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B Kinase 
Pathway in Response to Extracellular NaCl Content1
*
Institut National de la Santé et de la Recherche Médicale, Unité 514, Institut Fédératif de Recherche 53, Centre Hospitalier Universitaire, Maison Blanche, Reims, France;
Service de Pneumologie, Hôpital Cochin, Paris, France; and
Département de Chirurgie Cardio-Vasculaire, Hôpital Broussais, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B, we investigated whether an elevated
extracellular NaCl content in airway fluids significantly impaired the
regulation of the NF-B/IB
complex and the chemokine IL-8
production in primary non-CF and CF human bronchial gland epithelial
cells. Exposure of non-CF gland cells to hypotonic (85 mM) NaCl
solution, compared with isotonic (115 mM) NaCl and hypertonic (170 mM)
NaCl solutions, resulted in a significant decrease in IL-8 production
that was paralleled by a strong inhibition of activated NF-B
associated with an increased cytosolic expression of IB and a
decrease in the IB kinase protein level. In CF gland cells, we
demonstrated that, compared with the high IL-8 in an hypertonic
solution, the release of IL-8 was significantly reduced 2-fold in an
isotonic solution and 5-fold in a hypotonic solution. Strikingly,
exposure of CF bronchial gland cells to either hypotonic or isotonic
milieu did not result in a marked inhibition of the activated
NF-B/IB system. This is the first demonstration that primary
human CF bronchial gland cells exhibit abnormally high IL-8 production
through constitutively activated NF-B and high IB kinase
level, whatever the hypo-, iso-, and hypertonic NaCl
milieu. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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F508 CF human bronchial
gland (HBG) cells exhibited a selective up-regulation in chemokine IL-8
production (7) associated with constitutively activated
NF-B and a lack of cytosolic IB protein (9). The
constitutive and/or excessive production of IL-8 by CF bronchial
epithelial cells could implicate these cells in facilitating the
neutrophil, T lymphocyte, and monocyte migration associated with airway
inflammation in CF patients.
In CF HBG, the mechanism(s) directly responsible for constitutive
NF-B activation remains unknown. NF-B dimers exist as inactive
complexes in the cytoplasm of unstimulated cells due to their
interaction with a family of inhibitory proteins collectively
designated IBs. These IBs are phosphorylated by cellular kinase
complexes known as IB kinases (IKK)- and -ß (10, 11). The prototypic and best-studied member of the IB family,
IB, binds to the nuclear translocation sequence of p65 and
sequesters NF-B in the cytoplasm. The key regulatory steps in the
signal-induced control of NF-B include the phosphorylation and
degradation rates of IB. Cytosolic inhibitor factor IB
appears to be a major regulator of NF-B, as indicated by the
degradation of IB matching the translocation of NF-B to the
nucleus (11, 12). Elevated NaCl concentrations in CF
airway fluids have been noted by several investigators in both in vivo
and in vitro studies. For example, Gilljam et al. found a
Cl- concentration of 170 ± 80 mM in
bronchial fluid from CF patients vs 85 ± 54 mM in control
subjects (13). Two more recent publications have
demonstrated abnormally high Na+ and
Cl- concentrations (
180 mM) in airway surface
fluid recovered from CF-cultured epithelial cells and CF bronchial
xenografts and a significantly reduced ability of airway fluid to kill
bacteria, unless these fluids were diluted in water (14, 15). To date, it is not known whether the NF-B/IB
complex in airway epithelial cells plays a determinant role in airway
inflammatory processes in patients with CF or whether the NF-B
activation in airway epithelial cells is sensitive or not to variations
of extracellular NaCl concentration. Nor is it known whether a loss of
CFTR protein activity, i.e., loss of chloride transport in the human CF
airway gland serous cells, which is well recognized to express high
amount of CFTR protein (16, 17), alters the activity of
transcription factors such as NF-B and the subsequent induction of
IL-8 gene expression. Therefore, we address the question of whether
different extracellular NaCl concentrations would impair the regulation
of NF-B/IB complex and subsequent IL-8 production in human CF
compared with non-CF airway epithelial cells. Understanding such
mechanisms is of great interest, as it may lead to the development of
novel therapeutic strategies in CF airway disease.
