Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
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ABSTRACT |
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A tendency
toward excessive inflammation in cystic fibrosis (CF) patients often
accompanies lung infections with Pseudomonas aeruginosa. We
tested the cytokine response to P. aeruginosa in two pairs
of human airway epithelial cell lines matched except for CF
transmembrane conductance regulator activity. The 9/HTEo
CF-phenotypic cell line produced significantly more interleukin (IL)-8,
IL-6, and granulocyte-macrophage colony-stimulating factor but not
regulated on activation normal T cell expressed and secreted (RANTES)
in response to Pseudomonas than the 9/HTEo
control line, and the differences widened over time. Similarly, a 16HBE
cell line lacking transmembrane conductance regulator activity showed
enhanced IL-8 and IL-6 responses compared with the control cell line.
The pharmacology of the cytokine response also differed because
dexamethasone reduced cytokine production to similar levels in the
matched cell lines. The protracted proinflammatory cytokine response of
the CF-phenotypic cell lines suggests that the limiting mechanisms of
normal cells are absent or attenuated. These results are consistent
with in vivo observations in patients with CF and suggest that our
novel cell lines may be useful for further investigation of the
proinflammatory responses in CF airways.
cystic fibrosis; interleukin-8; interleukin-6; granulocyte-macrophage colony-stimulating factor; inflammation
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INTRODUCTION |
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IN CYSTIC FIBROSIS (CF), the chloride transport defect in airway epithelium and submucosal glands is somehow translated into chronic bacterial infection and excessive inflammation that are the proximate causes of lung destruction and, ultimately, the death of the patient. The mechanisms underlying this translation are probably multiple. Several hypotheses have been proposed to explain the propensity to bacterial infection in the lungs, including dysfunction of bactericidal molecules in the epithelial lining fluid of abnormal electrolyte composition (25), failure of epithelial cells to ingest and thereby inactivate bacteria (20, 21), and increased binding of specific bacteria to the surface of CF airway epithelial cells (31). Recently, however, it has become clear that in addition to the propensity toward infection in CF patients, there is also tendency toward excessive inflammation. Several pieces of data support this concept. Inhibition of inflammation by high-dose ibuprofen or steroids, far from allowing the infection to progress, is beneficial to the lung disease of patients with CF (8, 13). Increased interleukin (IL)-8, a chemoattractant cytokine, has been found in the airways of young children with CF compared with that in other young children without CF but with a comparable airway burden of bacteria (17). Inflammation has been detected very early in the life in patients with CF, even those in whom no infection can be documented. Moreover, CF mice die earlier and to a greater extent from chronic Pseudomonas infection than their non-CF littermates, and this increased death rate is accompanied by increased proinflammatory cytokines in bronchoalveolar lavage fluid but not by an increased bacterial burden (30).
One possible link between the tendency toward infection and the
excessive inflammation is the response of the airway epithelial cell to
contact with Pseudomonas aeruginosa, the most common
infecting organism in patients with CF. This organism has increased
binding to CF cells in culture and also induces greater release of IL-8 from CF cells than from normal cells (7, 10). It has been suggested that this excessive response may arise from the interaction of pilin on the surface of Pseudomonas with
asialo-GM1 on the surface of CF airway epithelial cells
(23). In addition, the increased binding of
Pseudomonas to CF airway epithelial cells has been
associated with the presence of the F508 CF transmembrane conductance regulator (CFTR) allele (31). To investigate
this issue further, we used an airway epithelial cell line with the CF
phenotype developed by overexpression of the regulatory (R) domain of
CFTR. At the appropriate phosphorylation conditions and relative
concentrations of R domain and native CFTR, this results in inhibition
of CFTR chloride transport activity but persistent expression of CFTR,
at least at the mRNA level (19). Thus these cells express
the CF chloride transport phenotype but lack mutant CFTR. Our
laboratory has shown that these cells have abnormal surface
properties as assessed by lectin binding (14) and also by
binding of Pseudomonas (2). We used these cells to investigate the cytokine response of normal and CF-phenotypic cells
to intact Pseudomonas and isogenic mutants lacking pilin or
pilin and flagellin. We found that the responses of CF-phenotypic cells
differ from those of normal cells and that these abnormalities may
contribute to the excessive inflammatory response observed in the lungs
of CF patients.
