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Department of Allergic Diseases, Novartis Research Institute, Vienna, Austria
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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could not act as inducer on its
own nor did it synergize with IL-4. The activities of eotaxin-3
promoter luciferase constructs were significantly increased by IL-4 and
IL-13 in human dermal fibroblasts. This effect was mediated by a
binding site for the transcription factor STAT6 in the eotaxin-3
promoter sequence. Mutations in the STAT6 binding site abrogated
up-regulation of eotaxin-3 promoter activity. In STAT6-defective human
embryonic kidney 293 cells, the wild-type luciferase construct, but not
the STAT6 binding mutant, was inducible by IL-4 only upon
cotransfection of STAT6 expression vector. In addition, eotaxin-3
protein was detectable in the supernatants of STAT6-transfected human
embryonic kidney 293 cells upon IL-4 or IL-13 stimulation. In the same
experiments, TNF- induced activation of the monocyte chemoattractant
protein-1/CCL2 gene was independent of STAT6 transfection. These
results indicate that IL-4 and IL-13 activate eotaxin-3 gene expression
in a STAT6-dependent fashion. Although both eotaxin-1 and -3 are
regulated by this transcription factor, the response of the eotaxin-3
gene to TNF- stimulation appears to be
different. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the most potent and efficient chemoattractants for eosinophils is the CC chemokine eotaxin-1. In vitro studies have demonstrated the generation of eotaxin-1 by lung and dermal fibroblasts following stimulation with the Th2 cytokines IL-4 and IL-13 (23, 24, 25, 26).
Up-regulation of eotaxin-1 gene expression in human lung
epithelial cells has been reported after treatment with the
proinflammatory cytokines TNF- and IL-1 (27).
Interestingly, IL-4 and TNF- appear to induce eotaxin-1 in human
dermal fibroblasts and airway epithelial cells in a synergistic fashion
(25, 28). The mechanism operative in regulating eotaxin-1
expression was recently investigated. It was shown that TNF-- and
IL-4-stimulated eotaxin-1 gene expression in epithelial cells was
mediated by activation of the transcription factors NF-
B and STAT6
(29). Our previous studies have demonstrated the
involvement of STAT6 in the regulation of IL-4 as well as
TNF--induced eotaxin-1 expression in human dermal fibroblasts
(30). The present study investigates the regulation of
eotaxin-3 gene activation in human dermal fibroblasts. The data
demonstrated that IL-4 and IL-13 but not TNF- induced eotaxin-3 gene
expression at the RNA and at the protein level in this cell type. The
stimulatory effect was mediated by the transcription factor STAT6 at
the level of transcription due to the presence of a binding site for
this factor in the promoter region of the eotaxin-3 gene. Despite the
dependence of eotaxin-1 and -3 on STAT6 for activation of gene
expression by IL-4, no role of TNF- in eotaxin-3 up-regulation was
apparent as opposed to eotaxin-1 gene induction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal human dermal skin fibroblasts from neonatal skin
(Clonetics, San Diego, CA) were cultured in FGM-2 medium (Clonetics).
Human embryonic kidney (HEK)293 cells were carried at 37°C with 5%
CO2 in DMEM containing 100 U/ml penicillin, 100
µg/ml streptomycin, and 10% heat-inactivated FCS (Life Technologies,
Grand Island, NY). Purified human rIL-4 was obtained from Novartis
(Basel, Switzerland) with a sp. act. of 0.5 U/ng. Recombinant human
TNF- (Bender Med Systems, Vienna, Austria) was used at a
concentration of 100 U/ml. Recombinant human IL-13 was purchased from
PeproTech (London, U.K.). Human eotaxin-3 was measured by ELISA
according to a standard streptavidin-HRP assay protocol (R&D Systems,
Minneapolis, MN). Briefly, eotaxin-3 was captured with an
anti-human eotaxin-3 Ab (PeproTech) and developed with biotinylated
anti-human eotaxin-3 (PeproTech). Recombinant human eotaxin-3 (R&D
Systems) was used as standard. The detection limit of this assay was 25
pg/ml. MCP-1 protein was quantitated using commercially available ELISA
kits (R&D Systems).
