Novartis Respiratory Research Centre, Horsham, West Sussex RH12 5AB, United Kingdom
Submitted 1 April 2003 ; accepted in final form 29 May 2003
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ABSTRACT |
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MUC5AC; inflammation; hypersecretion; cytokine
Interleukin (IL)-13 is a T helper (Th) 2 cytokine that has been implicated
as an important mediator of inflammation in respiratory diseases including
asthma (8). In animal models of
pulmonary inflammation, IL-13 has been demonstrated to play a key role in the
development of airway inflammation and a goblet cell metaplastic phenotype
(12,
14,
43,
44). The IL-13 receptor
(IL-13R) is a heterodimer of the IL-4R and the IL-13R
1 chains
(18,
34) and mediates both IL-4 and
IL-13 activities at least partially through phosphorylation of STAT6. IL-4 and
IL-13 signaling are impaired in both IL-4R
-deficient and
STAT6-deficient mice, and in these animals the development of an
allergen-induced mucus hypersecretory phenotype is likewise inhibited
(12,
23).
The question has therefore emerged as to whether IL-13 drives goblet cell metaplasia through direct interaction with the epithelium or indirectly through the recruitment of inflammatory cells? The IL-13R has been demonstrated to be expressed in human airway epithelial cells (1), and well-differentiated cultures of human bronchial epithelial cells (HBEs) respond to stimulation with IL-4 and IL-13 by switching from their normal fluid absorptive state to a hypersecretory phenotype, independently of a change in goblet cell density (GCD) (9, 11). The answer to the question as to whether IL-13 can also drive the human airway epithelium toward a goblet cell phenotype in vitro is presently unclear since literature data conflict (4, 6, 17, 19, 22, 25, 41) with reports of either IL-13-driven increases or decreases in mucin gene/protein expression in a variety of culture systems. The aims of the present study were therefore twofold: 1) to establish whether IL-13 could indeed elicit robust, quantifiable changes in the GCD in air-liquid interface (ALI) cultures of HBEs, and 2) to pharmacologically probe some likely signaling pathways contributing to these changes. To this end, we have treated HBE cultures with IL-13 (and IL-4) during the period of differentiation, and using histological analysis we demonstrate for the first time that treatment with IL-13 (and IL-4) can both increase and decrease the GCD in a concentration-dependent manner, a potential explanation for conflicting literature reports. Associated with the increase in goblet cells is the development of a fluid hypersecretory ion transport phenotype, perhaps representing an adaptive response of the epithelium to maintain adequate hydration of the increased mucin load. Using pharmacological tools, we have further demonstrated the involvement of ERK, p38 MAP kinase, and phosphatidylinositol (PtdIns) 3-kinase pathways in the regulation of the IL-13-stimulated goblet cell phenotype in the human airway epithelium and a lack of involvement of the epidermal growth factor receptor-tyrosine kinase (EGFR-TK) pathway.
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MATERIALS AND METHODS |
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HBEs (donors: nos. 0F2095, 0F2129, 1F1811; Clonetics) were cultured based on the method of Gray and colleagues (13). Each of these donors developed a well-differentiated mucociliary phenotype. The level of basal GCD was donor dependent. P2 HBEs were seeded into plastic T-162 flasks (Costar) and grown in bronchial epithelial cell growth medium (BEGM, BioWhittaker) supplemented with bovine pituitary extract (52 µg/ml), hydrocortisone (0.5 µg/ml), human recombinant EGF (0.5 ng/ml), epinephrine (0.5 µg/ml), transferrin (10 µg/ml), insulin (5 µg/ml), retinoic acid (0.1 µg/ml), triiodothyronine (6.5 µg/ml), gentamicin (50 µg/ml), and amphotericin B (50 µg/ml). Medium was changed every 48 h until cells were 90% confluent. Cells were then passaged and seeded (8.25 x 104 cells/insert) onto 1.1-cm2 Transwell inserts (Costar) in differentiation media containing 50% DMEM in BEGM with the same supplements as above but without amphotericin B or triiodothyronine and a final retinoic acid concentration of 50 nM (all-trans retinoic acid, Sigma). Cells were maintained submerged for the first 7 days in culture, after which time they were exposed to an ALI for the remainder of the culture period. Media were refreshed three times each week: Monday, Wednesday, and Friday, although in some initial studies media were refreshed daily for the duration of culture. At least once weekly the apical surface of the cells was rinsed with PBS (37°C) to remove accumulated mucus and debris. At all stages of culture, cells were maintained at 37°C in 5% CO2 in an air incubator. Under these conditions, HBEs formed a well-differentiated mucociliary phenotype with the classical ion transport phenotype associated with this tissue (3, 9). In some studies the effect of the removal of EGF from the differentiation media for the course of ALI culture was examined. Group sizes were six to eight in all studies.
