From the Center for Comparative Respiratory Biology
and Medicine and Division of Pulmonary and Critical Care Medicine,
University of California, Davis, California 95616, § Institute of Environmental Health Sciences, Wayne State
University, Detroit, Michigan 48201, and ¶ GlaxoSmithKline
Pharmaceuticals, King of Prussia, Pennsylvania 19406
Received for publication, October 11, 2002, and in revised form, March 6, 2003
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ABSTRACT |
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Mucus hypersecretion and persistent airway
inflammation are common features of various airway diseases, such as
asthma, chronic obstructive pulmonary disease, and cystic fibrosis. One
key question is: does the associated airway inflammation in
these diseases affect mucus production? If so, what is the underlying
mechanism? It appears that increased mucus secretion results
from increased mucin gene expression and is also frequently accompanied
by an increased number of mucous cells (goblet cell
hyperplasia/metaplasia) in the airway epithelium. Many studies on mucin
gene expression have been directed toward Th2 cytokines such as
interleukin (IL)-4, IL-9, and IL-13 because of their known
pathophysiological role in allergic airway diseases such as asthma.
However, the effect of these cytokines has not been definitely linked
to their direct interaction with airway epithelial cells. In our study,
we treated highly differentiated cultures of primary human
tracheobronchial epithelial (TBE) cells with a panel of cytokines
(interleukin-1 Chronic lung diseases such as asthma, chronic obstructive
pulmonary disease (COPD),1
and cystic fibrosis are all characterized by inflammation of the
airways and mucus hypersecretion (1). The mucus hypersecretion by
itself might increase morbidity and mortality in these conditions by
obstructing the airways and impairing gas exchange (2). One of the main
components of mucus secretion is the mucin protein. Mucins are a family
of large glycoproteins that have a molecular mass of several thousand
kilodaltons, and they are a major determinant of the viscoelasticity of
mucus secretion (3). There are currently 19 identified mucin genes
highly expressed in tissues, such as lung, nose, salivary glands, GI
tract, and uterus (4, 5). In the lung, synthesis and secretion of
mucins are restricted largely to the airway with little to no
expression in alveolar airspaces (1). Although at least eight mucin
genes (MUC 1, 2, 4, 5AC, 5B, 7, 8, 13) have been found to be
expressed in adult human lung (4), MUC5AC and
MUC5B appear to be the predominant genes expressed, and
their glycoprotein products are the most abundant in mucus secretions
(4, 6-9). MUC5AC appears to be produced mainly in the
airway epithelium by goblet cells (8, 10), while MUC5B is
mostly produced in the underlying submucosal glands (10). In contrast
to this normal distribution pattern, we previously showed that
MUC5B could be expressed by surface airway epithelial cells
in addition to the expression by submucosal gland cells in airway
tissue sections obtained from COPD and asthma (10), while
MUC5AC expression was still restricted at the surface epithelial cells in these tissue sections. These results suggest that
changes in MUC gene expression, especially MUC5B,
are associated with airway diseases. The source of increased mucus
production in diseases such as asthma and COPD is due at least
partially to an increased number of goblet cells in the airway
epithelium (11-13). Studies have linked goblet cell metaplasia to the
increase of mucin gene expression in airway epithelial cells (10,
14).
In recent years, inflammatory cytokines have been linked to increased
mucus production by their effects on mucin gene expression in the
airway epithelium (15). For example, tumor necrosis factor (TNF) It was later suggested that IL-13 was also important for the
development of asthmatic phenotypes such as airway hyperreactivity, eosinophilic infiltration, and mucous cell metaplasia (24, 25). In vivo models using transgenic mice (26) and intranasal
(14) or intratracheal (27) injections of IL-13 consistently showed increased goblet cells in the airways of mice. However, one limitation of these in vivo experiments was their inability to
determine the exact mechanism of how the cytokine affects mucin gene
expression. Does IL-13 interact directly with receptors on the airway
epithelium to induce mucin gene expression, or are its effects mediated
through inflammatory cell recruitment or the induction of local
mediator release from surrounding cells such as fibroblasts or smooth
muscle cells? For instance, significant infiltration of eosinophils and neutrophils within 4-8 h after the instillation of IL-13 was observed (28). Since products from both neutrophils and eosinophils can induce
mucin gene expression (29-31), one cannot determine convincingly whether it is IL-13 or the inflammatory cells and their products that
are responsible for the mucous cell metaplasia. In airway epithelial
cell cultures, IL-13 has been shown to enhance mucous cell
differentiation in human nasal (32) and pig tracheal (33) epithelial
cells. However, in these studies, the requirement of IL-13 treatment
for 10-14 days is difficult to understand. In another recent study,
IL-13 was also shown to inhibit MUC5AC gene expression in
nasal epithelial cells (34) and had no effect in NCI-H292 cells
(22).