RT-PCR
Total RNA was isolated using the TRIzol reagent (Life
Technologies) according to the instructions of the manufacturer. Three
micrograms of total RNA was reverse transcribed using avian
myeloblastosis virus reverse transcriptase (Roche Molecular
Biochemicals, Mannheim, Germany) in a total volume of 50 µl. A 260-bp
fragment of the eotaxin-1 transcript was amplified using the
intron-spanning PCR primers 5'-CATGAAGGTCTCCGCAGCACTTCT-3'
(upstream) and 5'-CCAGATACTTCATGGAATCCTGC-3' (downstream) from cDNA
corresponding to 20 ng of RNA. For the detection of a 290-bp eotaxin-3
transcript the intron-spanning primer pair
5'-GCCTGATTTGCAGCATCATGATGG-3' (upstream) and
5'-CGGATGACAATTCAGCTGAGTCAC-3' (downstream) was used. PCR was
performed for 30 cycles at 94°C, 30 s; 61°C, 30 s; and
72°C, 30 s. The PCR primer pair 5'-ATGGATGATGATATCGCCGCG-3'
(upstream) and 5'-AGTCCATCACGATGCCAGTGG-3' (downstream) was used to
amplify a 480-bp fragment of the 
-actin mRNA using the same reaction
conditions.
Cloning of eotaxin-3 reporter constructs
A DNA fragment containing the human eotaxin-3 promoter (970 bp) was amplified from genomic DNA (Roche Molecular Biochemicals) using the PCR primers 5'-AGTCAAGCTTCATCATGATGCTGCAAATCAGG-3' and 5'-CTGACTCGAGTCTGTTAGATCTCTCAAATGCC-3'. The PCR fragment was digested with XhoI and HindIII and cloned into pGL3-Basic (Promega, Madison, WI) to give pGL3-EOT. pGL3-EOT3S1 and -EOT3S2 were subcloned using the natural SacI and BamHI sites within the EOT3 fragment. The shortest eotaxin-3 luciferase reporter construct pGL3-EOT3S3 (150 bp) was amplified with the primer pair 5'-AGTCAAGCTTCATCATGATGCTGCAAATCAGG-3' and 5'-CAGTCTCGAGGTCAAAAGTGCTGCTTCTGT-3' and cloned as XhoI/HindIII fragment into the vector. A point mutation was introduced into the core region of the STAT6 binding site in the context of the pGL3-EOT3 construct as reported earlier (31) using the following oligonucleotides: pGL3-EOT3 M1, 5'-TGTTCCCAACCACAGAATAGTCTGGAATTGTTTTCAGGGCCGT-3' and 5'-GAGACGGCCCTGAAAACAATTCCAGACTATTCTGTGGTTGGGA-3'. The cloning of the STAT6 expression vector has been described earlier (32). Plasmids were analyzed by digestion with restriction endonucleases and DNA sequencing. Constructs used for transient transfections were purified by cesium chloride density gradients.
Transient transfection of HEK293 cells and human dermal fibroblasts
The day before transfection, 5 x 104 cells were seeded into 24-well culture plates in fresh medium. Transient transfection of HEK293 cells was achieved using calcium phosphate coprecipitation. Briefly, 1–2 µg of plasmid DNA was diluted in 42 µl of H2O, mixed with 7 µl of 2 M CaCl2 and added dropwise to 50 µl of 2x HeBS (280 mM NaCl, 1.5 mM Na2HPO4, and 50 mM HEPES, pH 7.05). After a 2-min incubation period at room temperature, the mixture was added to the cells. Human dermal fibroblasts were transfected using Effectene Transfection Reagent (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. After 24 h, cells were washed and cultured for 12 h in the presence or absence of 50 ng/ml IL-4 or 100 ng/ml IL-13 before luciferase assays were conducted in triplicate according to the instructions of the manufacturer using the Promega Luciferase Assay System (Promega). In some experiments, pRL-Tk (Promega) was cotransfected as internal control for normalization of differences in transfection efficiency. Lysates from these cells were quantitated for luciferase using the Dual-Luciferase Reporter Assay System (Promega).
