1Department of Internal Medicine, Shiga University of Medical Science, Seta-Tukinowa, Otsu 520-2192; and 2Department of Pharmacology, Graduate School of Medicine, Osaka City University, Osaka 545-8585, Japan
Submitted 29 January 2003 ; accepted in final form 14 May 2003
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ABSTRACT |
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interleukin-11; inflammatory bowel diseases; transforming growth factor-1; interleukin-1
In the gastrointestinal tract, it is of particular interest that IL-11 prevents or improves the development of acute and chronic intestinal inflammation in animal models. For example, recombinant human (rh)IL-11 downregulated the expression of mucosal proinflammatory cytokines and ameliorated the development of colitis in HLA-B27 transgenic rats (35). Recombinant IL-11 was also protective against trinitrobenzene sulfonic acid-induced colitis (37). Treatment with rhIL-11 reduced the mortality in a murine model of combined chemotherapy and irradiation, in which mortality was due to sepsis secondary to gastrointestinal mucosal damage (14, 32). These effects were explained in association with at least two distinct mechanisms. As mentioned above, IL-11 is a potent inhibitor of the activation of mucosal macrophages and T cells (4, 10, 45, 46). Another mechanism is that IL-11 enhances the integrity of the intestinal mucosa through trophic effects on the gastrointestinal epithelium (23, 32). On the basis of these studies, Sands et al. (38, 39) reported that rhIL-11 is safe and effective in inducing remission in mild to moderately active Crohn's disease. Thus IL-11 plays an important role in the pathophysiology of intestinal inflammation and repair process.
Subepithelial myofibroblasts (SEMFs) are present immediately under the
basement membrane in the normal intestinal mucosa, juxtaposed to the bottom of
the epithelial cells (24,
36). These cells are
specialized mesenchymal cells that exhibit the ultrastructural features of
both fibroblasts and smooth muscle cells and can be characterized by positive
immunoreactivity for -smooth muscle actin but negative immunoreactivity
for desmin. These cells play a central role in the regulation of a number of
epithelial cell functions, such as proliferation and differentiation and
extracellular matrix (ECM) metabolism affecting the growth of the basement
membrane (20,
24,
31,
36). Recent studies from our
laboratory (18) have suggested
that SEMFs might play a role in the pathogenesis of intestinal inflammation
via the secretion of proinflammatory cytokines, such as IL-6 and a variety of
chemokines.
Given the importance of IL-11 expression in intestinal inflammation, we
were interested in the physiological secretion of IL-11 in the human
intestine. Previously, IL-11 secretion has been reported in bone
marrow-derived stromal cells
(33,
47) and mesenchymal cells
derived from the skin (42),
joints (26), and lung
(15). A549 lung cancer cell
line also secretes IL-11 (44).
However, IL-11 secretion in the intestinal mucosa remains unclear. In this
study, we investigated IL-11 secretion in human intestinal SEMFs and assessed
the key signaling pathways in this response. This study demonstrated that
intestinal SEMFs are the local bio-synthetic site for IL-11. Among the various
cytokines and growth factors, IL-1 and transforming growth factor
(TGF)-
1 act as potent inducers of IL-11 secretion through the
transcription factor activating protein (AP)-1- and the mitogen-activated
protein (MAP) kinase-dependent pathways.
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MATERIALS AND METHODS |
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Culture of human colonic myofibroblasts. The primary cultures of SEMFs were prepared according to the method reported by Mahida et al. (24), and the characters of the cells have been described in our previous reports (18, 31). The cells were cultured in DMEM containing 10% FBS. All culture media were supplemented with 50 U/ml penicillin and 50 µg/ml streptomycin. The studies were performed on passages 2-6 of myofibroblasts isolated from three resection specimens.
Quantification of human IL-11. The amount of antigenic IL-11 in the samples was determined by sandwich ELISA kits purchased from TECHNE (Minneapolis, MN). The lower detectionlimit was 15.6 pg/ml for human IL-11.
