1 Novartis Respiratory Research Centre, Horsham, West Sussex RH12 5AB, United Kingdom; and 2 Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interleukin (IL)-13 has been associated with asthma, allergic rhinitis, and chronic sinusitis, all conditions where an imbalance in epithelial fluid secretion and absorption could impact upon the disease. We have investigated the effects of IL-13 on the ion transport characteristics of human bronchial epithelial cells cultured at an apical-air interface. Ussing chamber studies indicated that 48 h pretreatment with IL-13 or IL-4 significantly reduced the basal short-circuit current (Isc) and inhibited the amiloride-sensitive current by >98%. Furthermore, the Isc responses were increased by more than six- and twofold over control values when stimulated with UTP or forskolin, respectively, after cytokine treatment. The IL-13-enhanced response to UTP/ionomycin was sensitive to bumetanide and DIDS and was reduced in a low-chloride, bicarbonate-free solution. Membrane permeablization studies indicated that IL-13 induced the functional expression of an apical Ca2+-activated anion conductance and that changes in apical or basolateral K+ conductances could not account for the increased Isc responses to UTP or ionomycin. The results indicate that IL-13 converts the human bronchial epithelium from an absorptive to a secretory phenotype that is the result of loss of amiloride-sensitive current and an increase in a DIDS-sensitive apical anion conductance.
calcium-activated chloride channel; hypersecretion; asthma; interleukin-4
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE AIRWAY
EPITHELIUM acts as a barrier protecting the lung from inhaled
substances and has developed specifically for this purpose. It serves
to regulate airway surface liquid volume and composition, mucus
secretion, and cilia beat to maintain a sterile lung through effective
mucociliary clearance. The airway epithelium is also in the ideal
location to interact with the immune system when it becomes exposed to
potentially harmful substances (17, 26). The bronchial
epithelium is a tissue comprising a heterogeneous cell population,
including ciliated columnar cells, goblet cells, submucosal glands,
serous cells, and basal cells. There are only a few reports of the
effects of inflammatory stimuli on the functioning of the intact
epithelium (1, 7, 13, 25). With the exception of
submucosal glands, the bronchial epithelium can be modeled in vitro to
display a differentiated mucociliary phenotype with the ion transport
characteristics of the native tissue. To date, there is only one report
of the effects of inflammatory stimuli on the ion transport function of
the human airway epithelium (13). Galietta and colleagues
(13) described the effects of the T-helper (Th) 1 cytokines interferon- (IFN-
) and tumor necrosis factor-
(TNF-
) on the ion transport characteristics of human bronchial epithelial cells (HBECs) and demonstrated that TNF-
was without effect, although the basal amiloride-sensitive short-circuit current (Isc) was reduced by IFN-
and
agonist-stimulated anion-secretion was enhanced.
Currently, there are no published reports of the effects of Th2 cytokines on the ion transport characteristics of the human airway epithelium. In this study, we report the effects of the Th2 cytokine interleukin (IL)-13 on the ion transport phenotype of the human bronchial epithelium. Increased IL-13 production is recognized in asthma (atopic and nonatopic), chronic sinusitis, and allergic rhinitis (16, 18, 19, 22, 31), all conditions in which alterations in the volume and composition of secretions and the epithelial lining fluid could impact the normal functioning of the tissue. The effects of IL-13 on the ion transport characteristics of other epithelia have been reported. IL-13 has been demonstrated to decrease transepithelial resistance (RT) of cultured T84 monolayers (39), but in contrast to the related Th2 cytokine IL-4 was without effect on agonist-stimulated anion secretion. In cultured rat glomerular visceral epithelial cells, both IL-4 and IL-13 decreased RT, an effect that was attributed to an increase in transcellular conductance (35). In this paper, we demonstrate that IL-13 and IL-4 are able to convert the human bronchial epithelium from its normal absorptive state to a secretory phenotype. This phenomenon may represent a potential mechanism by which the atopic airway can become hypersecretory and could highlight novel therapeutic approaches to treat airway diseases associated with imbalances of fluid secretion and absorption (32).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture
HBECs (Biowhittaker) were cultured using a modification of the method described by Gray and colleagues (15). Cells were seeded in plastic T-75 flasks and were grown in bronchial epithelial cell growth medium (BEGM; Biowhittaker) supplemented with bovine pituitary extract (52 µg/ml), hydrocortisone (0.5 µg/ml), human recombinant epidermal growth factor (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), gentamycin (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 × 105 cells/insert) on polycarbonate Snapwell 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). Cells were maintained submerged for the first 7 days in culture, after which time they were exposed to an apical air interface for the remainder of the culture period. Cells were used between days 14 and 21 after establishment of the apical-air interface. At all stages of culture, cells were maintained at 37°C in 5% CO2 in an air incubator. HBECs from three donors were used for these studies.Isc Measurements