Preparation of nuclear extracts and EMSA
Nuclear extracts from unstimulated human neonatal dermal
fibroblasts or cells that had been stimulated for 30 min with 50 ng/ml
IL-4, 100 ng/ml IL-13, or 100 U/ml TNF- were prepared according to
the method described by Andrews and Faller (33). A
double-stranded oligonucleotide probe containing the STAT6 binding site
in the eotaxin-3 promoter between positions -86 and -45 was
end-labeled using [32P]dCTP (Amersham,
Little Chalfont, U.K.) and Klenow polymerase (Roche Molecular
Biochemicals). The nucleoprotein binding reaction was performed as
described (34) using 5 µg of nuclear extracts. For
oligonucleotide competition assays, a 50-fold molar excess of cold
oligonucleotide was added to the binding reaction 30 min before the
radiolabeled probe. For supershift experiments, extracts were
preincubated with 2 µg of Ab for 30 min before the radiolabeled probe
was added. All Abs used in supershift experiments were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To explore whether eotaxin-3 expression can be induced in other
cell types than endothelial cells (19), normal human
fibroblasts were stimulated with IL-4 or IL-13 for different times.
Eotaxin-3 mRNA was detected in total RNA preparations by RT-PCR. Both
cytokines stimulated eotaxin-3 expression within 2 h (Fig. 1
). After that, a continuous increase in
steady-state mRNA was observed. The relative potencies of the two
agents were very similar. Cell supernatants from similar experiments
were quantitated for eotaxin-3 protein by ELISA. Both cytokines induced
the expression of eotaxin-3 protein in a time-dependent manner. These
results supported the data obtained at the RNA level. The stimulatory
effect was also dependent on the cytokine concentration. Fig. 2 demonstrates that cultivation of
fibroblasts with increasing quantities of IL-4 or IL-13 for 8 h
led to a corresponding increase in eotaxin-3 mRNA expression. Maximal
chemokine expression was seen at 50 ng/ml IL-4. More than 100 ng/ml
IL-13 was needed to achieve optimal eotaxin-3 production.
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does not synergize with IL-4 to induce eotaxin-3 expression
It has been reported that IL-4 and TNF- led to synergistic
up-regulation of eotaxin-1 synthesis in fibroblasts and epithelial
cells (25, 30, 35). The question was asked whether the
same regulatory principle was operative for induction of the eotaxin-3
gene. Fibroblasts were cultivated for various times with IL-4, TNF-,
or the cytokine combination, and steady-state transcripts for both
eotaxins as well as secreted eotaxin-3 protein was quantitated as
above. IL-4 or TNF- induced transcription of eotaxin-1 mRNA, and the
combination had a synergistic effect as described (29, 30). In contrast, TNF- stimulation did not lead to eotaxin-3
expression, both at the RNA and protein level (Fig. 3). In addition, no additive or
synergistic effect of TNF- was apparent. These data showed that the
regulation of the eotaxin-3 gene was different from the one of
eotaxin-1 with respect to the role of TNF-.
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The mechanism by which IL-4 induces expression of eotaxin-1 at the
molecular level has been shown to depend on the transcription factor
STAT6 (29, 30). Visual inspection of the eotaxin-3
nucleotide sequence upstream of exon 1 revealed the presence of a
canonical STAT6 binding site between positions -62 and -71 relative
to the transcriptional start site (Fig. 4). This sequence differs from the
functional STAT6 binding site in the eotaxin-1 promoter by a single
nucleotide (Fig. 4). To test whether this motif was able to interact
with STAT6, EMSA experiments were conducted. Nuclear extracts prepared
from cytokine-stimulated cells were incubated with a radiolabeled
oligonucleotide probe containing the putative STAT6 binding sequence
(86/45). The results shown in Fig. 5
demonstrate that IL-4 and IL-13 but not TNF- induced the formation
of a nucleoprotein complex. No band was observed in control extracts.
Preincubation of the extracts with Abs directed against STAT6 (M20)
resulted in reduction of the complex and in appearance of two
supershifted complexes. This effect was specific because incubation
with Abs recognizing the NF-B family members p50 and p65 as well as
anti-PU.1 and anti-bcl-6 Abs had no effect on the original
IL-4-induced band. These data strongly suggest that the
cytokine-induced protein factor was STAT6. To corroborate these
results, the same extracts were incubated with the 86/45 M1
oligonucleotide probe. The sequence of this DNA differs from the
wild-type probe by two nucleotide substitutions in the inverted repeat
region of the STAT6 binding site (Fig. 4). Mutations at these positions
have been shown to abrogate STAT6 binding and function (30, 36). This probe could not interact with the cytokine-induced
protein factor (Fig. 6A).
