| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Laboratory of Clinical Investigation III,*
Department of Pediatrics, University Hospitals, Geneva, Switzerland;
the Division of Medical Genetics,
University
Medical School, Geneva, Switzerland; and Transgène
SA,
Strasbourg, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
F508 CFTR cDNA, or the
ß-galactosidase gene. The number of PMN migrating in response to
N-formyl-Met-Leu-Phe across inverted CF15
monolayers expressing ß-galactosidase was similar to that seen across
CF15 monolayers rescued with CFTR, whatever the
proportion of cells expressing the transgene. Moreover, PMN
migration across monolayers expressing various amounts of mutated CFTR
was not different from that observed across matched counterparts
expressing normal CFTR. Finally, PMN migration in response to
adherent or Pseudomonas aeruginosa was equivalent across
CF and corrected monolayers. The possibility that mutated CFTR may
exert indirect effects on PMN recruitment, via
an abnormal production of the chemotactic cytokine
interleukin-8, was also explored. Apical and basolateral
production of interleukin-8 by polarized CF cells expressing mutated
CFTR was not different from that observed with rescued cells,
either in baseline or stimulated conditions. CF15 cells
displayed a CF phenotype that could be corrected by CFTR-containing
adenoviruses, because two known CF defects,
Cl- secretion and increased P. aeruginosa
adherence, were normalized after infection with those viruses.
Thus, we conclude that the presence of a mutated CFTR does not
per se lead to an exaggerated inflammatory response of
CF surface epithelial cells in the absence or presence of a bacterial
infection.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To explore these questions, we developed an in vitro model
to measure PMN migration in the physiological direction, across a
tight-polarized human CF airway epithelial cell line, the
CF15 cells,8
corrected or not with
CFTR. Matched CF and non-CF monolayers were created by infecting the CF
epithelial sheets with adenoviral vectors containing either the
wild-type or F508 CFTR cDNA, or a ß-galactosidase gene reporter.
Our data show that a similar number of PMN migrated across the
different types of monolayers, and that no difference in production of
IL-8 was observed whatever the proportion of cells expressing the
transgenes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The CF15 human nasal airway epithelial cell
line, derived from a CF patient homozygous for the F508 mutation,
was transformed and characterized by Jefferson et al.8
CF15 cells were passaged once a week, plated in
flasks coated with human placental collagen IV (50 µg/ml) and
cultured in Dulbecco’s minimal essential medium/Ham F-12 (3:1)
supplemented with 10% fetal calf serum (FCS) and seven growth
factors.8
For culture on permeable filters, the cells were
seeded at a density of 0.6 x 106
on
1-cm2
polycarbonate 3-µm-pore filters
(Transwell inserts, Costar, Badhoevedorp, The Netherlands). When airway
cells were seeded on inverted inserts, 200 µl of medium containing
the cells were disposed on the lower surface of the filter and the
cells were allowed to attach overnight at 37°C, before turning the
inserts again. PMN migration and transepithelial electrical
measurements were performed on day 8 after seeding. The cells were
regularly tested for the presence of mycoplasma, and only pathogen-free
cells were used for this study. All tissue culture supplies were
obtained from Life Technologies, Inc. (Basel, Switzerland), FCS was
from SeraTech (Griesbach, Germany), epidermal growth factor was from
Collaborative Biomedical Products (Bedford, MA), and all other reagents
were purchased from Sigma Chemical Co. (Buchs, Switzerland).
Recombinant Adenoviruses
Transgenes Driven by Rous Sarcoma Virus (RSV) or Cytome Galovirus (CMV) Promoters
The replication-defective adenoviruses were derived from the human
adenovirus serotype 5, and contained either the CFTR cDNA controlled by
the RSV promoter (AdTG 6429) or the CMV promoter (AdTG 6418), the CMV
promoter-driven eGFP (enhanced green fluorescent protein) gene (AdTG
6297), or the RSV promoter-driven lacZ gene.9
All vectors were constructed as infectious plasmids by homologous
recombination in E. coli as described.10,11
The
vectors contain a deletion in E1 ( nucleotides 459-3327) and in E3
( nucleotides 28592–30470). All vectors have the transgene
incorporated in place of the viral E1 gene. For the generation of
viruses, the viral genomes were released from their respective plasmids
by PacI digestion and transfected into E1-complementing 293
cells as described.10
Viral stocks were prepared from the
transfected cells, purified, and stored in viral storage buffer (1
mol/L sucrose; 10 mmol/L Tris-HCl, pH 8.5; 1 mmol/L
MgCl2; 150 mmol/L NaCl; and 0.005% Tween 80).
