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Division of Immunology, Medical Institute of Environmental Hygiene at the University of Düsseldorf, Düsseldorf, Germany
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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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Although the exact mechanisms of the various toxic responses are not known, aberrant induction of genes has been assumed to be a major factor in toxicity. Upon ligand binding, the AHR releases its chaperoning heat shock protein 90, translocates into the nucleus, and dimerizes with AHR nuclear translocator (ARNT) (10). The heterodimeric AHR-ARNT complex binds to a short DNA motif termed DRE (dioxin response element, also known as AHRE or XRE) and alters the transcription of genes controlled by DREs. Potential DREs are found in many genes (11), and functional studies on cytochrome P-4501A1 have been pivotal in understanding the action of dioxins in gene induction (12, 13, 14).
It is conceivable that cytokines are important in mediating the biological outcome of AHR overactivation in the immune system, in particular the immunosuppressive effects and effects on thymus development. Expression of several cytokine mRNAs was shown to be inducible by TCDD (15, 16). IL-2, which is comparatively strongly inducible, is produced by T cells and has central functions for growth and differentiation of T cells, B cells, NK cells, and macrophages (17). IL-2 is mandatory for the homeostasis of T cells (18). However, the direct transcriptional control of a gene of the immune system by the AHR has not been shown until now. Here, we demonstrate that the TCDD-activated AHR can directly bind to and transduce the distal region of the IL-2 promoter, revealing its previously unknown enhancing function. Depending on the T cell maturation stage and, in some circumstances, costimulation, IL-2 will be expressed upon ligand activation of the AHR in thymus and spleen cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 mice (Zentralinstitut für Versuchstierkunde, Hannover, Germany) and AHR homozygous and heterozygous offspring of female B6,129AhRtmlbra (AhR-/-) and male C57BL6AhRtmlbra (AhR-/+) mice (The Jackson Laboratory, Bar Harbor, ME) bred in our animal facility were used. All animals were kept under specific pathogen-free conditions. For in vivo exposure, TCDD (purity >99%; Oekometric GmbH, Bayreuth, Germany) was dissolved in DMSO and diluted in sterile corn oil to 5 µg/ml. Mice were injected i.p. with a single dose of 50 µg TCDD/kg body weight. Control mice received DMSO/corn oil. At posttreatment days 3, 6, and 8, mice were killed by asphyxiation, and RNA was extracted from thymi and spleens with TRIzol according to the manufacturer’s instructions (Life Technologies, Gaithersburg, MD). RNA was reverse transcribed by standard procedures. Before RNA extraction, erythrocytes were depleted from spleen cells by hypotonic lysis.
Primary cell cultures
RPMI 1640 supplemented with 5% FCS, 5 x
10-4 M 2-ME, 100 U/ml penicillin, and 0.1 mg/ml
streptomycin was used as medium throughout. TCDD was diluted in
1,4-dioxane (Merck, Darmstadt, Germany) and added to the medium at a
final concentration of 10 nM in 0.1% solvent. Anti-CD3
was coated
onto six-well plates, and 10–13 x 106
thymocytes or 5–7 x 106 spleen cells from
adult animals were added in 2 ml medium with 10 nM TCDD or solvent
alone. After 4 h, RNA was isolated for RT-PCR. In other
experiments, supernatants were taken after the indicated incubation
times with anti-CD3, and IL-2 content was measured by ELISA
(Mouse ELISA kit; Endogen, Woburn, MA). Alternatively, fetal thymus
lobes from mice at gestation day 15 were excised, freed of adhering
tissue, and cultivated on nitrocellulose filters set in a well with 300
µl medium plus or minus 10 nM TCDD as described before
(19, 20, 21). Control lobes were cultivated in medium plus
0.1% solvent alone. At 4 or 5 days of culture, single-cell suspensions
were prepared, and RNA was extracted and reverse transcribed. For
analysis of thymocyte subpopulations,
CD4-CD8-,
CD4+CD8+,
CD4-CD8+, and
CD4+CD8- thymocytes were
sorted on a FACS440 cell sorter (Becton Dickinson, Mountain View, CA)
after staining with anti-CD4 and anti-CD8 Abs (PharMingen,
Hamburg, Germany).
RT-PCR assays
Semiquantitative RT-PCR assays were performed as previously
described (15). Total RNA (1 µg) was used for cDNA
synthesis with mouse mammary tumor virus (MMTV) reverse transcriptase.