Ussing Chamber Studies
HBEs were cultured as described above on Snapwell inserts (Costar) for the 14-day ALI culture. Inserts were mounted in vertical diffusion chambers (Costar) and were bathed with continuously gassed Ringer solution (5% CO2 in O2, pH 7.4) maintained at 37°C containing (in mM): 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 CaCl2, 1.2 MgCl2, and 10 glucose, as previously described (9). The solution osmolarity was always between 280 and 300 mosmol/kgH2O. Cells were voltage clamped to 0 mV (model EVC4000, World Precision Instruments). We measured transepithelial resistance (RT) by applying a 1-mV pulse at 30-s intervals and calculating RT by Ohm's law. Data were recorded on a PowerLab workstation (ADInstruments). The basal characteristics of the cells in addition to the amiloride-sensitive short-circuit current (ISC) (10 µM, apical side) and the subsequent response to UTP (30 µM, apical side) were measured. Salts, amiloride, and UTP were purchased from Sigma.
Cytokine/Compound Treatment
Cytokines and test compounds were added to the differentiation medium for the duration of ALI culture unless otherwise stated. IL-4 and IL-13 (both from Peprotech EC) were studied at a variety of concentrations (0.1-10 ng/ml) having been prepared as 1,000x stock solutions in PBS (0.1% BSA) and stored at -20°C. AG-1478, PD-98059, U-0126, SB-202190 (Tocris), and LY-294002 (Calbiochem) were prepared as 1,000x stock solutions in DMSO and stored at -20°C. Compounds were added directly to the differentiation media, and treatment started at the same time as the first administration of cytokine. The effect of each compound was studied against both the IL-13-driven goblet cell differentiation and also the basal level of differentiation. Concentration ranges were selected on the basis of literature reports of effective and noncytotoxic doses after prolonged exposure (>48 h where possible) (2, 15, 29, 30, 32, 33, 39). Furthermore, all cultured epithelia were examined histologically for any signs of compound-related toxicity. An additional marker of cytotoxicity was the normally dry apical surface of the ALI cultures. In ALI-cultured HBEs, compound-related toxicity results in a profound decrease in RT and the "leak" of media from the basolateral surface onto the apical side, resubmerging the culture.
Preparation of Inserts for Goblet Cell Quantification
After 14 days of differentiation at ALI, the apical surface of the cells was gently rinsed with PBS (37°C) to remove accumulated mucus and debris. Cells were subsequently fixed in 10% neutral buffered formalin and wax embedded. The inserts were then sectioned at 4-µm thickness and stained with either 1% Alcian blue (in 3% aqueous acetic acid, pH 2.5) to detect total mucins or 45M1 (Neomarkers, Fremont, CA), an anti-MUC5AC antibody. Finally all sections were counter-stained with Coles hematoxylin.
The GCD was assessed by two techniques: manual scoring of absolute numbers
of Alcian blue-stained goblet cells and by semiautomated image analysis for
the determination of 45M1 (MUC5AC)-stained area. For both techniques the
entire length of two to three sections of each insert (20-30 mm total
epithelial length) were examined under an Axio-plan 2 microscope (Zeiss).
Alcian blue-stained goblet cells were counted (x20 magnification) and
expressed as the percentage of the total numbers of epithelial cells lining
the apical surface. Alternatively, inserts were visualized at x10
magnification, and images were acquired with a charge-coupled device camera
interfaced with a personal computer. Image analysis software (KS400 version
3.0, Imaging Associates) under the command of a custom-written macro
quantified both the 45M1-stained area and the length of epithelium along which
the measurements were made. The ratio of the 45M1-stained area to the
epithelial length was used as a measure of MUC5AC-stained area. All samples
were blinded before being counted to remove the potential for operator
bias.