Transgenes Driven by the ß-Actin Promoter
These serotype-5-derived recombinant adenoviruses contained either
the wild-type CFTR (Ad CB CFTR) or the mutated CFTR (Ad CB F508)
cDNA controlled by a CMV enhancer/ß-actin promoter. They were
engineered by Yang et al12
and provided by the Vector Core
of the Institute for Human Gene Therapy of the University of
Pennsylvania Health System (Philadelphia, PA).
Viral Infections and 5-Bromo-4-Chloro-3-Indolyl-ß-D-Galactopyranoside (X-Gal) Staining
Confluent CF15 monolayers on inserts were infected with adenoviruses on the day 6 of culture for 16 hours (unless otherwise indicated) with 200 µl of OptiMeM (Life Technologies, Inc.) supplemented with 2.5% FCS and containing or not the viruses at a multiplicity of infection (MOI) of 1 to 1000 (for RSV- and CMV-based viruses, a MOI of 1 = 1 infectious unit/cell (~1/2 plaque-forming unit/cell), whereas for ß-actin-based viruses, a MOI of 1 = 1 plaque-forming unit/cell). For infection of inverted monolayers, the inserts were placed directly on a 200-µl drop of medium containing the viruses. The cells were then rinsed and cultured for an additional 24 hours in normal medium before the experiment. Expression of ß-galactosidase was detected by light microscopy as nuclear-localized blue staining using the X-Gal substrate. Infected cells were rinsed with phosphate-buffered saline (PBS), fixed with 0.5% glutaraldehyde for 10 minutes, and incubated for 6 hours at 37o with 1 mg/ml X-Gal, 5 mmol/L K+ ferricyanide, 5 mmol/L K+ ferrocyanide, and 1 mmol/L MgCl2 in PBS.
Transepithelial Electrical Measurements
Right-side-up or inverted CF15 monolayers on
inserts were placed in a modified and thermostatized Ussing chamber
(manufactured by J. Pahud, CHUV, Lausanne, Switzerland) containing
Hanks’ balanced salt solution (HBSS) supplemented with 1.3 mmol/L
Ca2+, 1 mmol/L Mg2+, and 10
mmol/L (N-[Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid])
(HEPES), pH 7.4 (HBSS+), heated to 37°C.
Transepithelial potential difference (V) and short
circuit current (Isc) were
measured with apical and basolateral agar bridges connected to Ag/AgCl
electrodes of a current/voltage clamp apparatus (VCC 600, Physiological
Instruments, San Diego, CA). Transepithelial electrical resistance
(TER) was calculated from Ohm’s law, after measuring the difference in
current induced by voltage pulses of 1 mV. The sequential electrical
responses to various drugs were determined: 100 µmol/L amiloride,
added to the mucosal side of the epithelium, mucosal
Cl- replacement with HBSS+
containing 3.6 mmol/L Cl- (chloride replaced by
gluconate), 50 µmol/L forskolin, and 200 to 500 µmol/L
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The TER of
CF15 monolayers varied from week to week (from
200 to 800 
·cm2), but all monolayers
displayed a similar TER at a given time in culture.