In this report, we investigated the effects of hypotonic (85 mM),
isotonic (115 mM), and hypertonic (170 mM) NaCl solutions on the
activation of the NF-B/IB system and subsequent IL-8
production in non-CF vs F508 homozygous CF bronchial gland
epithelial cells. We also focused our study on the phosphorylation and
degradation status of IB to gain further insight into the
NF-B-inducing kinase (NIK) and IKK- and -ß pathways in the
salt-dependent IL-8 production in human non-CF and CF bronchial gland
cells.
Our data demonstrate that extracellular fluid NaCl content modulates
the NF-B activity and subsequent IL-8 production in human non-CF
gland bronchial epithelial cells but not in CF bronchial gland cells.
On exposure to isotonic (115 mM) and hypertonic (170 mM) NaCl milieu,
primary human CF bronchial gland epithelial cells exhibit abnormally
high IL-8 production through constitutive NF-B binding activity and
high levels of IKK-, thus contributing to the heightened
inflammatory response seen in airways of CF patients
(4, 5, 6, 7, 8, 9).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell isolation and culture procedures of HBG cells were
performed on bronchial tissues collected from eight F508 homozygous
CF patients (four females and four males; mean age 17.3 years, range
9–27 years) and four non-CF patients (two males with primary pulmonary
hypertension, aged 28 and 29 years, respectively, and two males with
pulmonary idiopathic fibrosis, aged 40 and 61 years, respectively), as
described previously (7). Briefly, HBG cells were isolated
by enzymatic digestion from bronchial submucosa and grown onto type I
collagen-coated 25-cm2 tissue culture flasks in a
DMEM/Ham’s F12 mixture (50/50%, v/v) supplemented with 1% Ultroser G
(a serum substitute from Sepracor, Villeneuve-la-Garenne, France),
glucose (10 g/L), and sodium pyruvate (0.33 g/L). Penicillin G (100
U/ml) and streptomycin (100 U/ml) were also added. After 4 wk in
primary culture, second- and third-passage CF-HBG and non-CF HBG cells
had proliferated and exhibited characteristics of homogenous submucosal
epithelial and secretory gland cells, as previously described
(18, 19). Using the halide-sensitive fluorescent dye
6-methoxy-N-(3-sulfopropyl)-quinolinium, we have
previously shown a significant increase in Cl-
channel activity via the CFTR protein in non-CF HBG cells in response
to forskolin treatment (demonstrating a cAMP-dependent activation of a
Cl- efflux), which is not preserved in cultured
CF HBG cells (7).
Exposure of bronchial epithelial cells to different extracellular NaCl solutions
Before the exposure of bronchial epithelial cells to either low
(hypotonic, 85 mM NaCl), intermediate (isotonic, 115 mM NaCl), or high
(hypertonic, 170 mM NaCl) saline solutions, confluent cultures of
F508 homozygous CF and non-CF HBG cells were incubated for 16 h
in an Ultroser G-free RPMI 1640 medium in 95% air/5%
CO2 to ensure that cells were in a quiescent
state. At the end of the 16-h period, individual monolayers of primary
CF and non-CF HBG cells were exposed for a further 6-h period to
Cl- solutions containing either 85 mM, 115 mM,
or 170 mM Cl-, respectively. The three
chloride-containing solutions used in this study (85 mM
Cl-, 115 mM Cl-, and 170
mM Cl-) contained 1 mM
CaCl2, 20 mM KCl, and either 60 mM, 85 mM, or 148
mM NaCl, pH 7.4, respectively, as previously reported (7, 14). Immediately after each period of CF and non-CF cell
exposure, supernatants were collected and stored at -80°C until
testing for the presence of IL-8. All reagents were molecular biology
grade, and all buffers and solutions were prepared using pyrogen-free
grade water. In all culture supernatants, undetectable levels of
endotoxin (detection limit, 
5 pg/ml) were found using a quantitative
chromogenic limulus amebocyte lysate assay
(BioWhittaker, Emerainville, France).