IL-9 has also been shown to have the ability to stimulate mucous cell
hyperplasia in vivo (35) as well as mucin gene expression in vitro (36, 37). However, gene knockout mice of IL-9
showed that IL-9 was necessary for mucous cell hyperplasia in a
granuloma model of disease (38) but not in an allergic asthma model
(39). In cell cultures, stimulation of MUC5AC by IL-9 could
be seen (36, 37) but the cells used were undifferentiated TBE cells or
cancerous cell lines. To further define the roles of cytokines in mucin
gene expression, we used well differentiated primary cultures of human
tracheobronchial epithelial (TBE) cells (40, 41) to determine which
cytokines can stimulate MUC5AC and MUC5B expression. Our study has demonstrated that only IL-6 and IL-17, not
Th2 cytokines, can directly stimulate mucin gene expression in these
primary human TBE cells. A similar observation was extended to primary
TBE cells derived from monkey and mouse.
Primary Cell Culture from Human, Monkey, and Mouse Airway
Tissues--
Human tracheobronchial tissues were obtained from the
University of California at Davis Medical Center (Sacramento, CA) by patient consent. The University Human Subjects Review Committee approved all procedures involved in tissue procurement. In this study,
tissues were collected only from patients without diagnosed lung-related disease. Monkey tissues were obtained from the California Regional Primate Research Center at the University of California, Davis, CA. Transgenic mice were generated by the in-house transgenic animal facility. Primary cultures derived from these airway tissues have been established before (42). Normally, tracheobronchial epithelial (TBE) cells were plated on a collagen gel substratum-coated TranswellTM (Corning Costar, Corning, NY) chamber (25 mm) at 1-2 × 104 cells/cm2, in a Ham's F12/Dulbecco's
modified Eagle's medium (DMEM) (1:1) supplemented with insulin (5 µg/ml), transferrin (5 µg/ml), epidermal growth factor (10 ng/ml),
dexamethasone (0.1 µM), cholera toxin (10 ng/ml), bovine
hypothalamus extract (15 µg/ml), bovine serum albumin (0.5 mg/ml),
and all-trans-retinoic acid (30 nM). These primary TBE cultures, after a week in an immersed cultured condition, were transferred to an air-liquid interface (biphasic) culture condition. Under biphasic culture conditions, a mucociliary epithelium with the formation of cilia and mucus-secreting granules was observed (40).
Cytokine, Antibody, and Inhibitor Treatment--
Cytokines,
IL-1 RNA Isolation and Northern Blot Hybridization--
RNA was
isolated from the cultures by a single step phenol/chloroform
extraction (43). For Northern blot hybridization, an equal amount of
total RNA (20 µg/lane) was subjected to electrophoresis on a 1.0%
agarose gel in the presence of 2.2 mM formaldehyde and transblotted onto Nytran membranes as described above (44). For human
and monkey cells, single-stranded antisense oligonucleotides corresponding to the tandem repeat unit of human MUC5B and
MUC5AC, 5'-TGTGGTCAGCTTTGTGAGGATCCAGGTCGTCCCCGGAGTGGAGGAGGG-3' (423-376 nt, GenBankTM: U63836) and
5'-AGGGGCAGAAGTTGTGCTGGTTGTGGGAGCAGAGGTTGTGCTGGTTGT-3' (582-535
nt, GenBankTM: Z34277), respectively, were end-labeled with
[ RT-PCR Analysis of MUC5AC and MUC5B Gene Expression--
PCR
approach was carried out to examine MUC5AC and
MUC5B gene expression. cDNAs were generated from the RNA
mentioned above by oligo(dT) primer. MUC5AC and
MUC5B gene-specific primers were designed according to
sequences retrieved from GenBankTM. Specifically, for
MUC5AC, forward primer (5'-ACCCAGATCTGCAACACACACT-3') and reverse primer (5'- GAGCGAGTACATGGAAGAGCTG-3') were designed based
on MUC5AC sequence (AJ001403). For MUC5B, forward
primer (5'-ACATGTGTACCTGCCTCTCTGG-3') and reverse primer
(5'-TCTGCTGAGTACTTGGACGCTC-3') were designed based on
MUC5B sequence (Y09788). Each PCR reaction contained 10 µM primers for a total volume of 50 µl of PCR reaction solution. The initial denaturing step was 94 °C for 2 min, and the
last elongation step was 72 °C for 7 min. These PCR reactions were
all carried out the same way: denaturing at 94 °C for 30 s,
annealing at 55 °C for 45 s and extension (or polymerizing) at
72 °C for 1 min per cycle. Cycle number was determined by experiment with different dilutions of the cDNA samples to avoid saturation. Ultimately 25 cycles were chosen for the PCR. Generation of the MUC5B-Luciferase Transgenic Mouse and the
Luciferase Assay--
A chimeric construct containing the proximal
4169 bp of the human MUC5B promoter region (10), and a
luciferase reporter gene was prepared using pGL-3 vector. Transgenic
mice were generated using B6 mice from Targeted Genomics Laboratory
(University of California, Davis). The transgenic positive mice were
determined by Southern blot and RT-PCR. The expression profiles of the
human MUC5B promoter-driven luciferase were consistent with
mouse Muc5b gene expression in the mouse tissues. Two
different founder mice were used for this study. For the luciferase
assay, cell extracts were prepared from mouse TBE cultures and
incubated with Luclite plusTM (Packard Instrument, Meriden,
CT) according to the manufacturer's protocol. The relative luciferase
activity was expressed with the total protein concentration after
normalization. The results were averaged from triplicate dishes of two
separate cultures derived from two different founder mice.