Snapwell 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. The solution osmolarity was always between 280 and 300 mosmol/l for all physiological salt solutions used. Cells were voltage clamped to 0 mV (model EVC4000; WPI). RT was measured by applying a 2-mV pulse at 30-s intervals and calculating RT by Ohm's law. Data were recorded using a PowerLab workstation (ADInstruments). For low-chloride, bicarbonate-free studies, NaCl was replaced by equimolar sodium gluconate, and the solution was buffered with HEPES (10 mM). These solutions were gassed with air. In studies to evaluate the basolateral membrane K+ conductance (GK), the apical membrane was permeabilized with amphotericin B (10 µM; apical side only) with the apical solution containing potassium gluconate (120 mM) in place of NaCl and the basolateral solution containing sodium gluconate, again in the place of NaCl. Amphotericin B was added to the apical membrane 5-10 min after voltage clamping and was present throughout the experiment. In studies to evaluate the contribution of apical GK and chloride conductance (GCl), the basolateral membrane was permeabilized withCytokine Treatment and Compound Additions
Initially, HBECs were treated basolaterally with the cytokines IL-13 (10 ng/ml) or IL-4 (10 ng/ml) for 48 h. Cytokine or vehicle-containing medium was refreshed at 24 h. At 48 h, the basal characteristics of the cells in addition to the amiloride-sensitive Isc (10 µM; apical side) were recorded. The subsequent responses to UTP (30 µM; apical side), ionomycin (1 µM; apical and basolateral), and forskolin (0.6 µM; apical and basolateral) were also assessed. In additional experiments, the sensitivity of the responses to bumetanide (60 µM; basolateral) and DIDS (300 µM; apical) were examined. The effect of UTP on control and IL-13-treated HBECs was also assessed in the absence of amiloride.Effects of IL-13 on UTP-Stimulated Intracellular Ca2+
HBECs were seeded on clear-bottomed, black-walled 96-well tissue culture-treated plates (Costar) at 20,000 cells/well in differentiation media with or without IL-13 (10 ng/ml). At 48 h after seeding, cells were loaded with fluo 4-AM (0.7 µM in DMSO + 20% pluronic acid; Molecular Probes) in loading buffer containing differentiation media, HEPES (20 mM), and probenecid (2.5 mM) at 37°C (5% CO2) for 60 min. The final DMSO concentration did not exceed 0.1% vol/vol. The cells were then washed three times by rinsing with wash buffer containing Hanks' balanced salt solution (with Ca2+, Mg2+ without phenol red), HEPES (20 mM), and probenecid (2.5 mM), and the final volume was adjusted to 100 µl/well (Labsystems Cellwash Microplate Washer). Fluorescence intensity was then continuously measured before and after the addition of UTP (final concentration 0.1-100 µM) using FLIPR (FLuorescence Imaging Plate Reader; Molecular Devices) with excitation and emission wavelengths at 488 and 535 nm, respectively.Histology
HBECs were treated with vehicle or IL-13 (10 ng/ml; 48 h) as described above and were then fixed in 10% neutral-buffered formalin (pH 7.4; 24 h). Inserts were then processed and embedded in wax. Sections (3 µm) were mounted on glass slides and dried overnight before staining (Alcian blue and hematoxylin). The numbers of goblet cells on the epithelium were counted and expressed as the percentage of the total number of epithelial cells on the apical surface. A total of four sections was used from each insert, and the entire length of the insert was used for scoring. Each group consisted of six individual inserts.Expression of Results and Statistical Analysis
Results are expressed as absolute changes in Isc (mean ± SE). Measurements were taken either as peak changes or once responses had plateaued and were stable. Control inserts were run alongside all experiments for paired comparisons to be made because of the potential day-to-day and interbatch variability of the Isc. Student's t-test was used to compare between groups, with statistical significance assumed at P < 0.05. For the FLIPR studies, data are expressed as a percentage of the maximum response to UTP (mean ± SE).Reagents
HBECs obtained from postmortem specimens were purchased from Biowhittaker, as were all media. All other cell culture reagents were purchased from Life Technologies. Cytokines were purchased from (PeproTech). All other reagents were purchased from Sigma, unless stated otherwise. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The culture methods employed in these studies produced a multilayered bronchial epithelial tissue that had differentiated to the extent that ciliated and goblet cells were identifiable. Goblet cells typically accounted for 25-30% of the total number of cells at the apical surface (see Histology). All of the control cells used in these studies displayed an amiloride-sensitive Isc, although there was inevitable inter- and intradonor variability. Paired controls were used throughout.
IL-4 and IL-13 Inhibit Basal and Amiloride-Sensitive Isc but Enhance the Responses to UTP and Forskolin
Basal Isc.
Initial studies investigated the effects of IL-4 and IL-13 on the basal
and stimulated ion transport properties of HBECs (Fig. 1). Control cells displayed a basal
Isc of 34.7 ± 1.4 µA/cm2 and
RT of 846 ± 62 · cm2 (n = 6). Amiloride
inhibited 72.8 ± 3.2% of the basal current (n = 6). In contrast, cells that had been treated with IL-4 (10 ng/ml) or
IL-13 (10 ng/ml) displayed significantly reduced basal currents of
5.7 ± 0.3 µA/cm2 (P < 10
8; n = 6) and 5.3 ± 0.7 µA/cm2 (P < 10
8;
n = 6), respectively. IL-4- and IL-13-treated cells
also showed an increased RT of 1,391 ± 162
· cm2 (P < 0.02) and
1,580 ± 112
· cm2 (P < 10
4), respectively. Furthermore, <2% of the basal
current was amiloride-sensitive in both the IL-4- and IL-13-treated
cells (P < 10
7; n = 6).
|
UTP-stimulated Isc.