To test the hypothesis that CF-phenotypic epithelial cells respond to
Pseudomonas infection with an increase in proinflammatory cytokine response compared with that in normal cells, we tested another
pair of airway epithelial cell lines clonally derived from parental
16HBE14o cells (4). The cell line that
demonstrates a CF phenotype, confirmed by a lack of cAMP-stimulated
chloride secretion in 36Cl efflux assays and no increase in
short-circuit current when grown as monolayers and studied in an Ussing
chamber, disrupts CFTR function through expression of a 131-bp
antisense (AS) sequence to CFTR (16HBE-AS). The control cell line was
transfected with the same expression vector containing the
corresponding sense (S) strand to CFTR (16HBE-S) and maintained the
parental chloride efflux responses to cAMP agonists. We found that IL-8
and IL-6 secretion after Pseudomonas infection in these
CF-phenotypic cells is significantly greater than that in the control
cells. Our results in two matched airway epithelial cell lines in which
the CF phenotypes are engineered in very different ways suggest that it
is the absence of CFTR function that predisposes the CF epithelium to
respond to infection with exaggerated proinflammatory responses.
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MATERIALS AND METHODS |
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Cell Lines
pCEP and pCEP-R cell lines.
Human tracheal epithelial cells (9/HTEo) derived from
SV40-transformed human tracheal epithelial cells (kindly provided by Dieter Gruenert, University of California, San Francisco) were transfected with the LIPOFECTIN reagent (GIBCO BRL, Life Technologies, Gaithersburg, MD) with either empty vector (pCEP4, Invitrogen, San
Diego, CA) or vector cloned with the R domain of CFTR (pCEP-R) as a
CF-phenotypic cell line. Cells were grown in Dulbecco's minimal Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and
2.5 mM L-glutamine and maintained under selection with 40 µg/ml of hygromycin at 37°C in an atmosphere of 95% air-5%
CO2.
16HBE14o antisense and sense cell lines.
To establish the matched cell lines, plasmids containing the first 131 nucleotides of human CFTR were transfected into 16HBE14o
cells (provided by D. Gruenert) in the sense (16HBE-S) and antisense (16HBE-AS) orientations and maintained under selection. Both cell lines
polarized and formed tight junctions on a filter support. The
CF-phenotypic 16HBE-AS cell line did not respond to cAMP agonists with
increased chloride secretion, whereas the 16HBE-S cell line demonstrated a significant response (22). 16HBE-S and
16HBE-AS cells were maintained in DMEM supplemented with 10% fetal
bovine serum, 2 mM L-glutamine, and 200 µg/ml of G-418.
Bacteria
The nonmucoid laboratory isolate PAO1 and the isogenic derivative strains used for cytokine assays were kindly provided by Alice Prince (Columbia University, New York, NY). The phenotypic properties of an isogenic strain of PAO1 lacking pili (PA/NP) and an isogenic strain lacking both pili and flagella (PA/NP/fliACytokine Reagents
Tumor necrosis factor-Stimulation of Cytokine Production by Cells
9/HTEo16HBE14o cells transfected with the pBKCMV vector
expressing CFTR sense (16HBE-S) or antisense (16HBE-AS) were plated at
an original density of 1 × 106 cells on Millicell-HA
0.45-µm filters (12-mm diameter; Millipore, Bedford, MA) as inserts
in 24-well plates and grown for 12-14 days until tight monolayers
were established. Both apical and basolateral surfaces were bathed in
0.5 ml of medium. The cells were incubated in serum-free medium for
18 h before the experiments. The permeability of the monolayer was
determined at the beginning and end of the experiment by applying 20 µg/ml of FITC-inulin (Sigma) to the apical side of the filter and
measuring the percentage that passed through to the basolateral
solution in 2 h. Less than 1.5% of the amount applied to the
apical surface was recovered from the basolateral medium. In addition,
under experimental conditions, known concentrations of human IL-8
(Sigma) were added to unstimulated triplicate filters, and the change
in concentration of IL-8 in the basolateral compartment compared with
that in nonspiked control cells was also determined. Not more than
6-7% appeared in the basolateral compartment.