ELISA for IL-8
IL-8 ELISA in the culture supernatants and cell lysates of non-CF and CF HBG cells were conducted according to the manufacturer’s instructions in commercially available ELISA kits (Biosource International, Camarillo, CA). Cell lysates were prepared as follows. After each period of different saline treatments, CF and non-CF HBG cell monolayers were washed in PBS, pH 7.2, harvested by scraping, centrifuged (300 x g, 5 min, 4°C), and total protein was extracted (30 min, 4°C) in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl2, 1% Nonidet P-40 (Sigma), 0.5% deoxycholate (Sigma), and 0.1% SDS). Cell debris were centrifuged (12,000 x g, 30 min, 4°C), and supernatants were retained and stored at -80°C. The ELISAs for IL-8 were sensitive down to a level of 5 pg/ml. The uniformity of the CF and non-CF HBG cells in monolayer culture was determined by quantifying the cell number/well. Cell viability of CF and non-CF HBG cells exceeded 97%, as determined by trypan blue exclusion after all experimental interventions. All results were expressed as pg/ml/viable 106 cells/h.
Immunofluorescence
After each period of the different saline treatments described
above, CF and non-CF HBG cell monolayers were fixed in situ in cold
methanol for 10 min at -20°C, air dried, and rehydrated in 0.1 M PBS
at pH 7.4 before immunofluorescence detection. Cells were stained for
IB expression using rabbit antiserum to human IB (Santa
Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature
and a donkey anti-rabbit FITC-conjugated Ab for 45 min at room
temperature, as previously described (9). Negative
controls were obtained by using either nonspecific IgG as the primary
Ab (M7769; Sigma) or with FITC-conjugated Ab alone. Representative
fields of CF and non-CF HBG cells in different saline conditions were
recorded with a Zeiss Axiophot microscope (Zeiss, Le Pecq, France) and
employing epifluorescence and Nomarski differential interference
illumination.
Cell extracts and Western blot analysis
After each period of different saline treatments, CF and non-CF
HBG cell monolayers were washed in PBS, pH 7.2, harvested by scraping,
centrifuged (300 x g, 5 min, 4°C), and total protein
was extracted (30 min, 4°C) in RIPA buffer (50 mM Tris, pH 8.0, 150
mM NaCl2, 1% Nonidet P-40 (Sigma), 0.5%
deoxycolate (Sigma), and 0.1% SDS). Protein extracts were centrifuged
(12,000 x g, 30 min, 4°C), and protein
concentrations were measured using the Bradford assay (Bio-Rad,
Hercules, CA). Equal amounts of protein were boiled for 4 min in
Laemmli buffer, and electrophoresis was conducted under denaturing
conditions using 4–15% polyacrylamide gels (Pharmacia Biotech, Orsay,
France), which were then transferred onto a nitrocellulose membrane
(Millipore, Bedford, MA) by electroblotting. Membranes were blocked
with TBS containing Tween 20 (40 mM Tris, pH 7.6, 300 mM NaCl, 0.1%
Tween 20) containing 5% nonfat dry milk for 4 h at room
temperature before exposure to rabbit polyclonal anti- human
IB and anti-IBß (Santa Cruz Biotechnology). For analysis
of IB kinases and ß (IKK- and IKK-ß), membranes were
exposed to rabbit polyclonal anti-IKK- and IKK-ß Abs,
respectively (Santa Cruz Biotechnology). The level of phosphorylated
IB was analyzed by Western blot using a polyclonal
phospho-specific anti-IB (New England Biolabs, Beverly, MA)
Ab, which detects IB only when activated by phosphorylation at
Ser32. Proteins were visualized using
HRP-conjugated donkey anti-rabbit IgG (Boehringer Mannheim,
Mannheim, Germany) and the enhanced chemiluminescence detection kit
(Amersham Life Science, Arlington Heights, IL). Prestained m.w. markers
(Bio-Rad, Hercules, CA) were loaded on each gel to verify the effective
transfer of proteins to membranes and to determine the m.w. of
proteins. Densitometric analyses of Western blots were performed on a
Bio-Rad model GS-690 imaging densitometer using Molecular Analyst
software, version 1.4.1. The intensities of bands were compared on the
basis of adjusted volume (mean optical density x area in square
millimetres).