The subsequent addition of UTP (30 µM; apical) in the presence of
amiloride induced a biphasic1
increase in Isc in both control and
cytokine-treated cells (Fig. 1). In control cells, the current peaked
at an increase of 7.1 ± 0.3 µA/cm2
(n = 6). In IL-4- and IL-13-treated cells, the
Isc peaked at an increase of 50.9 ± 0.9 µA/cm2 (P < 1012;
n = 6) and 44.3 ± 2.2 µA/cm2
(P < 10
8; n = 6),
respectively. Changes in RT were also associated
with these Isc changes. In control cells, mean
RT decreased from 1,038 ± 31 to 824 ± 64
· cm2 (n = 6) upon the
addition of UTP. In IL-4- and IL-13-treated cells,
RT was likewise reduced after UTP stimulation
from 1,650 ± 267 to 487 ± 17
· cm2
(n = 6) and 1,655 ± 192 to 541 ± 18
· cm2 (n = 6), respectively. It
should, however, be noted that these values could only be calculated
after the peak response had reached a steady plateau and do not
necessarily represent the true value of RT at
the time of the peak increase in Isc.
Forskolin-stimulated Isc.
After the resolution of the UTP response, the baseline
Isc remained elevated over the pre-UTP level in
control (139 ± 6%; P < 0.02; n = 6) and IL-4 (199 ± 15%; P < 106; n = 6)- and IL-13 (199 ± 24;
P < 10
4; n = 6)-treated
cells. The subsequent addition of forskolin (0.6 µM; apical and
basolateral) induced a peak increase in Isc of 9.7 ± 1.1 µA/cm2 (n = 6) in control
cells that was significantly elevated in the IL-4- and IL-13-treated
cells to an increase of 15.0 ± 0.5 µA/cm2
(P = 0.001; n = 6) and 15.4 ± 1.2 µA/cm2 (P = 0.003; n = 6), respectively.
IL-13-Induced Effects on the HBEC Ion Transport Phenotype Are Apparent at 6 h
Initially, HBECs were treated with IL-13 (10 ng/ml; basolateral) for 24, 48, and 72 h to determine whether the phenomenology observed above was dependent on the duration of treatment. In this study, control cells (receiving fresh media at 0, 24, and 48 h) displayed a basal Isc of 20.2 ± 0.8 µA/cm2 (n = 6). In the IL-13-treated cells, the basal Isc was reduced to 9.4 ± 1.1, 5.8 ± 0.6, and 8.6 ± 0.5 µA/cm2 in the 24-, 48-, and 72-h treatment groups, respectively. The amiloride-sensitive current was also completely inhibited in all IL-13-treated groups (P < 10
|
Inhibitory Isc Response to UTP is Lost in IL-13-Treated HBECs
As previously observed, HBECs that had been treated with IL-13 (10 ng/ml; 48 h) had a significantly reduced basal Isc of 10.4 ± 0.3 µA/cm2 compared with 14.1 ± 0.3 µA/cm2 in the control cells (P < 10
|
IL-13 Enhances Forskolin-Stimulated Isc Under Basal Conditions
In a subsequent study, HBECs that had been treated with IL-13 (10 ng/ml; 48 h) again showed a reduced basal Isc of 6.9 ± 0.8 µA/cm2 compared with 15.9 ± 0.5 µA/cm2 in control cells (P < 10IL-13 Treatment Does Not Affect Goblet Cell Density
In control cells, goblet cells accounted for 26.3 ± 3.6% of the cells at the apical surface of the epithelium (n = 6). In paired cells treated for 48 h with IL-13 (10 ng/ml), 31.0 ± 3.3% of the cells at the apical surface of the epithelium were goblet cells (P = 0.36, n = 6).Sensitivity of the UTP-Stimulated Increase in Isc to Bumetanide and DIDS
We next investigated the nature of the increased responsiveness to UTP in IL-13-pretreated cells using bumetanide, a blocker of the basolateral Na+-K+-2Cl
|
For the studies with DIDS, ionomycin was used in place of UTP, as DIDS
has been demonstrated to block P2Y2 receptors
(33). In IL-13-treated cells, ionomycin (1 µM;
apical and basolateral) induced a biphasic increase in
Isc that peaked at 50.7 ± 6.9 µA/cm2, a significantly larger response than observed in
the control cells of 4.9 ± 0.5 µA/cm2
(P < 104; n = 6; Fig.
5, A and B). The addition of
DIDS before ionomycin did not affect the peak increase in
Isc in the control cells (4.1 ± 0.6 µA/cm2; P = 0.35; n = 6;
Fig. 5C). In the IL-13-treated cells, DIDS attenuated the
ionomycin-stimulated peak increase in Isc from 50.7 ± 6.9 to 21.4 ± 2.3 µA/cm2
(P = 0.002; n = 6; Fig. 5D).
