Departments of Biomolecular Screening and Protein Chemistry, Immunex Corporation, Seattle, Washington 98101
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ABSTRACT |
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To understand the
effects of cytokines on epithelial cells in asthma, we have
investigated the effects of interleukin (IL)-4, IL-13, and interferon
(IFN)- on barrier function and wound healing in Calu-3 human lung
epithelial cells. IL-4 and IL-13 treatment of Calu-3 cells grown on
Transwell filters resulted in a 70-75% decrease in barrier
function as assessed by electrophysiological and
[14C]mannitol flux measurements. In contrast, IFN-
enhanced barrier function threefold using these same parameters. Cells
treated concurrently with IFN-
and IL-4 or IL-13 showed an initial
decline in barrier function that was reversed within 2 days, resulting in barrier levels comparable to control cells. Analysis of the tight
junction-associated proteins ZO-1 and occludin showed that IL-4 and
IL-13 significantly reduced ZO-1 expression and modestly decreased
occludin expression compared with controls. IFN-
, quite unexpectedly
given its enhancing effect on barrier function, reduced expression of
ZO-1 and occludin to almost undetectable levels compared with controls.
In wound-healing assays of cells grown on collagen I, IL-4 and IL-13
decreased migration, whereas IFN-
treatment enhanced migration,
compared with control cells. Addition of IFN-
, in combination with
IL-4 or IL-13, restored migration of cells to control levels. Migration
differences observed between the various cytokine treatments was
correlated with expression of the collagen I-binding
2
1-integrin at the leading edge of cells
at the wound front;
2
1-integrin
expression was decreased in IFN-
-treated cells compared with
controls, whereas it was highest in IL-4- and IL-13-treated cells.
These results demonstrate that IL-4 and IL-13 diminish the capacity of
Calu-3 cells to maintain barrier function and repair wounds, whereas
IFN-
promotes epithelial restitution by enhancing barrier function
and wound healing.
asthma; tight junctions; cell migration
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INTRODUCTION |
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ASTHMA HAS TRADITIONALLY BEEN considered a disease of the immune system (35). T cells secreting Th2-type cytokines, such as interleukin (IL)-4 and IL-13, play a key role in disease initiation and propagation by activating and recruiting a variety of cells, such as mast cells, eosinophils, and B cells, to the lung (for review see Ref. 35). The resulting cellular infiltration and inflammation are believed to contribute to other changes associated with lung function, such as airway remodeling and airway hyperresponsiveness (AHR).
An alternate view of asthma suggests that the epithelium, along with the immune system, is required for the disease to develop (11, 12). In the absence of this epithelial response, simple atopy, and not asthma, would ensue. According to this hypothesis, damage to epithelial cells that occurs during the initial stages of asthma stimulates the proliferation of fibroblasts and muscle cells, which, in turn, increases the production and secretion of extracellular matrix proteins. This increased cellular proliferation and matrix deposition give rise to the thickening of the submucosa and the fibrosis characteristic of asthma.
One of the major roles of the epithelium in the lung is its function as a barrier between the lumen and the underlying submucosa, which contains cells of the immune system. Histological analysis of lung biopsies from asthmatic individuals shows a disruption in epithelial cells and, in some instances, complete loss of these cells, indicating that perturbation of barrier function occurs in vivo (17, 21). Loss of barrier function results in the uncontrolled flux of allergens and other noxious substances from the lumen into the submucosa and subsequent activation of the immune system, culminating in inflammation in the lung. Until the barrier is restored, this situation persists and progresses, leading to more inflammation and epithelial damage. Restoration of the barrier involves healing of the wound formed by a loss of the cells and a reestablishment of the specialized cell adhesion complexes, which are crucial to epithelial barrier function. Thus the study of epithelial barrier regulation in the lung during inflammation may be crucial for understanding the pathogenesis of asthma and, ultimately, for the development of therapeutics to treat asthma. Unfortunately, little is known about pulmonary epithelial barrier regulation and how proinflammatory cytokines, such as IL-4 and IL-13, affect epithelial barrier function and restitution in asthma. IL-4 and IL-13 have been shown to disrupt epithelial barrier function in intestinal model systems (37), but similar work with lung cells has not been done.