[
-32P]-labeled CTP was added during RT-PCR.
HPRT (hypoxanthine phosphoribosyltransferase; Ref.
22), a housekeeping gene, was amplified together with the
cDNA of IL-2 in the same reaction tube. To analyze the
amplification of both cDNAs in the linear range, the
"primer-dropping" method (23) was used to calibrate
for the different expression levels of HPRT vs IL-2 cDNAs. Briefly, the
cDNA for the IL-2 test were first amplified for several cycles
(depending on their previously established abundance), and then
HPRT primers were dropped into the reaction tubes followed
by an additional 25 cycles. PCR products were separated on 10%
polyacrylamide gels. Gels were dried and visualized by autoradiography.
The primer sequences, the annealing temperature, and cycles for RT-PCR
are shown in Table I. The autoradiographs
were analyzed with an OmniMedia scanner and the whole-band analysis
program (Bio Image system; Millipore, Bedford, MA). Quantification was
performed on autoradiographs in the linear range of radioactivity to
photosensitivity. PCR experiments for each gene expression were
performed at least two times with similar results; for data
presentation, one representative experiment is shown.
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Complementary single-stranded oligonucleotides were synthesized,
annealed, and end-labeled with [
-32P]ATP.
Sequences of oligonucleotides are shown in Table II. Nuclear and cytoplasmic proteins from
an AhRb/b mouse thymic epithelial cell
line, ET (a gift from Ronald Palacios, Basel Institute of Immunology,
Basel, Switzerland; Ref. 24), were prepared
(25) and EMSA were essentially performed as described
(26). Briefly, 15 µg of nuclear proteins were incubated
with 30,000 cpm end-labeled double-stranded oligonucleotides for 20 min
in a final volume of 15 µl. Binding reactions were conducted in 25 mM
HEPES, pH 7.5; 1 mM EDTA; 0.7 mM DTT; 0.5 mM PMSF; 10% glycerol; and
156 mM KCl. Samples were preincubated for 10 min in the presence of 1
µg poly d(I:C)d(I:C) and 0.5 µg salmon sperm DNA. A 200-fold excess
of unlabeled oligonucleotides was used in specific and nonspecific
competition experiments. AHR-DRE complexes were separated on 4% (w/v)
polyacrylamide gels in TGE buffer (50 mM Tris, 380 mM glycin, 2 mM
EDTA, pH 8.5). Gels were transferred to Whatman 3 MM paper (Bio-Rad,
Munich, Germany), dried, and autoradiographed. For supershift analyses,
the nuclear protein extract and labeled oligonucleotide were incubated
with 1 or 3 µl of anti-AHR Ab (Dianova, Hamburg, Germany) for 10
min at room temperature before gel electrophoresis.
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The plasmid constructs used in this study were as follows:
pGudLuc 1.0, a reporter plasmid containing the firefly
luciferase gene under the control of the MMTV promoter; pGudLuc
1.1, produced by cloning five DREs (-1301 to -819) isolated from
the CYP1A1 promoter of mouse into the MMTV promoter of
pGudLuc 1.0 (both plasmids were gifts from Michael Denison, University
of California at Davis; Ref. 29) and used as positive
control in all assays; pMMTV-IL-2-DRE(0.5), produced by
cloning the Esp3 I-SspI fragment spanning -1296
to -756 of the 5' region of the IL-2 gene and containing
the three DREs into the BglII site within the MMTV promoter
of the pGudluc 1.0 vector; pDRE(0.5)MutII/III and pDRE(0.5)MutIII, in
these plasmids, point mutations of the DREII/III or only DREIII had
been introduced into the core recognition sites of pMTV-IL-2-DRE(0.5);
pDRE I, pDRE II, and pDRE III, complementary single-stranded
oligonucleotides (50 bases) encoding two copies of each DRE of
IL-2 promoter and were synthesized, annealed, and cloned
into the BglII site of pGudluc 1.0 (Table III). The DNA sequences of the
oligonucleotides are shown in Table III (the core recognition sequence
for the DRE motifs is in bold print); pSV-IL-2-DRE(0.5) and
pTK-IL-2DRE(0.5), the IL-2 fragment -1296 to -756 was
cloned into the pGL3P vector in front of the SV40 promoter
or TK promoter (Promega, Madison, WI), respectively.
pSV-IL-2-DRE(0.1), the BstEII to NsiI
fragment (400 bp) between DRE I and DRE II of the IL-2 5' region, was
deleted from pSV-IL-2-DRE(0.5). All plasmids were examined by
sequencing.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In previous studies we had shown that IL-2, TNF-, and IL-1ß
mRNA are inducible in a dose-dependent fashion by TCDD in thymocytes.