Whole-Cell Patch-Clamp Recordings of CFTR-Dependent Cl- Currents
CF15 cells seeded on Petri dishes were infected for 16 hours with either Ad RSV CFTR or Ad RSV ß-galactosidase at a MOI of 500. Whole-cell patch-clamp experiments were performed as described.13 Briefly, the cells were superfused with a solution containing 136 mmol/L NaCl; 4 mmol/L KCl; 1 mmol/L CaCl2; 1 mmol/L MgCl2; 2.5 mmol/L glucose; and 10 mmol/L HEPES, pH 7.4, supplemented with 100 µmol/L amiloride. Patch electrodes were filled with a solution containing 1 mmol/L NaCl; 138 mmol/L KCl; 2.9 mmol/L CaCl2; 5.5 mmol/L EGTA; 3 mmol/L MgATP; 0.1 mmol/L GTP; and 10 mmol/L HEPES, pH 7.2. Cells were held at 0 mV and membrane capacitance was determined using the automatic compensation procedure of an EPC-9 patch-clamp amplifier (Heka Elektronic, Lambrecht, Germany). Every 5 seconds, cells were depolarized from the holding potential to -70 mV for 600 milliseconds. CFTR-dependent currents were then stimulated with 50 µmol/L forskolin (or the solvent ethanol) added to the superfusing solution.
Isolation of Neutrophils and Transmigration Experiments
PMN from buffy coats of citrated blood collected from healthy donors were isolated by dextran sedimentation followed by density gradient centrifugation in Ficoll-Hypaque (Amersham, Uppsala, Sweden).14 Contaminating red blood cells were lysed by hypotonic shock with cold water for 40 seconds, PMN were washed twice and resuspended at a concentration of 107/ml in HBSS+ (without phenol red). The inserts with monolayers were lifted from wells, drained of media by inverting, and gently rinsed by dipping in HBSS+ heated to 37°C. They were then placed in new 12-well plates with 1 ml HBSS+ containing the chemotactic factor N-formyl-Met-Leu-Phe (fMLP) or solvent in the lower compartment, before adding 5 x 106 PMN to the upper compartment. PMN were then allowed to transmigrate for various periods of time at 37°C. For migration experiments in response to adherent bacteria, 150 µl containing 5 x 107 colony-forming units (cfu) P. aeruginosa (strain PAO1, see culture details below) were disposed on the apical surface of inverted monolayers and allowed to adhere for 2 hours. The monolayers were then turned again and placed in 12-well plates, rinsed four times with HBSS+, and PMN were added to their basolateral side.
The number of PMN having migrated into the lower compartment was quantified by coloration of the PMN-specific azurophil granule marker, myeloperoxidase, with a modification of the technique of Madara et al.15 After stopping PMN migration by placing the 12-well plates on ice, the lower surface of the inserts was rinsed 10 times with the liquid present in the lower compartment to remove attached PMN. Myeloperoxidase was then solubilized after PMN lysis by 100 µl of 10% Triton X-100, and the remaining myeloperoxidase trapped within PMN-derived DNA was dissolved by adding 20 µl of 10 mg/ml DNase (200 µg/ml final). After 15 minutes of shaking on ice, 100 µl of 1 mol/L citrate, pH 4.2, was added before transferring 100 µl of each sample in a 96-well microtiter plate and adding 100 µl of substrate (2 mmol/L 2,2'-azino-bis[3-ethylbenzthiazoline 6-sulfonic acid] diammonium; [Sigma] and 0.06% H2O2 in 100 mmol/L of citrate buffer, pH 4.2). The colorimetric reaction was stopped by adding 25 µl of 5.5% sodium dodecyl sulfate (0.5% final) and read at 405 nm after centrifugation of the plates at 500 rpm for 5 minutes. Standards were made with serial dilutions of the same PMN, in 1 ml of HBSS+, and processed in the same way as described above. The assay was linear in the range of 4 to 600 x 103 PMN/ml.
IL-8 Production by Polarized Monolayers
To determine IL-8 production at the apical or basolateral side of
the CF15 monolayers, the cells were seeded on
1-cm2
inserts and infected for 16 hours on day 6.
Ten hours after the end of infection, the monolayers were incubated
overnight with culture medium without FCS and with 0.1% BSA, before
being challenged on their mucosal side with or without tumor necrosis
factor-
(TNF-) for 1 or 4 hours. Apical and basolateral
supernatants (500 µl each) were collected after 4 hours or 16 hours.
