(Received for publication, January 24, 1997)
From the Cell Biology and Inflammation Research, Pharmacia & Upjohn, Inc., Kalamazoo, Michigan 49007
Interleukin-4 (IL-4), an immunoregulatory
cytokine secreted from activated T-helper 2 lymphocytes,
eosinophils, and mast cells, stimulates the expression of a
number of immune system genes via activation of the transcription
factor, STAT6. However, IL-4 can concomitantly suppress the expression
of other immune-related gene products, including light chain,
Fc
RI, IL-8, and E-selectin. We demonstrate that IL-4 activates STAT6
in human vascular endothelial cells and that two STAT6 binding sites
are present in the promoter of the E-selectin gene. IL-4-induced STAT6
binding does not activate E-selectin transcription but instead
suppresses tumor necrosis factor
-induced expression of the
E-selectin gene. STAT6 was found to compete for binding to a region in
the E-selectin gene promoter containing overlapping STAT6 and NF-
B
binding sites, effectively acting as an antagonist of NF-
B binding
and transcriptional activation. This novel mechanism for IL-4-mediated
inhibition of inflammatory gene expression provides an example of a
STAT factor acting as a transcriptional repressor rather than an
activator.
IL-41 is a pleiotropic immunomodulatory cytokine secreted by T-helper 2 (TH2) lymphocytes, eosinophils, and mast cells (1, 2). IL-4 promotes the differentiation of premature lymphocytes to the TH2 subset, induces immunoglobulin class switching in B-lymphocytes, and is present at high levels in tissues of patients with chronic inflammatory diseases such as asthma, where it appears to play an important role in disease progression (3-6). STAT6 (IL-4 STAT) (7), is activated following ligand binding to the IL-4 receptor and has been directly implicated in the transcriptional activation of the low-affinity IgE receptor (FcRMIIb/CD23), the class II major histocompatability complex genes and the constant region of immunoglobulin heavy chain genes (8, 9). Mice lacking STAT6 are deficient in TH2 lymphocyte differentiation and immunoglobulin class switching to the IgE phenotype (10, 11). STAT proteins (Signal Transducers and Activators of Transcription) are a novel family of transcription factors which mediate the biological effects of many cytokines (12). Binding of cytokines to cell surface receptors, expressed on a wide variety of cells, activates receptor-associated Janus kinases (JAKS) resulting in tyrosine phosphorylation of cytoplasmic STAT monomers and subsequent homodimerization, nuclear translocation and transactivation of genes containing STAT regulatory elements (13). STAT6 phosphorylation and activation is associated with JAK1 and JAK3 activation (14, 15). The STAT6 DNA recognition site has been characterized as a GAS-like element with consensus sequence TTC(N3-4)GAA (16). The presence of STAT6 in endothelial cells has not been reported.
Paradoxically, IL-4 also suppresses the expression of many
immune-related proteins. For instance, by promoting the maturation of
TH2 lymphocytes, IL-4 indirectly inhibits the release of the TH1-derived inflammatory mediators, interferon- and IL-2 (1). IL-4
also directly inhibits the secretion of IL-1
, tumor necrosis factor
(TNF
), and IL-6 in monocytes (17) and antagonizes many of the
stimulatory actions of interferon-
and lipopolysaccharide including
metalloproteinase biosynthesis in macrophages (18), IgG receptor
(Fc
RI/CD64) expression in monocytes (19), and
light chain in
murine pre-B cells (20). The immunosuppressing character of IL-4 has
heightened interest in its use as a therapeutic agent, and clinical
investigations of this cytokine are in progress (2). However, the
molecular mechanisms by which IL-4 suppresses gene expression are not
currently understood. Such an understanding would not only provide a
mechanistic appreciation of the biologic action of this cytokine but
might also indicate opportunities for the rational design of
pharmacologic agents with enhanced biological specificity.