P. aeruginosa strains PAO1, PA/NP, and
PA/NP/fliA were grown in TSB for ~18 h until there was
an optical density at 600 nm of ~1.0 or ~1 × 109
colony-forming units (cfu)/ml (determined by dilution plating). Aliquots of the overnight cultures were sedimented and washed twice by
resuspension in HBSS (GIBCO BRL) to a final dilution in HBSS of the
appropriate colony-forming units per milliliter. Washed bacterial
aliquots (0.5 ml/well) were then immediately incubated for 60 min,
unless otherwise indicated, with the confluent monolayers of epithelial
cells at 37°C. The doses of bacteria indicated are per well of cells.
There was no growth of Pseudomonas in the serum-free culture
medium at 24 or 48 h after experimental treatment as tested by
plating aliquots of the medium on TSB plates. Nontreated control wells
were processed similarly. For 16HBE14o
cells on filters,
Pseudomonas and other treatments were applied in 0.5 ml of
HBSS to the apical surface only. As a positive control, the cells were
stimulated for 1 h with IL-1
(100 ng/ml) and TNF-
(100 ng/ml; both from Sigma). The cell monolayers were then washed three
times in HBSS followed by incubation for the indicated time points in
0.5 ml of serum-free cell culture medium containing 100 µg/ml of gentamicin.
To test for the ability of killed Pseudomonas to stimulate
cytokine production, 109 cfu/ml of PAO1, PA/NP, or
PA/NP/fliA were either killed by heating to 65°C for 30 min or lightly fixed in 0.5% glutaraldehyde for 30 min and washed.
Aliquots of the bacterial suspensions were prepared on grids for
negative staining and electron microscopy. Triplicate wells from each
experiment were also fixed in 1% paraformaldehyde after being washed
for visualization by electron microscopy.
Cytokine Assays
Collected medium was assayed for human cytokine IL-8, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and regulated on activation normal T cell expressed and secreted (RANTES) concentrations with enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN). Epithelial cell monolayers were lysed with 300 µl of 1× lysis buffer (Promega, Madison, WI), and protein concentration was determined with the bicinchoninic acid method (Pierce). The protein concentration displayed a linear relationship to cell number over the range of 105 to 2 × 106 cells. Each treatment was performed in triplicate wells, and duplicates of each sample were assayed. To test for cytokine release, IL-8 concentration was measured 24 h after PAO1 stimulation in the medium and epithelial cell lysates. To combine multiple experiments, the secreted cytokine concentration (in pg/mg protein) of 109 cfu of PAO1-stimulated pCEP-R cells at 24 h was set to 100% for each experiment, and other concentrations are expressed relative to this value.Cell Viability Assays
The viability of pCEP, pCEP-R, 16HBE-S and 16HBE-AS cells was assessed by trypan blue exclusion and by measuring the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) from cells with compromised membrane permeability before and after PAO1 or TNF-Data Analysis
Where applicable, statistical analysis of results and differences was determined by SigmaStat (SPSS, San Rafael, CA) with Student's t-test; if equal variance testing failed, the Mann-Whitney rank sum test was applied. Results shown are means ± SE. Results were considered significant when P ![]() |
RESULTS |
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Pseudomonas Strains and Cytokine Secretion From pCEP and pCEP-R Cells
We performed multiple experiments to measure the proinflammatory response of pCEP (control) and pCEP-R (CF-phenotypic) epithelial cells to the most common bacterial colonizer in CF lungs, P. aeruginosa, by incubating serial dilutions of three isogenic strains of the laboratory isolate PAO1 (PAO1, PA/NP, and PA/NP/fliAFigure 1 illustrates one representative
experiment (of two similar experiments) that measured the potent
proinflammatory chemokine for neutrophils, IL-8, 24 h after an
initial exposure of 1 h to various strains of
Pseudomonas. Both cell lines showed a dose-dependent release
of IL-8 in response to PAO1 (Fig. 1A) from 0.1 to ~100 bacteria/epithelial cell, a saturating concentration for the available asialo-GM1 receptors on the cell surface (2,
10). The IL-8 response of the CF-phenotypic or pCEP-R cells was
significantly greater than that of the control cell line at all
bacterial dilutions that elicit responses above background
(109 and 108 cfu, P 0.001;
107 cfu, P = 0.004; 106 cfu,
not significant). The Pseudomonas strain that lacks pili (PA/NP), a major adherence component to asialo-GM1 cell
surface receptors, also provoked IL-8 secretion in a dose-dependent
response to levels ~30-45% of that induced by intact PAO1 (Fig.