In this study, we have utilized Calu-3 lung epithelial cells as an in
vitro model to determine the effects of IL-4 and IL-13 on barrier
function and wound healing. We have also examined the effects of the
Th1 cytokine interferon (IFN)- because of studies showing that
asthma may be ameliorated by a Th1-type cytokine response
(31). Our results show that treatment of Calu-3 cells with
IL-4 and Il-13 decreases barrier function and reduces wound healing. In
contrast, IFN-
enhances barrier function and promotes wound healing.
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MATERIALS AND METHODS |
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Cell culture.
Calu-3 cells, derived from a human lung adenocarcinoma, were grown in
minimal essential medium (Life Technologies, Gaithersburg, MD)
containing 10% heat-inactivated fetal bovine serum, penicillin, streptomycin, and glutamine. Cells were passaged when they were ~80%
confluent. For barrier function experiments, cells were plated at a
density of 1 × 105 cells per 300 µl of medium and
added to the upper chamber of 6.5-mm Transwell filters (tissue
culture-treated polyester, 0.4-µm pore size; Costar, Cambridge, MA).
The bottom chamber contained 800 µl of complete medium. Cells were
maintained for 2-3 days before barrier function was assessed by
measurement of transepithelial electrical resistance (TER) using a
voltohmeter equipped with a chopstick electrode (World Precision
Instruments, Vero Beach, FL). Cells were used when TER values were
stable for 2 days and measured 300
· cm2. The
background resistance (filter + media without cells) was subtracted from the TER values.
Cytokine treatment and assessment of barrier function.
All cytokines were obtained from R & D Systems (Minneapolis, MN). Cells
were treated with IL-4 (5 ng/ml), IL-13 (5 ng/ml), or IFN- (50 ng/ml) added to the basolateral side of the Transwell filters for the
indicated period of time. Anti-IL-4 receptor-
antibody (10 µg/ml;
R & D Systems), soluble human IL-4 receptor-
(10 µg/ml), or murine
anti-human CD3 (10 µg/ml; isotype control antibody) was added at the
same time as the cytokines to the basolateral side of the filters.
Dose-response curves were done for all the cytokines and cytokine
receptor antagonists, and the concentration used was the lowest dose
that gave the maximal response in the barrier assay. TER was measured
at 24-h intervals. Mannitol flux was determined by adding 0.5 µCi of
[14C]mannitol (2.07 GBq/mmol; NEN, Boston, MA) to the
apical chamber at day 3 of the experiment and measuring the
amount of [14C]mannitol in the basolateral chamber
18 h later.
Immunoblotting of tight junction proteins and E-cadherin. For analysis of tight junction proteins and E-cadherin, Calu-3 cell lysates were prepared by differential detergent solubility (28, 36). Briefly, Calu-3 cells grown on filters were washed twice with PBS and then scraped into 750 µl of NP-40-IP buffer (25 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 4 mM EDTA, 25 mM NaF, 1 mM Na3VO4, 1% NP-40, 10 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 20 µM pepstatin A, and 2 mM o-phenanthrolene) and solubilized on ice for 90 min with frequent trituration. The lysate was centrifuged at 14,000 rpm for 30 min at 4°C in an Eppendorf microfuge. The supernatant contained the NP-40-soluble proteins. The pellet was resuspended in 75 µl of SDS-IP buffer (25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 4 mM EDTA, 25 mM NaF, 1 mM Na3VO4, 1% SDS, 10 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 20 µM pepstatin A, and 2 mM o-phenanthroline), transferred to a Dounce homogenizer, and disrupted with 10-12 strokes. NP-40-IP buffer (675 µl) was added, and the lysate was passaged through a 25-gauge needle 10-15 times to shear the DNA. The sample was left on ice for 90 min with frequent trituration and centrifuged as described above. This supernatant contained the NP-40-insoluble proteins. Protein fractions were assayed by the micro-bicinchoninic acid procedure (Pierce Chemicals, Rockford, IL).
