivity when the SBE was
specifically altered by mutagenesis. Importantly, STAT4 was required
for IL-12 induction of the IRF-1 promoter.
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D, stimulation of NK3.3 cells with IL-12 resulted in
formation of DNA binding complexes with the human IRF-1 promoter. These
complexes were specific because they were present in the stimulated
cell only (Fig. 5D, lane 4 vs lane 3),
were formed with the IRF-1 probe but not with the control probe (Ctl)
(Fig. 5D, lanes 1 and 2), and were
abolished with an excess of unlabeled IRF-1 probe (data not shown).
Supershift experiments (Fig. 5E) with anti-STAT4 Abs
showed that the EMSA signal detected after IL-12 stimulation contained
STAT4 complexes (lane 3). A control Ab (Ctl) had no
effect on the pattern of the complexes (Fig. 5E, lane
4), and none of the Abs had an effect on the unstimulated cells
(data not shown). IL-4 does not up-regulate IRF-1 expression and fails to inhibit IL-12-dependent up-regulation of IRF-1
The phenotype of IRF-1-/- mice indicates that IRF-1
is important for Th1 differentiation. In contrast to IL-12, IL-4
promotes Th2 responses; therefore, we next sought to determine whether
IL-4 also regulated IRF-1 expression. In addition, IL-4 has been shown
to inhibit IL-12-dependent induction of IFN- (32) and to inhibit
IFN--dependent up-regulation of IRF-1 (24). We therefore also
analyzed whether IL-4 blocked IL-12-dependent IRF-1 induction. As shown
in Fig. 6, whereas IL-12 up-regulated
IRF-1 (lane 2), no up-regulation of IRF-1 was found
in IL-4-stimulated cells (lane 3). Similar results
were found using T cells (data not shown). IL-4 was active, since it
induced the expression of IL-4R (data not shown) and inhibited the
IL-12 induction of IFN- gene (Fig. 6, lane 4).
Interestingly, IL-4 treatment did not block IL-12-dependent
up-regulation of IRF-1 (Fig. 6, lane 4). These data are
important because they not only indicate that a Th2 cytokine does not
induce IRF-1 but also because they confirm that IL-12 directly
up-regulates IRF-1. That is, although IL-4 inhibited IL-12-mediated
IFN- production, IL-12-dependent up-regulation of IRF-1 was
unaffected. This further demonstrates that the IL-12-mediated induction
of IRF-1 is independent of IFN-.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-12 signaling suggested that these cytokines
may regulate some genes in common. Moreover, the importance of IFN-
as an immunoregulatory cytokine has been suggested by several findings.
First, IFN- has been shown to increase the frequency of
IFN--secreting CD4 Th cells and therefore favors the induction of
Th1 cells (33). Second, IL-12R ß2 subunit, which is expressed only on
Th1 but not Th2 clones, is induced by IL-12 and IFN- during
differentiation of human naive cells along the Th1 but not Th2 pathway
(34). For these reasons we thought it important to begin cataloging
genes induced uniquely and commonly by IL-12 and IFN-.
We show here that IL-12 up-regulated some genes (MIP-1, MIP-1ß,
and IL-1RA) that had previously been shown to be up-regulated by
IFN-. Interestingly, it has been reported that Th1 but not Th2 cells
produce MIP-1, MIP-1ß (35), and IL-1RA (36), and that these
chemokines are efficient chemoattractants specifically for Th1 cells,
(37). Conversely, although granzyme B is recognized as an
IL-12-inducible gene, IFN- also up-regulated this gene.
Additionally, although RVP1 was not previously recognized as being
regulated by either IL-12 or IFN-, its expression was regulated
by both of these cytokines. In contrast, GBP, ISG15, and ISG54,
each of which contains IFN-stimulation response element in its
promoter region, were specific for IFN-.
We also report that IL-12, as well as IL-2, induced IRF-1 gene
expression. While IRF-1 knockout mice have a profound defect in Th1
development and cell-mediated responses, the mechanisms underlying this
defect are only partly understood. IL-12 is critical for cell-mediated
immunity, and IRF-1 likely contributes to the regulation of the
IL-12p40 gene. In addition, a major source of IFN- in the initial
phase of the Th1 response is thought to be NK cells (1), and
IRF-1-/- mice have impaired NK cell development. This may
be because IRF-1 contributes to IL-15 gene regulation and IL-15 is
important in NK cell differentiation (16). Thus, the poor IFN-
production in IRF-1-/- is related in part to the absence
of IL-12 and to the lack of NK cells.