1B). The CF-phenotypic cells still showed a significant increase over
control cells (109 cfu, P = 0.015;
108 cfu, not significant; 107 cfu,
P = 0.016; 106 cfu, not significant). When
both pili and flagella were eliminated in the isogenic
Pseudomonas strain PA/NP/fliA
(Fig.
1C), very little IL-8 was secreted (only 3-8% of the
PAO1-stimulated values), but still pCEP-R cells produced significantly
more (109 cfu, P = 0.037; 108
cfu, P = 0.016; 107 cfu, not significant).
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Figure 2 shows the results from the same
experiment for the pleiotropic inflammatory mediator IL-6. Similar to
the PAO1-stimulated IL-8 response, both cell lines exhibited a
dose-dependent increase in IL-6 secretion measured 24 h after the
initial exposure, with significant increases in the pCEP-R cell line
compared with the pCEP control cell line (109 and
108 cfu, P 0.001; 107 cfu,
not significant; Fig. 2A). The PA/NP strain also evoked a
dose-dependent response, which reached 25-45% of PAO1-stimulated values and was significantly higher in the CF-phenotypic cells at
109 cfu (Fig. 2B). There was no IL-6 secretion
above that in nonstimulated control wells for either cell line with the
PA/NP/fliA
(Fig. 2C). Although, for the
particular experiment shown in Figs. 1 and 2, it appears that the
constitutive IL-8 and IL-6 secretion (the "none" condition) was
greater from pCEP-R cells, combined results from 13 experiments showed
no significant difference between the cell lines in unstimulated
conditions (IL-8: pCEP, 3.6 ± 0.9 pg/mg protein; pCEP-R, 3.1 ± 1.0 pg/mg protein; IL-6: pCEP, 5.2 ± 1.7 pg/mg protein;
pCEP-R, 3.9 ± 0.9 pg/mg protein).
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To determine whether the differences in stimulated IL-8 secretion between the cell lines were due to differences in cytokine release rather than production, we compared intracellular and secreted concentrations of IL-8 before and after PAO1 stimulation. Intracellular IL-8 was 8-12% of the secreted IL-8 in both cell lines, indicating that there was no difference in the ability to secrete the cytokine after stimulation.
These results taken together demonstrate that the CF-phenotypic cells responded differently to the same Pseudomonas inoculum, and although pili were a major contributor to the IL-8 and IL-6 secretion in response to Pseudomonas stimulation, factors other than the asialo-GM1-pili interaction must also be important in eliciting the inflammatory response as well as the differential response between the CF-phenotypic and non-CF-phenotypic cells.
To examine the components of PAO1 necessary for this response, we
tested LPS, which stimulates nuclear factor-B activation in some
airway epithelial cell systems (6). Neither
Pseudomonas nor E. coli LPS in concentrations
from 10 µg/ml to 1 mg/ml stimulated IL-8 and IL-6 release in these
cell lines. The requirement for motile Pseudomonas with pili
for proper adherence and signaling was examined by killing the bacteria
by heating to 65°C for 30 min before exposure. Heat-killed PAO1 did
not evoke a proinflammatory cytokine response even at the highest
concentration applied, 109 cfu (data not shown).