Except TNF-, these cytokines contain consensus sequence of DREs in
the published upstream region of their respective genes
(11). The IL-2 gene has three potential DRE
sites at -1261, -851, and -815 bp. We decided to test the three DREs
of the IL-2 distal promoter region for direct interaction
and inducibility by the liganded AHR. Expression of AHR and its
dimerization partner ARNT, both necessary for TCDD-mediated gene
transactivation, was verified by RT-PCR in the ET thymic epithelial
cell line (data not shown). Also, expression of CYP1A1, a
known TCDD-responsive gene (3, 12), was inducible by TCDD
in this cell line, indicating the functionality of the endogenous AHR
and ARNT proteins (data not shown). Nuclear extracts of ET cells,
containing the AHR/ARNT complex, did bind to labeled
IL-2-DRE-oligonucleotides only when treated with TCDD but
not with solvent alone (Fig. 1A). No specific AHR binding
was observed with cytoplasmic protein, indicating that TCDD-dependent
dimerization of AHR and ARNT takes place in the nucleus (Fig. 1B).
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A, lanes 3 and
4). Binding was not inhibited with oligo-DNA of an unrelated
sequence (Fig. 1A, lane 5). Preincubation of the
mixture of nuclear extracts and DREs with anti-AHR Abs led to the
expected supershift of the complex (Fig. 1A, lanes
6 and 7). The three DREs differed in their binding
capacity. In particular, the binding of DRE I was much weaker than that
of DRE II and DRE III. This is in agreement with the deviation from the
published consensus sequence 5'-T G/C G/T CACGC N C/A-3'
(14), which is three base pair for DRE I, but only two for
DRE II and DRE III (see Table II). Still, the core sequence, to which
the AHR/ARNT/TCDD complex makes physical contact (14, 28),
is unchanged in all three DREs. Note that DRE I binds to proteins of an
unknown nature, albeit this complex is not shifted by TCDD
treatment. The IL-2 distal region contains enhancer elements responsive to TCDD
To test whether TCDD leads to transactivation of the AHR on the
IL-2-DREs, we transiently transfected hepatoma cells with
pMMTV-IL-2-DRE, a reporter plasmid containing luciferase under the
control of the -1296 to -756 region of the 5' IL-2 gene.
We then measured the induction of luciferase activity (Fig. 2). Hepatoma cells were used because of
unresolvable technical difficulties in efficiently transfecting primary
T cells or thymocytes; T cell hybridomas lost AHR expression during
prolonged cultivation. We observed that this distal IL-2
promoter region induced expression of luciferase in the presence of
TCDD. The induction of transcription increased 
13 times compared
with the background. In comparison, transcription from pGudluc 1.1
(29), which contains five DREs from CYP1A1, a
gene highly sensitive to TCDD, was induced 20-fold (Fig. 2A). Similar results were obtained when using vectors
containing either an SV40 or tk minimal promoter. These
vectors yielded different backgrounds, yet always easily detectable
TCDD induced luciferase expression (Fig. 2C). Thus, the far
distal region of the IL-2 promoter contains DNA sequences
highly responsive to TCDD. We then introduced single point mutations
into the core recognition sites of the DREs II and III in the
pMMTV-IL-2-DRE(0.5) plasmid, transfected cells, and measured luciferase
activity. When both DRE II and III sites are mutated the luciferase
induction by TCDD is abrogated. A mutation in the DRE III only reduced
the response but did not completely abrogate it (Fig. 2A).
Thus, the DREs are necessary elements and both elements are required
for a full response (Fig. 2). There is no other, hidden AHR-responsive
element in the 500-bp distal enhancer segment.
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, the distal
IL-2 promoter region has possible AP-1 binding sites, but they have not
been demonstrated to be used for the IL-2 enhancer/promoter.