The endothelium presents a critical barrier between blood and
surrounding tissues, thereby serving as a key component of the immune
system in regulating leukocyte and macromolecular trafficking and
maintaining a nonthrombotic surface (21). Proinflammatory mediators
such as lipopolysaccharide, IL-1, and TNF
stimulate the
endothelium to express cell adhesion molecules such as E-selectin and
vascular cell adhesion molecule-1 (VCAM-1) and cytokines such as IL-6
and IL-8 (22, 23). Together, cell adhesion molecules and cytokines
promote the adhesion and migration of leukocytes from the blood to
sites of inflammation in surrounding tissues (23). Lipopolysaccharide,
IL-1
, and TNF
treatment of endothelium results in the activation
of the transcription factor, NF-
B, which is a key regulator of cell
adhesion molecule and cytokine gene expression (24). The most well
studied form of NF-
B is a heterodimer composed of p50 and p65 (RelA)
subunits, although homodimers and complexes comprised of additional
family members are known to exist (25). NF-
B is sequestered as an
inactive form in the cytoplasm by the presence of an inhibitor protein, I
B, which masks the nuclear localization signal present on the p50
and p65 subunits. Activation of NF-
B requires both the
phosphorylation and proteolytic degradation of I
B and results in the
translocation of NF-
B to the nucleus where it can bind to specific
promoter sequence elements and induce gene transcription (26). In
vascular endothelium, IL-4 differentially regulates the expression of
cell adhesion molecules such as E-selectin and VCAM-1, and
proinflammatory cytokines such as IL-6 and IL-8 (16-18). IL-4 augments
the expression of VCAM-1 and IL-6 but concomitantly suppresses the
expression of E-selectin and IL-8 (22, 27, 28), thereby modulating leukocyte recruitment to sites of inflammation (19). Treatment with
IL-4 alone increases VCAM-1 protein levels via stabilization of the
VCAM-1 mRNA (30). However, the mechanisms by which IL-4 suppresses
E-selectin and IL-8 levels is unknown. The studies we describe in this
report were designed to explore a possible role for STAT6 in the IL-4
modulation of endothelial E-selectin gene expression.
Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords and cultured as described (29). Briefly, HUVECs were plated in tissue culture flasks pretreated with 0.1% gelatin and grown in medium M199 (Life Technologies, Inc.) containing 20% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 2 mM L-glutamine (Life Technologies, Inc.), 60 µg/ml endothelial cell growth supplement (Collaborative Research, Medford, MA), 10 units/ml heparin (Sigma), and 100 units/ml penicillin G with 100 µg/ml of streptomycin sulfate (Life Technologies, Inc.). Cells used in experiments were from passages 3 to 5.
Cell Adhesion Molecule ELISA AssaysHUVECs were grown to
confluence in 96-well microtitre plates. Cytokine was added as a
10-µl addition to the well medium. Treatments were either TNF (300 units/ml; specific activity, 2 × 109 units/mg) or
IL-4 (10 ng/ml) (Genzyme, Cambridge, MA) or both as described in the
text. At the end of the incubation period, cells were washed once with
phosphate-buffered saline (PBS) and incubated with freshly prepared 4%
paraformaldehyde solution, pH 7, for 60 min. Plates were then washed
once with PBS, blocked overnight at 4 °C with 2% bovine serum
albumin in PBS, washed once with PBS, and incubated with 1 µg/ml
primary antibody in 0.1% bovine serum albumin in PBS at 37 °C for
2 h. Monoclonal antibody to VCAM-1 (CL40) was from Pharmacia & Upjohn, whereas monoclonal antibody to E-selectin was from R & D
Systems (BBA1, Minneapolis, MN). After incubation with primary
antibody, the cells were washed three times with 0.05% Tween 20 in
PBS, incubated with alkaline phosphatase-conjugated goat anti-mouse IgG
(1:500) (Organon Teknika Corp., West Chester, PA) in 0.1% bovine serum albumin in PBS at 37 °C for 1 h, washed three times with 0.05% Tween 20 in PBS, and washed once with PBS. The cells were then incubated in chromogenic substrate (1 mg/ml
-nitrophenyl phosphate in 1 M diethanolamine, 0.5 mM
MgCl2, pH 9.8) at 37 °C for 10-30 min, and absorbance
was measured at 405 nm using a ThermoMax microplate reader (Molecular
Devices, Menlo Park, CA). The results are presented as mean ± standard deviation of quadruplicate samples. Statistical significance
was determined using two way analysis of variance.
HUVECs were treated with
TNF (300 units/ml; specific activity, 2 × 109
units/mg) or IL-4 (10 ng/ml) or both for 30 min. Cells were suspended by treatment with trypsin and pelleted by centrifugation at 1000 × g for 5 min. Nuclear proteins were isolated as described
previously (29).