Heat-killed PAO1 prepared for electron microscopy with negative
staining showed that the bacteria had lost most flagella and appeared
shriveled compared with non-heat-treated but lightly
glutaraldehyde-fixed bacteria (data not shown). Mild fixation of PAO1
preserved morphology, including the presence of flagella. Application
of 109 cfu of these fixed bacteria to the epithelial cells
for 1 h also did not elicit an IL-8 response (data not shown). In
contrast, even 10 min of exposure of epithelial cells to live PAO1
(109 cfu) was sufficient to elicit an IL-8 but not an IL-6
response in both pCEP and pCEP-R cells assayed 24 h after the
initial exposure. The IL-8 response was much less after 10 min of
exposure compared with a 1-h PAO1 exposure (~1,400 pg/mg protein
compared with up to 50,000 pg/mg protein for 1 h of exposure) and
was not significantly different between the cell lines (data not
shown). These data taken together indicate that the killing of the
Pseudomonas was effective and that any killed bacteria
retained on the monolayer had a much reduced effectiveness in
stimulating cytokine production.
Time Course of Cytokine Secretion in pCEP and pCEP-R Cells
We measured the kinetics of the secreted cytokines 6, 12, 24, 36, and 48 h after an initial incubation with 109 cfu of PAO1 or with TNF-
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We also examined the time course of production of GM-CSF, an epithelial cell-derived neutrophil chemoattractant that enhances neutrophil survival by delaying apoptosis. (3, 29). There was no increase in GM-CSF secretion after 6 h from normal cells, but there was a continuous secretion of GM-CSF from the CF-phenotypic cells over 24 h, accentuating the differences in the epithelial cell responses over time.
The mixture of TNF- and IL-1
, potent proinflammatory cytokines,
stimulated production of IL-8, IL-6, and GM-CSF in both cell lines. For
IL-8, the only significant difference between the cell lines occurred
at the 24-h time point (results of 13 combined experiments normalized
to pCEP-R PAO1-stimulated values: pCEP, 106 ± 11.037; pCEP-R,
189.87 ± 23.544; P = 0.013; Fig.
4A), and there was no
continued secretion from the CF cells between 24 and 48 h. IL-6
secretion from the CF-like cells was significantly increased at all
time points after TNF-
-IL-1
stimulation (6, 24, 36, and 48 h, P
0.001; 12 h, P = 0.010),
and there was no further accumulation of IL-6 from the normal cells
after 6 h (Fig. 4B). GM-CSF secretion continued to
increase between 6 and 24 h from pCEP-R cells, yet the pCEP cells
did not continue to secrete this cytokine 6 h after treatment,
resulting in a very significant difference at 24 h
(P
0.001; Fig. 4C).
RANTES is a potent chemoattractant produced by lung epithelial cells
for various important inflammatory cells, including eosinophils, and is
involved in the pathophysiology of inflammation-associated lung injury
(26). We found that neither the pCEP nor pCEP-R cell line
responded with RANTES secretion when stimulated with PAO1 (Fig.
5), yet TNF- and IL-1
elicited a
response that did not differ between the CF-phenotypic and non-CF cell
lines (n = 3 experiments).
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Glucocorticoid Inhibition of IL-8 and IL-6 Secretion of pCEP and pCEP-R Cells
Glucocorticoids exert multiple anti-inflammatory activities, including the inhibition of lymphocyte migration and inhibition of transcription of cytokines dependent on nuclear factor-TNF--IL-1
-stimulated IL-8 responses were inhibited by
dexamethasone at all time points observed (Fig. 4A),
inhibiting the secretion by both normal and CF-phenotypic cells to
approximately the same level, eliminating the excess secretion from
pCEP-R cells. Dexamethasone did not inhibit the IL-6 response of normal
cells to TNF-
-IL-1
stimulation but inhibited the IL-6 secretion
from the pCEP-R cells so that over time there was no increase in
secretion (Fig. 4B).
