1 Laboratorio di Genetica Molecolare and 3 Clinica Pediatrica, Istituto Giannina Gaslini, 16148 Genoa; 2 Istituto di Cibernetica e Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genoa; and 4 Telethon Institute for Gene Therapy, Ospedale San Raffaele, 20132 Milan, Italy
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human bronchial epithelial cells were
treated in vitro with interferon- or tumor necrosis factor-
to
assess their effect on transepithelial ion transport. Short-circuit
current measurements revealed that Na+ absorption was
markedly inhibited by interferon-
(10-1,000 U/ml). The cystic
fibrosis transmembrane conductance regulator was also downregulated by
interferon-
as evident at the protein level and by the decrease in
the cAMP-dependent current. On the other hand, interferon-
caused an
increase of the current elicited by apical UTP application, which is
due to the activity of Ca2+-dependent Cl
channels. Tumor necrosis factor-
caused few changes in ion
transport. Transepithelial fluid transport was measured in normal and
cystic fibrosis cells. At rest, both types of cells showed an
amiloride-sensitive fluid absorption that was inhibited by
interferon-
but not by tumor necrosis factor-
. Our results show
that interferon-
alters the transepithelial ion transport of
cultured bronchial cells. This effect may change the ion composition
and/or volume of periciliary fluid.
airway surface fluid; sodium absorption; cystic fibrosis; airway epithelium; chloride transport
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE THIN LAYER OF FLUID that covers the surface
of the airways plays an important protective role. Inhaled particles
and bacteria are trapped in the mucous layer covering the airway
surface fluid (ASF) and are propelled to the oropharynx by the beating
of cilia. Bacteria in the ASF are also neutralized by substances like
lysozyme, lactoferrin, and the recently discovered defensins (4, 17, 37). The volume and ion composition of the ASF are the result of the
balance between absorption and secretion of ions and water. Transepithelial ion transport is based on the activity of specific ion
channels and transporters. Water movement across
airway epithelium may be driven by osmotic forces as well as by
yet to be determined humoral or intracellular mechanisms. Absorption is
mainly controlled by the activity of the amiloride-sensitive epithelial
Na+ channel (ENaC) (10). Indeed, Na+
absorption, which is followed by Cl through
paracellular or transcellular pathways, drives water from the apical to
the basolateral side. The importance of this process is confirmed by
the finding that mice with nonfunctional Na+ channels die
at birth because they are unable to remove water from the airways (22).
Conversely, secretion on the surface epithelium or in the lumen of
submucosal glands is due to Cl
exit through ion
channels placed on the apical membrane. In cystic fibrosis (CF), a
cAMP-regulated anion channel termed the CF transmembrane conductance
regulator (CFTR) is mutated, and, therefore, Cl
transport is impaired (41). CF is also characterized by increased activity of the ENaC. These alterations may produce dehydration of
mucus secretions in the airways, thus causing airway colonization by
pathogenic bacteria (28). An alternative hypothesis proposes that the
altered ion concentration in the ASF of CF patients might impair the
bactericidal activity of defensins (44).
Under inflammatory conditions, the airway epithelium is exposed to a
series of cytokines and soluble mediators that are secreted by
lymphocytes, macrophages, and epithelial cells themselves (26, 37).
These mediators, which include interferon- (IFN-
) and tumor
necrosis factor-
(TNF-
), are involved in a complex cascade of
events such as expression of inducible nitric oxide synthase (iNOS)
(2), induction of intercellular cell adhesion molecule-1 (24),
secretion of interleukin-8 (6), and expression of cyclooxygenase (40).
It has been shown in intestinal epithelial cells that IFN-
and
TNF-
are able to downregulate CFTR expression (7, 31). These results
led us to hypothesize that proinflammatory conditions are also able to
affect ion transport in the airway epithelium. Accordingly, we have
investigated the effect of IFN-
and TNF-
on the activity of
Na+ and Cl
channels in bronchial
epithelial cells. We have also investigated the effect of these stimuli
in CF cells in which the transepithelial ion transport is already
compromised. Our results show that IFN-
, but not TNF-
, causes
profound changes in ion transport in both normal and CF cells,
resulting in an altered fluid transport.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture. Human bronchial epithelial cells
were cultured as previously described (16). Briefly, cells were
detached from the bronchi after overnight incubation at 4°C with
protease XIV. The cells were obtained from lung resections (non-CF
subjects) or lung transplants (CF patients). After detachment, the
cells were plated on petri dishes or culture flasks and cultured for three to six passages in a serum-free culture medium made by mixing LHC9 and RPMI 1640 media (1:1). Subsequently, the cells were plated at
high density on Transwell or Snapwell permeable supports (Corning Costar) that had diameters of 24 and 12 mm, respectively. Under these
conditions, the medium was DMEM-Ham's F-12 medium plus 2% fetal clone
II serum (HyClone) and various hormones including 20 nM hydrocortisone
(HC) (16). Where indicated, HC was removed. To monitor epithelial
differentiation, transepithelial resistance was measured daily with an
epithelial volt-ohmmeter (Millipore-ERS, Millipore) with chopsticklike
electrodes. After 4-8 days in complete medium, the bronchial
monolayers developed a transepithelial resistance in the range
1,000-4,000 · cm2 depending on
the day of culture and the type of support. Cells on Transwell cups
usually showed a higher resistance. Removal of HC decreased the
resistance by ~30%. After optimal differentiation, the resistance of
CF cells was always higher than that of normal cells. The difference
was significant (P < 0.01).