ELISA Measurement--
A human IL-6 QuantikineTM
ELISA kit (R&D systems Inc. Minneapolis, MN) was used to measure the
secreted IL-6 concentrations in both the apical and basal media of
biphasic cultured cells following the manufacturer's instructions.
Immunohistochemistry--
Anti-human IL-6R antibody (R&D systems
Inc., Minneapolis, MN) was used to characterize the expression of IL-6
receptors in these primary TBE cultures and various human tracheal
tissue sections. The staining was carried out by using FITC-conjugated
anti-mouse secondary antibody (Vector Laboratories Inc. Burlingame, CA)
and Vectashield mounting medium with propidium iodide (1.5 µg/ml), following the manufacturer's instructions. The staining pictures were
captured by a digital camera attached to a Zeiss fluorescent microscope.
Statistical Analysis--
Data are expressed as mean ± S.D. The number of repeats are described under "Results" and in the
figure legends. Group differences were calculated by analysis of
variance. When p < 0.01, the difference was considered significant.
Effects of Various Cytokines--
We treated primary
tracheobronchial epithelial cells grown in an air liquid interface with
a panel of cytokines (IL-1
A similar stimulation of MUC gene expression by these two
cytokines was also seen in primary TBE cultures derived from monkey and
mouse tissues (Figs. 2C and 4A, respectively).
However, both animal cultures required a longer treatment time than
human cells in order to see the stimulation by Northern blot analysis,
which was probably related to the fact that human TBE cell culture has many mucous cells (goblet cell) while primary monkey and mouse TBE cell
cultures have very
few.2
The significance of the stimulation by these two cytokines was further
supported by the dramatic increase of the Alcian blue-PAS stained cell
population in primary human TBE cultures (arrows in Fig.
3, A-C), suggesting the
elevation of mucous cell phenotypes in these cultures after IL-6 and
IL-17 treatment. It is noteworthy to point out that throughout these
studies, Th2 cytokine treatments had no stimulatory effect in these
cultures (data not shown).
Stimulation of MUC5B Promoter Activity by IL-6 and IL-17--
In
order to further clairfy the nature of mucin gene regulation, we looked
into its transcriptional regulation. Because of the difficulty in
transfecting well-differentiated primary human TBE cells, we examined
the effects of IL-6 and IL-17 on primary cultures of mouse TBE cells
derived from transgenic mice carrying multiple copies of a
MUC5B promoter-luciferase reporter gene construct (10).
These cells essentially acted as "stable-transfected" cells. We
grew mouse TBE cells from two independent transgenic lines in culture
and treated each of these cultures with 10 ng/ml of IL-6 and IL-17. As
shown in Fig. 4B, the
background luciferase activity was very low in these TBE cultures,
which was consistent with the observation that there are not many
mucous cells in mouse TBE cell culture. One day after treatment with
IL-6 and IL-17, there was a significant (1.6-fold) increase of the
luciferase activity. This increase continued to 9- and 8-fold at day 3, and it reached 223- and 96-fold by day 7 after IL-6 and IL-17
treatments, respectively. These increases were also consistent with the
Northern blot analysis of mouse Muc5b message in these
cultures (Fig. 4A).
IL-17-mediated IL-6 Autocrine/Paracrine Loop--
Since IL-17 is
known to stimulate IL-6 secretion in bronchial epithelial cells (46),
we suspected that part of the IL-17 stimulatory effect on
MUC expression was through the IL-6 autocrine/paracrine loop. Consistent with this notion, we found that IL-17 also stimulated IL-6 production in primary human TBE cultures (Fig.
5A). This secretion occurred
mostly at the apical side of the stimulation. By use of a neutralizing
anti-IL-6 antibody, IL-17-mediated MUC5B expression was
significantly attenuated in primary human TBE cells (Fig.
5B, lane 2). This effect was not observed in the
control treatment (Fig. 5B, lane 3) when a
nonspecific mouse antibody was used.
The predominantly apical secretion of IL-6 prompted us to examine if
the IL-6 receptor was also present apically. By using anti-IL-6R
antibody and FITC based fluorescent microscopy, we found a strong
staining on the apical surface membranes of both cultured cells (Fig.
5C) and human tracheal tissue sections (Fig. 5D).
These stains were apparently located in both secretory and non-secretory cell types, including a weak staining on the basal cell
type. These results support the functional relevance of the apical IL-6
secretion and IL-6-mediated MUC expression in
IL-17-treated cells.