E-cadherin expression was analyzed using the NP-40-soluble protein fraction, and ZO-1 and occludin expression were analyzed using the NP-40-insoluble protein fraction. Proteins (25 µg) were separated on a 10% Tris-glycine polyacrylamide gel (Novex, San Diego, CA), transferred to nitrocellulose (0.45 µm; Bio-Rad, Richmond, CA), and blocked for 4-6 h in PBS-0.1% Tween 20-5% dried milk (PTM). The blots were then incubated overnight at 4°C with primary antibodies (ZO-1, occludin, and E-cadherin; Zymed Laboratories, South San Francisco, CA) diluted 1:1,000-1:2,000 in PTM. The blots were then washed in PBS, incubated at room temperature for 90 min with biotinylated secondary antibody (Molecular Probes, Eugene, OR) diluted 1:1,000 in PTM followed by horseradish peroxidase-conjugated streptavidin (Sigma, St. Louis, MO) diluted 1:10,000 in PTM, and then processed for chemiluminescence detection (ECL, Amersham, Arlington Heights, IL).Wound assays. Calu-3 cells were plated on chamber slides (Nunc, Napierville, IL) coated with collagen I or fibronectin (10 µg/ml; both from Becton-Dickinson, Bedford, MA) and allowed to reach confluence. Wounds were made as previously described (5) by adding 1 µl of 1 N NaOH to the center of the well and then immediately rinsing with excess PBS. This treatment produced 3- to 6-mm-diameter circular wounds. Monolayers were allowed to recover for 24 h before the cytokine treatments. Cells were treated with various combinations of cytokines or cytokine receptor antagonists and photographed at 24-h intervals to record migration from the edge of the wound. Images were collected using a charge-coupled device camera (Sony) attached to an inverted phase contrast microscope (Nikon, Melville, NY). Images were analyzed using Metamorph Imaging series 4.5 (Universal Imaging, West Chester, PA) to measure the area of the wounds at the indicated times. The results are expressed as the area of the repaired region, which is defined as the difference between wound area at time 0 and wound area at 24 or 48 h.
Immunofluorescence and immunoblot analysis of integrin
expression.
Calu-3 cell monolayers from the wound assays were washed three times in
ice-cold complete medium and incubated with
anti-2
1-integrin antibody (10 µg/ml;
Chemicon, Temecula, CA) in complete medium at 4°C for 60 min. Cells
were washed in ice-cold PBS, fixed for 15 min in 4% paraformaldehyde
in PBS, washed twice in PBS, and incubated for 30 min in 50 mM
NH4Cl to quench unreacted aldehyde groups. The cells were
washed twice more with PBS and then permeabilized and blocked in PBS
containing 0.1% Triton X-100 and 5% BSA (PTB) for 30 min.
Subsequently, the cells were incubated for 60 min with Alexis
488-conjugated goat anti-mouse IgG (10 µg/ml; Molecular Probes) and
Alexis 568-conjugated phalloidin (10 µg/ml; Molecular Probes) diluted
in PTB. The cells were then washed three times with PBS, mounted in
Prolong (Molecular Probes), and sealed with a coverslip. The samples
were analyzed on a laser scanning microscope (model 2001, Molecular
Dynamics, Sunnyvale, CA) equipped with a Kr/Ar laser and a Nikon ×60
NA 1.4 oil lens. Images were collected using an SGI Iris Indigo 4000 Workstation (Silicon Graphics, Mountain View, CA). The final images
were prepared using Adobe Photoshop version 5.5 (Adobe Systems). All
images were treated identically in terms of exposure, background
subtraction, and contrast enhancement.
Statistical analysis. Values are means ± SD. Differences between groups were analyzed using the Student's t-test. P < 0.05 was considered to be significant.
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RESULTS |
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IL-4 and IL-13 treatment of Calu-3 cells decreased TER.
