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Regular Articles |
From INSERM Unité 314*
and Laboratoire
d'Immunologie,
CHU Maison Blanche, Reims,
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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F508
deletion expressed elevated levels of the endogenous chemokine
interleukin (IL)-8 but not the pro-inflammatory cytokines IL-1ß and
IL-6, compared with non-CF bronchial glands. Moreover,
basal protein and mRNA expression of IL-8 were constitutively
up-regulated in cultured F508 homozygous CF human bronchial gland
cells, in an unstimulated state, compared with non-CF
bronchial gland cells. Furthermore, the exposure of CF and
non-CF bronchial gland cells to an elevated extracellular
Cl- concentration markedly increased the release of
IL-8, which can be corrected in CF gland cells by reducing the
extracellular Cl- concentration. We also found
that, in contrast to non-CF gland cells, dexamethasone
did not inhibit the release of IL-8 by cultured CF gland cells. The
selective up-regulation of bronchial submucosal gland IL-8 could
represent a primary event that initiates early airway submucosal
inflammation in CF patients. These findings are relevant to the
pathogenesis of CF and suggest a novel pathophysiological concept for
the early and sustained airway inflammation in CF
patients.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The production of tumor necrosis factor (TNF)-
, IL-1ß, IL-6, and
IL-8 and other pro-inflammatory cytokines by airway epithelial cells
and lung macrophages along with the accumulation and activation of
neutrophils in the CF airways may underlie the early pathogenesis of CF
lung disease.4,5,8
Before lung infection, an excessive
release of pro-inflammatory cytokines and an increased number of
neutrophils have been reported in the bronchoalveolar lavage fluids of
CF patients.4,5
Moreover, Noah et al9
recently
demonstrated that the IL-8 levels in bronchoalveolar lavage fluids, in
comparison with other pro-inflammatory cytokine levels, are markedly
increased in children with CF compared with non-CF children with a
bacterial infection of the lower airways. Thus, it is possible that
excessive inflammation in the CF lung may be related to constitutive
abnormalities in the regulation of pro-inflammatory cytokine expression
by CF airway epithelial cells, independent of infectious stimuli. In
this context, we have recently shown in CF mutant mice10
that airway inflammation may be a direct consequence of mutant CFTR
(cystic fibrosis transmembrane conductance regulator) protein
expression, the gene product in CF disease, as evidence for an
increased number of inflammatory cells was observed in the lamina
propria of CF mice in the absence of any sign of infection. All of
these data favor the hypothesis that an endogenous pathway for airway
inflammation may exist in CF airway cells before the manifestation of a
bacteria-related infection. Submucosal gland epithelial cells are of
special interest in this regard, given their ability to express the
highest level of CFTR protein in comparison with other human airway
epithelial cell types11,12
and also their ability to
provide the bulk of the fluid component of airway secretions. Recently,
it was shown that CFTR Cl- channel dysfunction in CF
tracheal submucosal gland cells leads to abnormal transepithelial salt
and fluid secretion.13
Based on these observations it is
reasonable to postulate that intrinsic abnormalities related to the
mutant CFTR protein in the bronchial submucosal gland cell type could
lead to early inflammation in CF airways. At present, it is not known
whether CF human bronchial glands in vivo could represent a
major local source of pro-inflammatory cytokines and, in particular,
for the IL-8 chemokine. Nor is it known whether the abnormally elevated
Cl- concentration described for the airway secretions of
CF patients14,15
can modulate the inflammatory responses in
airways. The concept of bronchial gland epithelial cells as an
important component of airway inflammation in F508 homozygote CF
patients has been applied to understanding the pathogenesis of early
pulmonary inflammation in CF patients.4,5
We therefore
decided to address the following questions. 1) Is there evidence of
elevated levels of pro-inflammatory cytokines IL-1ß, IL-6, and IL-8
expressed by F508 homozygous CF human bronchial submucosal glands
compared with non-CF bronchial submucosal glands in vivo?