Except where indicated, IFN- (10-1,000 U/ml) and
TNF-
(40 ng/ml) were applied in the culture medium for 48 h before
Ussing chamber experiments were performed. IFN-
application always
caused an increase in the transepithelial resistance. For example,
normal cells on day 8 on Snapwell cups developed a resistance
of 1,228 ± 95
· cm2 (n = 32 monolayers) in complete medium and 3,468 ± 140
· cm2 with IFN-
(P < 0.01; n = 26 monolayers).
Ussing chamber experiments. After 6 (Transwell) or 8 days (Snapwell) in culture, the permeable supports were mounted in Ussing chamber-like systems: Trans-24 miniperfusion system (World Precision Instruments) for Transwell cups and Vertical Diffusion Chamber (Corning Costar) for Snapwell inserts. The apical and basolateral chambers were filled with a Krebs bicarbonate (KB) solution that contained (in mM) 126 NaCl, 0.38 KH2PO4, 2.13 K2HPO4, 1 MgSO4, 1 CaCl2, 24 NaHCO3, and 10 glucose. The solution was kept in both chambers at 37°C and bubbled with 5% CO2-95% air. The transepithelial potential difference was short-circuited with a voltage clamp (558-C5, Department of Bioengineering, University of Iowa, Iowa City, IA) connected to the apical and basolateral chambers via Ag-AgCl electrodes and agar bridges. The potential difference and fluid resistance between potential-sensing electrodes were compensated for.
After the voltage-clamp condition was established, amiloride
was applied to the apical solution to measure the fraction of the basal
short-circuit current due to ENaC activity. Amiloride-treated epithelia
were then stimulated with apical and basolateral 8-(4-chlorophenylthio) (CPT)-cAMP or apical UTP to determine the corresponding effects. In
some experiments, UTP was given after CPT-cAMP. The net effect of UTP
in the presence of CPT-cAMP was not statistically different from that
obtained in its absence. Therefore, the data obtained with UTP in both
conditions were pooled. The absolute effects of IFN- and TNF-
on
transepithelial currents were measured on Transwell supports. The time
course and dose response of cytokines and the effect of channel
blockers were studied on Snapwell cups. After normalization for the
different surfaces, the amiloride-sensitive (IAmil)
and the UTP-dependent (IUTP) currents were not
quantitatively different in the two types of supports. However, the
cAMP-dependent current (IcAMP) was larger in
Snapwell cups.
In some experiments, the basolateral membrane was permeabilized with amphotericin B (250 µg/ml). Membrane permeabilization was assessed by two criteria: 1) progressive decrease in the transepithelial potential measured in open-circuit conditions and 2) progressive decrease in epithelial resistance measured in short-circuit conditions by applying short voltage pulses. When permeabilization reached a stable level, we applied a transepithelial potential of +10 mV to provide a driving force for ion transport.
Immunodetection of CFTR. Human bronchial epithelial cells were
grown for 6 days on Transwell filters, detached, and lysed in a buffer
containing 20 mM HEPES, pH 7.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet
P-40, and protease inhibitors. Equal amounts of protein lysates (1 mg/sample) were immunoprecipitated with anti-CFTR monoclonal antibody
MATG 1105 (kindly provided by Dr. A. Pavirani, Transgene, Strasbourg,
France) directed against the R domain of CFTR. The
antibody (1 µg/sample) was incubated for 1 h at 4°C with the cell
lysate and precipitated with protein G Sepharose beads (Pharmacia
Biotech, Uppsala, Sweden). Immunoprecipitated proteins were then
phosphorylated for 1 h at 30°C by the addition of 10 µCi of
[-32P]ATP (3,000 Ci/mmol; NEN Life Science,
Boston, MA) with 5 U/sample of catalytic subunit of protein kinase A
(Sigma) and separated on 6% SDS-PAGE. Controls included HT-29 cells,
which have been previously shown to express the CFTR protein (45), and
A549 cells, which do not express the endogenous CFTR (32).
Measurements of intracellular
Ca2+. Optical measurements of
internal Ca2+ were performed as previously described (25).
Epithelial monolayers in Snapwell cups were incubated with 5 µM fura
2-AM for 30 min at 37°C in a standard physiological saline
containing (in mM) 135 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, with pH adjusted to 7.4 with NaOH. The
cells were then washed several times with the same solution in the
absence of the dye at room temperature. The specimen was then mounted
on the stage of an inverted microscope (IM35, Zeiss), and the apical
side was continually superfused with solutions fed by gravity. The
cells were viewed through a ×100 Nikon Fluor objective and
illuminated by a xenon lamp (PTI, Brunswick, NJ) equipped with a
rotating wheel with six interference filters, four filters centered at
340 nm and two centered at 380 nm. The emissions relative to each
series of filters were mediated, and the ratio (R) of the 340- to
380-nm emissions (E340/E380) was
calculated. Internal Ca2+ concentration
([Ca2+]i) was calculated
according to the function [Ca2+]i = KD[(R
Rmin)/(Rmax
R)], where R = E340/E380, Rmin = E340/E380 in 0 Ca2+,
Rmax= E340/E380 in
Ca2+ saturated solution,
= E380(0
Ca2+)/E380(saturated
Ca2+), and KD is the dissociation
constant of the dye (140 nM) (20). To obtain the parameter values,
after each experiment, the cells were incubated in 10 µM 4-bromo
calcium ionophore A-23187 for 20-40 min in a 0-Ca2+
bath (0 Ca2+ added plus 1 mM EGTA), then superfused with
the saturated Ca2+ solution. At the end of this procedure,
5 mM MnCl2 was added to the bath to quench the fluorescence
produced by the dye and determine the autofluorescence values.