Northern blot analysis. Total cellular RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform method (7). Northern blot analysis was performed according to the method previously described (2, 41). The hybridization was performed with a 32P-labeled human IL-11 probe, generated by a random primed DNA labeling kit (Amersham, Arlington Heights, IL), and evaluated by autoradiography. The human IL-11 cDNA probe was prepared from a monolayer of human umbilical vein endothelial cells by the RT-PCR method using the primers: 5'-CTGAGCCTGTGGCCAGATACA-3' corresponding to nucleotides 99-119 isolated by Paul et al. (33) and 5'-CTCCAGGGTCTTCAGGGAAGA-3' coresponding to nucleotides 434-414. The PCR products were ligated into the TA cloning vector (Promega, Madison, WI) and were sequenced by the dideoxynucleotide chain termination method. The human IL-6 and IL-8 cDNA probes were described in our previous reports (2, 41).
Nuclear extracts and EMSA. Nuclear extracts were prepared from the
cells exposed to IL-1 (10 ng/ml) and TGF-
1 (50 ng/ml) for 2 h by
the method of Dignam et al.
(12). Oligonucleotides from
the AP-1 binding motif in the IL-11 promoter
(5'-AGGGTGAGTCAGGATGTGTCAGGCC) were used
(29,
44). The consensus sequence
for the binding of AP-1 is underlined. The oligonucleotides were
5'-end-labeled with T4 polynucleotide kinase (Promega, Madison, WI) and
[
-32P]ATP (Amersham). The binding reactions were performed
according to the methods previously described. Supershift experiments were
performed as described above, except that 1 µl of antibody to each
transcription factor was added to the binding mixture in the absence of
labeled probe. Antisera specifically recognizing each transcriptional factor
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): anti-pan Fos,
sc-253x; anti-pan Jun sc-44x; anti-c-Fos, sc-7202x, anti-FosB, sc-48x;
anti-Fra-1, sc-605x; anti-Fra2, sc-13017x; anti-JunB, sc-46x; anti-c-Jun,
sc-1694x; and anti-JunD, sc-74x. The experiments with unlabeled
oligonucleotides used a 100-fold molar excess relative to the radiolabeled
oligonucleotide.
Adenovirus-mediated gene transfer. We used a recombinant
adenovirus expressing the dominant negative mutant of c-Jun (Ad-DN-c-Jun) and
a recombinant adenovirus containing bacterial -galactosidase cDNA
(Ad-LacZ), as described in a previous report
(48). The dominant negative
mutant c-Jun (TAM67) is lacking the transactivational domain of amino acids 3
to 122 of the wild-type c-Jun but retains the DNA-binding domain
(5). In preliminary
experiments, the Ad-LacZ infection of intestinal SEMFs at a
multiplicity of infection of 200 showed a maximal expression (85% positive)
for
-galactosidase. The recombinant adenovirus was transferred into the
intestinal SEMFs according to the methods previously described
(48). The cells were made
quiescent for 48 h before being assessed for the effects of the transferred
gene. The cells were then stimulated for 12 h with IL-1
(10 ng/ml) or
TGF-
1 (50 ng/ml), and the IL-11 mRNA expression was determined by
Northern blot analysis. The recombinant adenovirus containing the bacterial
-galactosidase gene (Ad-LacZ) was used as the negative control
for Ad-DN-c-Jun.
Nuclear run-on assays. Nuclear run-on assays, using nuclei from confluent SEMFs, were performed according to the method described previously (2). In this experiment, cells were exposed to stimuli for 8 h, scraped off, and lysed in buffer (10 mM Tris, pH 7.4, 10 mM NaCl2, 3 mM MgCl2, 0.5% Nonidet P-40). Empty plasmids of the TA cloning vector were also used to detect nonspecific background.
Western blot analysis. The cells were exposed to cytokines in the presence or absence of inhibitors for the indicated periods of time. The cells were then washed with PBS and lysed in SDS sample buffer containing 100 µM orthovana-date. Western blot analysis was performed according to the method previously described (41).
Measurement of radioactivity. The radioactivity of each band from the Northern blot analysis was determined by the Instant Imager electronic autoradiography system (model 2024/417257, Packard, Meriden, CT). For a comparison of the radioactivity in the stability study, each value was converted to a relative radioactivity based on the value at assay start.