Calu-3 cells form polarized, relatively impermeable monolayers when
grown on Transwell filters (34). These cells can be used
to model epithelial barrier function in the lung. The effect of IL-4
and IL-13 on barrier function in Calu-3 cells was assessed by TER
measurements. Calu-3 cells formed barriers with a TER of ~400
· cm2 and maintained that level throughout the
course of the assay (Fig. 1). Treatment
of the cells with 5 ng/ml of IL-4 or IL-13 resulted in a rapid,
pronounced decrease (~70-75%) in TER by day 1 and
remained at that level for the remainder of the assay. Treatment of the
cells with 50 ng/ml of IL-4 or IL-13 showed similar results, whereas
treatment with
0.5 ng/ml of either cytokine had no effect on barrier
function (data not shown). The cytokine concentrations used were the
lowest doses that gave the maximal response in the barrier assay. In
addition, these effects on barrier function were observed only when the
cytokines were added to the basolateral side of the cells (data not
shown). These results demonstrate that IL-4 and IL-13 caused a decrease
in Calu-3 barrier function.
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IFN- protects barrier function in IL-4- and IL-13-treated cells.
IFN-
was shown previously to disrupt the barrier function in an
intestinal epithelial barrier model (19, 36). To determine whether IFN-
possessed the same activity in lung epithelial cells, the Calu-3 cells were treated with 50 ng/ml of IFN-
, a concentration that was shown to be very effective at disrupting barrier function in
T84 colonic epithelial cells (36). Quite unexpectedly,
IFN-
treatment of the Calu-3 cells enhanced barrier function about threefold (Fig. 4). At early time points,
IFN-
-treated cells had barrier levels comparable to control cells,
but by day 2, a slight enhancement became apparent. By
day 3 of treatment, barrier function increased by greater
than threefold compared with control cells.
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Mannitol flux measurements confirm the TER results.
To confirm that the changes in TER induced by the various cytokine
treatments were a reflection of altered paracellular flow and not
changes in ion flux, [14C]mannitol flux was measured in
cells after cytokine treatment. Mannitol is not transported by cells,
so when added to the apical chamber in a barrier assay, its appearance
on the basolateral side is a measure of paracellular flow, i.e.,
barrier leakiness (19). Treatment of the cells with IL-4
and IL-13 resulted in a ~2.5-fold increase in mannitol flow into the
basolateral compartment compared with control cells (Fig.
5). The addition of anti-IL-4 receptor
antibody or soluble IL-4 receptor to IL-4-treated cells reduced
mannitol flux to the same level as controls. In the case of IL-13, only
the anti-IL-4 receptor antibody was able to achieve this effect; the
soluble receptor had no effect on mannitol flux. IFN- treatment of
the cells reduced mannitol flux to about half that of control cells.
These results confirm that barrier function is indeed decreased by IL-4
and IL-13 and increased by IFN-
, in agreement with the
electrophysiological measurements.
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Analysis of tight junction proteins.
To examine the effects of the cytokines on proteins associated with
tight junctions, ZO-1 and occludin were examined by immunblotting. ZO-1
is a cytoplasmic protein that plays a central role in tight junction
organization by linking the transmembrane components of tight junctions
to the actin cytoskeleton (7). Occludin is a tight
junction-associated transmembrane protein that binds, in a
homophilic manner, to occludin on apposing cells via its extracellular
domain and to actin through its association with ZO-1 (9).
In addition to these two proteins, the cell adhesion molecule
E-cadherin was also examined, because disruption of E-cadherin-mediated adhesion events has been shown to prevent tight junction formation (10). Cytokine treatment of the Calu-3 cells showed
unexpected effects on ZO-1 and occludin (Fig.
6). In control cells, prominent bands
corresponding to ZO-1, occludin, and E-cadherin were observed. In IL-4-
and IL-13-treated cells, ZO-1 levels were decreased by 60-70%,
whereas occludin was reduced by ~30%. The reduction in ZO-1 and
occludin is consistent with the fact that the epithelial barrier is
also reduced. The effect of IFN- on ZO-1 and occludin was even more
pronounced than the effect of IL-4 or IL-13, decreasing their
expression to almost undetectable levels (Fig. 6). This result is
surprising and counterintuitive in light of the fact that IFN-
enhanced barrier function yet dramatically reduced the expression of
two putatively important tight junction proteins. E-cadherin expression
was unaffected by any of the cytokine treatments.