And 2) If so, what are the levels and differential expression of these
cytokines, in particular for the IL-8 chemokine, according to the
extracellular Cl- concentration by subcultures of F508
homozygous CF human bronchial gland cells compared with levels measured
in non-CF bronchial gland cells in vitro?
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human CF bronchial tissue was obtained from eight recipients
undergoing lung transplant operations (the CF patients were all F508
homozygous; four females and four males; mean age, 17.3 years; age
range, 9 to 27 years). Tissue for control experiments was obtained from
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). For all
experiments, bronchial segments were prepared within 5 hours after lung
resection and were incubated in a serum-free medium composed of a 1:1
mixture of Dulbecco's modified Eagles medium (DMEM) and Ham's F-12
medium supplemented with the antibiotics colistin (200 U/ml),
penicillin G (100 U/ml), and streptomycin (100 µg/ml).
Histological Examination of Bronchial Submucosal Tissues
The upper lobar bronchi of six CF patients and three non-CF disease controls (one with primary pulmonary hypertension and two with pulmonary idiopathic fibrosis) were dissected form the pathological lung resected before transplantation, fixed with 10% formalin, and embedded in paraffin for light microscopy. Transverse bronchial sections were stained with a Giemsa solution. Areas of the submucosal connective tissue surrounding glands was selected and analyzed. A minimum of 24 microscopy fields (>450 mm2 of submucosal tissues) were examined at a magnification of x400, and the number of inflammatory cells was quantified using a computer-assisted analysis system (CAS-200; Becton-Dickinson, Oxford, UK). The total number of inflammatory cells was counted in each delineated submucosal area. Results were expressed as inflammatory cells per square millimeter. Polymorphonuclear neutrophils could be easily recognized by their lobular nuclei and were quantified in each area examined. The percentage of neutrophils was also calculated.
Immunohistochemical Staining
For the immunohistochemical analysis of bronchial tissues, frozen bronchial tissue samples were embedded in OCT (Miles Tissue Tek, Elkhart, IN), immersed in liquid nitrogen, and stored at -80°C. Bronchial cryosections (5 µm thick) deposited onto gelatin-coated glass slides were stored at -20°C after air drying and rehydrated in 0.1 mol/L PBS at pH 7.2. Sets of consecutively cryofixed sections were then blocked with PBS/1% bovine serum albumin for 10 minutes and stained for IL-1ß, IL-6, IL-8, and lysozyme, a specific protein marker of bronchial gland serous-type cells.16,17 Monoclonal antibodies against the cytokines IL-1ß, IL-6, and IL-8 (dilution, 1:100) were purchased from Biosource International (Camarillo, CA). Rabbit antiserum to human lysozyme (dilution, 1:500) was purchased from Dakopatts (Glostrup, Denmark). In all immunofluorescence experiments, bound antibodies were detected using the streptavidin-fluorescein isothiocyanate (FITC) system (Amersham International, Amersham, UK). Secondary antibodies of goat biotinyled anti-mouse and anti-rabbit IgG fractions (Boehringer Mannheim, Meylan, France) and streptavidin-FITC were used at a dilution of 1:50. Negative controls were performed using either nonimmune mouse or rabbit IgG fractions (Sigma Chemical Co., St Louis, MO).
The co-localization of the IL-8 chemokine and lysozyme was performed by indirect double immunofluorescence in cryofixed CF and non-CF bronchial sections. Sections were incubated for 60 minutes at room temperature in PBS/1% bovine serum albumin containing 1 µg/ml anti-human IL-8 antibody. After staining with the IL-8 monoclonal antibody, sections were incubated for 60 minutes in PBS/1% bovine serum albumin containing 2 µg/ml lysozyme polyclonal antibody, washed in three changes of PBS/1% bovine serum albumin for 5 minutes each time followed by incubation in PBS/1% bovine serum albumin containing 2 µg/ml of both donkey anti-mouse FITC-conjugated antibody and donkey anti-rabbit Texas-red-conjugated antibody for 45 minutes. After rinsing in three changes of PBS/1% bovine serum albumin for 10 minutes each time, all specimens were counterstained with Harris hematoxylin solution for 10 seconds, mounted in citifluor antifading solution (Agar Scientific, Stansted, UK), and observed by using a Zeiss Axiophot microscope (Zeiss, Le Pecq, France) employing epifluorescence and Nomarski differential interference illumination.