For IL-8 production in response to adherent P. aeruginosa,
the monolayers were exposed on their apical side to 5 x
107
cfu PAO1 for 2 hours. After rinsing four
times with HBSS+, the monolayers were incubated
for 4 hours with culture medium without FCS or antibiotics,
supplemented with 100 µg/ml cycloserine and 0.1% BSA, before
supernatant collection. IL-8 was measured using an ELISA kit (CLB,
Amsterdam, The Netherlands).
Pseudomonas aeruginosa Adherence
Confluent CF15 cells in 24-well plates were infected with adenoviruses for 16 hours on day 6, and bacteria were allowed to adhere 1 day after the end of infection. P. aeruginosa strain PAO1 was grown to a density of 5 x 108 cfu/ml and labeled with 35S-methionine (Amersham, Zürich, Switzerland) to a specific activity of ~5000 cfu/cpm for 15 minutes. After being washed in PBS, the bacteria were suspended in CF15 culture medium without FCS or antibiotics, supplemented with 100 µg/ml cycloserine. PAO1 was then added to confluent CF15 cells (5 x 107 cfu/well) for 2 hours at 37°C. Unbound bacteria were removed by rinsing the monolayers three times with PBS. CF15 cells and bacteria were solubilized in 0.5 ml sodium dodecyl sulfate 2% by shaking at 110 rpm and scintillations counted. The experiments were done in quadruplicates.
All data are means ± SEM and compared using a two-tailed unpaired Student’s t-test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To determine whether PMN migration across electrically-tight CF
airway monolayers is affected by the orientation of the epithelium,
CF15 cells were seeded on either side of the
permeable inserts, before being exposed to PMN that were allowed to
cross the monolayers in response to fMLP. Thus, PMN migrating across
right-side-up monolayers first encounter their apical membrane, whereas
those migrating across inverted monolayers move in the physiologically
relevant direction, ie, from the basolateral to the apical side of the
monolayer. Figure 1
shows that the number
of PMN having migrated across right-side-up monolayers was 27% to 54%
lower than that observed across inverted monolayers, in conditions of
similar initial transepithelial resistance (TER). Many experiments of
comparison were performed, but only those in which the TER of the
monolayers was not significantly different were interpreted and
reported.
|
To compare PMN migration in the physiologically relevant
direction, a pair of matched CF and non-CF airway monolayers was
created. For this purpose, inverted CF15
monolayers were infected with adenoviruses containing the CFTR (Ad RSV
CFTR) or ß-galactosidase (Ad RSV ßgal) gene. We determined whether
cyclic adenosine monophosphate (cAMP)-mediated
Cl- secretion was restored in the group of cells
infected with Ad RSV CFTR using various drugs (Figure 2A)
. This typical recording shows the
correction of defective transepithelial Cl-
secretion in a monolayer exposed to Ad CFTR, with an important increase
in Isc in the presence of a low
Cl--containing solution. The cAMP-stimulating
drug forskolin promoted a further increase of the response. In
contrast, the cells infected with Ad ßgal displayed only a small
increase of Isc in response to low
Cl-, without any change when challenged with
forskolin (Figure 2B)
. The fact that inverted monolayers maintained
their polarity when seeded on the lower side of the filter is shown by
comparing Figure 2A to 2C
. The latter shows the transepithelial
Cl- current obtained with a
CF15 right-side-up monolayer infected with Ad RSV
CFTR. The trace mirrored that obtained with inverted cells, because
Isc was of opposite sign. This
demonstrates that the direction of transport of ions across the
epithelium is maintained whatever the orientation of the monolayer. The
Cl- channel blocker
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid significantly reduced
the forskolin-mediated effect, indicating that the observed increase of
Isc was due to the activation of
Cl- channels.
|
shows a typical recording of
Cl- currents in a CF15
cell infected with Ad RSV CFTR or Ad RSV ßgal. The combined data
obtained with all cells tested (Figure 3B)
reveal that, in 5 out of 8
cells infected with Ad CFTR, Cl- current was
already increased at the basal level (-20.89 ± 7.4 pA/pF,
mean ± SEM, n = 8), whereas no increase was
observed in the Ad ßgal-infected cells (-1.36 ± 0.19,
n = 8). Likewise, when stimulated with forskolin, 6 out
of 8 cells displayed an increase in Cl-
conductance in the former group ( = 40.73 ± 24.5,
n = 8), whereas none responded to this agent in the
latter group ( = 0.5 ± 0.23, n = 8).