Data Analysis and Statistics
GCDs are expressed as either the percentage of goblet cells in the apical region of the epithelia (means ± SE) or as the ratio of 45M1-stained area to unit length of epithelium normalized to control cytokine/compound untreated cells (means ± SE). All experimental data presented represent one of at least two repeated studies. A Student's t-test with Bonferroni correction for multiple comparisons was used to test for differences between groups with significance taken when P < 0.05. Amiloride-sensitive current and UTP-stimulated current are expressed as absolute mean current changes (±SE). A Student's t-test was used to test for differences between groups with significance taken when P < 0.05.
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RESULTS |
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Initial pilot studies indicated that HBEs treated with a single
concentration of IL-13 (10 ng/ml) for the 14 days of ALI culture, with medium
refreshment every 24 h, failed to develop goblet cells (assessed by Alcian
blue staining, Fig. 1). In one
representative study, goblet cells accounted for 40% of the apical
surface cells in the control group, in contrast to the IL-13-treated cells
where no goblet cells were present (Fig.
1B). Furthermore, IL-13-treated cultures appeared to be
less well differentiated, looking flat and squamous rather than multilayered
as in the controls.
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The basal GCD varied from week to week and was also donor dependent. The donor used for these early studies generally developed a higher GCD than the subsequent donors (note 40% apical goblet cells in Fig. 1 controls vs. 10-15% in Figs. 2 and 4). Medium refreshment every 24 h (as opposed to 48-72 h) also appeared to increase the GCD.
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In subsequent studies and with medium refreshment limited to three times weekly (see MATERIALS AND METHODS), concentration-response relationships to both IL-13 and IL-4 were established. Under these conditions, IL-13 induced a concentration-dependent increase in GCD (and MUC5AC protein expression) when added to the differentiation media from the first day at ALI (Fig. 2). The GCD was assessed by both manual counting of Alcian blue positive-stained cells (Fig. 2, A and B) and also 45M1 (MUC5AC)-stained area (Fig. 2C). The data from both methods correlated closely (Fig. 2D), indicating that the semiautomated 45M1 (MUC5AC) counting technique also provided a reliable measure of GCD. Furthermore, the effects were donor independent (Fig. 2E). IL-13 at 1 ng/ml gave the largest increase in GCD irrespective of scoring method (P < 0.002 by both techniques) and donor. With these two donors IL-13 (1 ng/ml) produced a 5.47 ± 0.92-fold increase (donor 0F2129, Fig. 2C) and a 10.86 ± 1.25-fold increase (donor 0F2095, Fig. 2E) in GCD. The highest concentration of IL-13 studied (10 ng/ml) significantly reduced the GCD from the elevated level seen with 1 ng/ml in both donors irrespective of the counting method employed.
IL-4 induced a similar pattern of GCD changes to those observed with IL-13 (assessed by using MUC5AC protein expression) (Fig. 2F). The increase in GCD was maximal at 1 ng/ml and was absent at 10 ng/ml.
IL-13 Induces a Hypersecretory Ion Transport Phenotype
HBEs cultured for 14 days of ALI in the presence of IL-13 (1 ng/ml, high
GCD cultures) displayed a significantly reduced basal RT
(539 ± 79 · cm2 compared with 1,593 ±
164
· cm2 in controls; P = 0.0004) and
reduced basal ISC (7.1 ± 0.8 µA/cm2
compared with 12.6 ± 0.4 µA/cm2 in controls, P =
0.0002; Fig. 3). Furthermore,
the amiloride-sensitive Na+ absorptive component of the basal
ISC was reduced from 5.4 ± 0.7 µA/cm2
in controls to 0.3 ± 0.2 µA/cm2 (P = 0.0001) in
the IL-13-cultured cells. The subsequent secretory ISC
response to UTP was increased from 5.0 ± 0.1 µA/cm2 in
controls to 42.2 ± 1.0 µA/cm2 in the IL-13-cultured cells
(P < 10-10).
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EGFR Regulates Basal but not the IL-13-Enhanced GCD
The removal of EGF from the differentiation media throughout the course of the cellular differentiation at ALI significantly attenuated the GCD of the cultures (assessed by Alcian blue scoring) from 15.5 ± 1.5 to 6.7 ± 0.8% (P < 0.0005, n = 6; Fig. 4A). In the same study the removal of all differentiation medium supplements with the exception of retinoic acid resulted in a complete absence of goblet cells.