Nuclear extracts (125 µg) were subjected to electrophoresis on a 10% SDS-polyacrylamide gel, and the fractionated proteins were electrophoretically transferred to Immobilon-P membrane (Millipore, Bedford, MA) using a Multiphor II semi-dry blotting device (Pharmacia Biotech Inc.). The membrane was blocked with 4% nonfat milk powder in PBS with 0.05% Tween 20 (PBS-T), incubated with 0.1 µg/ml rabbit polyclonal antibody to STAT6 (Santa Cruz Biotechnology, Santa Cruz, CA) in PBS-T, washed, and then incubated with a polyclonal donkey anti-rabbit IgG antibody (1:2000) conjugated with horseradish peroxidase (Amersham Corp.). After extensive washing with PBS-T, chemiluminescent substrate was added (ECL detection system, Amersham Corp.), and the membrane was subjected to autoradiography with Hyperfilm MP (Amersham Corp.).
Electrophoretic Mobility Shift AssayOligonucleotides were
end-labeled with [-32P]ATP and T4 polynucleotide
kinase as single strands prior to annealing to form double stranded
target molecules. The NF-
B consensus DNA, 5
AGTTGAGGGGACTTTCCCAGGC
3
, was purchased as a double stranded oligonucleotide from Promega
(Madison, WI). The oligonucleotide sequences used for E-selectin and
Fc
RI are given under "Results." For each assay, 10 µg of
nuclear protein extract was incubated with 35 fmol of
32P-labeled oligonucleotide probe in binding buffer (4%
glycerol, 10 mM Tris.Cl, 50 mM NaCl, 1 mM MgCl, 0.5 mM EDTA, 0.5 mM
dithiothreitol, and 50 µg/ml poly(dI-dC), pH 7.5) for 30 min at room
temperature. Competition studies were performed by the addition of a
50-fold molar excess of unlabeled oligonucleotide to the binding
reaction. Supershifting experiments were performed by the addition of
antibody to the binding reaction for 20 min following the normal 20-min binding reaction. Rabbit polyclonal antibodies to p50 and p65 were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Resultant
protein-DNA complexes were resolved on native 6% polyacrylamide gels
using a 0.5 × TBE buffer (0.1 M Tris-Cl, 90 mM boric acid, 1 mM EDTA, pH 8.4). The gels
were prerun for 45 min at 150 V, and the samples were then
electrophoresed at 250 V for 2-3 h at 4 °C, dried onto paper under
vacuum, and subjected to autoradiography.
HUVECs were co-transfected with
pCMV (Clontech, Palo Alto, CA), a mammalian vector containing the
-galactosidase reporter driven by the cytomegalovirus promoter, and
pE-luc, a mammalian vector containing 861 base pair of the E-selectin
promoter (
9 to
870 base pairs from translation start site) inserted
upstream of the luciferase reporter plasmid pGL2 (Promega, Madison,
WI). Site-directed mutagenesis of pE-luc was performed using
Pfu DNA polymerase (Stratagene) extension of both plasmid
strands from mutated primers. The sense primers were;
5
-GCATCGTGGATA(T)(T)CCCGGGAAAG-3
where the Ts in parentheses were
changed to Cs to construct two separate point mutants. Clones were
sequenced and shown to contain only the introduced mutation. The
transfection procedure used Lipofectin reagent as outlined in the
manufacturer's instructions (Life Technologies, Inc.). Briefly, cells
were seeded into gelatin coated 6-well plates and allowed to grow to
60% confluency. Cells were transfected with 6 µl of Lipofectin and
500 ng of each vector in 800 µl of serum-free medium for 3.5 h
and then incubated in normal medium for 24-48 h. In experiments where
total cytokine treatment was for 5 h, the cytokine was added
48 h after transfection. In experiments involving cytokine
pretreatment, the cytokine was added 30 h after transfection.
Following cytokine treatment cells were washed once in ice-cold PBS,
solubilized by incubation in 200 µl of reporter lysis buffer for 15 min (Promega, Madison, WI), transferred to a 96-well plate, and
centrifuged to pellet cellular debris, and the supernatant was stored
at
80 °C. Luciferase activity was measured using the Luciferase
Assay System (Promega, Madison, WI), and
-galactosidase activity was
determined by the Luminescent
-galactosidase Genetic Reporter System
(Clontech, Palo Alto, CA). Both activities were quantified in a model
ML1000 Luminometer with version 3.993 software (Dynatech, Chantilly, VA). Final E-selectin-luciferase activities were normalized against
-galactosidase activity.