Isolation and Culture of Bronchial Submucosal Gland Cells
Human bronchial gland (HBG) cells were isolated from eight F508
homozygous CF patients and from four non-CF patients as described
above. Cell isolation and subcultivation procedures were performed as
described previously.17,18
In brief, 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 µg/ml) were also added. The culture
medium was replaced every 3 days. After 2 weeks in primary culture at
37°C under 5% CO2 in air, cells were treated with 0.25%
trypsin, 0.5 mmol/L EDTA in a Ca2+- and
Mg2+-free PBS solution. The removal of contaminated
fibroblasts from the primary culture was carried out using a selective
trypsination procedure as previously described.17
Cells
were then grown in 25-cm2
tissue culture flasks. After 4
weeks, third-passage HBG cells had proliferated and exhibited
characteristics of homogeneous submucosal epithelial and secretory
gland cells, including two protein markers specific to the glandular
serous-type cell, these being lysozyme and secretory leukocyte
proteinase inhibitor (SLPI) also known as
antileukoprotease.17,18
Functional Cl- Channel Activity of CFTR Protein
The phosphorylation-regulated Cl- channel activity of CFTR was assessed using the halide-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) as previously described.19 The CF and non-CF HBG cells grown on type I collagen-coated glass coverslips were loaded with 3.5 mmol/L SPQ in a hypotonic chloride buffer (1:1 mixture of distilled water and a chloride buffer containing 130 mmol/L NaCl, 2.4 mmol/L K2HPO4, 10 mmol/L D-glucose, 1 mmol/L CaSO4, 1 mmol/L MgSO4, and 10 mmol/L HEPES, pH 7.4) for 10 minutes at 37°C. Cells were then rinsed twice and incubated with the chloride buffer for 15 minutes at 37°C. Cells were placed in a temperature-controlled chamber (37°C) on the stage of an inverted microscope (Zeiss IM35) and incubated in a nitrate buffer in which NaCl was replaced by 103 mmol/L NaNO3. Cyclic AMP (cAMP) stimulation was achieved by exposing the cells to 25 µmol/L forskolin (Sigma Chemical Co.). The chloride secretion of approximately 60 to 80 cells was estimated by measurement of SPQ fluorescence variations obtained using an excitation light wavelength at 365 nm and emission light wavelength at >395 nm through a 32x planachromat objective. A software-driven shutter in the excitation light path was used to automatically illuminate cells for 2 seconds and simultaneously record the fluorescent image every minutes for 15 minutes. Mean variations in SPQ fluorescence after cAMP stimulation were plotted against time over the 15-minute period. The mean number of cells analyzed per non-CF and CF HBG cell monolayer culture was at least 65. Data are presented as the relative fluorescence, this being 100X (Ft/Fo), where Ft is the fluorescence intensity at time t and Fo is the fluorescence intensity at time 0.