In a subsequent study using standard differentiation media, IL-13 (1 ng/ml) induced a 4.85 ± 0.60-fold increase in GCD (P < 0.003). Treatment of these cells with AG-1478 (0.1-10 µM), a potent EGFR-TK inhibitor, did not significantly attenuate the IL-13-enhanced GCD (Fig. 4B). However, the basal GCD was signifi-cantly attenuated to 33.0 ± 7.2% of the untreated control by AG-1478 (10 µM) (P < 0.001).
MAP Kinase and PtdIns 3-Kinase Inhibitors Attenuate the IL-13-Driven Increase in GCD
To examine the potential role of MAP kinase pathways in the IL-13-driven increase in GCD, we treated HBEs with PD-98059 (a nonselective MEK inhibitor), U-0126 (a MEK1/2 selective inhibitor), or SB-202190 (a p38 MAP kinase inhibitor) throughout the course of IL-13 treatment.
PD-98059. IL-13 induced a 9.79 ± 0.43-fold increase in GCD (P < 10-10) that was attenuated in a concentration-dependent manner by PD-98059 (Fig. 5A). The maximum effect of PD-98059 was achieved at 2 µM, limiting the IL-13-induced rise in GCD to 4.66 ± 0.53-fold above control (P < 10-5).
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U-0126. IL-13 induced a 5.51 ± 0.66-fold increase in GCD (P < 10-4) that was attenuated in a concentration-dependent manner by U-0126 (Fig. 5B). U-0126 at 3 µM (the highest concentration studied) limited the IL-13-induced increase in goblet cells to 2.66 ± 0.32-fold above control (P < 0.002).
SB-202190. IL-13 induced an 8.55 ± 0.73-fold increase in GCD (P < 0.001) that was attenuated in a concentration-dependent manner by SB-202190 (Fig. 5C). The inhibitory activity of SB-202190 reached statistical significance at 10 µM, limiting the IL-13-induced increase in GCD to 1.21 ± 0.39-fold (P < 10-5).
There were no significant effects of PD-98059, U-0126, or SB-202190 on the GCD in the control, non-IL-13-treated cells. The concentration ranges examined for each of these agents have been previously demonstrated to be noncytotoxic in primary cell culture (see MATERIALS AND METHODS). Furthermore, during ALI culture, the apical surface of the HBEs remained dry, an indicator of epithelial integrity and permeability. Histological examination of the fixed sections during goblet cell scoring revealed no signs of toxicity with compound-treated cells forming differentiated, confluent, multilayered epithelia.
LY-294002. To determine whether PtdIns 3-kinase-dependent pathways were involved in the IL-13-driven increase in GCD, we added LY-294002, a nonselective inhibitor of the PtdIns 3-kinase family, to the differentiation media from the first day of ALI culture. During ALI culture, however, the apical surface of LY-294002 (20 µM)-treated HBEs became increasingly submerged in fluid. Histological examination of the HBEs following 14 days of treatment with LY-294002 (20 µM) revealed patches of epithelium that were flat, monolayered, or even absent from the culture insert (data not shown). In an attempt to minimize exposure to LY-294002 and to avoid any potential compound-related toxicity, we investigated alternative IL-13 treatment strategies. HBEs were treated with IL-13 (1 ng/ml) for days 1-8, 8-14, or the full 14 days of ALI culture (Fig. 6A). It was found that treatment of HBE cultures from days 8-14 ALI with IL-13 induced an equivalent increase in GCD to the full 14-day stimulation (days 8-14, 5.76 ± 0.52-fold increase; days 1-14, 5.88 ± 0.81-fold increase). We subsequently investigated the effects of LY-294002 using this revised day 8-14 IL-13 treatment strategy. LY-294002 treatment was started on the same day as the first exposure to IL-13 (day 8 ALI). In this study, IL-13 induced a 6.86 ± 1.01-fold increase in GCD that was reduced to a 2.19 ± 0.47-fold increase (P < 0.001) by LY-294002 (20 µM). There were no signs of any potential compound-related toxicity.