The forskolin-stimulated Isc responses were
unaffected by DIDS in both control (23.7 ± 1.1 vs. 22.3 ± 1.0 µA/cm2) and IL-13-treated cells (57.8 ± 2.9 vs.
50.8 ± 2.2 µA/cm2).
|
Under low-chloride and bicarbonate-free conditions, the
UTP-stimulated increase in Isc was attenuated in
both control cells and cells that had been pretreated with IL-13. In
control cells, the peak UTP responses were reduced from 10.6 ± 0.2 (normal Ringer) to 2.8 ± 0.3 (low chloride, bicarbonate free;
n = 3) µA/cm2. Likewise, in IL-13-treated
cells, the peak responses were reduced from 63.9 ± 1.5 (normal
Ringer) to 30.5 ± 1.2 (low chloride, bicarbonate free;
n = 3; Fig. 6)
µA/cm2. Under the low-chloride bicarbonate-free
conditions, the recovery of the Isc response
toward baseline was more rapid than in the paired control (Fig. 6).
|
IL-13-Induced Effects on Apical GCl
To determine whether IL-13 increased the UTP-stimulated apical GCl of HBECs, cells were treated in Ussing chambers with amiloride and then
|
IL-13-Induced Effects on Basal and Stimulated K+ Currents
The potential contribution of basolateral and apical K+ currents to the enhanced UTP response after IL-13 treatment was studied by selectively permeabilizing either membrane while under an established K+ gradient.Basolateral GK.
Under an applied apical-to-basolateral K+ gradient, the
addition of amiloride reduced the basal Isc by
3.5 ± 0.4 (n = 6) and 1.0 ± 0.2 (n = 6) µA/cm2 in the control and
IL-13-treated cells, respectively, indicating that the IL-13 treatment
had affected the ion transport phenotype, as previously seen. The
addition of amphotericin B (10 µM) to the apical surface induced a
slow and sustained increase in Isc (Fig.
8) that has previously been demonstrated
to be due to the basolateral GK. There was no
difference in GK between control and
IL-13-treated cells (control increased by 58.5 ± 6.1 µA/cm2, and IL-13-treated increased by 66.2 ± 6.9 µA/cm2, P = 0.42). The subsequent
addition of UTP induced a transient increase in
Isc in both control and IL-13-treated cells of
102.7 ± 5.9 and 95.5 ± 4.9 µA/cm2,
respectively (P = 0.39, n = 6), that
was followed by a reduction in the basal GK, as
has been previously described in HBECs (10).
|
Apical GK.
Under an applied basolateral-to-apical K+ gradient, the
addition of -toxin (200 U/ml) to the basolateral membrane induced a
biphasic reduction in Isc in control cells that
reached a plateau after ~30 min of
46.5 ± 7.3 µA/cm2 (Fig.
9A). In contrast, the current
decrease induced by
-toxin in the IL-13-treated cells was
significantly lower at
11.2 ± 2.9 µA/cm2
(P < 0.002, n = 6; Fig.
9B). The subsequent addition of UTP induced a further
decrease in Isc of
44.3 ± 5.0 µA/cm2 in the control cells and
49.5 ± 4.8 µA/cm2 in the IL-13-treated cells (P = 0.47, n = 6). In the control cells, the
Isc reached a steady baseline at
1.3 ± 1.6 µA/cm2 compared with
9.1 ± 2.1 µA/cm2 in the IL-13-treated cells (P < 0.02, n = 6).
|
IL-13 Does Not Affect Agonist-Induced Increases in Intracellular Ca2+ Concentration
HBECs cultured on plastic for 48 h either in the presence or absence of IL-13 (10 ng/ml) responded to UTP in a concentration-dependent manner with an increase in intracellular Ca2+ concentration. There were no differences in either the sensitivity or magnitude of the response induced by IL-13 (Fig. 10). IL-13 was likewise without effect on the ionomycin-induced increase in intracellular Ca2+ concentration (data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IL-13 and IL-4 are key mediators of Th2-type inflammatory responses in conditions such as asthma, allergic rhinitis, and chronic sinusitis (16, 18, 19, 22, 31). Elevated levels of these cytokines have been demonstrated in disease, and transgenic mice producing increased levels of these proteins present with an airway hypersecretory phenotype and goblet cell metaplasia (33, 38). To date, there are no published studies examining the effects of Th2 cytokines on the ion transport characteristics of the human airway epithelium. It is therefore striking that in this study the treatment of the human bronchial epithelium with either IL-13 or IL-4 led to the development of a secretory phenotype, as reflected by the complete inhibition of basal amiloride-sensitive Na+ absorption and the appearance of an enhanced anion secretory response to both Ca2+-mobilizing and cAMP-elevating stimuli. These effects were apparent in the absence of any change in the gross differentiation state of the epithelia, as assessed by quantification of the goblet cell density in the cultures.