It is known that the liganded AHR may induce c-jun and
c-fos, creating AP-1 (30). To supply further
evidence that DREs are responsible for the IL-2 induction, we cloned
each of the three DREs separately into the pGudluc vector (see Fig. 2B), and tested them for promoter activity by TCDD in HepG2
cells. The results shown in Fig. 2B demonstrate that each of
the three isolated IL-2-DREs can drive luciferase
transcription in the presence of TCDD, demonstrating the
transcription-enhancing capacity of these small DNA elements. The same
results were obtained with pSV-IL-2DRE(0.1) in which the putative AP-1
sites between DRE I and DRE II are deleted (Fig. 2C, Table IV).
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To verify that the induction is AHR dependent, we performed the same
assay in AHR- or ARNT-deficient cell lines Hepa
C12 and Hepa C4, and wild-type Hepa C1. The results shown in Table IV
demonstrate that functional AHR as well as ARNT are necessary for
induction driven by IL-2-DREs. Also, in
AHR-deficient mice, IL-2 was not induced by TCDD (see
below).
Induction of IL-2 mRNA by TCDD depends on the differentiation stage of T cells
Most eukaryotic promoters have various modules that cooperate
adaptively to generate differentiation- and situation-specific
responses. Consequently, modules that are inducible in in vitro assays
could be not accessible or not relevant in vivo. Therefore, we tested
induction of IL-2 by TCDD in several maturational stages of T cells and
in response to stimulation of the TCR. All cells described here could
respond to TCDD with CYP1A1 induction, which indicates a
functional AHR/ARNT system (data not shown). In a first series of
experiments, mice were injected with 50 µg TCDD, and their thymocytes
were analyzed 3, 6, or 8 days later by RT-PCR. We observed an IL-2 mRNA
induction in vivo, suggesting that the DREs in the IL-2
promoter can be addressed (Fig. 4A). To assess whether IL-2
induction is dependent on the differentiation stage of cells, we next
set up fetal thymus organ cultures (FTOCs). Fetal thymi were excised
and exposed to TCDD for 4 or 5 days. Thymocytes were isolated and
sorted to high purity by CD4/CD8 expression for very immature
(CD4-CD8-), immature
(CD4+CD8+), prospective
helper T cells (CD4+CD8-),
and prospective cytotoxic T cells
(CD4-CD8+). IL-2 mRNA
induction was measured by RT-PCR. As shown in Fig. 4B, TCDD
increased IL-2 mRNA in total thymocytes, confirming previously
published results (15). The overall increase was
attributable to the immature
CD4-CD8- cells and future
cytotoxic CD4-CD8+ cells,
both of which responded to TCDD exposure with a strong (3- and
5-fold) increase of IL-2 mRNA.
CD4+CD8- cells displayed a
constitutive expression of IL-2. However, they also increased IL-2 mRNA
by TCDD. CD4+CD8+
thymocytes, which constitute the majority of thymocytes, remained
refractory to TCDD. Moreover, IL-2 induction by TCDD could not be
detected in spleen cells, a source of naive, fully mature T cells (data
not shown). Thus, IL-2 induction by TCDD is dependent on the maturation
stage of thymocytes/T cells, and the responsiveness of the promoter can
be switched on and off during maturation of T cells.
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signaling
Induction of IL-2 in T cells classically requires two signals, one
from the TCR, and another from the surface receptor CD28. Their dual
triggering leads to a cascade of events, eventually generating and
activating components like c-jun and c-fos complex (AP-1),
NFAT, and NF-
B, which transactivate the promoter (2).
Because TCDD alone was not sufficient to induce IL-2 production in
mature splenic T cells, we stimulated thymus and spleen cells in
six-well plates with anti-CD3 Ab, mimicking TCR triggering. Like
spleen cells from in vivo exposed mice, spleen cells exposed in vitro
could not be induced by TCDD alone (Fig. 5A). IL-2 mRNA became
inducible by TCDD in spleen cells only upon cross-linking with
anti-CD3 Ab. Induction was consistently about 4 times over the
untreated controls, but there was a threshold of 100 ng of
anti-CD3 necessary for the stimulation. The induction of IL-2
mRNA was readily detectable within 4 h, i.e., no cell division was
necessary. Also, in thymocytes, where IL-2 mRNA had been induced by
TCDD alone in vivo (Fig. 4), additional signaling via the TCR augmented
IL-2 mRNA in vitro (Fig. 5B).
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-stimulated cultures by ELISA
for IL-2 protein (Fig. 6). Exposure of
the cells to TCDD increased IL-2 protein synthesis compared with the
solvent controls, but a higher concentration of anti-CD3
costimulation than for mRNA induction was needed to detect this. The
IL-2 protein production lagged several hours compared with IL-2 mRNA
induction, suggesting new protein synthesis. At least for spleen cells,
cell density during stimulation and age of mice seemed to be important
parameters for the strength of the response, as IL-2
induction varied depending on culture conditions (data not shown).