Thus, interaction of the cytokine-induced protein with the 86/45
sequence was dependent on the 5'-TC-3' dinucleotide sequence within the
canonical STAT6 binding motif. Confirmation of the nature of the
cytokine-induced protein was obtained in competition experiments.
Nuclear extracts were preincubated with a 50-fold excess of unlabeled
IgE 104/83 double-stranded oligonucleotide containing the functional
STAT6 binding site of the human IgE germline gene promoter. This
resulted in loss of factor binding to the labeled 86/45 probe (Fig. 6B). The same effect was observed with an excess of 86/45
double-stranded oligonucleotide but not with the 86/45 M1
competitor, which is unable to interact with the cytokine-induced
protein. These data collectively demonstrate that IL-4 or IL-13
stimulated the interaction of STAT6 with a binding site in the
eotaxin-3 promoter.
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The involvement of the STAT6 site in Th2 cytokine-induced
activation of eotaxin-3 gene expression was assessed in transient
transfection experiments using eotaxin-3 reporter gene constructs. Four
DNA segments containing different portions of the putative eotaxin-3
promoter including the STAT6 site were cloned upstream of the firefly
luciferase reporter gene (Fig. 4
). Primary fibroblasts were transfected
with these constructs and stimulated with different cytokines (Fig. 7). In the absence of the cytokines, all
four plasmids led to elevated luciferase expression compared with the
empty vector pGL3basic, demonstrating that the cloned DNA displayed
promoter activity. This activity was comparable in all plasmids.
Stimulation of the transfectants with TNF- did not increase promoter
activity, confirming that this factor does not induce eotaxin-3
expression (see also Fig. 3). IL-4 and also IL-13 stimulation led to an
increase of eotaxin-3 promoter activity in all four plasmids. These
data showed that the cytokine-responsive phenotype of the eotaxin-3
gene occurred at least partially at the level of transcription. Because
all plasmids contain the STAT6 binding site, a regulatory involvement
of this motif appeared likely. In the longest construct EOT3, IL-4
stimulated eotaxin-3 promoter activity by a factor of 4. Similar to the
RNA time-course experiments shown in Fig. 1, no difference between the
Th2 cytokines was noted. The cytokine inducibility of the EOT3S1 and
EOT3S3 plasmids was consistently 2-fold lower than that of EOT3,
whereas the response of EOT3S2-transfected cells was comparable to that
of EOT3. These data suggested the presence of two DNA elements between
positions -233 and -105, and position -928 and -783, which acted as
additional positive regulators of cytokine inducibility. The phenotype
of the EOT3S1 plasmid can be explained by a negative acting
cis-element between positions -783 and -233. To confirm
that the cytokine-inducible phenotype was mediated by the STAT6 binding
site, nucleotide substitutions in the STAT6 site were introduced in the
context of the EOT3 construct as described above (EOT3 M1). Transfected
cells carrying this plasmid were not inducible with the cytokines
anymore, demonstrating that Th2 cytokine inducibility of these reporter
gene constructs was dependent on the intact STAT6 binding site. In
addition, these data suggested that the two additional positively
acting sequences could not confer inducibility without an intact STAT6
binding site.
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). Upon STAT6 cotransfection, reporter
gene expression from the EOT3 plasmid was 7-fold higher in the presence
of IL-4 or IL-13 compared with unstimulated cells. Similar to the data
obtained in primary fibroblasts, the stimulation index of the plasmid
EOT3S1 and EOT3S3 was much lower than that of the longest construct.
Plasmid EOT3S2 was also less responsive than EOT3 but still
significantly better than EOT3S1 and EOT3S3. Again, the STAT6-defective
mutant plasmid did not respond to the cytokine stimulus. These data
confirmed the essential role of the STAT6 protein in activation of the
eotaxin-3 promoter in response to IL-4 or IL-13. In addition, these
data confirmed that the two additional positively acting
cis-acting elements could not induce promoter activation but
required a functional STAT6 binding site.