Effects of Inhibitors on Signaling Pathways--
Janus kinases
(JAKs) are a family of protein tyrosine kinases known to have a major
role in mediating cytokine receptor signaling (47). Because JAK/STAT
and MAP kinase pathways have been shown to be activated by IL-17 and
IL-6 (48-52), we want to determine if they were involved in regulating
mucin expression as well. Treatment of cells with AG490, a specific
JAK2 inhibitor, at 5 µM significantly diminished
IL-17-mediated IL-6 secretion by 50% (Fig.
6A). A similar inhibition by
AG490 was seen in IL-17-mediated MUC5B expression (Fig.
6B). In contrast, IL-6-mediated MUC5B expression was not attenuated by AG490 (Fig. 6B). These results suggest
that IL-17-mediated IL-6 secretion is at least partly mediated through JAK2 but that IL-6-mediated MUC5B expression is not. The
decrease in MUC5B induction by IL-17 in AG490-treated cells
further supports the notion that part of the IL-17-mediated effects on
MUC5B is through IL-6 action in an autocrine/paracrine loop.
In order to examine the role of MAP kinase pathways, we used two
inhibitors (U0126 and PD98059) of the ERK pathways. U0126 at 1 µM (Fig. 6C) significantly diminished
MUC5B gene expression by both IL-6 and IL-17, while PD98059
at 25 µM had similar effects (data not shown). These
inhibitors did not affect IL-17 induction of IL-6 secretion (Fig.
6A), but significantly attenuated MUC5B
expression in both IL-6- and IL-17-treated cells. Because the PI-3
pathway can also be activated by JAK kinases, we also treated cells
with wortmannin, an inhibitor of PI 3-kinase, and noted no inhibitory
effect on MUC5B expression or IL-6 secretion (data not
shown).
In order to determine the direct effect of the cytokines on mucin
expression and mucous cell differentiation, we chose
MUC5B/5AC as our markers for the screening
because of their predominant roles in airway secretion. MUC2
was not included due to its very small contribution to airway mucus
(53, 54) as well as the mucin secretion in cell culture (55). In
various chronic airway diseases, MUC5B molecules have been
shown to be present in the most tenacious portion of the mucus (7, 9,
54). Although MUC5B is believed to be expressed mainly in
the submucosal gland, we have previously reported that it can also be
expressed on the airway surface epithelial cells in patients with
chronic airway diseases (10) as well as in a mouse asthma model (42).
These observations suggest that the expression of MUC5B
might be a marker of goblet cell hyperplasia and mucus hypersecretion
associated with various airway diseases.
Using primary tracheobronchial epithelial cells grown in an air/liquid
interface, we treated them with a panel of 19 common cytokines and
found that IL-6 and IL-17 could directly stimulate MUC5B/5AC expression. This finding was reproduced
in cultures of human, monkey, and mouse airway cells although the
latter two required a longer treatment time. However, we could not find
a stimulation of MUC5AC or MUC5B from TNF It is unclear why a longer treatment time was needed for IL-17 and IL-6
stimulation of MUC5B/Muc5b in monkey and mouse
TBE cells. We suspect that it could be due to the fact that in the primary mouse and monkey TBE cultures, there was a paucity of mucous
cells as compared with the human cell culture. Therefore, at the time
of treatment, human cells may already have had a greater percentage of
cells with a mucous cell phenotype ready to express mucin genes. In
contrast, mouse and monkey cells may require a lag time so that the
treatment will make them more capable of expressing mucin genes.
Some of the cytokines in our initial screening panel appeared to have
inhibitory effects on MUC5AC and MUC5B
expression. Because our main purpose was to identify cytokines that
stimulated mucin gene expression, we did not further explore those that
had inhibitory effects. However, it is logical to believe that
physiologically, mucin gene expression may be regulated by both
stimulatory and inhibitory influences. In inflammatory diseases such as
asthma (58) and COPD (59), the local cytokine milieu in the airways and
the balance between the stimulatory and inhibitory influences may
determine the degree of mucous cell hyperplasia and mucus production.