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DISCUSSION |
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These observations are, however, in contrast to several published reports
where IL-13 either was without effect
(6) or even induced a
paradoxical decrease in the mucus hypersecretory phenotype in either ALI HBEs
(4,
17) or human nasal epithelial
cells (19). We have clearly
demonstrated that the IL-4/IL-13-driven goblet cell response is critically
dependent on concentration: 1 ng/ml increased the GCD, whereas 10 ng/ml
markedly reduced the mucus phenotype, often to a level below the control
values. Although differences in culture methods could account for the
discrepancies between our results and the literature, in all of the cited HBE
studies, IL-4 and IL-13 were used at single concentrations 5 ng/ml.
Although we have not studied IL-13 specifically at 5 ng/ml, it is clear from
our data that this concentration could induce a decrease in the mucus
phenotype. It is, however, difficult to reconcile the lack of effect of the
low (<5 ng/ml) concentrations of IL-13 observed by Kim and colleagues
(19), other than the fact that
this group used human nasal epithelial cells. In further contrast to our data,
Kondo and colleagues (22)
reported that despite IL-13 inducing a rise in GCD, IL-4 was without effect on
cultured guinea pig tracheal epithelium. The species differences or
cross-reactivity of human IL-4 with guinea pig could account for the
differences.
The mechanism/s by which IL-4 and IL-13 apparently reduced the GCD and
MUC5AC protein expression at the 10 ng/ml concentration are not presently
known and will require further study. Certainly our initial observations after
14 days of daily IL-13 (10 ng/ml) treatment
(Fig. 1) indicated that IL-13
profoundly influenced the development of the epithelium. Interestingly, IL-13
(10 ng/ml) has been reported to reduce ciliated cell differentiation, to
attenuate cytoskeletal protein expression, and to alter the apparent
polarization of cultured human nasal epithelial cells
(25). A similar observation
has been reported following culture of HBEs in high concentrations of EGF
(13), and, therefore, of note
is the recent report that IL-13 treatment of ALI-cultured HBEs induced the
production and secretion of the potent EGFR ligand transforming growth factor
(TGF)- (2). One
potential explanation that will require further investigation is that IL-13
could be inducing an EGFR-mediated toxicity at high concentrations.
For secreted mucins to possess the optimal viscoelastic properties to enable effective mucociliary clearance and particle interactions, they have to be suitably hydrated (40). It is therefore informative that the IL-13-treated high-GCD cultures displayed a hypersecretory ion transport phenotype as opposed to the reabsorptive phenotype of the untreated cells (Fig. 3). Under normal conditions, the human airway epithelium constitutively absorbs Na+ ions from the airway surface liquid through epithelial Na channels, Na+ channels that are blocked by the diuretic amiloride (3). Na+ is transported transcellularly, and water (from the airway surface liquid) necessarily follows by the paracellular route. This process is fundamental to the regulation of airway surface liquid volume and, therefore, mucus hydration and clearance. In the high-GCD cultures, the reabsorptive amiloride-sensitive current was, however, lost, and the ability of the cells to secrete anions (and therefore fluid) in response to a physiologically relevant secretagogue (UTP) was greatly enhanced. The combination of the loss of fluid absorption with an increase in fluid secretion would be expected to increase the volume of airway surface liquid that would presumably be required to adequately hydrate the extra mucin load during goblet cell secretion. It is not presently known whether this hypersecretory phenotype is as a result of the change in the cell populations or due to a change in the transport characteristic of a single cell type. We have previously demonstrated that a similar IL-13-induced absorptive to hypersecretory phenotype shift is independent of a change in GCD and is due, in part at least, to the functional expression of an apical Ca2+-activated chloride conductance (9).
A number of pathways have been demonstrated to play roles in both the
regulation of MUC gene expression and the propagation of IL-13 signaling.