RNA Isolation and Northern Blot Analysis
Total cellular RNA was extracted from 25-cm2
culture
flasks of confluent third-passage F508 homozygous CF HBG and non-CF
HBG cells using an acid guanidinium/phenol/chloroform method (Trizol,
GIBCO BRL, Gaithersburg, MD). For Northern analyses, aliquots of 15
µg of total RNA (determined by spectrophotometry, 260-nm wavelength)
were denatured and size fractionated by electrophoresis through a 1.0%
agarose/7.0% formaldehyde gel. The integrity of the RNA was confirmed
by observing under ultraviolet (UV) light the 28 S and 18 S ribosomal
bands after ethidium bromide staining. For Northern blots, the RNA was
transferred onto a nylon membrane (Hybond N; Amersham International) by
capillary transfer and UV cross-linked to the membrane. Filters were
hybridized at 50°C with a 32P end-labeled oligonucleotide
probe with the sequence 5'GTT-GGC-GCA-GTG-TGG-TCC-ACT-CTC-AAT-CAC-3'
using a random prime DNA labeling kit (Boehringer Mannheim). Membranes
were hybridized for 15 hours at 50°C, washed twice for 10 minutes in
2X SSC, 0.01% SDS, 10 minutes in 1X SSC, 0.1% SDS at room temperature
and 15 minutes in 0.1X SSC, 0.1% SDS at 50°C, and finally
autoradiographed for 1 week at -70°C. Autoradiogram signal strengths
of hybridized mRNA were quantified by scanning densitometry (GS 690
imaging densitometer and Molecular Analyst software; Bio-Rad, Richmond,
CA). Adjustments for small differences in loading were made using
densitometry of the ethidium bromide fluorescence of the 28 S band of
the gel photographed before blotting.
Exposure of CF and Non-CF HBG Cell Monolayers to Low and High Cl- Concentration
Before the exposure of cells to either low (85 mmol/L),
intermediate (135 mmol/L), or high (170 mmol/L) Cl-
concentration, third-passage confluent monolayers of F508 homozygous
CF and non-CF HBG cells were incubated for 16 hours 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-hour period, the
culture supernatants from an additional 6-hour period were collected
and stored at -80°C until tested for the presence of the cytokines
IL-1ß, IL-6, IL-8, and IL-10, as described below. In this way,
individual monolayers of CF and non-CF HBG cells were exposed for an
additional 6-hour period to Cl- solutions containing
either 85 mmol/L, 135 mmol/L, or 170 mmol/L Cl-,
respectively. Immediately after each period of cell exposure,
supernatants were collected and stored at -80°C until tested for the
presence of cytokines. The three chloride-containing solutions used in
this study (85 mmol/L Cl-, 135 mmol/L Cl-,
and 170 mmol/L Cl-) contained 1 mmol/L CaCl2,
20 mmol/L KCl, and either 60 mmol/L NaCl, 105 mmol/L NaCl, or 148
mmol/L NaCl, pH 7.4, respectively, as previously
reported.15
In another set of experiments, at the end of the first 16-hour period
in the Ultroser G-free RPMI 1640 medium, the production of IL-8 by
third-passage confluent cultures of CF and non-CF HBG cells was
analyzed in response to exposure to glucocorticoids previously
solubilized in Ultroser G-free RPMI 1640 medium (1, 5, and 10 µmol/L
dexamethasone; Sigma Chemical Co.). From individual monolayers of
F508 homozygous CF and non-CF HBG cells, IL-8 secretion was measured
over the subsequent 6-hour culture period in the absence or the
presence of the different concentrations of dexamethasone.
ELISA for Cytokines
Cytokine concentrations in the culture supernatants of
third-passage confluent F508 homozygous CF and non-CF HBG cells were
determined by following the manufacturer's instructions in
commercially available ELISA kits (Biosource International). The ELISAs
for IL-1ß, IL-6, IL-8, and IL-10 were sensitive to a level of 5
pg/ml. In experiments where defined amounts of particular recombinant
human cytokines (used as standards) were added to supernatants, the
total recovery of each of the cytokines was always close to 100%.
Analysis of TNF- was not performed, however, due to the lack of
supernatant samples after analysis of the cytokines described above.
Cell viability was confirmed by trypan blue exclusion after all
experimental interventions. The uniformity of the cell monolayer was
determined by quantifying the cell number per well. Total cellular
protein concentrations were measured using the Bradford method (Bio-Rad
Laboratories). All results are expressed as pg/ml/106
cells.