Nuclear protein extraction and EMSAs
Nuclear extracts were prepared and analyzed after the non-CF and
CF HBG cells were incubated in different saline conditions, as
described previously (9). Briefly, 5–6 x
106 cells were suspended in 1.5 ml of lysis
buffer (10 mM HEPES, pH 7.9, 1.5 mM, MgCl2, 10 mM
KCl, 0.5 mM DTT (Sigma) and 0.1% Nonidet P-40 (Boehringer Mannheim)).
The homogenate was centrifuged at 10,000 rpm, and the resulting pellet
was resuspended in 30 µl of lysis buffer (20 mM HEPES, 420 mM NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, 25% [v/v] glycerol,
and 0.5 mM DTT). This suspension was incubated for 20 min at 4°C
followed by centrifugation at 14,000 rpm for 10 min. To minimize
proteolysis, all buffers contained 0.5 mM PMSF, 5 µg/ml aprotinine, 1
µg/ml chymostatine, 4 µg/ml pepstatine, 5 µg/ml leupeptin, and
0.1 mg/ml -1 antitrypsin (Boehringer Mannheim). The consensus B
DNA sequence was used for the EMSA (5'-AGT TGA GGG GAC TTT CCC AGG
C-3'; Promega, Madison, WI). The oligonucleotide was radiolabeled by
the T4 polynucleotide kinase (Pharmacia Biotech, Paris, France) enzyme
with [-32P]ATP. Nuclear extracts (4 µg)
were incubated with 50 kcpm of 32P-labeled
NF-B oligonucleotide in binding reaction mixture (20% Ficoll, 175
mM NaCl, 300 mM KCl, 0.05% Nonidet P-40, pH 7.0) in a final volume of
15 µl. After 30 min on ice, the protein-DNA complexes were
electrophoresed on a nondenaturing 5% polyacrylamide gel in a 1x TBE
buffer (89 mM Tris-HCl, 89 mM boric acid, and 2 mM EDTA). Gels were
then dried under vacuum and exposed at -80°C with autoradiographic
film. In competition studies and supershift assays, a 100-fold molar
excess of unlabeled oligonucleotide or 1 µg of Abs was added to the
binding reaction mixture as indicated, before the addition of the
labeled B probe. Identification of the different NF-B
heterodimeric proteins was conducted by incubating the nuclear extracts
with polyclonal Abs against the NF-B proteins NF-B1 (p50) and the
Rel (p65) RelA (Santa Cruz Biotechnology) before the addition of the
labeled B probe. These Abs were added to the above reaction mixture
at a concentration of 10 µg/100 µl. All samples were then incubated
at room temperature for 1 h before gel loading.
Statistical analysis
Results were expressed as means ± SD. Each data point was confirmed in triplicate at least, and each cell culture experiment was performed at least three times. Differences in IL-8 levels were analyzed by the Student’s t test for paired and unpaired samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have shown that established CF respiratory
epithelial cell lines (20) as well as human CF bronchial
submucosal tissues in situ and primary cultures of CF bronchial gland
cells (9, 21) constitutively expressed significantly high
levels of proinflammatory cytokines, particularly the chemokine IL-8.
IL-8 is one of the most potent neutrophilic chemoattractants in human
CF airways (22, 23). To further mimic the in vivo
situation, in which a change in NaCl concentration occurs in CF
bronchial liquids (13, 14, 15) as a result of CFTR deficiency
(i.e., 85 mM in non-CF vs 120–170 mM in CF), we examined whether the
primary F508 homozygous CF and non-CF HBG cells displayed any
differential expression in the IL-8 production and release in response
to the relevant electrolyte concentration in the extracellular milieu.