But does this completely explain the Th1 defect in
IRF-1-/- mice? The answer appears to be no, because
IRF-1-/- mice have also been shown to have impaired
responses to IL-12 (18, 19). Stimulation of CD4+ T cells
from IRF-1-/- mice with IL-12 induces substantially less
IFN- than wild type, even though expression of IL-12Rß1 and
IL-12Rß2 is reportedly normal. Thus, it was suggested that impaired
IL-12 production in IRF-1-/- mice alone does not account
for the Th2-dominant response observed. Based on the present results,
it can be proposed that the IL-12-up-regulation of IRF-1 is important
for the action of this cytokine; the unresponsiveness of
IRF-1-/- mice to IL-12 may be related in part to the lack
of up-regulation of this transcription factor. It is important to
emphasize, however, that our findings in an NK cell line and peripheral
blood T cells may not directly relate to the process of Th1
development. In addition, while our results that IL-12 induces IRF-1
expression may shed light on the defective IL-12 responses in
IRF-1-/- mice, IL-12 target genes are still poorly
characterized, and it is not immediately clear how IRF-1 contributes to
IL-12 signaling. Nonetheless, our results clearly indicate that IL-12
and IFN- activate a number of genes in common, including the
transcription factor IRF-1. CHX did not inhibit IL-12 (nor
IL-2)-induced IRF-1 expression, which emphasize the direct role of
these cytokines in the up-regulation of IRF-1. Indeed, CHX enhanced
the IRF-1 induction mediated by IL-12. What this labile inhibitor
of IRF-1 expression is remains obscure. Whether members of the PIAS
family or the CIS/SOCS/JAB/SSI family contribute to this inhibition is
unknown but is presently under investigation. Another possible
interpretation would be that IL-12 mediates its effect on the IRF-1
gene by message by inhibiting a degradative enzyme. Since there is no
superinduction of IRF-1 by IL-2, it could postulated that IL-2 is
acting on the transcriptional level, while IL-12 acts on the
posttranscriptional level.
It has been shown that STAT4 is essential for many IL-12 effects and is
required for the Th1 response (30, 31). We show here that STAT4 appears
to be important for the IL-12-dependent regulation of IRF-1. We have
demonstrated that IRF-1 transactivation by IL-12 was mediated by STAT4
binding and that the SBE present on the IRF-1 promoter-enhancer region
was necessary (Fig. 5C) and sufficient (Fig. 5A)
for the IRF-1 induction. We used fibroblasts in our studies because
they lack STAT4 and thus provide a useful vehicle for testing STAT4
dependence of IL-12 transactivation of the IRF-1 promoter construct.
Clearly, though, these results need to be verified in a more
physiologic system. That STAT4 has the capacity to bind to the IRF-1
promoter has been recognized since the cloning of STAT4. In the initial
studies, STAT4 was shown to bind the IFN- activation site (GAS)
element of the murine IRF-1 promoter (38). Although the significance of
this result was not pursued at that time, our data confirm that STAT4
binds the human promoter. Whether IRF-1 and STAT4 function in concert
to effect gene regulation or in parallel by activating different sets
of target genes has yet to be determined. Identification of target
genes of IRF-1 and STAT4 after IL-12 stimulation will clarify this
issue and help us to understand the gene-regulatory events mediating
Th1/Th2 differentiation. Comparing and contrasting IL-12-dependent gene
expression in IRF-1-/- and STAT4-/- mice
will be of considerable interest in this regard. It should be noted
that the IL-2-mediated induction of IRF-1 was, predictably, not
dependent on STAT4. Similarly, although IFN- can activate STAT4, we
have no data indicating that STAT4 is important for IFN--dependent
IRF-1 regulation; STAT1 is likely to be more relevant.
In contrast to IL-12, IL-4 promotes Th2 responses. IL-4 has been shown
to inhibit IL-12-dependent induction of IFN- (32) and to inhibit
IFN--dependent up-regulation of IRF-1 (24). Based on the phenotype
of IRF-1-/- mice, IRF-1 appears to participate in Th1
differentiation, and interestingly, no up-regulation of IRF-1 was found
in IL-4-stimulated cells. Importantly, IL-4 treatment did not block
IL-12-dependent up-regulation of IRF-1, and this contrasts with the
induction of IFN- by IL-12. The experiment thus substantiates our
claim that IL-12-dependent up-regulation of IRF-1 was not indirectly
mediated by IFN- production, as IL-4 blocked IFN- induction but
not IRF-1.
In conclusion, we have demonstrated that IL-12 and IFN- up-regulate
a number of genes in common. One important gene known to be regulated
by IFN- is IRF-1, but it too is directly regulated by IL-12 and
IL-2, but not by IL-4. Thus, IRF-1 can also be viewed as a gene that is
regulated by Th1 cytokines. The demonstration that IL-12 induces IRF-1
expression may help to clarify the defects found in
IRF-1-/- mice; loss of this transcription factor not only
interferes with IL-12 and IL-15 synthesis but may interfere with some
of the actions of IL-12 and IL-2. To what extent IRF-1 serves as an
IL-12-dependent transcription factor will be better appreciated as we
learn more about the genes induced by these cytokines.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: IRF-1, IFN regulatory factor-1; MIP, macrophage-inflammatory protein; SBE, STAT binding element; CHX, cycloheximide.
Received for publication August 14, 1998. Accepted for publication March 29, 1999.
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in human bone marrow stromal cells: inhibition of hematopoietic growth factors and induction of interleukin-1 receptor antagonist. Blood 84:4142. interferon activation site-binding protein expressed in early myeloid differentiation. Mol. Cell. Biol. 14:4342.This article has been cited by other articles:
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