Statistical analysis. The data are expressed as means ± SD. The statistical significance of the changes was determined by Mann-Whitney U-test. Differences resulting in P values <0.05 were considered to be statistically significant.
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RESULTS |
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The effects of IL-1 and TGF-
1 were also observed at the mRNA
levels (Fig. 1B). The
cells were incubated for 12 h, and the IL-11 mRNA expression was assessed by
Northern blot analysis. Both IL-1
and TGF-
1 induced a marked
increase in the IL-11 mRNA expression. The IL-11 mRNA consisted of two
transcripts of 1.5 and 2.5 kb, which differ at their 3'-polyadenylation
sites, yet encode the same function
(3). Compared with the
induction of IL-6 mRNA, IL-1
was a common inducer of IL-11 and IL-6, but
TGF-
did not stimulate IL-6 mRNA expression. In contrast, TNF-
was a strong inducer of IL-6 mRNA, but its effect on IL-11 mRNA expression was
modest. Thus it became clear that among the various factors, IL-1
and
TGF-
1 were the most potent inducers of IL-11 secretion in intestinal
SEMFs.
Induction of IL-11 by IL-1 and
TGF-
1. Intestinal SEMFs were incubated for 12 h with
increasing concentrations of IL-1
, and the IL-11 mRNA expression was
analyzed by Northern blot analysis (Fig.
2A). IL-1
and TGF-
induced a dose-dependent
increase in IL-11 mRNA expression. The effect of IL-1
was detected at as
low as 0.01 ng/ml and reached a maximum at 10 ng/ml. The effect of TGF-
1
was observed at as low as 1.0 ng/ml and reached a maximum at 100 ng/ml.
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These effects were also observed at the protein level
(Fig. 2A,
right). Incubation with IL-1 or TGF-
1 for 48 h dose
dependently induced IL-11 protein secretion.
Kinetics of IL-11 induction by IL-1 and
TGF-
1. The kinetics of IL-1
- and TGF-
1-induced
IL-11 mRNA expression were evaluated in human SEMFs
(Fig. 2B). The cells
were stimulated with IL-1
(10 ng/ml) or TGF-
1 (50 ng/ml), and the
sequential changes in IL-11 mRNA expression were determined by Northern blot
analysis. IL-1
induced an increase in the accumulation of IL-11 mRNA,
and this reached a maximum at 12 h after stimulation. Thereafter, the
induced-IL-11 mRNA levels decreased. TGF-
1 also induced an increase in
the accumulation of IL-11 mRNA. This reached a maximum at 12 h after
stimulation and remained at the same level for a further 12 h.
The effects of IL-1 and TGF-
1 on IL-11 protein secretion were
also evaluated (Fig.
2B, right). Each factor induced a time-dependent
increase in IL-11 levels in the supernatant. The effects of IL-1
were
noted within 24 h, and TGF-
1 induced a continuous increase in IL-11
secretion.
Modulation of transcription factor activation. Nuclear run-on
assay demonstrated that IL-1 and TGF-
1 induced an increase in the
transcription activity of the IL-11 gene
(Fig. 3A), indicating
that transcriptional activation of the IL-11 gene is involved in
IL-1
- and TGF-
1-induced IL-11 mRNA expression in intestinal
SEMFs.
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Two AP-1 motifs are located in the promoter region of the IL-11
gene (29,
44). To elucidate the
mechanisms underlying the response to IL-1 and TGF-
1 in intestinal
SEMFs, we evaluated AP-1 activation. As demonstrated in
Fig. 3B, stimulation
with either IL-1
(10 ng/ml) or TGF-
1 (50 ng/ml) for 2 h induced an
increase in AP-1-DNA binding activity. The effect of TGF-
1 was stronger
than that induced by IL-1
. The specificity of this reaction was
confirmed by the addition of cold oligo-DNA, which abolished the reactive
band. The addition of anti-pan Jun and anti-pan Fos antibodies induced
supershifts of the binding complexes, thus indicating that AP-1 complexes were
heterodimers consisting of Jun and Fos subunits.