As shown in Fig. 1
A, when the
extracellular NaCl content increased (from 85 mM to 170 mM), we
observed a significant 2-fold increase (p <
0.01) in IL-8 release from non-CF HBG cells. Interestingly, exposure of
CF HBG cells to isotonic (115 mM) and hypertonic (170 mM) NaCl resulted
in a statistically (p < 0.001) significant
3.0- and 5.0-fold increase in IL-8 release compared with similarly
treated non-CF HBG cells. We also observed that the increased IL-8
release in CF HBG cells was paralleled by an increased intracellular
IL-8 levels according to the extracellular NaCl content (Fig. 1B). Taken together, these findings clearly show that CF HBG
cells are characterized by a higher susceptibility to induce IL-8
production and secretion in response to extracellular iso- and
hypertonic NaCl changes when compared with that obtained from similarly
treated non-CF HBG cells.
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B
expression
Therefore, it was of interest to determine whether or not
variations in extracellular NaCl concentration affected NF-B
activation in the primary non-CF and CF HBG cell cultures. Nuclear
extracts of non-CF and CF HBG cells previously incubated either in
isotonic (85 mM) or hypotonic (115 mM) or hypertonic (170 mM) NaCl
solutions were prepared and incubated with an end
32P-labeled DNA oligonucleotide containing the
recognition site of NF-B. As demonstrated by EMSA (Fig. 2), no evidence of activated NF-B was
found in primary non-CF HBG cells treated with the hypotonic (85 mM)
NaCl solution (Fig. 2, lane 3). In contrast, when increasing
the extracellular NaCl concentration from hypotonic (85 mM) to isotonic
(115 mM) and hypertonic (170 mM), we noted a marked NF-B-DNA binding
activity in a gradual manner in non-CF HBG cells (Fig. 2, lanes
4 and 5). Surprisingly, high amounts of activated
NF-B were always detected in CF HBG cells for each of the hypo-,
iso-, and hypertonic salt solutions (Fig. 2, lanes 6–8).
Compared with non-CF HBG cells maintained in either isotonic (115 mM)
or hypertonic (170 mM) NaCl solutions (Fig. 2, lanes 4 and
5), a higher NF-B-DNA binding activity was demonstrated
in the nuclear protein extracts from similarly treated CF HBG cells
(Fig. 2, lanes 7 and 8), with a mean increase of
2.5- and 3.0-fold, respectively, as evaluated by densitometric analyses
(data not shown). The specificity of NF-B-DNA binding was confirmed
in competition experiments with a 100-fold excess of (cold B)
NF-B oligonucleotide, which led to a complete inhibition of binding
activity (Fig. 2, lane 1). Moreover, supershift assays
confirmed the presence of p65 subunits of NF-B (Fig. 2, lane
2). A similar result was observed with the addition of Abs to the
p50 subunit of human NF-B (data not shown).
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B and IBß
protein in non-CF and CF HBG cells
Activation of NF-B occurs via phosphorylation of two serine
residues of IB at positions 32 and 36 (24). This has
been shown to enable conjugation with ubiquitin followed by
proteasome-mediated degranulation of IB, resulting in the release of
active NF-B (25). To measure IB phosphorylation,
we used a phospho-specific anti-IB-Ab that detects IB
only when activated by phosphorylation at Ser32.
In a set of experiments, we examined the phosphorylation status of
IB over the hypo-, iso-, and hypertonic salt conditions in
similarly treated non-CF and CF HBG cells (Fig. 3). In hypotonic (85 mM) NaCl solution,
little or no phosphorylated IB was detected in non-CF HBG cells
(Fig. 3A, lane 1). In contrast, when we increased
the extracellular NaCl content from 85 mM to 115 mM and 170 NaCl mM, we
noted a marked increase of phosphorylated IB in non-CF HBG cells
(Fig. 3A, lanes 2 and 3). Image
analysis of digitized Western blots demonstrated that 115 mM and 170 mM
NaCl solutions increased the phosphorylated IB level by 25 and
75%, respectively (p < 0.05), compared with
that observed with the 85 mM NaCl solution (Fig. 3B,
lanes 2 and 3, compared with lane 1).