HUVECs
were grown to confluency on gelatin-coated coverslips, treated with
TNF (300 units/ml; specific activity, 2 × 109
units/mg) and/or IL-4 (10 ng/ml) for 30 min and then fixed with 4%
paraformaldehyde for 20 min. Fixed cells were washed in PBS, permeabilized in 100% methanol
20 °C for 6 min, washed again in
PBS, and incubated with polyclonal antibody to STAT6 or p65 (Santa Cruz
Biotechnology, Santa Cruz, CA) (1 µg/ml) in PBS with 0.1% bovine
serum albumin for 60 min. Cells were then incubated with goat
anti-rabbit IgG antibody conjugated to biotin (Sigma) for 60 min, washed in PBS, and incubated with 20 µg/ml fluorescein isothiocyanate-labeled avidin (Sigma) for 20 min in
the dark. Coverslips were washed and fixed face down onto microscope
slides in the presence of 1 mg/ml o-phenylenediamine
dihydrochloride (Sigma) dissolved in PBS-glycerine
(1:5, v/v). Cells were viewed at 400× magnification with a Nikon
Optiphot-2 microscope equipped with epiillumination and a fluorescein
excitation filter and photographed on Kodak Gold 400 print film.
Total RNA was
isolated from HUVECs using the reagent RNAzol (Tel-Test, Inc.,
Friendswood, TX) based on the procedure of Chomczynski and Sacchi (30).
RNA was resuspended in 15 µl of 1 mM EDTA and quantified
by absorbance at 260 nm. Total RNA was size fractionated on a 1%
agarose 0.6 M formaldehyde gel (31). After electrophoresis the gel was blotted onto Hybond-N+ membrane (Amersham Corp.) using the
manual capillary blot transfer system (Life Technologies, Inc.), and
the RNA was cross-linked to the membrane by UV irradiation in a
Stratalinker 2400 (Stratagene, La Jolla, CA). cDNAs of
approximately 500 base pairs in length were isolated by reverse
transcription-polymerase chain reaction from HUVECs using the primer
pair 5-TGGTCTTACAACACCTC-3
and 5
-CAGGCTTCCATGCTCAG-3
for human
E-selectin (32) and 5
-TTGCAGCTTCTCAAGCT-3
and 5
-ATCCTCAATGACAGGAG-3
for human VCAM-1 (33). A glyceraldehyde-3-phosphate dehydrogenase
cDNA was isolated from an amplimer kit (Clontech, Palo Alto, CA).
cDNA probes were prepared by random prime labeling with
[
-32P]dCTP (3000 Ci/mmol) using the Prime-It II
labeling kit (Stratagene, La Jolla, CA).
Group means and standard deviations were analyzed using one-way analysis of variance.
To establish that IL-4 can differentially modulate
cytokine-induced E-selectin and VCAM-1 levels, we examined the cell
surface expression of E-selectin and VCAM-1 in HUVECs stimulated with TNF in the presence or the absence of IL-4 (Fig. 1).
Treatment with IL-4 alone for 5 or 23 h had no effect on
E-selectin expression (Fig. 1A, lanes 2 and
3). However, consistent with previous reports (28),
treatment with IL-4 alone significantly increased VCAM-1 levels above
control (Fig. 1B, lanes 2 and 3).
Treatment with TNF
resulted in significant increase in E-selectin
and VCAM-1 cell surface levels (Fig. 1, lanes 4). IL-4
treatment in conjunction with TNF
resulted in an augmentation of
VCAM-1 protein levels and a significant, although not complete,
inhibition of E-selectin expression. Of note, although no pretreatment
was necessary to observe the IL-4 enhancement of VCAM-1 expression,
pretreatment was required for inhibition of E-selectin.