In previous reports, the AP-1 complex was induced in a stimulation- and
cell-specific manner (3,
19,
21). In intestinal SEMFs, we
analyzed the IL-1- and TGF-
1-induced AP-1 complexes using
antibodies specific for distinct Fos isoforms (c-Fos, FosB, Fra-1, and Fra-2)
and Jun isoforms (JunB, c-Jun, and JunD). As shown in
Fig. 3C, the
supershift analysis demonstrated that in unstimulated and stimulated cells,
the AP-1 complex consisted of FosB, Fra2, c-Jun, and JunD. Weak bindings of
c-Fos, Fra1, and JunB were observed, but these seemed to be equivocal.
Effects of adenovirus-mediated transfer of Ad-DN-c-Jun gene. It
has been reported that c-Jun is a major positive regulator of AP-1-mediated
gene-transcription (19,
27), although its role in
IL-11 induction remains unclear. To assess the role of the c-Jun molecule in
our system, we initially assessed the activation of JNK, a member of MAPK
family and a directly upstream activator of c-Jun
(30). As demonstrated in
Fig. 4A, IL-1
and TGF-
1 rapidly (within 5 min after stimulation) induced a
phosphorylation of JNK, indicating that IL-1
and TGF-
1 induced a
rapid activation of JNK. Next, we evaluated the effects of a recombinant
adenovirus containing Ad-DN-c-Jun lacking the transactivation domain of the
wild-type c-Jun to specifically inhibit AP-1 activity. As shown in
Fig. 4B, a truncated
c-Jun protein (
30 kDa) was strongly expressed in intestinal SEMFs infected
with Ad-DN-c-Jun at 48 h after infection, indicating a successful gene
transfer. Endogenous wild-type c-Jun protein was also detected at a low level
and was not suppressed by DN-c-Jun. As shown in
Fig. 4C, Ad-DN-c-Jun
markedly inhibited the IL-1
- and TGF-
1-induced IL-11 mRNA
expression. These inhibitory effects were not induced by Ad-LacZ. The
specificity was confirmed by the responses of IL-8 gene. Induction of
IL-8 gene has been reported to depend on NF-
B activation, but
AP-1 plays a minor role (2,
18,
31). As shown in
Fig. 4C, inhibitory
effects of Ad-DN-c-Jun were modest in IL-1
-induced IL-8 mRNA expression,
indicating specific inhibitory effects of Ad-DN-c-Jun on IL-11 mRNA
expression. Similarly, weak effects of TGF-
1 on IL-8 mRNA expression
were not affected by Ad-DN-c-Jun. Thus the activation of c-Jun AP-1 plays a
major role in the induction of the IL-1
- and TGF-
1-induced IL-11
mRNA expression in these cells.
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MAPK activation and MAPK inhibitors. The MAPK family has been
shown to play an important role in regulating gene expression in response to
inflammatory mediators (8,
17,
30). To assess whether similar
responses are involved in our system, we evaluated the effects of IL-1
and TGF-
1 on the p42/44 (ERK1/2) and p38 MAPK phosphorylation in human
SEMFs. As shown in Fig. 5, A and
C, IL-1
and TGF-
1 induced the phosphorylation
of p42/44 (ERK1/2) MAPKs as early as 15 min after the stimulation. IL-1
induced a rapid phosphorylation of p38 MAPK within 15 min
(Fig. 5A), but
TGF-
1 induced it at 60 min after the stimulation
(Fig. 5C). These
results indicate that the MAPK pathways are activated by IL-1
and
TGF-
1in human SEMFs, but the kinetics of the p38 phosphorylation were
different between IL-1
and TGF-
1.
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To investigate the role of MAPK activation in the induction of IL-11
secretion in intestinal SEMFs, we evaluated the effects of inhibitors of the
p42/44 MAPKs (PD-98059 and U-0216)
(1,
9) and an inhibitor of the p38
MAPK (SB-203580) (16). As
shown in Fig. 5, B and
D, these inhibitors induced specific inhibitory effects
on IL-1- and TGF-
1-induced phosphorylation of ERK1/2 and p38
MAPKs. Inhibition by U-0216 of ERK1/2 MAPKs was stronger than that induced by
PD-98059.