In striking contrast to non-CF HBG cells in which levels of
phosphorylated IB were salt dependent, high levels of
phosphorylated IB were found in CF HBG cells even in hypotonic
(85 mM) NaCl solution and did not change in accordance to the hypo-,
iso-, and hypertonic salt conditions (Fig. 3A, lanes
4–6). Image analysis of digitized Western blots of phosphorylated
IB showed at least a 220% increase of phosphorylated IB in
CF HBG cells regardless of the hypo-, iso-, and hypertonic salt
conditions, compared with the value obtained with the hypotonic (85 mM)
NaCl solution in non-CF HBG cells (Fig. 3B, lanes
4–6, compared with lane 1). Consistent with the
results of the phosphorylation state of IB, the increase of NaCl
content from 85 mM to 170 mM resulted in a marked decrease of IB
protein in the cytoplasm of non-CF HBG cells, as determined by
immunofluorescence analysis (Fig. 3C). In similarly treated
CF HBG cells, little or no cytosolic IB protein was found in
either the hypotonic (85 mM) or hypertonic (170 mM) NaCl treatment
groups (Fig. 3C), confirming our previous study in which we
reported a constitutive lack of cytosolic IB protein expression
in CF HBG epithelial cells in vivo and in vitro (9). To
assess whether extracellular NaCl content might directly modulate the
expression and/or degradation of the cytoplasmic NF-B regulatory
protein, IBß, in non-CF and CF HBG cells, Western blots of
IBß protein and image analysis of digitized Western blots were
conducted (Fig. 4). Surprisingly, in
contrast to the lack of IB protein expression in CF HBG cells
(Fig. 3C and Ref. 9), IBß protein was
detected in CF HBG cells. We showed that the IBß protein
levels in CF HBG cells in the hypotonic (85 mM) NaCl solution were
>250% higher than in similarly treated non-CF HBG cells and decreased
(by a 30% reduction) in the hypertonic (170 mM) NaCl solution (Fig. 4, lane 6 compared with lane 4).
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B kinases and ß
expression in non-CF and CF HBG cells
To determine whether NaCl-induced increases in phosphorylated
IB and IB degradation were related to enhanced IKK- and
IKK-ß, Western blot analyses of both IKK- and IKK-ß were
performed from cytoplasmic extracts of CF and non-CF HBG cells
similarly treated with the hypo-, iso-, and hypertonic NaCl solutions.
In good agreement with the results of phosphorylated IB levels
described in Fig. 3
, A and B, the NaCl-induced
IKK- protein level increased in a gradual manner with increasing
extracellular NaCl content in non-CF HBG cells (Fig. 5, A and C,
lanes 1–3). Image analysis of digitized Western blots of
the IKK- protein demonstrated that both isotonic (115 mM) and
hypertonic (170 mM) NaCl solutions increased the IKK- levels by 25
and 75%, respectively, in non-CF HBG cells (p
< 0.05), respectively, compared with the IKK- level observed in
hypotonic (85 mM) NaCl solution, (Fig. 5C, lanes
2 and 3 compared with lane 1). In agreement
with the results reported in Fig. 3, in which we showed a high level of
phosphorylated IB in CF HBG cells, we concomitantly observed high
levels of IKK- kinase in CF HBG cells, regardless of the salt
conditions (Fig. 5A, lanes 4–6). Image analysis
of digitized Western blots of IKK- demonstrated that the IKK-
levels in CF HBG cells were increased by >300% in all salt conditions
compared with the IKK- level obtained in non-CF HBG cells treated
with hypotonic (85 mM) NaCl solution (Fig. 5C, lanes
4–6 compared with lane 1). We further investigated
whether variations in extracellular NaCl content directly affected the
IKK-ß levels in non-CF and CF HBG cells. In striking contrast to the
high IKK- levels that were found to be salt sensitive in non-CF and
insensitive in CF HBG cells, we detected low IKK-ß levels in both
non-CF and CF HBG cells and found no significant alteration in IKK-ß
levels, regardless of the saline conditions (Fig. 5, B and
D, lanes 7–12).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we have now found evidence that increased NaCl concentrations induce an increase of IL-8 release (up to 5-fold) by CF bronchial gland cells compared with similarly treated non-CF bronchial gland cells. This is the first study to report the ability of extracellular dose-dependent NaCl to regulate IL-8 production by human non-CF and CF airway epithelial cells. In the presence of hypotonic (85 mM) NaCl concentration, the IL-8 production by CF bronchial gland cells was already higher than that by non-CF bronchial gland cells. More importantly, our data show that, compared with non-CF bronchial gland cells, CF bronchial gland cells were highly susceptible to induce increased IL-8 production with both isotonic (115 mM) and hypertonic (170 mM) NaCl solutions. Therefore, in in vivo situations, isotonic and hypertonic airway fluids, when present in CF patients, may represent a stimulus for bronchial gland IL-8 production, in addition to other