|
Comparison of PMN Migration across Inverted CF15 Monolayers Corrected or Not with CFTR
We determined whether the presence of a functional CFTR affects
cellular processes involved in PMN migration. Inverted
CF15 monolayers were infected with various MOI of
two different pairs of viruses containing either the RSV (Figure 4,A and B)
or the CMV (Figure 4, C and D)
promoter, and assayed for chemotactic migration. Figure 4A
shows that a
similar number of PMN migrated across monolayers of cells infected with
Ad RSV CFTR or Ad RSV ßgal, at all of the viral MOI tested. Moreover,
no significant difference of migration could be observed between the
monolayers infected with increasing MOI of a given virus. Ad RSV
CFTR-infected cells exhibited a response to low
Cl- and forskolin that increased with the virus
load (Figure 4B)
, whereas this response remained almost undetectable in
monolayers infected with Ad ßgal. The level of transgene expression
in the latter group also varied with the MOI, because the number of
blue cells increased from ~20% to 90%. PMN migration experiments
were also performed with monolayers expressing higher levels of
transgenes (Figure 4, C and D)
. Likewise, the number of PMN having
migrated across monolayers infected with Ad CMV CFTR was not different
from that having crossed their counterpart infected with Ad CMV eGFP.
The group of cells expressing CFTR displayed a MOI-dependent increase
of response to low Cl- and forskolin ~2.5- to
3-fold higher than that seen with the same MOI of Ad RSV CFTR (Figure 4, D and B
, respectively). Monolayers expressing eGFP did not respond
to those challenges, but their fluorescence increased with the viral
load (data not shown).
|
Comparison of PMN Migration across CF15 Monolayers Expressing Various Amounts of Normal or Mutated CFTR
To determine whether the amount of mutated CFTR plays a role in
cellular events involved in the migratory process, inverted
CF15 epithelia were infected with increasing MOI
of matched adenoviruses containing either the normal or F508 CFTR
cDNA (Ad CB CFTR and Ad CB F508, respectively). As shown in Figure 5
, no significant difference in the
number of transmigrated PMN was observed across monolayers expressing
various quantities of either normal or mutated CFTR. The data were also
similar to those obtained across uninfected monolayers
(n = 2 experiments, data not shown). These
adenoviruses also displayed a very efficient level of infection,
because the response to low Cl- and forskolin
showed a dose-dependent increase when the Ad CB CFTR viral load was
increased; indeed, over a MOI range of 25 to 500,
Isc increased from 22 to 243
µA/cm2.
|
To determine whether PMN migration in the physiological direction
in response to adherent P. aeruginosa is influenced by the
presence of a functional CFTR, experiments were done with monolayers
rescued or not with CFTR and challenged with PAO1 (Figure 6)
. Our data show that the presence of
adherent bacteria on the apical surface of monolayers expressing CFTR
does not induce a difference in PMN migration, as compared to that seen
across monolayers either uninfected or expressing ßgal.
|
Polarized CF15 monolayers corrected or not
with CFTR were tested for IL-8 secretion from their apical or
basolateral side (Figure 7)
. IL-8 present
in the supernatant collected from monolayers infected or not with Ad
RSV CFTR or Ad RSV ßgal showed a dose-dependent increase in response
to TNF-, in both the apical (Figure 7A)
and basolateral (Figure 7B)
compartment. However, no significant difference in IL-8 secretion was
detected between cells expressing CFTR or ß-galactosidase, in the
presence or absence of TNF-, in either compartment. The presence of
the adenoviral vector did not significantly affect IL-8 production in
cells challenged with 10 nM TNF-, as compared to parental uninfected
cells. The adenoviruses did not either affect baseline IL-8 production
(n = 2 experiments done in quadruplicate, data
not shown). Other experiments of IL-8 secretion done with monolayers
infected with adenoviral MOI ranging from 0 to 1000 demonstrated that
the production of this cytokine in Ad CFTR-infected cells was similar
to that observed in Ad ßgal-infected cells, with no increase
paralleling the increasing adenoviral load (data not shown).