Background fluorescence determined in this way differed by <5% from
the fluorescence measured in similar untreated cultures.
NO and NOS determination. The concentration of nitrite, a stable product of nitric oxide (NO), in the medium of cell cultures was determined with the Griess reaction (18). The NO synthase (NOS) enzymatic activity in cellular extracts was assessed by measuring the conversion of L-arginine to L-citrulline with a commercial kit (Stratagene, La Jolla, CA). CaCl2 was included in the reaction buffer to allow detection of both constitutive NOS and iNOS. Enzymatic activity is expressed as picomoles of arginine converted in 1 h. Activity was normalized for the amount of protein in the cellular extract.
Transepithelial fluid transport. The cells were plated on Transwell cups and cultured with complete serum-containing medium as explained in Cell culture. After 5 days, the apical medium was removed, and the apical side was washed twice with KB solution and then with 1 ml of KB solution containing [3H]inulin (1 µCi/ml). This solution was discarded and replaced with another 220 µl of the same radioactive solution. A 20-µl aliquot was collected immediately from the apical side to determine the radioactivity at time 0. The apical solution was then covered with 1 ml of mineral oil to prevent evaporation. In part of the experiments, amiloride (10 µM) was also included in the inulin-containing solution. The cells were kept in the incubator for 24 h with the complete culture medium on the basolateral side. After this time, the apical aqueous fluid was removed, and the radioactivity was determined. Inulin is poorly permeable through tight junctions. In fact, <3% of apical inulin passed to the basolateral side in 24 h. Thus we assumed that changes in inulin concentration should reflect the transport of fluid across the epithelial monolayer. We also directly measured the volume of the aqueous fluid recovered from the apical side. These values were consistent with those calculated from the inulin concentration.
Statistics. Data are presented as representative traces or as arithmetic means ± SE. Unpaired groups of data were compared with Student's t-test to assess significance.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When mounted in Ussing chambers, human normal bronchial epithelia
showed a short-circuit current that was largely inhibited by 10 µM
amiloride (Fig. 1A). The mean
amplitude of the IAmil, which reflects activity of
the ENaC, was 5.05 ± 0.36 µA/cm2 (n = 27 monolayers). After incubation of bronchial cells with 1,000 U/ml of
IFN- for 48 h, IAmil decreased to 1.93 ± 0.35 µA/cm2 (P < 0.001; n = 11 monolayers;
Fig. 1, B and C). The medium utilized for the culture
of polarized bronchial cells contained 20 nM HC (16). Because
inflammatory stimuli are often antagonized by glucocorticoids (14, 15,
29), we wanted to assess the effectiveness of IFN-
in the absence of
this hormone. Removal of HC from the culture medium caused a strong
reduction in IAmil (Fig. 1C). This result
was not unexpected because ENaC is regulated by glucocorticoids in the
lung (11). Under these conditions, treatment with IFN-
almost
completely abolished the residual IAmil (from 0.95 ± 0.14 to 0.09 ± 0.03 µA/cm2; P < 0.001).
Similar results were also obtained with bronchial epithelial cells from
a CF patient (Fig. 1D). Indeed, IFN-
downregulated IAmil in CF cells in either the presence or absence
of HC. The size of IAmil in normal and CF cells was
not statistically different. We tested also TNF-
(40 ng/ml). This
cytokine inhibited IAmil only in normal cells grown
in the absence of HC (Fig. 1, C and D).
|
The activity of CFTR was determined by looking at the response to a
membrane-permeable cAMP analog. On application of CPT-cAMP (500 µM),
the short-circuit current of non-CF cells increased by 3.22 ± 0.25 µA/cm2 (n = 20 monolayers; Fig.
2A). After treatment with IFN-
for 48 h, the IcAMP was only 0.96 ± 0.14 µA/cm2 (P < 0.01; n = 5 monolayers; Fig. 2, B and C). Removal of HC did not
affect these results. Indeed, IcAMP was unaltered
in HC-free medium, and the inhibition caused by IFN-
was similar to
that found in the presence of the hormone. TNF-
did not
significantly change IcAMP in either condition
(Fig. 2C). The current activated by CPT-cAMP was completely
blocked by glibenclamide (500 µM) and was absent in CF cells (data
not shown). These results are consistent with the assumption that
IcAMP is due to CFTR activity. CFTR protein levels
were assessed by immunoprecipitation and in vitro phosphorylation. This
assay revealed the presence of three bands of 135, 145, and 165 kDa,
which represent different stages of CFTR processing. The band with the
highest molecular mass should correspond to the
mature protein. Stimulation with IFN-
(1,000 U/ml) caused a
significant decrease in the CFTR bands (Fig.