IL-17 is a proinflammatory cytokine that is secreted primarily by T
cells (60) while IL-6 is secreted by a wide variety of cells including
inflammatory (e.g. T-cells, macrophages), stromal (e.g. fibroblast, smooth muscle), and epithelial cells
(e.g. airway, renal tubular) (61). IL-17 is a 20-30-kDa
polypeptide that exists as a homodimer (60). Its normal physiological
role may involve recruitment of neutrophils to sites of infection (62,
63). Mice carrying knockout of the IL-17 receptor show impairment of neutrophil influx and clearance of bacteria in a Klebsiella pneumonia model (64). IL-17 may also play a role in allergic inflammatory responses. IL-17 is found in high level in a mouse model of asthma and
its inhibition by an anti-IL-17 antibody reduces granulocyte influx
(65). In asthmatic patients, more IL-17 positive cells have been found
in sputum compared with control subjects (66). Targeted disruption of
the IL-17 gene in mice shows a reduction in both pulmonary
inflammation and airway hyperreactivity in an asthma model (67). IL-17
has also been implicated in the development of rheumatoid arthritis and
organ rejection (68-70). IL-6 is a 22-27-kDa polypeptide with a wide
variety of physiological functions. IL-6 has pleiotrophic effects. In
the lungs, IL-6 may play a role in causing subepithelial fibrosis and
airway remodeling in asthma (71). To our knowledge, no studies have
previously shown that IL-6 or IL-17 can directly induce mucin gene
expression in cell cultures of airway epithelial cells. Our results add
to the growing body of evidence of the important role cytokines play in
regulating mucin gene expression. We believe that the ability of IL-6
and IL-17 to induce mucin gene expression could play a role in the mucous cell metaplasia as seen in various airway diseases characterized by profound inflammation. IL-17 in particular is most notable for its
ability to recruit neutrophils to sites of inflammation. It would be
tempting to speculate whether it may play a role in diseases
characterized by neutrophilic infiltration such as COPD and cystic
fibrosis. Furthermore, because IL-17 is secreted mainly by T-cells, it
may also be involved in regulation of mucus secretion in diseases such
as asthma where sensitivity and reaction to environmental antigens
occur. Future studies focusing on measuring this cytokine in clinical
samples or animal models might shed some light on these issues.
In this study, we further studied MUC5B to determine how
IL-17 and IL-6 could effect mucin gene expression. We decided to focus
on MUC5B for two reasons. First, its expression could be elevated dramatically in the epithelium of the human chronic airway disease (10) and mouse disease model (42), and second, its induction by
cytokines was surprising and a novel finding to us. Mucin gene
expression can be regulated either by the rate of its transcription or
the stability of its RNA. The use of MUC5B-luciferase transgenic mouse tracheal epithelial cells clearly demonstrated that
both IL-6 and IL-17 could directly regulate MUC5B expression at the promoter level. Since mucin RNA messages are relatively stable
in cultured cells (72), our studies showing the early time of
stimulation of the MUC5B gene in human cells as well as the
stimulation of its promoter in transgenic mouse cells support a
transcriptional mechanism for the role of IL-6 and IL-17 in mucin gene expression.
Since IL-17 is known to be capable of causing IL-6 secretion in many
cell types (46), it was possible that IL-17's affect on
MUC5B gene expression was mediated through locally secreted IL-6 acting in a autocrine/paracrine manner. Indeed, we showed that
IL-17 could induce IL-6 secretion in human airway epithelial cells.
Moreover, treatment of IL-17-treated cells with an anti-IL-6 antibody
blocked MUC5B gene expression suggesting that IL-17's effect was partly mediated through IL-6 (Fig.
7).
, 1
, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, and tumor necrosis factor
). We found that IL-6 and
IL-17 could stimulate the mucin genes, MUC5B and
MUC5AC. The Th2 cytokines IL-4, IL-9, and IL-13 did not
stimulate MUC5AC or MUC5B in our experiments. A
similar stimulation of MUC5B/Muc5b expression by IL-6 and
IL-17 was demonstrated in primary monkey and mouse TBE cells. Further investigation of MUC5B expression demonstrated that
IL-17's effect is at least partly mediated through IL-6 by a
JAK2-dependent autocrine/paracrine loop. Finally, evidence
is presented to show that both IL-6 and IL-17 mediate MUC5B
expression through the ERK signaling pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
has been shown to induce expression of MUC2 (16) and MUC5AC (17) in NCI-H292 cells. More recently, IL-1
has
also been shown to induce MUC2 and MUC5AC
expression in NCI-H292 cells (18, 19). From studies of asthma, evidence
suggests that Th2 cytokines IL-4, 9, and 13 can also affect mucin gene
expression. Transgenic overexpression of IL-4 in murine lungs causes
mucous cell metaplasia and an induction of MUC5AC expression
in the airway epithelium (20). However, direct treatment of IL-4 on
airway epithelial cell culture has produced conflicting results. One group reported a decrease in MUC5AC expression (21), another reported no change (22), and a third reported an increase of MUC2 expression by IL-4 (23).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, 1
, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, and TNF
were purchased from R&D systems Inc. (Minneapolis, MN). They
were dissolved in phosphate-based saline with 1% bovine serum albumin
and added directly to the primary TBE cultures at concentrations of 10 and 50 ng/ml. For additional dosage study (as indicated under
"Results"), cytokine concentration was gradually increased up to
200 ng/ml. For the IL-6 neutralizing antibody study (R&D systems Inc.
Minneapolis, MN), the antibody was added to culture at 0.05, 0.1, and
0.2 µg/ml at the time of IL-17 treatment and its continuous presence
was maintained until the time of harvest. For the inhibitor study,
AG490, PD98059, U0126, and wortmannin were purchased from
Calbiochem-Novabiochem Corporation (San Diego, CA), and they were
dissolved in Me2SO. The dose for each of these
selected inhibitors was AG490 (5 µM), U0126 (1 µM), PD98059 (25 µM), and wortmannin (10 µM). Each dose was determined to be optimal in the
initial literature search and the following experimental trials.