Experiments of the same type were done with monolayers expressing
similar amounts of either normal or mutated CFTR (Figure 7, C and D)
.
The profile of IL-8 secretion by CF15 cells
infected with either Ad CB CFTR or Ad CB F508 was comparable,
demonstrating that the production of this cytokine is not affected by
the presence or amount of either type of CFTR. To measure IL-8
production in response to a bacterial stimulus, polarized monolayers
infected with Ad RSV CFTR or ß-galactosidase (MOI 500) were
challenged with PAO1 on their apical surface. The data obtained with
two experiments show that, after 4 hours, no difference in IL-8
production in either the apical or basolateral supernatants was
observed between both groups of monolayers (data not shown). These
preliminary data do not confirm those obtained by DiMango et
al.7
This may be due to the fact that IL-8 production has
already reached a plateau in CFTR-corrected monolayers, and is
therefore not further stimulated in uncorrected ones. Alternatively,
there may be conditions in which a difference in IL-8 production
between both groups occurs, but this would necessitate a thorough
investigation that is beyond the scope of this project.
|
To confirm that CF15 cells indeed displayed
a CF phenotype, experiments were done to test the adherence of P.
aeruginosa to the plasma membrane, a function reported to be
increased in CF.16-18
Figure 8
shows that there was no difference of
adherence between uninfected cells or cells infected with Ad RSV
ßgal. However, a significant decrease of adherence was observed with
the cells rescued with CFTR, in each of the three experiments
performed. This demonstrates that the CF15 cells
display a CF phenotype that can be corrected by CFTR.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
F508 mutation, the production of endogenous mutated CFTR
mRNA, as well as the lack of response to forskolin measured on
Isc, have been reconfirmed (data not
shown). In our model, PMN migration was shown to be greater in the physiological direction than in the apical-to-basolateral direction. This is in agreement with other studies suggesting that the polarity of the epithelium plays a role in PMN migration across intestinal monolayers such as T84 epithelial cells21-23 and airway epithelial barriers.24-26 However, no comparisons of the initial TER of inverted and right-side-up monolayers were done in the former studies, whereas only qualitative TER measures were provided in the latter ones. To address this issue, we compared PMN migration across right-side-up and inverted CF15 monolayers with similar initial TER and showed that the increase of migration observed in the physiological direction still persisted. This increase may be related to a difference in the polarity of epithelial receptors. Alternatively, CF15 cells may also have features similar to those of T84 intestinal epithelial cells, which were shown to display a luminal retention signal influencing PMN migration asymmetrically by cytoskeletal reorganization.23
The fact that airway inflammation was observed in infants and children with CF even in the absence of detectable pathogens4-6 has led to the suggestion that the CFTR defect may play a direct role in the initiation of PMN immigration into CF airways. To compare PMN migration across CF and control monolayers, the use of ex vivo primary CF airway epithelial cultures makes it difficult to distinguish between the responses due to the primary defect or secondary to the microenvironment to which the cells were formerly exposed. To address this issue, we set up an in vitro model of a matched pair of CF and control monolayers differing only by the presence or absence of a normal CFTR. With this model, it was also possible to create matched pairs expressing increasing amounts of transgene by varying the adenoviral load. Moreover, within a given experiment, the monolayers displayed a comparable tightness, because they originated from the same source. Our data demonstrated that a similar number of PMN migrated across inverted monolayers of CF15 cells expressing wild-type CFTR or ß-galactosidase, whatever the level of transgene expression. In the group of monolayers infected with Ad RSV ßgal, a MOI of 500 generally yielded ~50% of blue cells when detected by X-Gal staining. This value is certainly underestimated, because the sensitivity of detection with this method was reported to be relatively low.27 In the group of monolayers infected with Ad RSV CFTR, the defective Cl- current was corrected to levels observed in non-CF airway monolayers at a MOI of ~100 to 500, depending on the experiment. PMN migration experiments were therefore always performed with various MOI. This allowed us to confirm the lack of difference in PMN migration across monolayers displaying from 0 to >90% of transgenic cells. We therefore conclude that correction with CFTR does not lead to a difference in PMN migration across CF airway epithelial cells.