16HBE14o Antisense and Sense Cell Lines and
Cytokine Production
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Apical application of TNF--IL-1
stimulated similar amounts
of IL-8, IL-6, and GM-CSF secretion from the sense and antisense lines
measured 24 h after the original induction, although this stimulated release was less than that from PAO1 stimulation. However, the TNF-
-IL-1
stimulation of RANTES was significantly greater in
normal cells (P
0.001; Fig. 6B).
Glucocorticoid Inhibition of Cytokine Secretion
Dexamethasone significantly inhibited IL-8, IL-6, and GM-CSF release from 16HBE14oPolarity of Cytokine Secretion
We did not observe consistent excess cytokine secretion to either surface before or after stimulation. Ratios of apical to basolateral secretion did not differ significantly from 1. However, one might speculate that in the native condition in vivo, where apical surface fluid volume is very small, higher local concentrations of cytokines might result on the apical surface if the same amount of cytokine was secreted in both directions. Because, in our culture model, tight monolayers do not permit transepithelial passage of cytokines, one might speculate that such high concentrations in airway surface liquid might be sustained. ![]() |
DISCUSSION |
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Airway epithelial cells of the CF phenotype produce significantly more IL-8, IL-6, and GM-CSF in response to several different isogenic mutants of the laboratory strain of Pseudomonas PAO1, including those lacking pilin and both pilin and flagellin, than comparable normal cells. The time course of response differs between the normal and CF-phenotypic cells. For these three cytokines, the CF-phenotypic cells continued to increase production long after the normal cells had ceased or slowed production. With a low-level or brief stimulus or at early time points, the CF phenotype cells had a similar cytokine production compared with that in the normal cells. However, with more sustained stimuli or longer response times, the CF-phenotypic cell lines continued to produce large quantities of cytokines well after the normal cells had ceased or slowed production.
One possible explanation for the differences we observed is that the CF
cell lines bind Pseudomonas to a greater extent than normal
cells via pilin-asialo-GM1 ligation, and it is the
activation of signal transduction through this pathway that produces
the excessive response to Pseudomonas in the CF-phenotypic
cell lines. Indeed, stimulation of either normal or CF-phenotypic cell
lines with mutants of PAO1 lacking pilin produces less than half the cytokine response of intact PAO1, and the mutants lacking pilin and
flagellin produce still less (3-8%) IL-8 and no IL-6 at all. However, if the pilin-asialo-GM1 interaction were the sole
cause of the excess cytokine production in CF-phenotypic cells, then stimulation with PAO1 mutants lacking pilin, which bind to pCEP and
pCEP-R cells to the same extent (2), should stimulate
cytokine production to a similar extent in normal and CF-phenotypic
cells. Actually, pilin-negative and pilin- and flagellin-negative
mutants stimulate production of greater amounts of IL-8 and IL-6 in the CF-phenotypic cells compared with normal cells. Thus there must be
factors in addition to excessive asialo-GM1 stimulation
that account for the excess cytokine production in response to
Pseudomonas in the CF cells. Because the CF-phenotypic and
control cell lines do not differ with respect to cytokine production
without stimulation, the differences must reside in the response to
stimulation. Although the differences are most evident (for IL-8 at
least) in the response to Pseudomonas, significant
differences are also observed in the cytokine responses to
TNF--IL-1
, indicating that pilin-asialo-GM1 ligation
is not the only reason for the excessive cytokine response of the CF cells.
The temporal pattern of cytokine production is different in the normal and CF-phenotypic cell lines. Although at early time points normal and CF-phenotypic cell lines produce similar amounts of IL-8, by 24 h, the CF-phenotypic cell lines are clearly producing more. At later time points, the IL-8 accumulation in CF-phenotypic cells continues, but it levels off in the normal cells. This phenomenon is not due to increased intracellular retention of IL-8 in the normal cells because intracellular IL-8 represents ~10% of the total in both cell lines. Similarly, IL-6 and GM-CSF production in normal cells is maximal by only 6 h but continues to accumulate in CF cells over 24-48 h.