Statistical Analysis
Results are expressed as means ± SD. Each data point was performed in triplicate at least, and each cell culture experiment was performed at least three times. Differences in cytokine 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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The mean number of the inflammatory cells and, in particular, the
number of polymorphonuclear neutrophils found in six F 508
homozygous CF patients and in three non-CF disease controls are
reported in Table 1
. In CF patients, the
mean number of inflammatory cells was higher (1856 ± 721
cells/mm2; n = 6) as compared with non-CF
disease controls (910 ± 352 cells/mm2;
n = 3), but the difference was not significant between
the two groups. In the submucosal periglandular areas examined, a
predominance of mononucleated cells was observed in the two groups.
Although the mean number of neutrophils in the CF patient group
(116 ± 65 cells/mm2) was higher than that obtained
from non-CF disease control group (65 ± 16
cells/mm2), the difference was not significant.
Interestingly, when expressed in percentage of total inflammatory
cells, the percentage of neutrophils was low and similar in the two
groups of subjects analyzed (6 ± 2% in CF and 8 ± 4% in
non-CF disease controls).
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To evaluate the possibility that the endogenous expression of
pro-inflammatory cytokines is up-regulated in CF bronchial submucosal
gland epithelial cells, we monitored the immunoreactivity of cytokines
IL-1ß, IL-6, and IL-8 in consecutive serial bronchial tissue sections
obtained from eight F508 homozygous CF patients and from four non-CF
disease patients. Figure 1
provides a
representative set of the eight CF and four non-CF cryofixed bronchial
sections and shows the expression and localization of IL-8 protein by
immunofluorescence. The surface epithelium of CF bronchial tissues
(Figure 1, a and b)
and of non-CF bronchial tissues (data not shown)
demonstrated a notable absence of immunoreactivity to the IL-8
antibody.
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compared with non-CF bronchial tissues (Figure 1d)
. Immunostaining for IL-8 was observed exclusively in the submucosal
glands, with gland cells displaying strong immunoreactivity
interspersed with unstained gland cells. The distal portion of
bronchial submucosal glands contains a branching network of tubules
that contain serous cells. At higher magnification (Figure 1, e–h)
,
the expression of IL-8 protein in CF bronchial gland structures was
analyzed by double immunofluorescence using antibodies to both
lysozyme, a specific marker for the gland serous-type cell (Figure 1f)
,
and IL-8 (Figure 1, g and h)
. In CF bronchial submucosal glands, both
the lysozyme-positive gland cells (serous-type cells) and
lysozyme-negative gland cells (mucous-type cells) exhibited a
high-level reaction to the IL-8 antibody.
In a second set of experiments, we examined whether pro-inflammatory
cytokines other than the IL-8 chemokine were concomitantly up-regulated
in the same F508 CF bronchial submucosal tissue under investigation.
Immunohistochemical analyses were performed on serial consecutive
sections of the eight CF and four non-CF cryofixed bronchial tissues
with specific antibodies to IL-1ß and IL-6, respectively. Both of
these cytokines are known to be markedly increased in cultured airway
epithelial cells after exposure to viral20,21
and bacterial
products.22-24
Sections showed a very low and similar
level of staining for IL-1ß and IL-6 in both CF and non-CF bronchial
submucosal gland structures, respectively (data not shown).
Functional Activity of CFTR Protein in CF and Non-CF Bronchial Gland Cells in Culture
The CFTR defect in CF epithelial cells manifests itself as a
defective chloride secretion in response to stimulation to cAMP
agonists. To investigate the functional activity of the CFTR protein in
both cultured CF and non-CF HBG cells, we made measurements of
Cl- efflux in tight confluent third-passage HBG cell
monolayers. We examined the Cl- secretion of non-CF and CF
HBG cells after their exposure to 25 µmol/L forskolin. Results shown
in Figure 2
are typical of subcultures of
HBG cells isolated from all of the eight CF patients and all four
non-CF control patients. Cultured HBG cells from non-CF patients
demonstrated significant (P < 0.001) increases
in Cl- efflux in response to exposure to forskolin
compared with cultured HBG cells from F508 homozygous CF patients.