The IL-4-dependent increase in VCAM-1 levels is known to
result from a stabilization of VCAM-1 mRNA (28). We therefore
explored whether treatment with IL-4 also affected E-selectin mRNA
stability. IL-4 treatment augmented the levels of VCAM-1 mRNA after
4 h and stabilized VCAM-1 transcripts over the following 12 h
as previously reported. In contrast, IL-4 treatment had no effect on
the half-life of E-selectin mRNA (t1/2 = 5.5 h, t1/2 + IL-4 = 6 h) but did
result in a 22% decrease in the levels of steady state E-selectin
mRNA at 4 h (data not shown). Therefore,
IL-4-dependent stabilization of mRNA is specific for
VCAM-1 and was not found to occur with the mRNA for another cell
adhesion molecule, namely E-selectin. The observed decrease in
E-selectin mRNA levels suggested that the IL-4 effect on E-selectin
expression was being mediated at the level of transcription. To
demonstrate this further we examined the effect of IL-4 on the
cytokine-induced transcriptional activation of the E-selectin promoter,
using an E-selectin promoter-luciferase reporter gene construct. HUVEC
monolayers were co-transfected with a constitutively expressed
-galactosidase reporter gene construct and 861 base pairs of the
E-selectin promoter fused to the luciferase reporter gene. After
48 h, cells were treated with either TNF
or IL-4 alone, TNF
and IL-4 added simultaneously, or IL-4 added as a pretreatment of 20 min prior to TNF
(Fig. 2). Cells in growth medium
alone (lane 1) or treated with IL-4 for 5 h (lane
2) showed equal luciferase activity, demonstrating that IL-4
treatment alone cannot activate transcription from the E-selectin
promoter above levels observed in untreated cells. TNF
treatment
(lane 3) resulted in a 4.5-fold increase in luciferase activity, an observation consistent with studies showing a key role for
TNF
-induced signal transduction mechanisms in E-selectin transcriptional activation (34). Cells treated simultaneously with both
TNF
and IL-4 (lane 4) showed a pronounced decrease in
E-selectin promoter activity as compared with cells treated with TNF
alone. If cells were treated with IL-4 for 20 min (lane 5)
prior to TNF
addition, the inhibitory effect of IL-4 was enhanced, with E-selectin promoter activity being inhibited by 50% compared with
TNF
-treated cells (p < 0.001). This result
indicated that IL-4 inhibition of TNF
-induced expression of
E-selectin was likely due to inhibition of transcriptional activation
of the E-selectin gene.
In leukocytes, IL-4 has been shown to activate STAT6, a key component
of the IL-4-dependent transcription factor, IL-4 STAT (7).
We therefore examined whether STAT6 was present in endothelial cells
and activated in response to IL-4 treatment. Nuclear extracts from
HUVECs treated with TNF or IL-4 were examined for immunoreactive STAT6 protein (Fig. 3A). An immunoreactive
species of a molecular weight identical to STAT6 was observed in
nuclear extracts from IL-4-treated cells but not in extracts from
quiescent or TNF
-treated cells. Immunofluorescence studies with
HUVECs treated with TNF
and IL-4, either individually or in
combination, were used to examine in situ the activation and
nuclear translocation of STAT6 and p65, a subunit of the
TNF
-inducible transcription factor NF-
B (35) (Fig.
3B). Quiescent cells exhibited diffuse cytoplasmic staining
but no specific nuclear staining for NF-
B or STAT6. Treatment with
TNF
resulted in the nuclear localization of NF-
B. STAT6 staining
was no different from that observed with untreated cells. In contrast,
IL-4 treatment had no effect on NF-
B activation but resulted in the
nuclear translocation of STAT6 to the nucleus. Treatment with both
TNF
and IL-4 resulted in the simultaneous activation and nuclear
localization of both NF-
B and STAT6. These results demonstrate that
STAT6 is present in endothelial cells and is specifically activated by
IL-4 and not by TNF
.
A previous report has shown that IL-4 directly inhibits the activation
of NF-B in monocytes but not fibroblasts (36). To investigate this
mechanism we treated HUVECs with TNF
for 20 min, with and without a
20-min IL-4 pretreatment, and examined nuclear extract DNA binding
activity to STAT6 and NF-
B oligonucleotides (Fig. 4).
STAT6 binding activity was only observed in cell extracts from
IL-4-treated cells. We observed a minor decrease in NF-
B DNA binding
activity from cells treated with TNF
and IL-4 compared with cells
treated with TNF
alone (see legend to Fig. 4). Therefore, IL-4 did
not appear to markedly inhibit NF-
B activation in HUVECs.