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The reporter assays described above strongly
suggested that the IL-2 promoter is activated via the
TCDD-liganded AHR at its distal DREs. To rule out a gene-inducing
mechanism of TCDD not involving the AHR, we injected
AHR-deficient mice with 50 µg TCDD/kg body weight. After 6
days, thymus cells were harvested and total RNA was isolated. IL-2 mRNA
was measured by RT-PCR. As shown in Fig. 7, IL-2 mRNA induction by TCDD is
detected in thymus cells from AhR+/+ and
AhR+/- but not in thymus cells from
AhR-/- mice. Thus, the IL-2 mRNA
induction by TCDD is mediated by the Ah receptor.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, and AP-1, which cooperate and thus confer
the lineage specificity and external regulation of IL-2.
Others and we had shown that IL-2 expression could be induced by TCDD,
i.e., the AHR (15, 31, 32). Extending these findings, our
analysis of IL-2 induction in subpopulations of thymocytes showed that
TCDD can induce IL-2 mRNA in
CD4+CD8-,
CD4-CD8+, and
CD4-CD8-, but not in
CD4+CD8+ cells. Some
transcription factors for IL-2 expression, Oct-1, Sp-1, NF-B, and
cAMP response element binding protein (CREB), can be expressed or
induced in thymocytes of all developmental stages. In contrast, two
essential transcription factors, AP-1 and NFAT, are absent in
CD4+CD8+ cells. IL-2 is
shut off during this maturational phase and only becomes accessible
again later on (33, 34). TCDD cannot override this
complete inaccessibility. Note that in the same cells, CYP1A1 mRNA
remained inducible by TCDD (data not shown).
In contrast to thymocytes, TCDD alone was not sufficient to induce IL-2
in naive T cells, but needed stimulation via the TCR as an additional
signal. Again, this is in agreement with findings in the rat, where
TCDD could increase serum levels of IL-2 only after synergistic
injections of staphylococcus enterotoxin B injections
(31). In contrast to thymus, most spleen T cells are
resting and thus are unable to express IL-2. Stimulation via the
TCR/CD3 pathway alone triggers the mitogen-activated protein kinase
pathways (Raf-1, extracellular signal-related kinase). Together with
CD28 costimulation, different pathways are activated and the signal
cascades merge and eventually lead to AP-1 formation and IL-2
expression (35). Activation of the AHR can bypass the need
for CD28 cosignaling, probably in that an alternative signal
transduction pathway downstream of the TCR/CD3 is used. Apart from the
direct transcriptional activation shown here, other possibilities of
the AHR to interfere with the "normal" cell signaling exist. A
recent study by Tian et al. (36) demonstrated physical
interaction of the AHR with NF-B, leading to mutual repression in
their system. Upon exposure to TCDD, c-jun, c-fos, and c-Jun
N-terminal kinase induction has been observed in hepatoma cell lines
(37, 30) and in Jurkat cells (38). Thus,
indirect up-regulation of IL-2 by TCDD via these factors is possible.
However, we found that in thymocytes c-jun is not inducible by TCDD
(data not shown), favoring the direct transcriptional activation of
IL-2 in these cells. Thus, the AHR is not a strong transcription factor
in itself, but rather a modulator that may need the correct cellular
set-up for its function. Although it can act in thymocytes without
coactivation, in peripheral T cells it needed additional stimuli, i.e.,
TCR/CD3 triggering. It is interesting to note that the AHR is
particularly abundant in thymus epithelium and thymocytes
(39), pointing to a physiological role in this tissue.
Also, the fact that TCDD did not induce IL-2 mRNA in
AHR-deficient mice clearly demonstrated that the AHR
mediated this effect.