After we established conditions where IL-13 induced robust increases in the
GCD of HBEs, the second component of this study was to pharmacologically probe
four of these pathways. The pathways were chosen on the basis of the potential
overlap between roles in IL-13 signaling and MUC gene expression. The
IL-4R/IL-13R
1 complex would be expected to signal through JAK1,
JAK2, and TYK2 (18,
34), initiating the
phosphorylation of a variety of signaling cascades, in particular STAT6 and
insulin receptor substrate (IRS)-1. IRS-1 is then able to initiate signaling
through the Ras/Raf and PtdIns 3-kinase pathways, resulting in activation of
MAP kinases and p70S6K/Akt, respectively. We have demonstrated that
the MEK inhibitors PD-98059 and U-0126 were able to attenuate the IL-13-driven
increase in HBE GCD. Recent data from other nonhematopoietic cells have
likewise implicated a role for the ERK pathway downstream of the
IL-4R
/IL-13R
1 complex
(10,
15,
26,
33). Ras, Raf, and ERK have
also been demonstrated to regulate MUC2 expression in response to IL-1
(20), PMA
(27), and Pseudomonas
aeruginosa lipopolysaccharide
(28). Stimulation of
epithelial cells with cytoplasmic proteins derived from nontypeable
Haemophilus influenzae (NTHi) has been demonstrated to increase
MUC5AC transcription that is p38 MAP kinase sensitive
(42). Our results with
SB-202190 also support a role for p38 MAP kinase in the induction of an
IL-13-driven goblet cell phenotype. Furthermore, p38 MAP kinase dependence has
been implicated in IL-13-stimulated responses in additional nonhematopoietic
cells (5,
10,
15). PtdIns 3-kinase has
likewise been demonstrated to play a role in the regulation of
IL-1
-stimulated MUC2 gene expression in a human airway epithelial cell
line (20). In contrast, Wang
and colleagues (42) observed
that activation of PI3-kinase-Akt pathway downregulated NTHi-induced MUC5AC
transcription. Our data indicate that the IL-13-driven increase in GCD/MUC5AC
protein expression involves an LY-294002-sensitive pathway.
The EGFR has been demonstrated to play a central role in epithelial repair
processes and mucin synthesis
(35,
36,
39). In accordance with Gray
and colleagues (13), we have
demonstrated that EGF plays a key role in the basal level of epithelial
differentiation and that its removal from the culture media
(Fig. 4A) or
pharmacological inhibition of the EGFR-TK with AG-1478
(Fig. 4B) results in a
reduction of GCD. Furthermore, basal MUC5AC protein expression is sensitive to
AG-1478 (10 µM) in the mucoid epidermal cell line NCIH292
(39). The potential
involvement of the EGFR-TK pathway with IL-13 responses in HBEs has recently
been reported. As discussed above, IL-13 has been demonstrated to induce the
synthesis and secretion of the potent EGFR ligand TGF-
(2), resulting in enhanced HBE
proliferation. The lack of effect of AG-1478 against the IL-13-driven goblet
cell differentiation, at concentrations demonstrated to effectively attenuate
EGFR signaling in the same experiment (Fig.
4) and in a variety of airway epithelial cells
(2,
35,
36,
39), indicates that the
EGFR-TK pathway is not a major component of the IL-13 signaling leading to the
mucus hypersecretory phenotype in our model.
The question remains, however, as to whether IL-13 can induce goblet cell metaplasia in vivo through a direct interaction with the airway epithelium. Shim and colleagues (37) observed that the direct instillation of IL-13 into rat airways induced a goblet cell metaplasia that was dependent on the recruitment of neutrophils into the airways. In this study, inhibition of neutrophil recruitment completely abolished the IL-13-induced goblet cell metaplasia, arguing against a direct interaction of IL-13 with the rat airway epithelium leading to an increase in GCD. In contrast, it was recently reported that IL-13 could interact directly with the airway epithelium in vivo (24). In this elegant study, selective reintroduction of STAT6 into the airway epithelium of STAT6-/-/IL-13 transgenic mice recovered the otherwise absent goblet cell metaplastic phenotype, indicating that IL-13 could interact directly with the epithelium.
In conclusion, our data support the concept that IL-13 is able to induce a mucus hypersecretory phenotype through direct interaction with the human airway epithelium, a process involving ERK, p38 MAP kinase, and PtdIns 3-kinase signaling pathways. The observation of the concentration-dependent effects of the cytokines may also go some way to explaining the conflicting reports in the literature as to the relationship between IL-4, IL-13, and the development of a mucus hypersecretory phenotype in the airway epithelium. It, however, remains to be determined where the balance lies in vivo between the direct and indirect interactions of inflammatory mediators with the airway epithelium that ultimately lead to the goblet cell metaplasia. An understanding of this balance and of the mediators/signaling pathways contributing to the development of the mucus phenotype will potentially lead to the discovery of specific anti-mucus hypersecretory therapies.
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ACKNOWLEDGMENTS |
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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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