3). On the contrary, TNF-
caused an
increase in CFTR protein.
|
|
Airway epithelial cells possess Cl channels other
than CFTR (1, 27) that can be activated by various stimuli such as the
application of extracellular UTP or ATP. These compounds act by binding
to P2Y2 purinergic receptors and evoking an intracellular Ca2+ increase (27). We asked whether the activity of these
channels was also affected by IFN-
incubation. Apical application of
100 µM UTP to bronchial cells resulted in a transient stimulation of
short-circuit current (Fig. 4A).
Incubation with IFN-
for 48 h in the absence of HC resulted in an
increase in the peak of the IUTP both in non-CF and
CF bronchial epithelia (2.3-fold and 5-fold, respectively; Fig. 4). The
difference between normal and CF cells was significant (P < 0.001). IFN-
also induced a prolongation of the UTP response as
shown by the current measured after 15 min of UTP application (Fig. 4,
C and D). After 30 min, the current in IFN-
-treated
epithelia was still elevated (3.05 ± 0.13 µA/cm2;
n = 5 monolayers), whereas in untreated cells, it was already decreased to prestimulation levels (data not shown). TNF-
significantly increased the IUTP peak only in CF
cells (Fig. 4D). The upregulation of the
IUTP by IFN-
was not modified by HC (data not
shown). On the contrary, the small TNF-
effect observed in CF
epithelia was inhibited by HC.
|
IFN- (1,000 U/ml) was applied for 3, 6, 24, and 48 h to determine
the time course of IAmil,
IcAMP, and IUTP changes. As
shown in Fig. 5, the downregulation of
ENaC- and CFTR-dependent currents was already observed after 24 h of
incubation. At this time, the IUTP began to be more
sustained (data not shown). Conversely, the IUTP
peak was significantly upregulated only after 48 h (Fig. 5).
|
Lower concentrations of IFN- were also tested, namely 10 and 100 U/ml. The dose-response relationship shows that
IAmil is markedly reduced even at the lowest
IFN-
concentration tested, whereas IcAMP and
IUTP required 100 U/ml to obtain a maximal effect (Fig. 6).
|
To mimic the UTP effect, we stimulated bronchial cells with the
Ca2+ ionophore ionomycin (Fig.
7, A and B). Also in this
case, the currents were larger and more sustained in cells exposed to
IFN-. For example, in CF cells, the mean currents activated by
ionomycin were 1.10 ± 0.37 (n = 3 monolayers) and 6.83 ± 0.31 (n = 3 monolayers) µA/cm2 in control and
IFN-
-treated cells, respectively (P < 0.01).
|
We measured the effect of apical UTP application (100 µM) on
intracellular Ca2+ levels. There was a transient
Ca2+ increase that lasted for 2-3 min. The resting
levels of Ca2+ and the increase elicited with UTP were not
significantly affected by the long-term stimulation with IFN- (Fig.
7, C and D). In normal cells, the net increase in
intracellular Ca2+ reached 164 ± 44 (n = 5 monolayers) and 107 ± 28 (n = 4 monolayers) nM for
untreated and treated cells, respectively. This difference was not
significant. In CF cells, the Ca2+ peak was 277 ± 53 nM
(n = 3 monolayers) for control cells and 138 ± 25 nM
(n = 3 monolayers) for IFN-
-treated cells (P > 0.05).
To remove the contribution of basolateral channels and transporters, we
permeabilized the basolateral membrane with amphotericin B (Fig. 7,
E and F). Under this condition, apical UTP evoked a peak current of 1.36 ± 0.32 µA/cm2 (n = 3 monolayers) in control cells and 7.14 ± 0.68 µA/cm2
after IFN- incubation (P < 0.01). The response to UTP was
more transient in permeabilized cells than in intact epithelia.
We asked whether the production of NO could be one of the mechanisms
underlying IFN- effects. Actually, it has been reported that NO is
an inhibitor of ENaC in alveolar epithelium (21). To test this
hypothesis, we measured nitrite concentration in the supernatant of
normal bronchial cells. After 24 and 48 h of stimulation with IFN-
in the absence of HC, nitrite levels were very low and not increased
with respect to untreated cells. Indeed, at 48 h, nitrite concentration
was 1.1 ± 0.3 vs. 2.0 ± 0.6 µM in treated and nontreated normal
cells, respectively (P > 0.05). The addition of HC to the
culture medium did not change these results. CF cells also showed low
nitrite levels. To confirm these data, we measured NOS activity in
cellular extracts. The arginine-to-citrulline conversion rate was very
low in nontreated normal cells (3.3 ± 2.7 pmol · mg
1 · h
1)
and was not significantly changed by IFN-
treatment (1.7 ± 0.4 pmol · mg
1 · h
1).
Positive controls were murine macrophages (ANA-1) stimulated with 1 µg/ml of LPS for 24 h and rat cerebellar extracts (362.4 ± 9.2 and
329.3 ± 68.3 pmol · mg
1 · h
1,
respectively). To further investigate the possible involvement of NO,
we treated the cells with N-nitro-L-arginine methyl
ester (500 µM), an inhibitor of iNOS, for the entire period of
IFN-
incubation. Under these conditions, the effect of IFN-
on
IAmil, IcAMP, and
IUTP was unaltered (data not shown). We also
treated the bronchial monolayers acutely with NO donors, namely
S-nitroso-N-acetylpenicillamine (200 µM) or spermine
NONOate (200 µM), on both sides of the epithelium. The size of
IAmil, IcAMP, and
IUTP was not changed by exposure to NO (data not shown).