As shown in Fig. 6, each
inhibitor dose dependently blocked the IL-1- and TGF-
1-induced
IL-11 mRNA expression and significantly reduced IL-11 protein secretion. These
results indicate that p42/44 and p38 MAPKs play a role in the IL-1
- and
TGF-
1-induced IL-11 secretion.
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Posttranscriptional regulation of IL-11 mRNA accumulation. The
3'-untranslated region of the IL-11 mRNA contains multiple repeats of
the AUUUA sequence, which has been known to play a role in mRNA
destabilization (47).
Furthermore, it has been reported
(11,
25) that the p38 MAPK is
involved in the stabilization of mRNAs from various genes. To evaluate the
role of p38 MAPK in the posttranscriptional regulation of IL-11 mRNA
expression, we investigated the effects of IL-1- and TGF-
1-induced
IL-11 mRNA stability in the presence and absence of the p38 MAPK inhibitor
SB-203580. The cells were stimulated with cytokines for 12 h, and then a
chasing approach using the transcription inhibitor actinomycin D and a
Northern blot analysis were performed. As shown in
Fig. 7, in the unstimulated
cells, IL-11 mRNA decreased immediately, and both IL-1
and TGF-
1
increased the stability of the IL-11 mRNAs. The addition of SB-203580 caused a
destabilization of the IL-1
- and TGF-
1-induced IL-11 mRNAs, thus
indicating that posttranscriptional mechanisms mediated by p38 MAPK activation
were involved in the IL-1
- and TGF-
1-induced IL-11 mRNA expression
in intestinal SEMFs.
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Combined effects of IL-1 plus TGF-
1.
The combined effects of IL-1
plus TGF-
1 were evaluated at the mRNA
and protein levels (Fig. 8, A and
B). The cells were incubated with stimulators for 12 h,
and the IL-11 mRNA levels were determined by Northern blot analysis
(Fig. 8A). These
combinations increased the IL-11 mRNA abundance compared with the effects of
individual cytokines. Furthermore, similar effects were observed for IL-11
protein secretion (Fig.
8B), and this seemed to be due to an additive effect of
each cytokine. As shown in Fig.
8C, the combination of IL-1
and TGF-
1 induced
additive effects on MAPK and AP-1 activation, supporting the responses in
IL-11 mRNA expression and protein secretion.
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DISCUSSION |
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We investigated the molecular mechanisms responsible for the IL-1-
and TGF-
1-induced IL-11 expression in intestinal SEMFs. Previous studies
have identified two AP-1-binding motifs between -100 and -82 in the IL-11
promoter that are essential for the transcriptional activation of the
IL-11 gene (42,
44). AP-1 exists as a
homodimer of Jun isoforms or as a heterodimer of Jun/Fos isoforms, both
members of the immediate early gene family
(3,
21). Increasing gene
expression by AP-1 is mostly attributed to c-Jun-containing complexes
(19). In our experiments, both
IL-1
and TGF-
1 induced AP-1 DNA-binding activity in intestinal
SEMFs, and the supershift analyses revealed that the induced-AP-1 complexes
were composed of FosB, Fra-2, c-Jun, and JunD. The AP-1 activation by
IL-1
and TGF-
1 was accompanied by the phosphorylation of JNK, a
direct upstream activator of c-Jun
(30). Furthermore, the forced
expression of a dominant-negative c-Jun lacking the transactivation domain of
the wild-type c-Jun markedly reduced both IL-1
- and TGF-
1-induced
IL-11 mRNA expressions. These observations suggest that c-Jun-IL-11 promoter
binding might play a major role in the IL-1
- and TGF-
1-induced
IL-11 mRNA expression in intestinal SEMFs.
MAPK activation is an important signaling event in response to
proinflammatory stimuli (8,
17,
30), but its role in IL-11
induction has not been clarified. The MAPK family consists of three groups,
and all are phosphorylated on tyrosine and threonine residues by upstream
kinases, the MAPK kinases (8,
17,
30). As demonstrated above,
JNK, one of MAPK family, was phospohorylated by IL-1 and TGF-
1 in
our system. The p44 and p42 ERK1/2 and the p38 MAPK
(8,
17) were also activated by
IL-1
and TGF-
1 in human SEMFs. Stimulation by each factor induced
a rapid activation of ERK1/2 MAPK within 15 min. However, the apparent
activation of p38 by TGF-
1 was observed at 60 min after stimulation,
whereas IL-1
induced p38 activation within 15 min, thus indicating that
different mechanisms exist between IL-1
and TGF-
1 in the induction
of p38 MAPK phosphorylation.