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Wound assays.
Wound healing is a measure of cell migratory activity and is required
to restore integrity to the epithelium as a result of cell loss. To
assess the effects of various cytokine treatments on cell migration, an
in vitro wound-healing assay of Calu-3 cells was performed. The wounds
were generated by NaOH-induced damage to the monolayers
(5), resulting in ~3- to 6-mm-diameter circular wounds.
Cytokines were administered 24 h after the wounds were generated,
and the rate of cell migration was measured subsequently at 24-h
intervals for 2 days. The amount of wound repair was expressed as the
difference between the area of the wound at time 0 and that
at 24 or 48 h. Migration was measured on fibronectin- or collagen
I-coated slides, because the expression of these two matrix components
is elevated in the asthmatic lung (14). When these assays
were performed on fibronectin-treated slides, no difference was seen
between any of the treatments (data not shown). However, when the wound
assays were performed on collagen I-coated slides, differences in
migration rate between the various cytokine treatments were observed
(Fig. 7). Untreated cells showed a
time-dependent increase in the area of the repaired region from 3,211 at 24 h to 5,691 at 48 h. IFN--treated cells showed an
even greater amount of repair (6,400 at 24 h to 14,117 at 48 h), indicating that IFN-
stimulated cell migration and wound repair
compared with control cells. In contrast, in IL-4- and IL-13-treated
cells, wound repair was reduced by 55-65% compared with control
cells at 24 and 48 h. Coincubation of the cells with IL-4 or IL-13
and IFN-
stimulated the cells to migrate at rates comparable to
untreated cells, but less than with IFN-
alone. Similarly, addition
of anti-IL-4 receptor antibody or soluble IL-4 receptor-
to the
IL-4-treated cells produced wound repair similar to control cells. As
was the case in the barrier assay, only anti-IL-4 receptor antibody,
and not soluble IL-4 receptor-
, had an effect on IL-13-treated
cells, restoring the rate of wound repair to control levels. Treatment of the cells with anti-IL-4 receptor antibody or soluble IL-4 receptor
alone had no effect on cell migration, inasmuch as the rate of wound
repair was comparable to control cells (Fig. 7). These results
demonstrate that IFN-
stimulates cell migration and wound healing,
whereas IL-4 or IL-13 inhibits these processes. Furthermore, treatment
of the cells with IFN-
and IL-4 or IL-13 overcomes the inhibitory
effects of IL-4 and IL-13 on cell migration.
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Expression of integrins on cells in the wound assay.
To understand why cells treated with the various cytokines showed
differences in migration rate, the expression of the integrins that
mediate attachment to fibronectin and collagen I was examined by
immunofluorescence and immunoblotting. Lung epithelial cells utilize
v
6-integrin to bind fibronectin
(3) and
2
1-integrin to bind
collagen I (16). In addition to these adhesion receptors, the organization of actin was examined by immunofluorescence because of
the critical role of the cytoskeleton in cell migration. Calu-3 cells
plated on fibronectin-coated slides and stained with
anti-
v
6 antibodies showed a similar
staining pattern regardless of cytokine treatment (data not shown). The
staining was of uniform intensity on all cells in the monolayer,
including those at the leading edge of the wound (data not shown). In
contrast, expression of
2
1-integrin on
the cells plated on collagen showed differences in staining patterns
depending on the cytokine treatment (Fig. 8, A-D). In IL-4- and
IL-13-treated cells, staining for
2
1-integrin was uniformly bright
throughout the monolayer (Fig. 8, B and C). In
particular, the leading edge of some cells at the wound interface showed moderate to intense staining for
2
1-integrin (arrowheads, Fig. 8,
B and C). Control cells also showed staining of
all the cells in the monolayer (Fig. 8A); however, staining
intensity on the leading edge of cells at the wound front was reduced
compared with IL-4- and IL-13-treated cells (cf. Fig. 8A
with Fig. 8, B and C). Cells in this region
showed faint to moderate staining, and in some instances, no
2
1 staining was detectable.