SPQ-loaded non-CF HBG cells exhibited a significant response to cAMP
stimulation (ie, a CFTR functionality), as demonstrated by a
significant increase (27%) in the fluorescence signal 6 minutes after
the onset of the stimulation. In contrast, no significant
Cl- efflux in response to forskolin stimulation was
detected after the same period of time in CF HBG cell cultures from
eight CF patients.
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Consistent with our results of immunofluorescence
detection of IL-8 protein in CF bronchial submucosal gland cells
in situ, Northern blot analyses revealed that resting
F508 homozygous CF HBG cells constitutively produce high levels of
IL-8 mRNA transcripts. Under similar culture conditions (ie, in an
unstimulated resting state), there was no evidence of endogenous IL-8
mRNA transcripts in non-CF HBG cells (Figure 3a)
. Moreover, the spontaneous production
of IL-8 by subcultures of CF HBG cells was 13-fold higher compared with
non-CF HBG cells (Figure 3b)
.
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, similar and low
levels of cytokines IL-1ß and IL-6 were found in the supernatants
from both CF and non-CF HBG cell cultures. As normal human bronchial
surface epithelial cells were recently reported to constitutively
produce the anti-inflammatory cytokine IL-10, which appeared to be
down-regulated in CF human bronchial surface epithelial
cells,25
we also compared cultured CF and non-CF HBG cells
for their ability to produce this cytokine. Negligible levels of IL-10
were found in the supernatants of both the CF and non-CF HBG cell
cultures (Figure 3b)
. Reduction of the Extracellular Cl- Concentration Allows CF HBG Cells to Markedly Decrease Their IL-8 Secretion Level
To further mimic the in vivo situation, in which a
change in NaCl concentration occurs in CF bronchial
secretions14,15
as a result of CFTR deficiency (ie, 85
mmol/L in non-CF versus 120 to 170 mmol/L in CF), we
examined whether both CF and non-CF HBG cells display differential
expression in their release of IL-8 according to the electrolyte
concentration in the extracellular medium. As shown in Figure 4
, the exposure of cell cultures to a
high Cl- concentration (170 mmol/L) for 6 hours resulted
in a 6.5-fold increase in IL-8 production in CF HBG cells compared with
similarly treated non-CF HBG cells. More importantly, in the presence
of low (85 mmol/L) and intermediate (135 mmol/L) Cl-
concentration, F508 homozygous CF HBG cells behaved similarly to
non-CF HBG cells. Lowering the extracellular Cl-
concentration from 170 mmol/L to 85 mmol/L resulted in a significant
decrease (P < 0.001) in IL-8 production by CF
HBG cells down to the levels of IL-8 produced by non-CF HBG cells
exposed to the same low electrolyte content. These data indicate that
the extracellular Cl- concentration markedly regulates the
IL-8 release by CF HBG cells.
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In a final set of experiments, we tested the capacity of different
concentrations of glucocorticoid (1, 5, and 10 µmol/L dexamethasone)
to affect the release of the IL-8 chemokine by F508 homozygous CF
and non-CF HBG cell cultures. Over a 6-hour culture period, the
spontaneous IL-8 production of untreated CF and non-CF HBG cells was
1485 pg/ml/106
cells and 114 pg/ml/106
cells,
respectively. Figure 5
shows that CF HBG
cells were refractory to glucocorticoid treatment at lower
concentrations (1 and 5 µmol/L) whereas the high concentration (10
µmol/L) of dexamethasone decreased by 25% the basal level of IL-8
production by CF HBG cells. In contrast to CF HBG cells, dexamethasone
(1 and 5 µmol/L) significantly (P < 0.001)
reduced the spontaneous release of IL-8 in a dose-dependent fashion in
non-CF HBG cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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F508
homozygous CF HBG cells appeared selective. In this way, secretion
levels of other cytokines, such as IL-1ß, IL-6, and the
anti-inflammatory cytokine IL-10, by CF HBG cells in culture were low
and similar to non-CF HBG cells in the unstimulated resting state,
indicating no generalized disturbance of the F508 homozygous
genotype of CF HBG cells.