To measure fluid transport, a fixed volume of saline solution was
applied to the apical side of epithelial cells. After 24 h, we measured
the volume of the remaining fluid as explained in METHODS.
In control conditions, the apical fluid was reduced by 24-35%
(Fig. 8). This corresponds to a fluid
absorption of 14.7 ± 1.1 and 10.1 ± 0.9 µl · cm2 · 24 h
1 in normal and CF cells, respectively. This
difference was significant (P < 0.05). Fluid absorption was
significantly reduced by apical application of 10 µM amiloride in
both types of cells (Fig. 8). However, in CF cells, this effect was
less marked. When IFN-
was applied for 48 h, fluid absorption was
significantly reduced to 5.4 ± 1.7 µl/cm2 in normal
cells and 6.4 ± 1.2 µl/cm2 in CF cells (Fig. 8).
Conversely, TNF-
did not modify fluid transport. We also measured
fluid transport in the presence of apical UTP. In untreated epithelia,
UTP caused a small and not significant fluid absorption reduction (17 and 19% in normal and CF cells, respectively). This effect was not
significantly modified by IFN-
treatment (data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IFN- and TNF-
are important mediators that modulate the local
immune response against infective agents. IFN-
is secreted by T
lymphocytes and is believed to be involved in the response to viral
pulmonary infections (5, 30, 33, 38). IFN-
is not normally increased
in CF patients (9), but it is presumable that it might increase in
these subjects during respiratory viral infections. Local IFN-
levels also increase in the lungs after administration of viral and
nonviral vectors used in gene therapy studies (34, 39). TNF-
is
produced by activated macrophages and neutrophils and is elevated in CF
(9) and other inflammatory conditions (26, 43).
The binding of IFN- and TNF-
to their corresponding receptors
elicits a complex cascade of events that is based mainly on the
translocation to the cell nucleus of factors like nuclear factor-
B
for TNF-
and signal transducers and activators of transcription for
IFN-
(3, 8, 35) where they induce the transcription of specific
genes. Previous studies from other laboratories (7, 31) showed that
IFN-
and TNF-
downregulate the activity of CFTR in intestinal
epithelial cells. We asked whether these cytokines modulate CFTR and
possibly other ion transport mechanisms in airway epithelial cells. We
have found that IFN-
decreases CFTR-related ion transport in
bronchial epithelial cells. Immunodetection experiments indicate that
this effect is due to the lowering of CFTR expression and not to more
indirect mechanisms. We have also observed, for the first time, that
IFN-
downregulates ENaC-mediated currents and upregulates the
response to UTP, i.e., the Ca2+-dependent
Cl
transport. This latter effect is a useful
indication that IFN-
does not merely cause a general reduction in
the ion transport ability in epithelial cells, but, rather, it has a
more specific effect.
Inflammatory stimuli are often antagonized by glucocorticoids (14, 15,
29). Therefore, we removed HC from the culture medium in part of the
experiments. We found that the IFN- effects are largely HC insensitive.
To observe a significant effect on ion transport, IFN- has to be
applied for several hours. This relatively slow response suggests that
it is based on gene expression changes that could involve the
expression of the proteins forming the channels (i.e., ENaC subunits,
CFTR, Ca2+-dependent Cl
channels) or the
expression of regulatory proteins.
Our findings show for the first time that the
Ca2+-dependent Cl transport can be
regulated by extracellular factors. Actually, both UTP- and
ionomycin-dependent responses were similarly upregulated by IFN-
.
This result suggests that IFN-
affects a process that is placed
downstream of the intracellular Ca2+ elevation, thus
excluding mechanisms based on upregulation of the nucleotide receptor
or phospholipase C. Moreover, the experiments carried out with the fura
2 probe further demonstrate that IFN-
does not increase the amount
of Ca2+ mobilized by UTP. We considered the possibility
that IFN-
might indirectly increase Cl
transport
by upregulating the activity of basolateral channels or transporters
that provide the driving force for Cl
exit across
the apical channels. However, UTP was still able to activate a current
that was larger in IFN-
-treated cells when the basolateral membrane
was permeabilized. This result suggests that IFN-
modulates the
expression of Ca2+-dependent Cl
channels
or the activity of regulatory mechanisms that control channel activity.
It is known that Ca2+-dependent Cl
channels are inhibited by feedback mechanisms. This process seems to be
due, at least in part, to the intracellular accumulation of
D-myo-inositol 3,4,5,6-tetrakisphosphate (42).
IFN-
could affect this or a similar process. Such a mechanism could
be responsible for prolongation of the UTP response in IFN-
-treated cells.
In contrast to IFN-, TNF-
did not produce dramatic changes in ion
transport. This contrasts with immunodetection experiments in which
CFTR protein levels were increased by this cytokine. The lack of a
clear effect at the functional level could be due to the induction of
inhibitory pathways (e.g., phosphatases) that lower CFTR-channel
activity. The differences between IFN-
and TNF-
are not
surprising because the two cytokines use different signaling pathways.