DNA motifs, which are targets for the AHR/ARNT complex, have been early identified, and a consensus sequence was inferred from a number of DREs in different genes and species. A core sequence within the consensus sequence, which is indispensable for AHR/ARNT contact, has been mapped (14). The DREs, which we identified in the upstream IL-2 region by computer analysis, were functionally tested with EMSA and a luciferase reporter gene assay. Band shift analysis showed that the TCDD-activated AHR binds to DRE II and III, and only very weakly to DRE I. Likewise, in the transfection assay, luciferase induction was higher with DRE II and DRE III than with DRE I. Most likely this reflects the similarity to the consensus sequence. DRE I differs from this functional consensus sequence in three base pairs, whereas DRE II and III have only two base pair deviations. Moreover, DRE II and III have bidirectional core sequences, which are reportedly particularly responsive to TCDD (14). It is less likely that the broader context of other DNA sequences is important, as our luciferase experiments with the bare DRE sequences imply. Mutation of the DREs abrogated the response, demonstrating that there is no other hidden DRE in the 500-bp distal region we analyzed. Also, both DRE II and III are needed for a full response. Even mutating just one of them stopped the induction of the reporter gene; only a small rest activity remained. Generally, very little is known about synergistic or antagonistic effects of spatial distribution of transcription factor binding elements, which may be crucial in promoter regulation.
Most functionally investigated DREs are located between -1200 and
-800 bp in the upstream regions of the CYP1A1 from mice,
rats, and humans (40, 41, 42). Interestingly, the
IL-2-DREs were also located between -1200 and -800 bp in
the upstream region, and many potential DREs in a number of genes are
located in very distal positions (11). Possibly, the
distance is functionally important, such that chromatin structure is
changed and serves as a stable nucleation site for enhancer-promoter
communication and chromatin remodeling, as has been suggested for other
genes (43). Ward and colleagues have analyzed this by
mapping of DNaseI hypersensitivity sites for the
IL-2 promoter, albeit only until -730. They demonstrated
the importance of upstream regions for chromatin remodeling and gene
accessibility (44). It would be interesting to perform
similar studies on the even further upstream region and to analyze
remodeling under the influence of the AHR/ARNT complex in a
tissue-specific setting. This could also help to understand data
conceived from cat reporter assays in EL4E1 cells, indicating a
negative regulatory role of the -700 to -1000 region (Fig. 3). In the
context of AHR/ARNT activation, this negative regulation might be
inverted. Also in humans a few DRE consensus sequences can be found in
the very far 5' region (e.g., at -7324, -3528, -3398, -2735,
-2354); however, no functional data are available. The human and mouse
distal IL-2 promoter have many features in common, but expression
levels differed in the careful analysis of isolated sequences in
chloramphenicol acetyltransferase (CAT) assays done by Novak et al.
(2). The 500-bp stretch we analyzed here has only little
homology to the human IL-2 upstream region (2). It is
impossible to say at this time whether the human IL-2
promoter would also be responsive to AHR.
ARNT is the dimerization partner of the AHR; the complex forms in the
nucleus, and both proteins participate in DNA binding
(10). Our data are consistent with this because TCDD
cannot induce IL-2 expression in ARNT-deficient cell lines.
Interestingly, when we searched for a suitable cell line for the
transfection experiments, we could not find any T cell line that
expresses high levels of AHR. In contrast, all tested T cell lines
constitutively expressed ARNT. Thymocytes and thymic epithelium
contained AHR in abundance. ARNT is another member of the bHLH-PAS
family, and it can dimerize not only with AHR but also with yet other
bHLH-PAS proteins, for instance, hypoxia-inducible factor 1
(HIF-1), a protein shown to be involved in organogenesis by response
to oxygen pressure in tissues. Competition for ARNT in a cell may
be another control mechanism for transcriptional regulation, i.e., ARNT
is the central regulator in this protein family (5, 45).
Is there a physiological or pathological relevance for IL-2 induction by TCDD? TCDD causes thymus atrophy and immune suppression at low doses in many animal species (6, 7, 8, 9). The finding that TCDD induces IL-2 production seems paradoxical, as IL-2 is an established growth and survival factor for T cells. However, more recently, the role of IL-2 in T lymphocyte death has been discovered. In mice genetically deficient for IL-2, autoreactive lymphoid cells accumulated due to a failure of activation-induced cell death (18, 46, 47). Also, in FTOC, the addition of exogenous IL-2 inhibited proliferation and differentiation of T cells and diminished total cell yields (48, 49, 50). Interestingly, Fas ligand mRNA in CD4-CD8+ thymocytes becomes up-regulated upon TCDD exposure of FTOCs (M.-S. Jeon, unpublished observation), suggesting a possible mechanism for TCDD-induced thymus atrophy.