Although part of the cellular manifestations of the CF phenotype has
been attributed to the absence of a functional CFTR in airway
epithelial cells, other ones have been suggested to be the consequence
of the presence of a mutated CFTR. For example, an abnormal activation
of nuclear factor-
B in CF bronchial epithelial cells has been
reported by DiMango et al7
and suggested to be a
consequence of cell stress caused by the accumulation of mutant CFTR in
the endoplasmic reticulum. To determine whether the amount of F508
CFTR affects intracellular pathways involved in the interactions of
epithelial cells with PMN, the migratory process was done across
CF15 monolayers expressing various amounts of
either normal or F508 CFTR. Because no significant difference was
observed in the number of PMN having crossed the different types of
monolayers, we suggest that there is no direct link between the CF
genetic defect and the process of PMN migration across airway
epithelial barriers, in the absence of pathogens.
The fact that adherent P. aeruginosa promoted no difference in PMN migration across monolayers rescued or not with CFTR further strengthens the results of migration we obtained in response to fMLP. Indeed, those bacteria were chosen as a specific stimulus encountered in CF patients. The data acquired with our model suggest that the combined presence of a mutated CFTR and of P. aeruginosa is not enough to explain the excessive amount of PMN found in CF airways colonized by these bacteria. This also further underlines the complexity of the CF inflammatory response in vivo.
We then explored the possibility that CFTR may exert indirect effects
on PMN recruitment, via an abnormal production of the potent
chemotactic cytokine IL-8. Whereas some studies have reported high
concentrations of this cytokine in bronchoalveolar lavage fluid from
uninfected infants and children with CF, as compared to
controls,4-6
another study found no difference of IL-8 in
bronchoalveolar lavage from uninfected infants with or without CF, both
levels being very low.28
Contradictory data were also
published with in vitro experiments. Indeed, subcultures of
primary human CF bronchial gland cells were recently shown to
spontaneously release much higher levels of IL-8 than non-CF
ones,29
although no difference of baseline or stimulated
IL-8 production was observed in primary or immortalized airway
epithelial cells, in other studies.30-32
Massengale et
al33
recently even reported a defective IL-8 secretion by
CF airway cells. We addressed this issue with polarized
CF15 cells expressing various amounts of either
normal or mutated CFTR cDNA, or the ß-galactosidase transgene. The
cells were grown as tight monolayers on filters because differentiation
of epithelial cells was shown to regulate CFTR
expression,34
and because it is possible that part of the
effects of CFTR dysfunction on intracellular processes are not revealed
unless cell polarization occurs. Our data demonstrate that correction
of CF15 monolayers with CFTR did not induce a
change in the pattern of IL-8 secretion in either the basolateral or
apical compartments. The fact that monolayers overexpressing F508
CFTR displayed a similar profile of IL-8 production as matched
counterparts rescued with normal CFTR provides hitherto unreported
evidence that the accumulation of mutated CFTR in the ER does not lead
to an aberrant synthesis of this cytokine in polarized airway surface
epithelial cells.
To confirm the validity of our model, in particular the fact that CF15 cells display a biological dysfunction (other than the Cl- secretion defect) that can be corrected by CFTR, parallel experiments were done as a positive control. We tested the adherence of P. aeruginosa, which has been shown to be increased at the surface of epithelial CF cells,16-18 and observed that the number of bacteria bound to CF15 cells was indeed corrected by CFTR-containing adenoviruses.
In summary, our data suggest that there is no intrinsic defect of the airway surface epithelial cells predisposing to PMN infiltration into the lung. Although these results are consistent with the studies of Armstrong et al,28 they do not exclude other possibilities. For example, the inflammation observed in CF airways4-6 may have been initiated by poorly cleared airborne particles and/or members of the respiratory flora. Alternatively, CF cells other than surface epithelial cells may deliver a signal mediating PMN transmigration.29 We conclude that the presence of a mutated CFTR does not per se lead to an exaggerated inflammatory response of CF surface epithelial cells.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by Swiss National Foundation for Scientific Research grants 32–36338.92 and 32–45845.95 .