TNF-
acts through the nuclear factor-
B transcription factor (3,
35), the activity of which can be antagonized by the glucocorticoid
receptor (14, 15, 29). This fact may explain the ability of TNF-
to
change IAmil and IUTP only in the absence of HC.
We have considered the possibility that IFN- might affect ion
transport by inducing NO synthesis because iNOS can be elicited by
different cytokines and bacterial lipopolysaccharides (2). This
hypothesis is based on the recent finding that NO inhibits Na+ absorption in alveolar epithelium (21). Therefore, we
assumed that the possible induction of iNOS by IFN-
could similarly
decrease IAmil in our cells and possibly also
affect IcAMP and IUTP. Several lines of evidence seem to rule out this hypothesis. First, the treatment with IFN-
does not increase the nitrite concentration in
the cell supernatant and does not induce NOS activity in cellular extracts. Second, the NOS inhibitor
N-nitro-L-arginine methyl ester does not prevent
the effect of IFN-
. Finally, cell stimulation with NO donors does
not mimic the effect of IFN-
.
We compared the effects of IFN- and TNF-
in normal and CF cells.
This study is interesting insofar as it explores whether stimuli that
mimic proinflammatory conditions are able to alter ion transport in CF
patients and whether this alteration attenuates or further emphasizes
the CF basic defect. We have found that IFN-
also downregulates
Na+ absorption in CF cells. The effect seems to be
quantitatively similar to that observed in normal cells. Regarding
CFTR, we have used cells from a
F508 homozygote that have virtually
no cAMP-dependent Cl
-channel activity. However, it
is presumable that IFN-
might also downregulate CFTR in those
patients with milder mutations who have a residual channel activity,
thus worsening the basic functional defect. The upregulation of
IUTP is higher in CF cells. A higher activity of
Ca2+-dependent Cl
channels has been also
observed in CF mice (13, 19). A peculiarity of our observations is that
ENaC activity was not higher in CF with respect to normal cells as
evidenced by other investigators (12). Our findings may suggest that
the upregulation of ENaC in CF is influenced by factors other than the
simple interaction between ENaC and CFTR. These factors could be
variably affected in vitro by cell culture conditions and in vivo
by other unknown factors. Actually, it has been shown that the
amiloride-sensitive Na+ transport in the tracheae of
F508 and CFTR knockout mice is lower than in normal animals (13,
23). Measurements of fluid transport showed that bronchial epithelia
have a fluid absorption that is based on the amiloride-sensitive
Na+ transport. IFN-
treatment significantly decreased
fluid absorption in normal and CF cells as expected from the inhibition
of Na+ currents in short-circuit conditions. In CF cells,
the amount of amiloride-sensitive fluid transport was not elevated with
respect to normal cells, confirming data obtained by others (36).
Two models are presently proposed to explain the physiological NaCl
absorption and the pathogenesis of CF. In one model, Na+
goes through the ENaC and Cl follows passively
through the paracellular route (28). Because ENaC is hyperactive,
dehydration of the apical epithelial surface might impair mucociliary
clearance favoring airway infections. The other model postulates that
rather than moving through the paracellular pathway,
Cl
is transported through the CFTR (44). In CF
epithelia, effective NaCl absorption would be impeded, and,
consequently, periciliary fluid would have a high salt concentration
that inactivates the defensins. The effect of IFN-
in vivo would be
quite different depending on the model. In the former case, the
inhibition of Na+ transport elicited by this cytokine would
increase apical volume and, therefore, would facilitate mucociliary
clearance. In the latter model, the inhibition of Na+
transport would further increase the apical salt concentration, thus
inactivating the defensins and facilitating bacterial infection. Whatever the correct model is, the finding that ion transport in airway
epithelia can be modulated by an extracellular mediator has interesting
implications. First, it indicates that some inflammatory stimuli may
change airway ion transport, leading to possible alterations in
mucociliary clearance. Furthermore, assessment of the molecular mechanisms underlying the regulation of ENaC, CFTR, and other Cl
channels could lead to pharmacological tools
useful in manipulating transepithelial ion transport. This could be of
extreme importance in the treatment of CF and other chronic diseases of
the airways.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by a grant from the North American Cystic Fibrosis Foundation, Telethon Italy Grant E.593, and a grant from the Associazione Lombarda for Cystic Fibrosis.
![]() |
FOOTNOTES |
---|
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: L. J. V. Galietta, Lab. di Genetica Molecolare, Istituto Giannina Gaslini, L.go G. Gaslini 5, 16148 Genoa, Italy (E-mail: galietta{at}unige.it).
Received 6 October 1999; accepted in final form 1 January 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderson, MP,
Sheppard DN,
Bergev HA,
and
Welsh MJ.
Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia.
Am J Physiol Lung Cell Mol Physiol
263:
L1-L14,
1992
2.
Asano, K,
Chee CBE,
Gaston B,
Lilly CM,
Gerard C,
Drazen JM,
and
Stamler JS.
Constitutive and inducible nitric oxide synthase gene expression, regulation, and activity in human lung epithelial cells.
Proc Natl Acad Sci USA
91:
10089-10093,
1994
3.
Baldwin, AS.
The NF-B and I
B proteins: new discoveries and insights.
Annu Rev Immunol
14:
649-683,
1996[ISI][Medline].
4.