-32P]ATP by T4 polynucleotide kinase. For mouse
Muc5b gene detection, the clone corresponding to the 3'-end
sequence of mouse Muc5b (42) was labeled with
[
-32P]dCTP by a ready-to-goTM random
labeling kit (Amersham Biosciences). All blots were exposed overnight
to a phosphor screen and read by the STORMTM system
(Molecular Dynamics, Sunnyvale, CA). The relative abundance of
MUC5B/MUC5AC message in Northern blots was
normalized with the 18 S ribosomal RNA (rRNA) band.
-actin band was used
as an internal control. The PCR products were separated by electrophoresis on a 1.2% agarose gel and visualized by ethidium bromide post-staining.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, 1
, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, and TNF
) for 24 h. As shown in Fig.
1, our cytokine panel at 10 ng/ml showed that only IL-6 and IL-17 could induce a dramatic increase in expression of MUC5AC and MUC5B. The Th2 cytokines IL-4,
IL-9, and IL-13 did not stimulate either MUC5AC or
MUC5B. Treatment with a higher dose of 50 ng/ml yielded
identical results (data not shown). MUC5B/5AC mRNA signals from three independent primary TBE cultures from different donors were normalized to 18 S rRNA signals and quantified. A
3-4-fold stimulation of both MUC5B and MUC5AC
messages was consistently observed after an overnight (16 h) treatment
by IL-6 and IL-17 (Fig. 1, C and D). The effects
of IL-6 and IL-17 on mucin gene expression were
dose-dependent (Fig. 2,
A and B). Concentrations as low as 2 ng/ml of
IL-6 or 10 ng/ml of IL-17 elicited a significant stimulation of
MUC5B gene expression after 16 h of treatment on human
primary TBE cultures. To ensure that our failure to find any induction
by IL-4, IL-9, or IL-13 was not due to the generally lower sensitivity
of northern blots (45), we repeated the experiments for them as well as
IL-6 and IL-17 with reverse transcription polymerase chain reaction
(RT-PCR). In addition, we used concentrations of up to 200 ng/ml to
rule out that an inadequate cytokine concentration was a factor. As
shown in Fig. 2B, even at 200 ng/ml, no effects on
MUC5AC and MUC5B were detected in the IL-4, IL-9,
or IL-13 treatments. In contrast, IL-6 showed a
dose-dependent mucin-inducing activity at all
concentrations up to 200 ng/ml, while IL-17 induced mucin genes at
lower doses (10 and 50 ng/ml) but not at higher doses(100 and 200 ng/ml). The reason why lower levels of MUC5AC and
MUC5B were seen at the higher concentrations of IL-17 is
unclear. But it was apparently not due to toxicity because we routinely checked cell viability by the trypan blue dye exclusion test and found
no evidence that higher levels of IL-17 were harmful to the
cultures. For IL-4, IL-9, and IL-13, it was unlikely that a low level
induction of MUC5AC or MUC5B occurred that could
not be detected with the more sensitive RT-PCR (45). IL-2, 3, 5, 7, 8, and 18 appeared to have inhibited the expression of MUC5AC and MUC5B. These effects were not related to the
cytotoxicity of the cytokines since the viability of the cells (greater
than 95%) was routinely checked by the trypan blue dye exclusion test with consistently positive results.
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Fig. 1.
Northern blot analysis of MUC5AC and MUC5B
mRNA levels in primary human TBE cells after cytokine
treatment. Primary cultures were carried out under air-liquid
interface culture condition as described in the text. At day 21 after
plating, cytokines (10 ng/ml) were added to both the apical and basal
sides of the culture. Total RNA was collected after overnight
incubation (16 h). Cytokines: IL-1 , IL-1
, IL-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, TNF
. C, control.
A and B, representative Northern blots for
MUC5AC and MUC5B as well as 18 S ribosomal RNA,
respectively, from one of these primary cultures. C and
D, cumulative quantification of Northern blot analyses of
MUC5AC and MUC5B mRNA levels, respectively,
from three independent primary TBE cultures derived from different
donors. The intensity of mucin mRNA was normalized to the intensity
of the 18 S ribosomal RNA band, and the relative intensity was further
normalized with the control. C, vehicle (phosphate-buffered
saline/1% bovine serum albumin)-treated cultures. n = 3; *, p < 0.01.
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Fig. 2.
Dose- and time-dependent elevation of
MUC5B gene expression by cytokines. A,
dose-dependent elevation of MUC5B message by IL-6 and
IL-17, examined by Northern blot. IL-6 or IL-17 of various
concentrations, as indicated, was used to treat primary human TBE cells
overnight (16 h), as described in the legend to Fig. 1.