IL-4 and IL-13 both decreased the basal Isc and
amiloride-sensitive Na+ absorption in addition to
increasing the RT. A similar effect has been
described with IFN- (13), where the concurrent
transepithelial fluid transport was also attenuated. The mechanism(s)
underlying the IL-4- and/or IL-13-induced inhibition of the
amiloride-sensitive Isc are unknown. However,
because it is the apical epithelial Na+ channel (ENaC) that
is the rate-limiting step for amiloride-sensitive Na+
absorption in this tissue, it is likely that it is the result of a loss
of this apical Na+ conductance. A loss of ENaC function
could be because of a direct reduction in expression of one or more of
the ENaC genes or alternatively to an increase in the expression of a
negative regulator of ENaC function, such as CFTR (24). An
alternative mechanism behind the loss of the amiloride-sensitive
Isc in this study could be the inhibition of the
apical GK identified in the
-toxin
permeabilization experiments (Fig. 9). A higher apical
GK in control cells would tend to hyperpolarize
the apical membrane and likely increase the driving force for
Na+ entry. Conversely, an inhibition of the apical
GK by IL-13 would tend to depolarize the apical
membrane and thereby decrease the driving force for Na+
entry and inhibit Na+ absorption. This reduction in apical
GK may also contribute to the IL-13-induced
increase in RT that was observed. However, an IL-13-induced effect on the basolateral or paracellular resistances cannot be ruled out, although the former is unlikely, as no effect of
IL-13 on basolateral GK was observed (Fig. 8;
see below). Few studies have examined the effects of inflammatory
mediators on the amiloride-sensitive current in the airway. It has been
demonstrated that TNF-
and growth factors such as keratinocyte
growth factor can increase amiloride-sensitive currents in airway
epithelial cells both in vitro and in vivo (3, 11, 36).
Conversely, studies have also demonstrated that inflammatory stimuli
can decrease amiloride-sensitive currents in both culture and ex vivo
tissue samples (20, 23). The mechanism(s) underlying the
IL-4- and IL-13-mediated inhibition of the amiloride-sensitive
Isc in this study will require further investigation.
The most striking effects of IL-4 and IL-13 in this study were the enhancement of the UTP and ionomycin-stimulated increases in Isc. UTP was chosen as a Ca2+-mobilizing agonist for these studies, since the effects of nucleotide triphosphates have been widely characterized in the human airway epithelium. Ionomycin was used to demonstrate that the IL-13-induced enhanced UTP response was a receptor-independent effect. Furthermore, we have demonstrated that IL-13 does not affect the mobilization of intracellular Ca2+ induced by UTP. However, it should be considered that the Ca2+ studies were performed on HBECs cultured on plastic and could potentially behave differently to the polarized epithelia. Ionomycin was also used in the studies involving DIDS, since this chloride channel-blocking compound has been demonstrated to antagonize P2 receptors (33). The initial experiments (Figs. 1 and 2) demonstrated that, in the presence of amiloride, UTP induced an increase in Isc that was significantly larger after IL-13 treatment. It was therefore necessary to determine whether a similar effect was apparent in a more physiologically relevant, amiloride-free situation. In the absence of amiloride, UTP induced a transient increase in Isc in control cells that was followed by a sustained inhibitory phase, consistent with the observations of Devor and Pilewski (10; Fig. 3A). In contrast, cells that had been treated with IL-13 developed an enhanced UTP-stimulated increase in Isc similar to that observed in the presence of amiloride (Fig. 3B). This is of relevance, since Ca2+-mobilizing agonists appear to only inhibit Na+ absorption in the healthy airway, but these same agonists can clearly cause an anion secretory response in inflamed airways. All further characterization of the UTP-induced secretory Isc was performed in the presence of amiloride to remove the potential complication of the effect on the Na+ current.
The mechanisms responsible for Isc changes
induced by Ca2+-mobilizing stimuli in the airway epithelium
are not fully understood but are likely to involve the concerted
effects of apical CFTR and an as-yet-unidentified apical
Ca2+-activated GCl combined with the
apical and basolateral GK (9, 30,
37). A recent study reported by Paradiso and colleagues (30) demonstrated that UTP was able to activate both a
Ca2+-activated GCl and CFTR, the
latter through a protein kinase C-mediated effect. The study by
Paradiso et al. (30) also demonstrated that the transient
nature of the Isc changes induced by UTP was mirrored by the transient increase in intracellular Ca2+
concentration and that manipulations designed to attenuate rises in
intracellular Ca2+ concentration also reduced the
Isc changes. UTP has also been reported to
stimulate two independent GCl using the
perforated-patch technique with HBECs (37). It is apparent
that an effect of IL-4 or IL-13 on either the apical
GCl and/or basolateral GK
could manifest as an increase in an anion secretory response. An
immune-mediated increase in a Ca2+-activated
GK is not without precedent, as an anti-CD3
antibody has been demonstrated to increase the expression of hIKCa1 in human T cells (14). However, in this study, the apical
permeabilization experiments (Fig. 8) showed that IL-13 had no effect
on the basolateral GK under basal or
UTP-stimulated conditions. Galietta and colleagues (13)
recently reported that IFN- treatment enhanced the secretory response to Ca2+-mobilizing agonists in their HBEC model
and that the response was independent of the basolateral membrane.