Bals, R,
Weiner DJ,
and
Wilson JM.
The innate immune system in cystic fibrosis lung disease.
J Clin Invest
103:
303-307,
1999
5.
Baumgarth, N,
and
Kelso A.
In vivo blockade of gamma interferon affects the influenza virus-induced humoral and the local cellular immune response in lung tissue.
J Virol
70:
4411-4418,
1996[Abstract].
6.
Becker, S,
Koren HS,
and
Henke DC.
Interleukin-8 expression in normal nasal epithelium and its modulation by infection with respiratory syncytial virus and cytokines tumor necrosis factor, interleukin-1, and interleukin-6.
Am J Respir Cell Mol Biol
8:
20-27,
1993[ISI][Medline].
7.
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
8.
Boehm, U,
Klamp T,
Groot M,
and
Howard JC.
Cellular responses to interferon-.
Annu Rev Immunol
15:
749-795,
1997[ISI][Medline].
9.
Bonfield, TL,
Panuska JR,
Konstan MW,
Hilliard KA,
Hilliard JB,
Ghnaim H,
and
Berger M.
Inflammatory cytokines in cystic fibrosis lungs.
Am J Respir Crit Care Med
152:
2111-2118,
1995[Abstract].
10.
Canessa, CM,
Schild L,
Buell G,
Thorens B,
Gautschi I,
Horisberger J-D,
and
Rossier BC.
Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits.
Nature
367:
463-467,
1994[ISI][Medline].
11.
Champigny, G,
Voilley N,
Lingueglia E,
Friend V,
Barbry P,
and
Lazdunski M.
Regulation of expression of the lung amiloride-sensitive Na+ channel by steroid hormones.
EMBO J
13:
2177-2181,
1994[Abstract].
12.
Chinet, TC,
Fullton JM,
Yankaskas JR,
Boucher RC,
and
Stutts SJ.
Mechanism of sodium hyperabsorption in cultured cystic fibrosis nasal epithelium: a patch-clamp study.
Am J Physiol Cell Physiol
266:
C1061-C1068,
1994
13.
Colledge, WH,
Abella BS,
Southern KW,
Ratcliff R,
Jiang C,
Cheng SH,
MacVinish LJ,
Anderson JR,
Cuthbert AW,
and
Evans MJ.
Generation and characterization of a F508 cystic fibrosis mouse model.
Nat Genet
10:
445-452,
1995[ISI][Medline].
14.
De Bosscher, K,
Schmitz ML,
Vanden Berghe W,
Plaisance S,
Fiers W,
and
Haegeman G.
Glucocorticoid-mediated repression of nuclear factor-kappaB-dependent transcription involves direct interference with transactivation.
Proc Natl Acad Sci USA
94:
13504-13509,
1997
15.
De Vera, ME,
Taylor BS,
Wang Q,
Shapiro RA,
Billiar TR,
and
Geller DA.
Dexamethasone suppresses iNOS gene expression by upregulating I-B
and inhibiting NF-
B.
Am J Physiol Gastrointest Liver Physiol
273:
G1290-G1296,
1997
16.
Galietta, LJV,
Lantero S,
Gazzolo A,
Sacco O,
Romano L,
Rossi GA,
and
Zegarra-Moran O.
An improved method to obtain highly differentiated monolayers of human bronchial epithelial cells.
In Vitro Cell Dev Biol Anim
34:
478-481,
1998[ISI][Medline].
17.
Goldman, MJ,
Anderson GM,
Stolzenberg ED,
Kari UP,
Zasloff M,
and
Wilson JM.
Human -defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis.
Cell
88:
553-560,
1997[ISI][Medline].
18.
Green, LC,
Wagner DA,
Glogowski J,
Skipper PL,
Wishnok JS,
and
Tannenbaum SR.
Analysis of nitrate, nitrite and 15N nitrate in biological fluids.
Anal Biochem
126:
131-138,
1982[ISI][Medline].
19.
Grubb, BR,
Vick RN,
and
Boucher RC.
Hyperabsorption of Na+ and raised Ca2+-mediated Cl secretion in nasal epithelia of CF mice.
Am J Physiol Cell Physiol
266:
C1478-C1483,
1994
20.
Grynkiewicz, G,
Poenie M,
and
Tsien RY.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem
260:
3440-3450,
1985[Abstract].
21.
Guo, Y,
DuVall MD,
Crow JP,
and
Matalon S.
Nitric oxide inhibits Na+ absorption across cultured alveolar type II monolayers.
Am J Physiol Lung Cell Mol Physiol
274:
L369-L377,
1998
22.
Hummler, E,
Barker P,
Gatzy J,
Beerman F,
Verdumo C,
Schmidt A,
Boucher RC,
and
Rossier BC.
Early death due to defective neonatal lung liquid clearance in ENaC-deficient mice.
Nat Genet
12:
325-328,
1996[ISI][Medline].
23.
Hyde, SC,
Gill DR,
Higgins CF,
Trezise AEO,
MacVinish LJ,
Cuthbert AW,
Ratcliff R,
Evans MJ,
and
Colledge WH.
Correction of the ion transport defect in cystic fibrosis transgenic mice by gene therapy.
Nature
362:
250-255,
1993[ISI][Medline].
24.
Look, DC,
Rapp SR,
Keller BT,
and
Holtzman MJ.