IFN-
-treated cells showed staining similar to the control and IL-4-
and IL-13-treated cells in the monolayer distal to the wound edge but
differed considerably in cells at the leading edge of the wound (Fig.
8D). The cells at the wound front showed very low to
nondetectable staining for
2
1-integrin at
their leading edge. The fact that reduced
2
1-integrin expression was confined to
the leading edge of IFN-
-treated cells suggests that downregulation
or altered targeting of this receptor may contribute to the increased
migration rate and wound healing observed. To begin to address this
issue, the levels of
2
1-integrin were
examined semiquantitatively by immunoblotting extracts of cells from
the wound assays using antibodies specific for each subunit of
2
1. The results showed that the levels of
2- and
1-subunits were similar to or only
modestly different from the control (Fig.
9, A and B). Thus
the reduction in
2
1-integrin seen in the
IFN-
-treated cells was limited to the leading edge and does not
represent a global effect on the expression of this integrin.
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DISCUSSION |
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Epithelial cells in the lung form a restrictive barrier between
the lumen and the underlying submucosa. In asthma, this barrier is
disrupted, possibly contributing to the initiation and/or exacerbation of the inflammatory process that occurs in this disease
(12). Although the effects of proinflammatory cytokines in
modulating immune cell function and activation are well characterized,
the role they play in regulation of the lung epithelial barrier is poorly understood. In this report, using the Calu-3 cells as an in
vitro model of barrier function, we show that Th1 and Th2 cytokines have distinct effects on lung epithelial cell function. The Th1 cytokine IFN- has beneficial effects on barrier function and wound
healing. In contrast, IL-4 and IL-13, Th2 cytokines with elevated
expression in asthma, have detrimental effects on these same biological processes.
Epithelial barrier function is mediated by a complex of proteins,
including ZO-1 and occludin, which constitute the tight junctions (for
reviews see Refs. 1 and 20). ZO-1 plays a pivotal role in
tight junction formation and organization by linking the transmembrane
protein occludin to other cytoplasmic components of the tight junctions
and to the actin cytoskeleton (7). Occludin binds to cells
on apposing cells and is linked to ZO-1 and the cytoskeleton
(9). The regulation of epithelial barrier function by
cytokines has been studied most extensively in the gut using the T84
intestinal epithelial cell line. In these cells, IL-4, IL-13, and
IFN- induce barrier breakdown in a rapid, dose-dependent fashion
(19, 36, 37). In IFN-
-treated T84 cells, disruption of
barrier function correlates with a loss of ZO-1 and reorganization of
the apical actin cytoskeleton (36). The effects of
IFN-
, IL-4, and IL-13 on Calu-3 barrier function are the first such analysis using lung epithelial cells. IL-4 and IL-13 treatment of
Calu-3 cells shows reduced barrier function similar to that observed in
T84 cells treated with these cytokines and with IFN-
(19, 36,
37). However, IFN-
treatment of Calu-3 cells enhances barrier
function, while it decreases it in T84 cells. The effects of IFN-
on
Calu-3 and T84 cells are a striking example of how epithelial cells
from different tissues respond differently to an identical stimulus.
Treatment of the Calu-3 cells with IL-4 and IL-13 only modestly affects
occludin expression, whereas IFN- dramatically reduces it. Given the
important role of occludin in tight junction formation, the effects of
IL-4 and IL-13 on occludin expression are consistent with a reduction
in barrier function. What has been unexpected, however, is the increase
in barrier function induced by IFN-
, despite the profound reduction
in expression of occludin. One explanation may be the redundancy of
function provided by claudins, a new family of transmembrane proteins
that also localize to tight junctions (32). There are
20
known claudins, several of which interact with many of the same
proteins as occludin (32). The existence of proteins with
functional redundancy to occludin has been inferred from studies with
occludin-deficient embryonic stem cells that retain the ability to form
polarized epithelia possessing normal barrier function
(27). This occludin-like activity is mediated by the
claudins. In fact, it is becoming apparent that claudins are the major
transmembrane components of tight junctions and that the permeability
properties of an epithelium are the product of claudin composition
within those cells (20).