Thus, it cannot be excluded that IL-2 induction by TCDD may be causally connected to the various immunosuppressive effects of this toxic compound, although it needs further investigation. Here we demonstrated for the first time a direct interaction between AHR and DRE in the promoter region of the IL-2 gene, which is an important gene in the immune system. Whether TCDD-induced IL-2 expression or the induction of other genes results in immune suppression has to be investigated in further studies.
In summary, the TCDD-liganded AHR is capable of inducing transcription of IL-2 in mouse thymocytes and T cells. The natural ligand of the AHR is not yet known. We are tempted to speculate that the natural ligand is a player in IL-2 induction and thus important for cell homeostasis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Charlotte Esser, Division of Immunology, Medical Institute of Environmental Hygiene, Auf’ m Hennekamp 50, 40225 Duesseldorf, Germany.
3 Abbreviations used in this paper: AHR, arylhydrocarbon receptor; ARNT, AHR nuclear translocator; DRE, dioxin response element; FTOC, fetal thymus organ culture; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; MMTV, mouse mammary tumor virus; HPRT, hypoxanthine phosphoribosyltransferase; ET, mouse thymic epithelial cells.
Received for publication February 24, 2000. Accepted for publication September 11, 2000.
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References |
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 O. J. BOWERS, K. B. SOMMERSTED, R. T. SOWELL, G. E. BOLING, W. H. HANNEMAN, R. G. TITUS, and G. K. DEKREY 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) REDUCES LEISHMANIA MAJOR BURDENS IN C57BL/6 MICE. Am J Trop Med Hyg, October 1, 2006; 75(4): 749 - 752. [Abstract] [Full Text] [PDF]
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 M. N'Diaye, E. Le Ferrec, D. Lagadic-Gossmann, S. Corre, D. Gilot, V. Lecureur, P. Monteiro, C. Rauch, M.-D. Galibert, and O. Fardel Aryl Hydrocarbon Receptor- and Calcium-dependent Induction of the Chemokine CCL1 by the Environmental Contaminant Benzo[a]pyrene J. Biol. Chem., July 21, 2006; 281(29): 19906 - 19915. [Abstract] [Full Text] [PDF]
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C. J. Funatake, N. B. Marshall, L. B. Steppan, D. V. Mourich, and N. I. Kerkvliet Cutting Edge: Activation of the Aryl Hydrocarbon Receptor by 2,3,7,8-Tetrachlorodibenzo-p-dioxin Generates a Population of CD4+CD25+ Cells with Characteristics of Regulatory T Cells J. Immunol., October 1, 2005; 175(7): 4184 - 4188. [Abstract] [Full Text] [PDF]
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 M. T. Fisher, M. Nagarkatti, and P. S. Nagarkatti 2,3,7,8-Tetrachlorodibenzo-p-dioxin Enhances Negative Selection of T Cells in the Thymus but Allows Autoreactive T Cells to Escape Deletion and Migrate to the Periphery Mol. Pharmacol., January 1, 2005; 67(1): 327 - 335. [Abstract] [Full Text] [PDF]
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 J. Adachi, Y. Mori, S. Matsui, and T. Matsuda Comparison of Gene Expression Patterns between 2,3,7,8-Tetrachlorodibenzo-p-dioxin and a Natural Arylhydrocarbon Receptor Ligand, Indirubin Toxicol. Sci., July 1, 2004; 80(1): 161 - 169. [Abstract] [Full Text] [PDF]
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C. Yang, F. Boucher, A. Tremblay, and J. L. Michaud Regulatory Interaction between Arylhydrocarbon Receptor and SIM1, Two Basic Helix-Loop-Helix PAS Proteins Involved in the Control of Food Intake J. Biol. Chem., March 5, 2004; 279(10): 9306 - 9312. [Abstract] [Full Text] [PDF]
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S. Tomita, H.-B. Jiang, T. Ueno, S. Takagi, K. Tohi, S.-i. Maekawa, A. Miyatake, A. Furukawa, F. J. Gonzalez, J. Takeda, et al. T Cell-Specific Disruption of Arylhydrocarbon Receptor Nuclear Translocator (Arnt) Gene Causes Resistance to 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Induced Thymic Involution J. Immunol., October 15, 2003; 171(8): 4113 - 4120. [Abstract] [Full Text] [PDF]
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, 1 µ g 
, 0.5 µg
•
). Supernatants were taken after 4, 6, 8, 10, and 24 h, and
IL-2 protein was measured by ELISA.