stimuli provided by the presence of bacteria and/or bacterial exoproducts (20, 21). Consequently, isotonic and hypertonic airway fluids could increase the IL-8 amounts produced by CF bronchial gland cells and at least partially account for the recent findings that IL-8 levels and neutrophils in bronchoalveolar lavage fluids from infected children with CF were significantly increased compared with those from infected children with other chronic respiratory diseases, even when patients were matched for the pathogen present and for bacterial colony counts (27). In CF airway fluid, the effects of abnormal NaCl concentration on bronchial gland functions could provide another causal link between loss of CFTR function and CF airway disease. If abnormal ionic composition is present in CF airway fluid, as a primary abnormality occurring because of loss of CFTR function, then the effects on bronchial gland functions that we have observed could contribute to the early and sustained inflammatory pathogenesis of CF disease, even before development of bacterial infections.
The correlation between the levels of IL-8 production, the inhibitor
factor IB degradation, and nuclear translocation of NF-B
suggests a causal relationship in normal but not in CF bronchial gland
epithelial cells. Several molecules are involved in NF-B activation
such as a network of kinases (IKK-, IKK-ß, NIK, additional
proteins) constituting several hierarchical modules that ultimately
lead to the phosphorylation of IB (24, 25).
Stimulus-induced degradation of IBs by the proteasome requires the
phosphorylation of these inhibitor proteins at specific residues
(25). For example, IB is phosphorylated following
activation at the amino acids Ser32 and
Ser36 (24). This was the reasoning
behind our examination of whether elevated NaCl concentrations
interfered with the phosphorylation of IB. Indeed, using a
phospho-specific Ab, we were able to clearly demonstrate that isotonic
(115 mM) and hypertonic (175 mM) NaCl solutions induced the
phosphorylation of IB in a dose-dependent manner in normal but
not CF bronchial epithelial cells. Surprisingly, we observed that the
hypotonic (85 mM) NaCl solution significantly decreased the IL-8
production by CF bronchial gland cells with no significant inhibition
of activated NF-B. At this point, it should be mentioned that our
studies showed that even at the lowest (85 mM) NaCl concentration
IBß protein levels in CF bronchial gland cells were higher than
those observed in non-CF HBG cells, suggesting that the decrease of
IL-8 production by CF HBG cells in the hypotonic (85 mM) NaCl solution
may occur by preventing the degradation of the IBß protein. One of
the critical steps in the activation of the NF-B pathway is the
phosphorylation of the IB protein by two recently discovered IB
kinases, IKK- and IKK-ß, which leads to its degradation (10, 11, 24). Numerous studies on the role of the IKK complex in the
NF-B regulation have been limited to immortalized cell lines, and
there is little information on their function in normal cells or cells
derived from the site of human inflammatory diseases. The IKK complex
represents a potential convergence point for multiple signaling stimuli
that activate NF-B. We hypothesized that IKK is a key signal
transduction kinase that coordinates this process in human airway
epithelial cells. Our results support the conclusion that extracellular
milieu NaCl content selectively increases the IKK- level in a
concentration-dependent manner (but not IKK-ß) in non-CF bronchial
gland cells. Surprisingly, higher levels of IKK- were found in CF
bronchial gland cells at any extracellular NaCl concentration,
suggesting the constitutive lack of IB protein that we had
previously reported in CF bronchial gland epithelial cells in vivo and
in vitro (9) may be mediated in part by the high
constitutive IKK- protein expression (not for IKK-ß) that we
demonstrate in the present study. Therefore, the IKK complex,
especially IKK- seems to be responsible, in part, for the
constitutive IL-8-mediated NF-B activation in CF bronchial gland
cells. The constitutive activation of NF-B associated with the lack
of IB protein in CF HBG cells contrasts with the inducible
feature of this pathway reported in many epithelial cell types
(25). Therefore, it is reasonable to speculate that
inhibition of IL-8 secretion through the overexpression of a
nondegradable form of IB in airway epithelial cells could prevent
neutrophil recruitment and transepithelial inflammatory cell migration
in CF airways, as recently suggested for human intestinal epithelial
cells (28). Jobin et al. (28) have recently
reported that viral-delivered mutant IB, transferred into both
transformed HT-29 cells as well as primary human colonic epithelial
cells, blocked the production of multiple proinflammatory molecules,
including IL-8, by inhibiting the NF-B activation pathway.