IFN- treatment of the Calu-3 cells also dramatically reduces ZO-1
expression, yet barrier function is enhanced. This result, too, was
unexpected because of the importance ascribed to ZO-1 in the regulation
of tight junction function. In IFN-
-treated T84 cells, ZO-1 levels
are significantly reduced, with a concomitant loss of barrier function,
supporting the concept that this protein plays a crucial role in tight
junctions. The reduction in ZO-1 levels and the associated decrease in
epithelial barrier observed with IL-4 and IL-13 treatment of Calu-3
cells are also consistent with a critical role of ZO-1 in barrier
function. Although no adequate explanation can be provided to account
for the paradoxical effect of IFN-
on barrier function and ZO-1
expression, one intriguing possibility that we are exploring is that
Calu-3 cells express a protein with functional redundancy to ZO-1 that
may show differential regulation by IFN-
and IL-4 and IL-13.
Although the tight junction is the major cellular component of barrier
permeability, an intact epithelial monolayer is required for barrier
function at the tissue level. Damage and loss of epithelial cells that
occur in asthma result in denuded areas of epithelium and exposure of
the submucosa to the external environment (17, 21). The
process of epithelial repair may be abnormal in the asthmatic lung,
resulting in prolonged exposure of the submucosa to antigens and
irritants found in the lung lumen (12). In this study, we
have shown that IL-4 and IL-13 decrease lung epithelial cell migration,
which, if it occurs in vivo, would reduce the rate of repair and
restitution of the epithelium. As is the case with barrier function,
IFN- has the opposite effect of IL-4 and IL-13 and enhances
migration of the Calu-3 cells. The effects of the cytokines on
migration are specific for collagen I and do not affect migration on
fibronectin. These observations may be significant in an in vivo
setting, since increased collagen I deposition, leading to fibrosis, is
a hallmark of the asthmatic lung (6). The migration
effects correlate with altered expression of the collagen I-specific
integrin,
2
1, one of the major
collagen-binding integrins expressed in the lung (16).
Previous studies have suggested that
v
6,
the fibronectin-binding integrin in lung epithelial cells, modulates
inflammation in a murine asthma model (13). However, in
the Calu-3 cells, there is no difference in migration rate of the cells
on fibronectin, nor is there any change in
v
6-integrin expression with any of the
three cytokines examined. Migration of the Calu-3 cells on collagen I
is associated with changes in the levels of
2
1-integrin expression on the leading edge of the migrating cells; cells distal to the wound edge show no
differences in
2
1-integrin expression
regardless of cytokine treatment. The rate of migration is inversely
related to
2
1-integrin expression;
IFN-
-treated cells migrate the fastest and have no detectable
2
1-integrin on their leading edge,
whereas IL-4- and IL-13-treated cells migrate the slowest and have the
highest levels of
2
1-integrin on their
leading edge. This result is somewhat unexpected, since migration has
been associated with integrin expression on the leading edge of cells
(18). It is unclear how IFN-
-treated Calu-3 cells
migrate on collagen I if they have reduced levels of
2
1-integrin on their leading edge, although it is possible that they still possess enough of the receptor
for migration. Alternatively, high levels of integrin expression at the
leading edge of IL-4- and IL-13-treated cells may decrease motility,
because the cells become too adhesive to detach from the matrix. In
addition to changes in
2
1-integrin expression, actin at the leading edge of IL-4- and IL-13-treated Calu-3
cells was brightly stained by rhodamine-phalloidin, possibly indicating
increased stability of the cytoskeleton in this region of the cells.
Because lamellipodial extension involved in cell migration requires
actin reorganization (26), the increased actin staining at
the leading edge of IL-4- and IL-13-treated cells may also be a
reflection of the increased adhesiveness induced by these cytokines. In
contrast to IL-4 and IL-13, IFN-
-treated cells showed no detectable
actin at the leading edge, suggesting that it may be in a more dynamic
state required to promote lamellipodial extension and motility.