It is possible to limit the accumulation of inflammatory cytokines
pharmacologically. Dexamethasone inhibits TNF--stimulated IL-6,
GM-CSF, and IL-8 accumulation in both normal and CF cell lines, but
when the cytokines are stimulated with PAO1, dexamethasone has a
greater effect on CF-phenotypic cells. These data suggest that the
pharmacology of the TNF-
-IL-1
response is similar in normal and
CF-phenotypic cells but that the pharmacology of the PAO1 response is
different. Different signaling pathways may be activated or the mix of
pathways may be different in normal and CF cell lines.
Our observations in vitro are consistent with several clinical observations. Our finding that the increased cytokine production in response to Pseudomonas is sustained much longer in CF-phenotypic cells than in non-CF cells is consistent with a report (12) that infants with CF, even those with negative bacterial cultures, have elevated IL-8 in bronchoalveolar lavage fluid. It is possible that these patients' cytokine response to a previous infection persists for a very long time, well after the stimulus is removed (12, 18), just as we observed in vitro. Our observation that bacterial stimulus induces a much greater cytokine response from CF epithelial cells than from normal epithelial cells is consistent with the observations of Noah et al. (18) that CF children have higher IL-8 in bronchoalveolar lavage fluid for any given lung burden of organisms. The excess response of both the chemoattractant IL-8 and the neutrophil survival factor GM-CSF in CF airway epithelial cells might contribute to the exuberant neutrophil accumulation in the CF airway, and the relative lack of RANTES contributes to the relative paucity of eosinophils at this site.
In vitro studies of inflammatory cytokine production in normal and CF
airway epithelial cells have had mixed results. Although many studies,
especially those with cells from submucosal glands (11,
27), reported increased IL-8 production in response to various
stimuli (or even at the basal state) in CF compared with normal
samples, another study (24) found similar cytokine
profiles in normal and CF cell lines, and in a recent report
(16), CF cells are reportedly deficient in cytokine
production. Our results may shed some light on these differing results
because they show that distinguishing between normal and CF-phenotypic
cell lines depends on the specific stimulus, the intensity of the
stimulus, and the time after the stimulus is applied as well as the
specific cytokine under study. For IL-8, there is no significant
difference between normal and CF-phenotypic cells in the response to
PAO1 at early time points, but by 24 h and later after exposure,
the CF-phenotypic cells clearly have a marked increase in IL-8
production compared with normal cells. A briefer duration of PAO1
exposure and lower concentrations of bacteria applied to the cells also make it more difficult to reliably detect differences between normal
and CF-phenotypic cells. For IL-8, differences between CF and non-CF
cell lines in response to TNF--IL-1
are significant only at the
24-h time point under the conditions of the experiment. In contrast,
for IL-6 and GM-CSF, differences between normal and CF-phenotypic cells
are apparent at the 6-h time point for both PAO1 and TNF-
-IL-1
stimulation. In our studies, RANTES was not stimulated by exposure to
PAO1 but was stimulated by TNF-
-IL-1
in both normal and
CF-phenotypic cells. Although the CF cell lines produced less, the
difference was significant only for 16HBEo
cells. These
data are in general agreement with the report of Schweibert et al.
(24).
Another possible explanation for the variability of the earlier results
is the use of cell lines that differ in more than just CFTR expression
or function to make the comparisons. Our cell lines were constructed
from a SV40-transformed normal human tracheal epithelial cell line,
9/HTEo, or a human bronchial epithelial cell line,
16HBEo
. For the 9/HTEo
cells, transfection
with empty vector (pCEP4) did not alter the chloride transport
properties of the cells, although the clonal selection process did make
them more uniform. Transfection with pCEP-R, an episomal expression
vector containing the R domain of CFTR, completely eliminated
cAMP-stimulated chloride transport (which is also eliminated by
antisense to CFTR) (19). In the planar lipid bilayer
system, the R domain interacts with wild-type CFTR to prevent chloride
transport, which seems the mostly likely mechanism for this effect in
the 9/HTEo
cells (15). The mRNA for CFTR
continues to be expressed in R domain-transfected cells, and
cotransfection experiments in a heterologous system indicate that CFTR
processing is unaffected by free R domain protein (14,
19). Because pCEP4 is an episomal, nonintegrating vector,
insertional activation or inactivation of particular genes is unlikely.