We hypothesized that STAT6 binding sites may be present in the
E-selectin gene promoter and that STAT6 binding may mediate the
IL-4-induced inhibition of E-selectin expression. Computer analysis of
the E-selectin promoter revealed two potential STAT6 recognition
sequences (TTCN3-4GAA) (16) at positions 112 to
121
(E-selectin A) and
41 to
50 (E-selectin B) relative to the
transcription initiation site (Fig. 5A).
Oligonucleotides corresponding to these sites were synthesized, and the
ability of nuclear proteins to interact with these two putative
sequence elements was examined (Fig. 5B). IL-4-inducible
complexes of identical electrophoretic mobility were observed binding
to both of the E-selectin oligonucleotides and to an oligonucleotide
containing a known STAT6 binding site from the Fc
RI gene (7). The
IL-4-inducible complex formed with the Fc
RI oligonucleotide was
diminished in the presence of STAT6 antibody, confirming that the
DNA-protein complex contained STAT6 (Fig. 5B, lane
3). The presence of excess Fc
RI oligonucleotide resulted in the
complete loss of the IL-4-induced E-selectin A and B complexes,
demonstrating that the factor interacting with the E-selectin sites was
the same as that binding to the Fc
RI sequence (lanes 6 and 10). Addition of STAT6 antibody to the binding reactions
inhibited complex formation similar to that observed with Fc
RI
(lanes 7 and 11). Therefore, STAT6 specifically recognizes two sites within the E-selectin gene promoter, and STAT6
binding to these sites is induced by IL-4 treatment.
To determine whether IL-4-induced activation of STAT6 correlated
directly with the observed inhibition of E-selectin expression, we used
EMSA and ELISA analyses to compare the dose dependence of IL-4
activation of STAT6 (Fig. 6A) and inhibition of
E-selectin cell surface protein levels (Fig. 6B). The
dose-dependent concentration range of STAT6 nuclear
localization and DNA binding activity to the E-selectin A site was
0.1-10 ng/ml IL-4. Similarly, IL-4 inhibition of TNF-induced
E-selectin expression was observed at concentrations between 0.1 and 10 ng/ml IL-4, with maximal inhibition at 10 ng/ml.
Inspection of the sequences surrounding the E-selectin A site revealed
that this STAT6 recognition sequence overlaps two adjacent, well
characterized NF-B binding sites critical for TNF
-induced transcriptional activation (Fig. 7A) (34).
This observation suggested that binding of STAT6 to this site might
antagonize NF-
B binding and account for the observed IL-4
suppression of TNF
-induced E-selectin expression. To explore this
hypothesis, we synthesized an extended oligonucleotide containing both
the NF-
B sites and the STAT6 site, as shown in Fig. 6A,
and examined the binding of TNF
- and IL-4-activated nuclear proteins
to this sequence (Fig. 7B). Consistent with earlier
experiments, nuclear extracts from cells treated with IL-4 induced the
formation of a complex that was inhibited by the addition of unlabeled
Fc
RI oligonucleotide or STAT6 antibody (Fig. 7B,
lanes 2-4). Nuclear extracts from cells treated with TNF
alone induced a distinct complex of different mobility than that
identified as containing STAT6 (Fig. 7B, lane 5).
Formation of this complex was inhibited by the addition of unlabeled
NF-
B binding oligonucleotide or antibodies to the NF-
B component
proteins, p50 and p65 (Fig. 7B, lanes 6 and
7). When nuclear extracts from cells treated with both
TNF
and IL-4 were analyzed, both the NF-
B and STAT6 complexes were present in the same binding reaction (Fig. 7B,
lane 8). No novel complexes were observed, suggesting that
NF-
B and STAT6 cannot bind to the same site simultaneously. Addition
of unlabeled Fc
RI exclusively diminished STAT6 binding, whereas
unlabeled NF-
B oligonucleotide prevented formation of NF-
B
complex only (Fig. 7B, lanes 9-10). Similarly,
addition of STAT6 antibody resulted in a loss of the STAT6 containing
complex without affecting the NF-
B complex, whereas antibody to p50
and p65 resulted in the loss of the NF-
B complex without affecting
the STAT6 complex (Fig. 7B, lanes 11-12).
Therefore, NF-
B and STAT6 appear to bind a shared region of the
E-selectin promoter in a manner that is mutually exclusive and
consequently antagonistic.