Devor et al. (9) also observed an apical UTP-stimulated
secretory GK in their HBEC model that could also
influence the net current observed in response to UTP. A secretory
K+ current could mask the magnitude of any anion secretory
current or alternatively enhance an anion secretory response, since an increase in apical GK would be predicted to
hyperpolarize the cell and thereby increase the driving force for anion
secretion. The basolateral permeabilization study (Fig. 9) indicated
that UTP stimulated an apical secretory K+ current, as
previously described (9). The peak increase in this
current was unaffected by IL-13 pretreatment (Fig. 9). These observations therefore pointed to an IL-13- and/or IL-4-induced increase in an apical anion secretory conductance that was further supported by the bumetanide sensitivity and anion-dependent nature of
the current. The DIDS sensitivity of the ionomycin-stimulated response
(~60% inhibition of the Isc response at 300 µM) further indicated that a significant proportion of the current
was mediated through a conductance other than CFTR. It is also unlikely
that an increase in CFTR expression would account for the increased UTP
or ionomycin-stimulated currents as the forskolin response was
increased by approximately two- to threefold while the UTP/ionomycin responses were increased by more than sixfold. Finally, the observation of an enhanced UTP-stimulated increase in Isc in
HBECs under a chloride gradient (basolateral to apical) with the
basolateral membrane permeabilized was conclusive evidence of an
IL-13-induced functional Ca2+-activated anion conductance
in the apical membrane.
The only reports of Th2 cytokine-mediated effects on epithelial ion
transport function have used T84 cells and glomerular visceral
epithelial cells. In the T84 study, both IL-4 and IL-13 attenuated
RT, although only IL-4 affected chloride
secretion through an inhibition of CFTR expression (39).
In the glomerular visceral epithelial cells, both IL-4 and IL-13
increased basal Isc; however, the ionic basis
and mechanisms were not investigated (35). The molecular
identity of the Ca2+-activated GCl
in the airway epithelium are, however, unknown. Evidence is emerging
that a family of putative Ca2+-activated chloride channels
(12) that include the murine gene gob-5
(mCLCA3) may play a role in epithelial inflammation; gob-5 has recently been demonstrated to be upregulated and to play a key role
in the development of an asthma phenotype in vivo in the airways of
allergen-challenged mice (28). It remains to be determined
whether the IL-13-induced Ca2+-activated
GCl reported here is indeed a member of this
family. The effects of inflammatory stimuli on the expression of CFTR in epithelia have been studied more widely. Evidence exists for both
up- and downregulation of CFTR by inflammatory stimuli in various
epithelia (2, 5, 6, 13, 27). In HBECs, IFN- decreased
CFTR expression (13). In Calu-3 cells, IL-1
has been demonstrated to increase CFTR expression through an nuclear
factor-
B-mediated pathway (5), whereas in the gut
epithelial cell lines T84 and HT-29 CFTR expression can be
differentially regulated by IFN-
and IL-1
(2, 6).
These data all lead to the conclusion that, during both Th1 and Th2 inflammatory responses in the airway, the bronchial epithelium can convert from an absorbing to a secretory phenotype. The purpose of this phenotype shift can only be speculated upon at present but may represent a "flushing" response to rinse particulate and secreted mucus out of the airway lumen to both prevent congestion and to remove the inflammatory stimuli. Cystic fibrosis also underlines the importance of the balance between fluid and secreted mucus in the airway, and it may be that the epithelium becomes secretory to balance the increase in mucus secretion that is evident during these inflammatory events. Furthermore, in pseudohypoaldosteronism type II, ENaC is dysfunctional, and patients have a fluid hypersecretory phenotype in the airways that is evident as rhinitis (21). What is surprising is that, in these patients, the rate of mucociliary clearance is upregulated by up to fivefold, and it may be that the bronchial epithelium converts to a hypersecretory phenotype during inflammatory events to elicit a pseudohypoaldosteronism type II clearance response. These observations may have consequences for both the treatment of hypersecretory diseases of the lung and potentially cystic fibrosis, where an enhanced anion secretory response that is independent of CFTR could serve to address the imbalance of airway fluid transport.
![]() |
FOOTNOTES |
---|
1 For clarity we have quantified only the peak increase in Isc throughout.
Address for reprint requests and other correspondence: H. Danahay, Novartis Horsham Research Centre, Wimblehurst Rd., Horsham, West Sussex RH12, 5AB, UK (E-mail: henry.danahay{at}pharma.novartis.com).
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.
10.1152/ajplung.00311.2001
Received 7 August 2001; accepted in final form 10 October 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alpert, SE,
and
Walenga RW.
Ozone exposure of human tracheal epithelial cells inactivates cyclooxygenase and increases 15-HETE production.
Am J Physiol Lung Cell Mol Physiol
269:
L734-L743,
1995
2.
Besancon, F,
Przewlocki G,
Baro I,
Hongre AS,
Escande D,
and
Edelman A.
Interferon- downregulates CFTR gene expression in epithelial cells.
Am J Physiol Cell Physiol
267:
C1398-C1404,
1994
3.