Thus the cell lines we studied are likely well matched except for the
function of CFTR, and the CF-phenotypic cell line continues to express
and process its native CFTR normally. The 16HBEo
cell
lines were transfected with sense or antisense to CFTR and selected for
presence of the plasmid by PCR, Southern blot, appropriate CFTR
physiology by 36Cl efflux, and Ussing chamber experiments.
Thus they are likely to be quite similar except for CFTR activity. One
study (24) used a CF cell line (IB-3) corrected with CFTR
with an NH2-terminal truncation (C38 cells). Although this
mutant restored chloride transport, protein interactions involving the
NH2 terminus were lost. Another study (1) has
been done with corrected CF cell lines that may overexpress CFTR and
not be representative of the native state, with cell lines immortalized
from different people whose genetic complements outside the CFTR
genotype may differ profoundly or with primary cell lines from
different people. Indeed, polymorphisms in the promoter regions of some
cytokine genes, which cause profound changes in the amount of cytokine
produced, have been proposed as modifiers of the CF phenotype
(9). It is thus especially important to control the other
genes in the cell lines to be compared if the impact of CFTR activity
is to be assessed.
Although our two cell line pairs have advantages as CF models, they
also illustrate that cytokine responses may be cell line specific. For
example, although there is a brisk GM-CSF response in the
9/HTEo cell lines and the CF-phenotypic cell line
outstrips the normal in the 16HBEo
lines, the response is
quantitatively less, and there is no difference between CF and non-CF
cell lines. Model systems can be selected for their suitability to
study relevant activities; concordance between different models gives
more confidence in the conclusions.
Our results in these two cell lines also suggest that it is not misprocessing of mutant CFTR that entrains the excess cytokine production in CF airway epithelial cells. In neither of these cell lines is mutant CFTR present. Expression of the R domain does not increase endoplasmic reticulum retention of CFTR in a heterologous system (14). Thus our data suggest that the excess cytokine responses are a consequence of impaired cAMP-dependent chloride transport.
We conclude that two different airway epithelial cell lines in culture, remote from the inflammatory milieu of the CF airway, which have been converted to CF-phenotypic cells either by overexpression of the R domain or by expression of antisense constructs, have excessive cytokine responses to bacterial stimulation. These observations in engineered cell lines with no mutant CFTR make it likely that the excessive cytokine responses are related to impaired CFTR activity and not to a particular CF genotype. Moreover, one CF-phenotypic cell line expresses wild-type CFTR, making it unlikely that the absence of protein interactions with wild-type CFTR is critical for excess cytokine response. The pattern of response suggests that mechanisms that limit the inflammatory response in normal cells are absent or attenuated in the CF cell lines but that anti-inflammatory agents such as steroids, which are moderately effective in patients with CF in limiting the inflammatory response, are effective in this cell model as well. These well-matched cell systems may be useful for further dissection of the proinflammatory responses in CF airways and for evaluating potential therapeutic agents.
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ACKNOWLEDGEMENTS |
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We thank Alice Prince for the Pseudomonas aeruginosa
strains, Aura Perez for the 9/HTEo cell lines, and our
Cystic Fibrosis Center Inflammatory Mediator Core for expert technical services.
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FOOTNOTES |
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This work supported by National Heart, Lung, and Blood Institute Grants HL/DK-49003 and HL-60293 and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-27651.
Address for reprint requests and other correspondence: D. Kube, CWRU Dept of Pediatrics, BRB 835, 2109 Adelbert Rd., Cleveland, OH 44106-4948 (E-mail: dmk8{at}po.cwru.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 February 2000; accepted in final form 13 October 2000.
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