To demonstrate this transcription factor antagonism in vivo,
we performed site-directed mutagenesis on the E-selectin
promoter-reporter construct. Two point mutant constructs were generated
that changed the STAT6 binding site but retained the NF-B binding
consensus sequence of the upstream site. According to a previous study, mutant-1 should abolish STAT6 binding, and mutant-2 should retain the
STAT6 consensus sequence (8). In a promoter-reporter assay (Fig.
8) the control E-selectin luciferase construct showed
enhanced luciferase activity in the presence of TNF
that was
inhibited 50% with an IL-4 pretreatment (lanes 1-3) as
shown in an earlier experiment (Fig. 2). Mutant-1 showed an unexpected
partial loss of TNF
stimulation, suggesting that this point mutation
does affect NF-
B binding even though the introduced mutation
conformed to the theoretical consensus sequence. However, no additional inhibition was observed with IL-4, suggesting that STAT6 binding at
this site is responsible for the inhibitory effect observed (Fig. 8,
lanes 4-6). A second mutant-1 isolate exhibited the same activity profile (data not shown). Mutant-2 showed TNF
stimulation similar to that observed for the control and was likewise inhibited by
IL-4, confirming that this mutation does not abolish STAT6 binding
(Fig. 8, lanes 7-9).
IL-4 has been shown in vivo to retard the infiltration
of neutrophils and monocytes and to enhance the recruitment of
lymphocytes and eosinophils to sites of inflammation (3, 37). Selective leukocyte recruitment is a feature of inflammatory diseases such as
asthma, which are characterized by an eosinophilic infiltrate and high
levels of IL-4 (6). Evidence suggests that the mechanisms whereby IL-4
exerts this effect are diverse but include the augmentation of VCAM-1,
an important ligand for eosinophil adhesion but not required for the
recruitment of neutrophils (38); the inhibition of IL-8 secretion, a
major neutrophil chemoattractant (39); and the suppression of
E-selectin, an essential endothelial adhesion molecule for the initial
attachment and rolling of neutrophils (40). Intravital microscopic
evaluations of leukocyte rolling in E-selectin-deficient mice show
changes in initial tethering of leukocytes to the endothelial surface
(41). Indeed, monoclonal antibodies to E-selectin inhibit neutrophil
attachment to activated endothelium but have little effect on
eosinophil adhesion (42). E-selectin expression is activated by
proinflammatory mediators such as TNF and IL-1
released by
activated macrophages and mast cells at sites of inflammation. The
molecular pathways by which TNF
and IL-1
promote the
transcriptional activation of E-selectin expression are under intensive
investigation but have highlighted the necessity for activation of the
transcription factor, NF-
B.
We present evidence for a mechanism whereby IL-4 suppresses E-selectin
protein levels via the transcription factor STAT6. These experiments
demonstrate that NF-B and STAT6 are activated by distinct signal
transduction pathways in endothelial cells and that treatment with
TNF
and IL-4 results in the concurrent nuclear localization of both
NF-
B and STAT6. EMSA analysis demonstrates that STAT6 can compete
for binding to a dual NF-
B enhancer element previously shown to be
crucial for maximal E-selectin expression (34). The presence of STAT6
in the nucleus corresponds with repressed E-selectin promoter activity
and the suppression of E-selectin mRNA and cell surface protein.
STAT6 therefore acts as an antagonist of NF-
B, blocking its binding
and ability to transactivate the E-selectin gene.
The property of STAT6 acting as a transcriptional repressor may not be
unique to the E-selectin gene. The antagonistic action of STAT6 that we
have identified may also occur in an analogous manner within the
FcRI/CD64 gene in monocytes (43). Transcriptional activation of this
gene is regulated by the interferon-
-activated transcription factor,
GAF, but is inhibited by treatment with IL-4. STAT6 and GAF can both
bind the recognition sequence TTCN3GAA, which is present in
the Fc
RI gene promoter. IL-4-activated STAT6 may compete for binding
to this site, thereby preventing GAF-mediated transcription of the
Fc
RI gene. Further work is necessary to determine if similar STAT6
antagonistic mechanisms are responsible for the IL-4 suppression of
other genes.