B, IL-4, 6, 9, 13, 17 of various concentrations, as
indicated, were used to treat primary human TBE cells overnight (16 h),
as described in the legend to Fig. 1. Dose-dependent
elevation of MUC5AC and MUC5B messages by IL-6
and IL-17 examined by RT-PCR. IL-17 had no induction at higher doses
(100 ng/ml and 200 ng/ml). Notably, no inductions of MUC5B
message were seen in cultures treated with higher levels (up to 200 ng/ml) of IL-4, 9, and 13. C,
time-dependent elevation of MUC5B message in
monkey TBE cells after IL-6 (10 ng/ml) and IL-17 (10 ng/ml)
treatments.
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Fig. 3.
Increase of mucous cell population by IL-6
and IL-17. A-C, Alcian blue/PAS staining of primary human
TBE cultures after treatment with vehicle (phosphate-buffered
saline/1% bovine serum albumin), IL-6, and IL-17, respectively.
The arrows indicate "purple" cells stained
positively with Alcian blue/PAS.
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Fig. 4.
Effects of IL-6 and IL-17 on primary mouse
tracheal epithelial cells in culture. Primary tracheal epithelial
(TE) cells were isolated from transgenic mice carrying
multiple copies of MUC5B promoter-luciferase construct as
described in the text. These TE cells were cultured under an air-liquid
interface culture condition as described in the text. At day 14 after
plating, cells were treated with vehicle (phosphate-buffered saline/1%
bovine serum albumin) or 10 ng/ml each of IL-6 and IL-17, respectively.
At day 1, 3, and 7 after cytokine treatments, cultures were harvested
for luciferase activity assay, protein quantitation, and RNA isolation.
A, representative RNA Northern blot analysis from TE
cultures 7 days after cytokine treatments. Lane 1, control;
lane 2, IL-6; lane 3, IL-17. B,
promoter-reporter gene activity after IL-6 and IL-17 treatments. The
luciferase activity was standardized to the total protein
concentration. Triplicate culture chambers were used for each
treatment. And the whole experiment was repeated using the TBE cells
derived from another founder mouse. n = 6; *,
p < 0.01.
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Fig. 5.
IL-17-mediated IL-6 secretion and MUC5B
expression. A, ELISA analysis of IL-6 secretion by
human TBE cells in primary culture after IL-17 (10 ng/ml) treatment.
Apical and basal media were collected after an overnight treatment (16 h). Empty bars, apical secretion of IL-6. Streaked
bars, basal secretion of IL-6. Each point represents the average
of triplicate dishes and a similar experiment has been repeated in
three primary cultures from different donors. n = 9; *,
p < 0.01. B, effects of IL-6 neutralizing
antibody (200 ng/ml) on IL-17-mediated MUC5B expression.
Lane 1, control cultures without IL-17 and antibody
treatments; lane 2, cultures treated with both IL-6
neutralizing antibody and IL-17; lane 3, cultures treated
with control, nonspecific serum and IL-17. C and
D, immunofluorescent staining with IL-6 receptor antibody in
primary human TBE culture and human tracheal tissue section,
respectively. Anti-IL-6R antibody was used and stained with
FITC-conjugated secondary antibody. Nuclei were stained with
Vectashield mounting medium with propidium iodide (1.5 µg/ml).
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Fig. 6.
Effects of JAK2 and ERK inhibitors on IL-6
secretion and MUC5B expression. A, ELISA analysis of
IL-6 produced in primary human TBE cultures after IL-17 treatment.
Cultured human TBE cells were treated with vehicle, AG490, and UO126,
2 h before IL-17 treatment (lanes 4-6, respectively).
Controls (lanes 1-3, respectively), without IL-17
treatment, were treated with the same inhibitors. Media were collected,
and IL-6 secretion was measured as described in the legend to Fig. 5.
Only the apical secretions were quantified. Each point represents the
average of triplicate dishes and a similar experiment has been repeated
in three primary cultures from different donors. n = 9;
*, p < 0.01. B, effects of AG490
(JAK2-specific inhibitor) on cytokine-induced MUC5B
expression. Primary cultures were pretreated with AG490 (lanes
1, 3, and 5), then treated with IL-6
(lanes 3 and 4) and IL-17 (lanes 5 and
6). Lane 2, C, control without any
treatment. C, effects of ERK-specific inhibitor, UO126, on
cytokine-induced MUC5B expression. Primary cultures were
pretreated with U0126 (lanes 1, 3, and
5) and then treated with IL-6 (lanes 3 and
4) and IL-17 (lanes 5 and 6).
Lane 2, C, control without any treatment. Similar
results treated with PD98059 were obtained (data not included).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
IL1
, and Th2 cytokines such as IL-4, IL-9, and IL-13, which have all
been previously shown by other investigators to stimulate
MUC5AC expression. We believe the discrepancy between our
findings and those of other investigators may be due to the different
cell culture systems used. Many other studies looking at cytokines and
their effects on mucin gene expression in cell cultures made use of
undifferentiated primary TBE cells and immortalized or cancerous cell
lines such as NCI-H292 or A549 cells. We believe one limitation of
using such cells is that morphologically, they bear very little
resemblance to airway epithelial cells in the intact organism. For
example, normal airway epithelial cells in animals typically have cilia with tight junctions separating the cells as well as apical and basolateral polarity (56, 57). These characteristics are present in
airway epithelial cells grown under our conditions (40, 41) but are
absent in the undifferentiated TBE cells grown in culture dishes and
those immortalized or cancerous cell lines. For these reasons, we
believe that our primary cell cultures, under an air-liquid interface
culture condition, may better represent the native airway epithelium in
animals for in vitro studies.