Accepted for publication December 21, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B by adherent Pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells. J Clin Invest 1998, 101:2598-2605[Medline]
in a highly polarized fashion. J Cell Biol 1993, 120:785-798 and Respiratory Syncytial Virus stimulation. Am J Respir Cell Mol Biol 1998, 19:210-215This article has been cited by other articles:
 |
|
 |
|
  K J Mackerness, G R Jenkins, A Bush, and P J Jose Characterisation of the range of neutrophil stimulating mediators in cystic fibrosis sputum Thorax, July 1, 2008; 63(7): 614 - 620. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Hybiske, Z. Fu, C. Schwarzer, J. Tseng, J. Do, N. Huang, and T. E. Machen Effects of cystic fibrosis transmembrane conductance regulator and {Delta}F508CFTR on inflammatory response, ER stress, and Ca2+ of airway epithelia Am J Physiol Lung Cell Mol Physiol, November 1, 2007; 293(5): L1250 - L1260. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. E. Machen Innate immune response in CF airway epithelia: hyperinflammatory? Am J Physiol Cell Physiol, August 1, 2006; 291(2): C218 - C230. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Tabary, H. Corvol, E. Boncoeur, K. Chadelat, C. Fitting, J. M. Cavaillon, A. Clement, and J. Jacquot Adherence of airway neutrophils and inflammatory response are increased in CF airway epithelial cell-neutrophil interactions Am J Physiol Lung Cell Mol Physiol, March 1, 2006; 290(3): L588 - L596. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Solic, J. Wilson, S. J. Wilson, and J. K. Shute Endothelial Activation and Increased Heparan Sulfate Expression in Cystic Fibrosis Am. J. Respir. Crit. Care Med., October 1, 2005; 172(7): 892 - 898. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. P. Hurley, D. Siccardi, R. J. Mrsny, and B. A. McCormick Polymorphonuclear Cell Transmigration Induced by Pseudomonas aeruginosa Requires the Eicosanoid Hepoxilin A3 J. Immunol., November 1, 2004; 173(9): 5712 - 5720. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N. Becker, M. S. Sauer, M. S. Muhlebach, A. J. Hirsh, Q. Wu, M. W. Verghese, and S. H. Randell Cytokine Secretion by Cystic Fibrosis Airway Epithelial Cells Am. J. Respir. Crit. Care Med., March 1, 2004; 169(5): 645 - 653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Saba, G. Soong, S. Greenberg, and A. Prince Bacterial Stimulation of Epithelial G-CSF and GM-CSF Expression Promotes PMN Survival in CF Airways Am. J. Respir. Cell Mol. Biol., November 1, 2002; 27(5): 561 - 567. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Aldallal, E. E. McNaughton, L. J. Manzel, A. M. Richards, J. Zabner, T. W. Ferkol, and D. C. Look Inflammatory Response in Airway Epithelial Cells Isolated from Patients with Cystic Fibrosis Am. J. Respir. Crit. Care Med., November 1, 2002; 166(9): 1248 - 1256. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. H. Schroeder, M. M. Lee, P. W. Yacono, C. L. Cannon, A. A. Gerceker, D. E. Golan, and G. B. Pier CFTR is a pattern recognition molecule that extracts Pseudomonas aeruginosa LPS from the outer membrane into epithelial cells and activates NF-kappa B translocation PNAS, May 14, 2002; 99(10): 6907 - 6912. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Chanson, P.-Y. Berclaz, I. Scerri, T. Dudez, K. Wernke-Dollries, L. Pizurki, A. Pavirani, M. A. Fiedler, and S. Suter Regulation of Gap Junctional Communication by a Pro-Inflammatory Cytokine in Cystic Fibrosis Transmembrane Conductance Regulator-Expressing but Not Cystic Fibrosis Airway Cells Am. J. Pathol., May 1, 2001; 158(5): 1775 - 1784. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||