Complete inhibition of E-selectin expression by IL-4 was not observed,
probably because a third NF-B site, located downstream from those
present in the E-selectin A site, has been shown to promote up to 50%
of the total NF-
B-mediated transcriptional activation of the
E-selectin gene (34). This site is not in close proximity to either of
the STAT6 sites identified in the study. The function of the second
STAT6 site (E-selectin B), which is located downstream of the A site,
is at this time not clear. This site does not appear to overlap any
binding sites for known transcription factors. The presence of STAT6
alone is not sufficient to induce transcription of the E-selectin gene,
presumably because it is unable to promote the formation of an
effective transcriptional complex. This is interesting because a
STAT6-containing transcription factor has been shown to activate the
FcRMIIb/CD23, class II major histocompatability complex, and mouse C
domain of immunoglobulin heavy chain genes in leukocytes (8, 9). It
will be important to compare the composition of STAT6 transcription
factor complexes in endothelial cells and monocytes. It will also be
valuable to identify genes that are transcriptionally activated by
STAT6 in endothelial cells so as to define additional nuclear factors
required for STAT6-mediated transcription.
Activation and translocation of STAT6 by IL-4 is rapid, leading to
STAT6 DNA binding activity within 30 min of exposure to cytokine.
Although some inhibition of TNF-induced E-selectin promoter activity
was observed when TNF
and IL-4 were added simultaneously, the
inhibitory effect of IL-4 was more pronounced when HUVECs were
incubated with IL-4 for 20 min prior to TNF
treatment. This suggests
that either the pathway of NF-
B activation is more rapid than that
of STAT6 or that protein-DNA binding kinetics at the E-selectin A site
in vivo favor binding of NF-
B so that elevated levels of
STAT6 in the nucleus are required to effectively compete with NF-
B
for binding. Our observation that the E-selectin A site is completely
integrated within two adjacent NF-
B sites presented the possibility
that conservation of the STAT6 recognition sequence might be a
secondary consequence of conservation of the NF-
B sites. Although
the decameric NF-
B consensus sequence varies considerably in many
genes shown to be transcriptionally activated by NF-
B (26), we were
unable to destroy STAT6 activity without affecting NF-
B activity.
Therefore, conservation of the STAT6 site within two potentially
variable NF-
B sites could represent the selective retention of an
important biological role for IL-4 on expression of the E-selectin
gene. In this regard, Schindler and Baichwal (34) have shown that
nucleotide changes to the key residues comprising the STAT6 binding
site result in significant inhibition of NF-
B-mediated promoter
activity. Therefore, the conservation and interaction of these three
sites appears closely linked.
The mechanisms whereby IL-4 modulates the expression of E-selectin and VCAM-1 in endothelium highlights the fact that an individual cytokine may activate different intracellular signal transduction pathways. Although IL-4 treatment resulted in STAT6 activation and the consequent suppression of E-selectin gene transcription, we found no STAT6 binding sites in the VCAM-1 promoter. IL-4 augmentation of VCAM-1 expression is instead mediated via a poorly characterized pathway that results in the stabilization of VCAM-1 mRNA. This mechanism appears highly selective because it had no effect on E-selectin mRNA or glyceraldehyde-3-phosphate dehydrogenase mRNA stability (28). In addition, IL-4 has been shown to result in the phosphorylation of a 170-kDa substrate called 4PS (44). This protein is related by homology to insulin receptor substrate-1 and interacts with phosphatidylinositol-3 kinase. Therefore, IL-4 may activate at least three signal transduction pathways in a single cell type. Drug discovery efforts that focus upon exploiting such discrete signaling pathways may yield therapies with enhanced biological specificity and reduced side effects.
The purpose of this study was to examine the molecular mechanism
whereby an immunomodulatory cytokine, IL-4, may act on E-selectin expression and thereby alter the inflammatory profile of diseases such
as asthma. That E-selectin suppression is mediated by an antagonism of
NF-B reiterates the importance of this transcription factor in
inflammatory disease. NF-
B has been shown to be activated in
conditions as diverse as allergic airway inflammation (45), atherosclerosis (46), endotoxic shock (47), ischaemia-repurfusion injury (48), rheumatoid arthritis (49), restenosis (50), and sunburn
(51). Furthermore, it is notable that many agents that exhibit
anti-inflammatory properties have been shown to inhibit NF-
B action
including glucocorticoids (52, 53), antioxidants (54), salicylates
(55), gliotoxin (56), flavinoids (57), as well as endogenous mediators
such as nitric oxide (58) and the immunomodulatory cytokine, IL-10
(59). We now show that IL-4 also exerts some of its immunomodulatory
action by inhibiting the actions of NF-
B.