View larger version (26K):
[in a new window]
Fig. 7.
A summary of the IL-6 and IL-17 signalings on
the regulation of MUC5B gene expression. JAK?
represents the unidentified AG490 insensitive JAK(s). Blue
lines delineate the IL-6 autocrine/paracrine loop. Red
lines delineate the mucin synthesis pathway. X
represents the unidentified STAT that transduces the receptor-JAK
signal into the nucleus and is involved in the transcription of
MUC5B genes.
To determine what signal transduction mechanisms were involved in IL-17 or IL-6 receptor binding, we did some studies looking specifically at the JAK/STAT and the MAP kinase pathways. Four JAK family kinases, JAK1, 2, 3, and Tyk2 have been reported (73). IL-17 has been reported to activate JAK/STAT proteins (48) as well as MAP kinase (49, 50) in U937 and renal epithelial cells. The IL-6 receptor has been shown to interact with JAK1, 2, and Tyk2 in various cell types (74). We demonstrated that IL-17 could stimulate IL-6 secretion and the stimulation was attenuated by AG490, a JAK2 inhibitor, suggesting a JAK2-dependent pathway is involved in the regulation of IL-6 production. In addition to the IL-6 secretion effect, AG490 also suppressed IL-17-stimulated MUC5AC/MUC5B expression. This effect is consistent with the treatment of IL-6 neutralizing antibody on IL-17-treated cells. Taking these data together, it is suggested that a part of IL-17-stimulated MUC5AC/MUC5B expression is through a JAK2-dependent IL-6 autocrine/paracrine loop (Fig. 7).
Stimulation of MUC5B by IL-6 was not affected by AG490, suggesting it uses a different signaling pathway. To determine if MAP kinase pathways may have been involved, we used U0126 and PD98059, specific inhibitors of ERK signaling. Both inhibitors reduced IL-17 and IL-6 stimulation of MUC5B but neither had an effect on IL-17-mediated IL-6 secretion. This suggests that ERK signaling is required distal to IL-6 signaling but is not required for IL-17's effect on IL-6. We also looked to see if the PI-3 pathway could be involved as it has been shown to act distally to JAK kinases. However, wortmannin, a PI-3 pathway inhibitor, had no effect on either IL-6 secretion by IL-17- or IL-6-mediated MUC5B expression. Thus IL-6 and IL-17's effect is likely independent of the PI-3 pathway. The signaling pathway is summarized in Fig. 7. Since other JAK and MAP kinase pathways exist that were not evaluated in this study, further studies will be needed to define their potential roles in IL-6- and IL-17-mediated mucin gene expression.
In summary, after screening 19 cytokines, we have identified two
cytokines (IL-6 and IL-17) that have direct stimulatory effects on
MUC5AC and MUC5B gene expression in primary
airway epithelial cells. We further demonstrated that IL-17 mediates
its effect on MUC5B partly through IL-6 by acting in an
autocrine/paracrine manner and that JAK2 may be involved in the
signaling events. Furthermore, IL-6 and IL-17's effect on
MUC5B may depend on ERK signaling pathways. Future studies
should address whether other MAP kinase and JAK/STAT pathways are also
involved in IL-6- or IL-17-mediated gene expression in airway
epithelial cells. More experiments will also be needed to determine
whether these two cytokines play a role in the mucous cell metaplasia
seen in chronic airway diseases.
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ACKNOWLEDGEMENTS |
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We thank Dr. Cheryl Soref for her critical review of this manuscript prior to submission. Andrew Last is thanked for his editing work on this manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL35635, ES06230, ES09701, AI50496, ES00628, ES04699, and ES05707, California Tobacco-Related Disease Research Program 10RT-0262, and a grant from Glaxo/Smith Kline/Welcome Inc.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.
To whom correspondence should be addressed: Center for
Comparative Respiratory Biology and Medicine, Surge 1 Annex, Room 1121, University of California at Davis, One Shields Ave., Davis, CA 95616. Tel.: 530-752-2648; Fax: 530-752-8632; E-mail: rwu@ucdavis.edu.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M210429200
2 R. Wu, Y. Chen, and Y. Zhao, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: COPD, chronic obstructive pulmonary disease; TBE, tracheobronchial epithelial cells; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; MAP, mitogen-activated protein; PI, phosphatidylinositol; ELISA, enzyme-linked immunosorbent assay; nt, nucleotides; STAT, signal transducer and activator of transcription; FITC, fluorescein isothiocyanate; IL, interleukin; TNF, tumor necrosis factor.
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