These apparently contradictory results in the regulation of NIK
signaling pathway between the non-CF and CF bronchial gland cells
suggests that, in CF gland cells, the regulation of IL-8 production may
induce other mechanisms involving distinct triggering molecular
entities. For example, p38 mitogen-activated protein kinase (MAPK)
pathways have been demonstrated to play an important role in the
control of IL-8 production mediated by high salt concentrations in
human PBMC and THP-1 monocyte-like cells (29, 30). High
salt concentrations induced IL-8 at the transcriptional level, and this
induction of IL-8 was inhibited by the p38 MAPK inhibitor SB 20508
(30), indicating the involvement of the MAPK transduction
pathway. These investigators also found that IL-8 was not increased by
the addition of glycerol, which achieved extracellular osmolarity
equivalent to those of the media containing Na+,
Cl- dissociable ions. The authors further
concluded that hyperosmolarity itself is not a sufficient signal to
activate IL-8. With respect to the mechanism by which extracellular
NaCl content affects CF bronchial gland cells, further investigations
are required to determine whether variations in tonicity and/or ionic
imbalance differently affect the IL-8 production in CF and non-CF
bronchial gland cells through differential activation of NIK and/or p38
MAPK pathways. It is also possible that a loss of CFTR function in CF
bronchial gland cells may modify the NF-B binding activity.
Recently, Schwiebert et al. (31) suggested that
complementation of CF respiratory surface epithelial cells with
wild-type CFTR to correct the CFTR defect restored cytokine induction
of RANTES but not IL-8 expression via a NF-B-mediated pathway. In CF
bronchial gland epithelial cells, investigation of the mutant
CFTR-dependent mechanisms related to the signal transduction pathways
for IL-8 expression will provide a better understanding of the
inflammatory responses to the changes of the NaCl content in CF human
airways.
In summary, our data show that isotonic and hypertonic airway fluids
are capable of stimulating and/or maintaining high levels of NF-B
activation through the IKK- pathway and subsequent high IL-8
production in HBG epithelial cells. We also demonstrate for the first
time that primary CF HBG epithelial cells exhibit constitutive NF-B
binding activity and high levels of IKK- at any extracellular hypo-,
iso-, and hypertonic NaCl content. Given the proinflammatory functions
of NF-B, the IKK complex could represent a potential therapeutic
target for CF human airway disease. Although our findings remain to be
investigated in in vivo models, they may be informative with respect to
inflammatory processes in which excessive NaCl-induced IL-8 secretion
by airway epithelial cells plays a determinant role in the onset of
early and chronic airway mucosal inflammation in CF patients.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jacky Jacquot, Institut National de la Santé et de la Recherche Médicale, Unité 514, Institut Fédératif de Recherche 53, Hôpital Maison Blanche, 45 rue Cognacq-Jay, 51092, Reims Cedex, France. E-mail address:
3 Abbreviations used in this paper: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; HBG, human bronchial gland; F508 homozygous CF HBG cells, CF human bronchial submucosal gland cells expressing the cystic fibrosis transmembrane conductance regulator that contains a deletion of phenylalanine at position 508; IKK, IB kinase; NIK, NF-B-inducing kinase; MAPK, mitogen-activated protein kinase.
Received for publication August 30, 1999. Accepted for publication January 11, 2000.
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