The role of MAPKs in IL-1- and TGF-
1-induced IL-11 secretion
was investigated by using specific inhibitors. The imidazole compound
SB-203580 is a specific inhibitor of p38 MAPK
(9). In intestinal SEMFs,
SB-203580 specifically blocked the activation of IL-1
-and
TGF-
1-induced p38 phosphorylation and caused a significant decrease in
both IL-1
- and TGF-
1-induced IL-11 mRNA expression and secretion.
These results indicate that p38 activation was involved in the IL-1
- and
TGF-
1-induced IL-11 secretion. In addition, we addressed the role of
ERK1/2 in our system. PD-98059 is a specific inhibitor of MEK1
(1), the kinase directly
upstream to ERK1/2, and U-0216 is a specific inhibitor of MEK1 and MEK2
(16). U-0216 blocked the
phosphorylation of ERK1/2 more potently than PD-98059 in intestinal SEMFs.
PD-98059 and U-0216 blocked IL-1
- and TGF-
1-induced IL-11 mRNA
expression and caused a significant inhibition against IL-1
- and
TGF-
1-induced IL-11 secretion. Thus we concluded that ERK1/2 MAPKs also
participate in the IL-11 secretion induced by both IL-1
and TGF-
1
in intestinal SEMFs.
In the mRNA expression of many genes, p38 MAPK also plays an important role
at the posttranscriptional steps
(11,
25). Furthermore, the
3'-untranslated region of the IL-11 mRNA contains multiple repeats of
the AUUUA sequence, which has been shown to play a role in mRNA
destabilization (29,
47). To assess the involvement
of p38 MAPK activation in the IL-1- and TGF-
-induced IL-11 mRNA
expression, we evaluated the changes in IL-11 mRNA stabilities. As shown in
Fig. 7, the inhibitor of p38
MAPK SB-203580 markedly decreased the stabilities of IL-1
- and
TGF-
1-induced IL-11 mRNAs. Thus p38 MAPK plays a role via the induction
of IL-11 mRNA stabilization in the effects of IL-1
and TGF-
1.
The induction of IL-11 may potentially be advantageous in the reduction of
intestinal inflammation, because IL-11 exerts anti-inflammatory actions via an
inhibition of NF-B activation in monocytes/macrophages and the
induction of Th2 polarization in CD4+ T cells. In previous studies,
IL-1
and TGF-
1 have been established as representative pro- and
anti-inflammatory cytokines, respectively
(13,
22). Increased expression of
these factors has been reported in inflammatory bowel diseases (IBD)
(6,
28). The IL-11 induced by
IL-1
may be an autonegative regulation mechanism preventing
IL-1
-induced mucosal inflammation. On the other hand, some parts of the
anti-inflammatory effects of TGF-
1 may be mediated by the secondarily
induced IL-11. Furthermore, previous studies
(23,
32) have demonstrated that
IL-11 plays a role in mucosal repair via its trophic effects on the
epithelium. IL-1
and TGF-
1 have been reported to be as inducers of
ECM synthesis (10,
20,
22), which is an important
factor in mucosal repair. Taken together, IL-1
and TGF-
1 are
involved in a complex network of IL-11 induction, which promotes both
anti-inflammatory responses and mucosal repair processes.
In conclusion, this study demonstrated for the first time that IL-11 is
secreted by intestinal SEMFs in response to IL-1 and TGF-
1. These
responses were mediated by c-Jun AP-1- and MAPK-dependent pathways. Although
the pathogenesis of IBD is becoming increasingly apparent, mucosal IL-11
secretion has not been fully investigated in IBD patients. Further
investigations evaluating the in vivo IL-11 secretion of the mucosa of normal
individuals and IBD patients will clarify the mechanisms responsible for the
efficacy of rhIL-11 administration in IBD patients.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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