Selective induction of intercellular adhesion molecule-1 by interferon-gamma in human airway epithelial cells.
Am J Physiol Lung Cell Mol Physiol
263:
L79-L87,
1992
25.
Marchetti, C,
Amico C,
and
Usai C.
Functional characterization of the effect of nimodipine on the calcium current in rat cerebellar granule cells.
J Neurophysiol
73:
1169-1180,
1995
26.
Martin, LD,
Rochelle LG,
Fischer BM,
Krunkosky TM,
and
Adler KB.
Airway epithelium as an effector of inflammation: molecular regulation of secondary mediators.
Eur Respir J
10:
2139-2146,
1997
27.
Mason, SJ,
Paradiso AM,
and
Boucher RC.
Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium.
Br J Pharmacol
103:
1649-1656,
1991[Abstract].
28.
Matsui, H,
Grubb BR,
Tarran R,
Randell SH,
Gatzy JT,
Davis CW,
and
Boucher RC.
Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease.
Cell
95:
1005-1015,
1998[ISI][Medline].
29.
McKay, LI,
and
Cidlowski JA.
Cross-talk between nuclear factor-kappa B and the steroid hormone receptors: mechanisms of mutual antagonism.
Mol Endocrinol
12:
45-56,
1998
30.
Mo, XY,
Sarawar SR,
and
Doherty PC.
Induction of cytokines in mice with parainfluenza pneumonia.
J Virol
69:
1288-1291,
1995[Abstract].
31.
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].
32.
Reiner, M,
Tamanini A,
Nicolis E,
Rolfini R,
Imler J-L,
Pavirani A,
and
Cabrini G.
Use of a membrane potential-sensitive probe to assess biological expression of the cystic fibrosis transmembrane conductance regulator.
Hum Gene Ther
6:
1275-1283,
1995[ISI][Medline].
33.
Sarawar, SR,
Sangster M,
Coffman RL,
and
Doherty PC.
Administration of anti-IFN- antibody to
2-microglobulin-deficient mice delays influenza virus clearance but does not switch the response to a T helper cell 2 phenotype.
J Immunol
153:
1246-1253,
1994
34.
Scheule, RK,
George JA,
Bagley RG,
Marshall J,
Kaplan JM,
Akita GY,
Wang KX,
Lee ER,
Harris DJ,
Jiang C,
Yew NS,
Smith AE,
and
Cheng SH.
Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung.
Hum Gene Ther
10:
689-707,
1997.
35.
Schutze, S,
Wiegmann K,
Machleidt T,
and
Kronke M.
TNF-induced activation of NF-B.
Immunobiology
193:
193-203,
1995[ISI][Medline].
36.
Smith, JJ,
Karp PH,
and
Welsh MJ.
Defective fluid transport by cystic fibrosis airway epithelia.
J Clin Invest
93:
1307-1311,
1994[ISI][Medline].
37.
Thompson, AB,
Robbins RA,
Romberger DJ,
Sisson JH,
Spurzem JR,
Teschler H,
and
Rennard SI.
Immunological functions of the pulmonary epithelium.
Eur Respir J
8:
127-149,
1995
38.
Tishon, A,
Lewicki H,
Rall G,
von Herrath M,
and
Oldstone MBA
An essential role for type 1 interferon- in terminating persistent viral infection.
Virology
212:
244-250,
1995[ISI][Medline].
39.
Van Ginkel, FW,
McGhee JR,
Liu C,
Simecka JW,
Yamamoto M,
Frizzell RA,
Sorscher EJ,
Kiyono H,
and
Pascual DW.
Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene.
J Immunol
159:
685-693,
1997[Abstract].
40.
Watkins, DN,
Garlepp MJ,
and
Thompson PJ.
Regulation of the inducible cyclo-oxygenase pathway in human cultured airway epithelial (A549) cells by nitric oxide.
Br J Pharmacol
121:
1482-1488,
1997[Abstract].
41.
Wine, JJ.
The genesis of cystic fibrosis lung disease.
J Clin Invest
103:
309-312,
1999
42.
Xie, W,
Kaetzel MA,
Bruzik KS,
Dedman JR,
Shears SB,
and
Nelson DJ.
Inositol 3,4,5,6-tetrakisphosphate inhibits the calmodulin-dependent protein kinase II-activated chloride conductance in T84 colonic epithelial cells.
J Biol Chem
271:
14092-14097,
1996
43.
Xing, Z,
Jordana M,
Gauldie J,
and
Wang J.
Cytokines and pulmonary inflammatory and immune diseases.
Histol Histopathol
14:
185-201,
1999[ISI][Medline].
44.
Zabner, J,
Smith JJ,
Karp PH,
Widdicombe JH,
and
Welsh MJ.
Loss of CFTR chloride channels alters salt absorption by cystic fibrosis airway epithelia in vitro.
Mol Cell
2:
397-403,
1998[ISI][Medline].
45.
Zeitlin, P,
Crawford I,
Lu L,
Woel S,
Cohen ME,
Donowitz M,
Montrose MH,
Hamosh A,
Cutting GR,
Gruenert DC,
Huganir R,
Maloney P,
and
Guggino WB.
CFTR protein expression in primary and cultured epithelia.
Proc Natl Acad Sci USA
89:
344-347,
1992[Abstract].