Borok, Z,
Danto SI,
Dimen LL,
Zhang XL,
and
Lubman RL.
Na+-K+-ATPase expression in alveolar epithelial cells: upregulation of active ion transport by KGF.
Am J Physiol Lung Cell Mol Physiol
274:
L149-L158,
1998
4.
Boucher, RC.
Human airway ion transport.
Am J Respir Crit Care Med
150:
271-281,
1994[ISI][Medline].
5.
Brouillard, F,
Bouthier M,
Leclerc T,
Clement A,
Baudouin-Legros M,
and
Edelman A.
NF-B mediates up-regulation of CFTR gene expression in Calu-3 cells by interleukin-1
.
J Biol Chem
276:
9486-9491,
2001
6.
Cafferata, EG,
Guerrico AM,
Pivetta OH,
and
Santa-Coloma TA.
NF-B activation is involved in regulation of cystic fibrosis transmembrane conductance regulator (CFTR) by interleukin-1
.
J Biol Chem
276:
15441-15444,
2001
7.
Chang, MM,
Wu R,
Plopper CG,
and
Hyde DM.
IL-8 is one of the major chemokines produced by monkey airway epithelium after ozone-induced injury.
Am J Physiol Lung Cell Mol Physiol
275:
L524-L532,
1998
8.
Christoffersen, CR,
and
Skibsted LH.
Calcium ion activity in physiological salt solutions: influence of anions substituted for chloride.
Comp Biochem Physiol A Physiol
52:
317-322,
1975[ISI].
9.
Devor, DC,
Bridges RJ,
and
Pilewski JM.
Pharmacological modulation of ion transport across wild-type and F508 CFTR-expressing human bronchial epithelia.
Am J Physiol Cell Physiol
279:
C461-C479,
2000
10.
Devor, DC,
and
Pilewski JM.
UTP inhibits Na+ absorption in wild-type and DeltaF508 CFTR-expressing human bronchial epithelia.
Am J Physiol Cell Physiol
276:
C827-C837,
1999
11.
Fukuda, N,
Jayr C,
Lazrak A,
Wang Y,
Lucas R,
Matalon S,
and
Matthay MA.
Mechanisms of TNF- stimulation of amiloride-sensitive sodium transport across alveolar epithelium.
Am J Physiol Lung Cell Mol Physiol
280:
L1258-L1265,
2001
12.
Fuller, CM,
and
Benos DJ.
Electrophysiological characteristics of the Ca2+-activated Cl channel family of anion transport proteins.
Clin Exp Pharmacol Physiol
27:
906-910,
2000[ISI][Medline].
13.
Galietta, LJV,
Folli C,
Marchetti C,
Romano L,
Carpani D,
Conese M,
and
Zegarra-Moran O.
Modification of transepithelial ion transport in human cultured bronchial epithelial cells by interferon-.
Am J Physiol Lung Cell Mol Physiol
278:
L1186-L1194,
2000
14.
Ghanshani, S,
Wulff H,
Miller MJ,
Rohm H,
Neben A,
Gutman GA,
Cahalan MD,
and
Chandy KG.
Up-regulation of the IKCa1 potassium channel during T-cell activation. Molecular mechanism and functional consequences.
J Biol Chem
275:
37137-37149,
2000
15.
Gray, TE,
Guzman K,
Davis CW,
Abdullah LH,
and
Nettesheim P.
Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells.
Am J Respir Cell Mol Biol
14:
104-112,
1996[Abstract].
16.
Hamilos, DL,
Leung DY,
Wood R,
Bean DK,
Song YL,
Schotman E,
and
Hamid Q.
Eosinophil infiltration in nonallergic chronic hyperplastic sinusitis with nasal polyposis (CHS/NP) is associated with endothelial VCAM-1 upregulation and expression of TNF-alpha.
Am J Respir Cell Mol Biol
15:
433-450,
1996[Abstract].
17.
Holgate, ST,
Lackie P,
Wilson S,
Roche W,
and
Davies D.
Bronchial epithelium as a key regulator of airway allergen sensitisation and remodelling in asthma.
Am J Respir Crit Care Med
162:
S113-S117,
2000
18.
Huang, SK,
Siao HQ,
Klein-Tebbe J,
Paciotti G,
Marsh DG,
Lichtenstein LM,
and
Liu MC.
IL-13 expression at the sites of allergen challenge in patients with asthma.
J Immunol
155:
2688-2694,
1995[Abstract].
19.
Humbert, M,
Durham SR,
Kimmitt P,
Powell N,
Assoufi B,
Pfiser R,
Menz G,
Kay AB,
and
Corrigan CJ.
Elevated expression of messenger ribonucleic acid encoding IL-13 in the bronchial mucosa of atopic and nonatopic subjects with asthma.
J Allergy Clin Immunol
99:
657-665,
1997[ISI][Medline].
20.
Iwase, N,
Sasaki T,
Shimura S,
Fushimi T,
Okayama H,
Hoshi H,
Irokawa T,
Sasamori K,
Takahashi K,
and
Shirato K.
Signature current of SO2-induced bronchitis in rabbit.