Our findings of a nonsignificant increase of neutrophils surrounding
submucosal secretory glands in CF bronchial tissues as compared with
non-CF periglandular tissues may appear surprising. A possible
explanation for the relatively low number of neutrophils in CF
bronchial submucosa and high number of neutrophils found in
bronchoalveolar lavage in CF patients4,5,9
could be their
low resident time in the bronchial submucosal connective tissue and
their rapid migration across the tissues into the airway lumen. To
date, the primary cause initiating recruitment of neutrophils in CF
lungs remains unknown. Studies with human recombinant IL-8 in SCID
mice6
have shown that IL-8 is a strong chemoattractant for
neutrophils and activates neutrophils to release prestored chemotactic
products mediating T lymphocyte and monocyte accumulation at sites of
inflammation. In airway epithelial cells, the CFTR protein can be
detected in subcellular components, such as the endoplasmic reticulum
(ER) and vesicles,26-29
and in the membrane of secretory
granules of submucosal glands.12
The different locations of
CFTR protein suggest the existence of intracellular mechanisms whereby
a mutated CFTR in CF HBG cells could lead to abnormalities in IL-8
secretion, as recently demonstrated by Moss et al30
who
showed a reduced IL-10 secretion by human CD4+ T
lymphocytes expressing mutant CFTR. Recently, it has been suggested
that the CFTR protein, depending on its level of expression, is a
multifaceted molecule with multiple roles in epithelial
cells.28
Combined with our previous
observation10
showing airway inflammation in mutant CF mice
raised in pathogen-free conditions, the present study reinforces our
idea that CF submucosal gland secretory cells may be the first and
predominant source of IL-8, which initiates the early airway mucosal
inflammation in CF patients. Although it is tempting to speculate that
the selective up-regulated expression of chemokine IL-8 by CF HBG cells
could be related to a CFTR-dependent mechanism, the relationships
between the mislocalization of mutated CFTR protein, the regulation of
secretory processes, and the up-regulated IL-8 production in CF HBG
cells remain open to debate. Irrespective of the mechanisms involved in
the selective up-regulation of IL-8 expression by CF HBG cells, our
observations support the notion that abnormal constitutive secretion of
IL-8 by CF HBG cells is primarily due to a dysregulated endogenous
pathway rather than a consequence of a general response to airway
inflammation. Consequently, neutrophils activated by locally secreted
IL-8 release elastase and oxidants that stimulate airway surface
epithelial cells to produce other chemotactic factors, such as TNF-,
IL-1ß, IL-6, and IL-8,22,31
which may generate and
perpetuate an inflammatory vicious cycle in CF airways. This
inflammation can be thereafter greatly amplified after
Pseudomonas aeruginosa infection, as recently demonstrated
by Heeckeren et al32
in CF mice. In CF, it is known that
the airway epithelium is exposed to P. aeruginosa products
that have previously been shown to actively increase the production of
most of the pro-inflammatory cytokines by airway epithelial
cells.31,33
It has been suggested that a feedback mechanism
exists, involving activated neutrophils, phagocytosis of bacteria, and
epithelial IL-8, resulting in a persistent inflammation and infection
in CF airways.34
One of the most striking results that we obtained in the present study is the NaCl-dependent production of IL-8 by cultured CF HBG cells. The finding that the level of IL-8 release from CF bronchial submucosal gland cells is significantly increased during exposure to high (170 mmol/L) and intermediate (135 mmol/L) Cl- concentration is of particular significance in CF where the NaCl concentration in bronchial secretion liquids has been shown to be higher than that found in normal subjects.14,15 Recent data have shown that high NaCl concentrations in CF airway surface fluid contribute to the diseased state by impairing the neutrophil killing of P. aeruginosa35 