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-stimulated HEK293 cells that had
been transiently transfected with a STAT6 expression vector. Cells
transfected with empty vector did not produce eotaxin-3 upon
stimulation (Fig. 9). Importantly, the
secretion of the known TNF--inducible chemokine MCP-1 measured in
the same supernatants was very similar irrespective of the presence of
cotransfected STAT6, demonstrating that HEK293 cells are not generally
defective in TNF- signal transduction and that the effect seen with
eotaxin-3 was specific.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An important regulatory role of STAT6 upon IL-4 stimulation has been
recently described for the eotaxin-1 gene (29, 30). The
molecular mechanisms operative after IL-4 induction appear to be very
similar. In both genes, a functional STAT6 binding site is responsible
for mediating IL-4 or IL-13 activation of the respective promoter. This
leads to eotaxin-1 or -3 protein production in HEK293 cells ectopically
expressing STAT6. However, there exist significant differences in the
regulation of the two eotaxin genes in response to TNF-. Eotaxin-1
expression can be stimulated in fibroblasts by TNF- alone, whereas
eotaxin-3 production could not be triggered by this cytokine. In
addition, no synergistic effect of IL-4 plus TNF- could be measured
for eotaxin-3, in contrast to eotaxin-1 (25, 30). The
synergistic effect may be related to the presence of an NF-B binding
site in the eotaxin-1 promoter, which overlaps with the STAT6 site. No
such structural feature was observed around the STAT6 site in the
eotaxin-3 promoter. In addition, eotaxin-3 reporter gene constructs did
not respond to TNF- stimulation in transient transfection
experiments in contrast to eotaxin-1 promoter plasmids (29, 30). Finally, STAT6 expressing HEK293 cells did not synthesize
eotaxin-3 protein upon TNF- stimulation even in the presence of
functional STAT6, whereas eotaxin-1 was readily produced under these
conditions (30). In summary, TNF- does not appear to
play a role in regulation of the eotaxin-3 gene, whereas it is an
important regulator of eotaxin-1 gene expression. This difference may
have consequences in vivo. It is possible that eotaxin-1 is produced
early during the onset of an allergic immune response through the
action of the proinflammatory cytokine TNF-. In contrast, eotaxin-3
may only be synthesized later in the face of a robust Th2 response
characterized by high levels of IL-4 and IL-13. Support for this theory
comes from studies with eotaxin-1-deficient mice, which demonstrated a
role of eotaxin-1 for early recruitment of eosinophils into
allergically challenged lungs but not for later stages when other
inflammatory cells are present (40).
Our studies showed that eotaxin-3 cannot only be produced by endothelial cells (19) but also by fibroblasts. This has potential implications on the function of this chemokine in allergic inflammatory reactions. It can be speculated that eotaxin-3 may not only be important for trans-migration of eosinophils through vascular walls by endothelial derived eotaxin-3 but also for attraction of these leukocytes to the sites of inflammation by fibroblast-derived eotaxin-3. The relative importance of the two eotaxins (and of course also of the other eosinophilic chemokines) for migration of eosinophils within the tissue remains to be explored.
The dependence of eotaxin-3 and eotaxin-1 synthesis on STAT6 upon Th2 cytokine stimulation may also help to explain the phenotype observed in STAT6-deficient mice. In mouse models of allergic lung inflammation (41, 42, 43), contact hypersensitivity (44), and allergic diarrhea (45), STAT6 deficiency was accompanied by a lack of eosinophil influx into the sites of allergic inflammation. This effect may be at least partially due to defective eotaxin-1 and -3 gene activation in these animals.
In summary, this study adds another member to the list of immediate early genes that are induced by STAT6. Another recent example may be the eotaxin-2 gene. Zimmermann et al. (46) demonstrated strong induction of eotaxin-2 mRNA in the lungs of mice transgenic for IL-4. This effect was shown to be dependent on STAT6, because induction of eotaxin-2 mRNA could not be observed in mice transgenic for IL-4 but genetically deficient for STAT6. The fact that most of STAT6-regulated genes are associated with an allergic immune response underscores the importance of STAT6 as therapeutic target in allergic diseases.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: MCP, monocyte chemoattractant protein; HEK, human embryonic kidney.
Received for publication March 26, 2001. Accepted for publication July 10, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induce the migration and activation of normal human eosinophil granulocytes. J. Exp. Med. 176:1489.B and STAT6 in human airway epithelial cells. J. Immunol. 163:6876. induced fibroblasts. J. Immunol. 166:4507.B or PU.1 to mediate IL-4-induced activation of IgE germline gene transcription. J. Immunol. 163:4383.
and Fc receptor II (CD23b) genes activated via binding of an interleukin-4-induced transcription factor to a novel responsive element. Eur. J. Immunol. 23:3066.[Medline]
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