J Clin Invest
99:
1651-1661,
1997
21.
Kerem, E,
Bistritzer T,
Hanukoglu A,
Hofmann T,
Zhou Z,
Bennett W,
MacLaughlin E,
Barker P,
Nash M,
Quittell L,
Boucher R,
and
Knowles MR.
Pulmonary epithelial sodium-channel dysfunction and excess airway liquid in pseudohypoaldosteronism.
N Engl J Med
341:
156-162,
1999
22.
Kotsimbos, TC,
Ernst P,
and
Hamid QA.
Interleukin-13 and interleukin-4 are coexpressed in atopic asthma.
Proc Assoc Am Physicians
108:
368-373,
1996[ISI][Medline].
23.
Kunzelmann, K,
Beesley AH,
King NJ,
Karupiah G,
Young JA,
and
Cook DI.
Influenza virus inhibits amiloride-sensitive Na+ channels in respiratory epithelia.
Proc Natl Acad Sci USA
97:
10282-10287,
2000
24.
Kunzelmann, K,
Schreiber R,
Nitschke R,
and
Mall M.
Control of epithelial Na+ conductance by the cystic fibrosis transmembrane conductance regulator.
Pflügers Arch
440:
193-201,
2000[ISI][Medline].
25.
Martin, LD,
Norford D,
Voynow J,
and
Adler KB.
Response of human airway epithelium in vitro to inflammatory mediators (Abstract).
Chest
117:
267S,
2000
26.
Martin, LD,
Rochelle LG,
Fischer BM,
Krunkosy TM,
and
Adler KB.
Airway epithelium as an effector of inflammation: molecular regulation of secondary mediators.
Eur Respir J
10:
2139-2146,
1997
27.
Nakamura, H,
Yoshimura K,
Bajocchi G,
Trapnell BC,
Pavirani A,
and
Crystal RG.
Tumor necrosis factor modulation of expression of the cystic fibrosis transmembrane conductance regulator gene.
FEBS Lett
314:
366-370,
1992[ISI][Medline].
28.
Nakanishi, A,
Morita S,
Iwashita H,
Sagiya Y,
Ashida Y,
Shirafuji H,
Fujisawa Y,
Nishimura O,
and
Fujino M.
Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma.
Proc Natl Acad Sci USA
98:
5175-5180,
2001
29.
Nettesheim, P,
and
Bader T.
Tumor necrosis factor alpha stimulates arachidonic acid metabolism and mucus production in rat tracheal epithelial cell cultures.
Toxicol Lett
88:
35-37,
1996[ISI][Medline].
30.
Paradiso, AM,
Ribeiro CM,
and
Boucher RC.
Polarized signaling via purinoceptors in normal and cystic fibrosis airway epithelia.
J Gen Physiol
117:
53-67,
2001
31.
Pawankar, RU,
Okuda M,
Hasegawa S,
Suzuki K,
Yssel H,
Okubo K,
Okumura K,
and
Ra C.
Interleukin-13 expression in the nasal mucosa of perennial allergic rhinitis.
Am J Respir Crit Care Med
152:
2059-2067,
1995[Abstract].
32.
Pilewski, JM,
and
Frizzell RA.
Role of CFTR in airway disease.
Physiol Rev
79:
S215-S255,
1999[Medline].
33.
Schultz, BD,
Singh AK,
Devor DC,
and
Bridges RJ.
Pharmacology of CFTR chloride channel activity.
Physiol Rev
79:
S109-S144,
1999[Medline].
34.
Temann, UA,
Prasad B,
Gallup MW,
Basbaum C,
Ho SB,
Flavell RA,
and
Rankin JA.
A novel role for murine IL-4 in vivo: induction of MU5AC gene expression and mucin hypersecretion.
Am J Respir Cell Mol Biol
16:
471-478,
1997[Abstract].
35.
Van den Berg, JG,
Aten J,
Chand MA,
Claessen N,
Dijkink L,
Wijdenes J,
Lakkis FG,
and
Weening JJ.
Interleukin-4 and interleukin-13 act on glomerular visceral epithelial cells.
J Am Soc Nephrol
11:
413-422,
2000
36.
Wang, Y,
Folkesson HG,
Jayr C,
Ware LB,
and
Matthay MA.
Alveolar epithelial fluid transport can be simultaneously upregulated by both KGF and -agonist therapy.
J Appl Physiol
87:
1852-1860,
1999
37.
Zegarra-Moran, O,
Sacco O,
Romano L,
Rossi GA,
and
Galietta LJ.
Cl currents activated by extracellular nucleotides in human bronchial cells.
J Membr Biol
156:
297-305,
1997[ISI][Medline].
38.
Zhu, Z,
Homer RH,
Wang Z,
Chen Q,
Geba GP,
Wang J,
Zhang Y,
and
Elias JA.
Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production.
J Clin Invest
103:
779-788,
1999
39.
Zünd, G,
Madara JL,
Dzus AL,
Awtrey CS,
and
Colgan SP.
Interleukin-4 and interleukin-13 differentially regulate epithelial chloride secretion.
J Biol Chem
271:
7460-7464,
1996