and by inhibiting the bactericidal properties of CF airway fluid associated with a decreased activity of ß-defensin-1 peptides produced by airway cells.36 Our study shows, in addition, that high NaCl concentrations may contribute to and sustain an exaggerated and prolonged inflammatory state by releasing high amounts of IL-8 from CF airway submucosal glands. This aberrant secretion of IL-8 by CF gland cells can be fully corrected by reducing the extracellular NaCl concentration. Therefore, it will be of importance to define the ionic composition of fluid secreted by CF and non-CF submucosal gland cells more precisely and to characterize the contribution of these ions to surface airway fluids. Although a recent work revealed no differences in the ionic composition of airway surface liquids from bronchial regions of CF and non-CF patients,37 the ionic composition of liquids secreted by bronchial submucosal gland cells from CF and non-CF patients is still unknown.
A possible explanation for our findings of the NaCl dose-dependent
release of IL-8 by CF bronchial glands could involve abnormal dynamic
changes in intracellular Ca2+ mobilization in response to
the external ionic composition, as it has recently been demonstrated
that a reduction in the extracellular Na+ concentration
causes a rapid release of Ca2+ from internal stores in
normal human airway epithelial cells.38
Furthermore, an
abnormal CFTR protein accumulation in the ER of CF gland secretory
cells might result in a reduced intracellular Ca2+ handling
associated with a defect in the process of exocytosis.39
The amplitude and the duration of intracellular Ca2+
signals were recently shown to be directly responsible for the
differential activation of several Ca2+-sensitive
transcriptional regulators, including the transcription nuclear
factor-
B (NF-B), c-JunN-terminal kinase (JNK), and
NFAT, which are required for the transcriptional activation of
diverse pro-inflammatory cytokines, including IL-8.40
In CF
gland cells, a possible mechanism of action could involve the effect of
the accumulation of mutant CFTR protein in the ER41,42
on
the synthesis and secretion of cytokines, as overload of the ER by
resident proteins activates NF-B by causing a rapid Ca2+
release from the ER.43
In the present study, we have also shown that dexamethasone treatment
of CF HBG cells failed to block the IL-8 production and release but, in
contrast, significantly blocked the IL-8 release from non-CF HBG cells.
In this way, a defective inhibition by dexamethasone in B lymphocytes
in CF patients was reported.44
The action of
glucocorticoids is very complex and appears to be via the inhibition of
NF-B complex activation.45
Whether or not the
differential IL-8 response to dexamethasone treatment between F508
homozygous CF and non-CF HBG cells is due to variability in the
specific NF-B complexes or to direct protein-protein interactions
with mutated CFTR protein involved in regulating IL-8 gene expression
requires further investigation.
In summary, we have demonstrated that CF bronchial submucosal glands constitutively produce high levels of IL-8 in the resting (unstimulated) state, which may represent the primary signal that initiates the early and sustained mucosal inflammation observed in CF lungs.4,5 We suggest that the basic genetic defect in CF patients may be a contributory factor to the selective up-regulated IL-8 expression observed. Even so, the molecular events related to the up-regulation of the IL-8 gene in CF airway gland epithelial cells together with variations of the extracellular ion concentrations in airway fluids remain to be elucidated.
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Acknowledgements |
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Footnotes |
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Supported by a grant from the Association Française de Lutte contre la Mucoviscidose. The GOEMAR Laboratories (St. Malo, France) fund O. Tabary.
Accepted for publication June 29, 1998.
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