The differential effects of Th1 and Th2 cytokines on Calu-3 cells may
have important implications in how we consider treatments for asthma.
Because asthma is associated with an increased Th2-type immune profile,
one therapeutic strategy being considered is the stimulation of a Th1
cytokine response to inhibit the Th2 response (31). This
approach has been validated in a number of experimental models of
asthma using IL-12 (15, 29) or CpG oligonucleotides (30) to enhance the Th1 character of the immune system.
The general interpretation of these studies is that skewing the
cytokine profile from Th2 to Th1 affects the activation and recruitment of mast cells, eosinophils, and B cells and that these changes in
immune cells subsequently alter disease progression. However, as the
results of this study with the Calu-3 cells showed, lung epithelial
cells can also alter their biological responses to Th1 and Th2
cytokines. More important is the fact that a Th1 cytokine (IFN-) can
inhibit the effects of Th2 cytokines (IL-4 and IL-13) in the Calu-3
cells, resulting in the maintenance of barrier function and a normal
rate of wound healing. By extrapolation to the in vivo setting, a Th2
cytokine-rich environment in the lung would result in stimulation and
activation of immune cells and epithelial cells, the latter
contributing to disease exacerbation through disruption of the
epithelial barrier and reduction in wound healing. Conversely, the net
result of Th1 cytokine induction in an asthmatic lung would be a
reduction in Th2 immune cell response and additional dampening of
inflammation by enhanced epithelial barrier function and wound healing.
Therapeutic agents that are able to affect epithelial cell function may
prove to be of great benefit for both lung inflammation and AHR
associated with asthma. In the case of inflammation, restitution of the
epithelial barrier would eliminate a source of stimulatory antigens
from further activating the immune system, thus helping to decrease
tissue inflammation (24). AHR, which is associated with
permanent alterations in lung architecture, results in enhanced sensitivity to noxious agents that enter the lung (6). As
with inflammation, a restoration of the epithelial barrier could act to
eliminate contact between these irritants and responding cells in the
submucosa. In addition, the restitution of the epithelial cells may
restore the levels of relaxing factors that can inhibit bronchoconstriction associated with AHR (8). In this
study, IFN- showed very potent epithelial restitution activity and, more importantly, was able to inhibit the deleterious effects of IL-4
and IL-13, suggesting that it may prove beneficial in a clinical
setting. Recently, however, the results of a clinical trial of IL-12, a
stimulator of IFN-
production, on AHR in asthmatic patients showed
no clinical efficacy, although it is unclear whether IFN-
levels in
patients were increased as a result of the treatment (4).
Soluble IL-4 receptor-
was also able to inhibit the effects of IL-4
in the barrier and wound-healing assays. Although soluble IL-4
receptor-
is ineffective against IL-13, it is possible that inhibition of IL-4 in vivo may indirectly reduce IL-13 levels by
decreasing the number of IL-13-producing cells (33).
Soluble IL-4 receptor is currently in clinical trials for asthma, and the results of phase I/II trials indicate that patients with moderate asthma showed improvement in respiratory parameters (2).
In summary, the results of this study demonstrate that Th1 and Th2
cytokines have distinct activities in lung epithelial cells that are
consistent with the role of these cytokines in asthma. IL-4 and IL-13
disrupt epithelial barrier function and wound healing, both of which
would be expected to exacerbate inflammation. In contrast, IFN-
enhances epithelial barrier function and wound healing, which would
contribute to a reduction in inflammation.
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ACKNOWLEDGEMENTS |
---|
We thank Joel Tocker and Kathy Anderson for many helpful discussions, Lori Whittaker, Helen Hathaway, Stewart Chipman, and Douglas Williams for critical reading of the manuscript, Ann Aumell for editorial assistance, and Gary Carlton for assistance with graphics.
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FOOTNOTES |
---|
Address for reprint requests and other correspondence: A. Youakim, Dept. of Protein Chemistry, Immunex Corp., 51 University St., Seattle, WA 98101 (E-mail: Ayouakim{at}immunex.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.
Received 12 March 2001; accepted in final form 31 July 2001.
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