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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Mutations in cystic fibrosis transmembrane conductance regulator (CFTR), particularly the common
F508 mutation, have been associated with alterations in glycolipid sialylation and the availability of receptors for Pseudomonas aeruginosa binding. The surface properties of 9HTEo
tracheal epithelial cell
lines transfected with plasmids that overproduce the regulatory (R) domain of CFTR (pCEP-R) and lack cyclic adenosine monophosphate-stimulated Cl conductance were compared with control cell lines with
normal CFTR function. There was increased bacterial adherence to the mutant cell lines with abnormal
CFTR activity. Cell lines with overexpression of the R domain had surface properties similar to cells expressing the common F508 mutation in CF. P. aeruginosa adherence correlated with the increased numbers of asialoGM1 residues available on the surface of the epithelial cells with altered CFTR function; and
antibody to asialoGM1, a P. aeruginosa pilin receptor, was able to compete with piliated bacteria for epithelial binding sites. The pCEP-R cell lines with increased bacterial binding were also associated with increased production of interleukin-8 in response to adherent P. aeruginosa compared with cells transfected with the empty vector pCEP. P. aeruginosa pil mutants that lack the adhesin specific for the asialoGM1
receptor did not discriminate between epithelial cells with normal or deficient CFTR function. These results confirm a direct relationship between aberrant CFTR function and increased levels of apical
asialoGM1, and support the role of these asialylated glycolipids as P. aeruginosa receptors that initiate an
epithelial proinflammatory response in response to bacterial ligands.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Pseudomonas aeruginosa infection of the respiratory tract
is a characteristic finding in cystic fibrosis (CF) patients of all ages. Colonization with environmental strains of P. aeruginosa typically precedes the chronic disease associated with
the mucoid forms of the bacteria, which heralds the onset
of the decline in pulmonary function in these patients (1-
3). Although normal airway defense mechanisms usually
prevent inhaled bacteria from coming into direct contact
with the epithelial cell itself, organisms that elude the activity of antimicrobial peptides (4) and are able to broach
the glycocalyx barrier that protects the mucosal surface
may access epithelial receptor sites. P. aeruginosa can adhere to epithelial cells via pili which recognize a GalNAc
1-4Gal disaccharide exposed in asialylated glycolipids such
as asialoGM1 (5, 6). Although such asialylated receptors
are not particularly numerous on the normal epithelial surface, they are increased in areas of regenerating epithelium
(7) and are relatively more abundant in cells expressing
mutant CF transmembrane conductance regulator (CFTR)
(5, 8, 9).
Pilin-mediated attachment to asialoGM1 elicits the epithelial expression of interleukin (IL)-8 (10), an important neutrophil chemokine contributing to the airway inflammation characteristic of the early stages of CF lung disease (11). It is this polymorphonuclear leukocyte-dominated inflammatory response that is associated with much of the morbidity of CF pulmonary disease. Clinical studies suggest that excessive secretion of proinflammatory cytokines in CF airways is common (14) and may precede overt bacterial infection (12). Bacterial induction of epithelial cytokine expression is likely to contribute significantly to the overall inflammatory milieu associated with CF airway disease (15).
The increased number of P. aeruginosa receptors on the CF epithelium is related to the level of apical sialylation (9). Although deficiencies in sialylation and altered carbohydrate composition of mucin glycoconjugates have been documented in CF by several independent investigators (9, 16), exactly how defective CFTR function is related to sialylation is less well established. The sialylation defect may be due to abnormalities in the distribution of the 2,6, sialyltransferase (J. Barasch, unpublished data) as well as decreased sialyltransferase activity (16), possibly due to less acidic (and therefore suboptimal) pH in the compartment in which it is active. However, this acidification defect has not been detected by all investigators (21), and the precise mechanism remains uncertain.
CFTR is an adenosine triphosphate (ATP)-regulated
chloride channel which consists of two membrane-spanning regions, two nucleotide-binding domains that are
likely to hydrolyze ATP, and a regulatory (R) domain (22).
Phosphorylation of the R domain by a cyclic adenosine monophosphate (cAMP)-dependent protein kinase allows
channel opening (23, 24). The Cl channel is closed unless
the R domain is phosphorylated at any of several sites.
The phosphorylated protein may assume an altered conformation to allow Cl transport or the channel may open
through an electrostatic interaction (25, 26). Epithelial
cells with homozygous mutations F508 have several abnormalities related to this dysfunctional Cl channel, including aberrant trafficking of the mutant CFTR, and accumulation in the endoplasmic reticulum (ER) (22).
Human airway epithelial cells transfected with episomes that constitutively overexpress the R domain of
CFTR lack cAMP-responsive Cl channel activity (27).
Because cells with mutations in the CFTR coding sequence, such as F508, have diminished Cl channel activity and abnormalities in sialylation, we postulated that epithelial cell lines with increased expression of the R domain
might also have similar abnormalities of superficial sialylation with concomitant increase in the number of receptor
sites available for bacterial adherence. To explore further
the relationship between CFTR dysfunction and the availability of asialylated receptors for P. aeruginosa binding,
the properties of epithelial cells that overproduce the R
domain of CFTR were compared with those of control
cells transfected with empty vector.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Epithelial Cell Culture
The 9HTEo epithelial cell lines used are derived from
SV40 transformed human tracheal epithelial cells (D. Gruenert, University of California, San Francisco) (28).
The lines were transfected by lipofection with plasmids
containing inserts in a pCEP-4 vector, and their electrophysiologic properties have been well characterized (28).
9HTEo cells were also transfected with pCEP-F508, a
plasmid that expresses the F508 CFTR complementary
DNA under cytomegalovirus (CMV) promoter control. The
epithelial cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2.5 mM L-glutamine in the presence
of 40 U/ml hygromycin (ICN, Aurora, OH) in 5% CO2 at
37°C as described by Gruenert and colleagues (28). Assays
were performed on cell cultures at the same degree of confluence. Chemicals were obtained from Sigma (St. Louis,
MO) unless otherwise specified.
Bacterial strains and culture conditions. The nonmucoid laboratory isolate PAO1 used for adherence studies was grown in M9 minimal media, and cultures in late log phase were routinely used in adherence assays. PAO/NP is a pil mutant of PAO1 constructed by replacement of the wild-type pilA gene with the pilA gene derived from P. aeruginosa strain PAK, interrupted with a tetracycline cartridge (29). The phenotypic properties of PAO/NP, unrelated to the expression of pilA, should be the same as the parental strain.
Flow cytometry analysis of asialoGM1 and GM1 on epithelial cells.
Monolayers of epithelial cells, matched for
degree of confluence, were incubated with rabbit polyclonal antibody to asialoGM1, 27 mg/ml (Wako Chemical
Co., Richmond, VA) in a series of dilutions, at 4°C for 30 min. After washing with 0.5 ml of phosphate-buffered saline
(PBS) plus 0.1% bovine serum albumin (BSA), bound antibody was detected using goat antirabbit IgG (H+L[ab'2])::
fluorescein isothiocyanate(FITC)-conjugated (Caltag, San
Francisco, CA) at a concentration of one-twentieth at 4°C
for 30 min. GM1 was identified by incubating the monolayers with dilutions of the B subunit of cholera toxin 0.5 mg/ml (CTB) conjugated to FITC (List Biologicals, Campbell, CA). Anti-cadherin (Sigma) antibodies were used in
similar dilutions as controls. The incubations were done in
24-well plates in a 200 µl volume of PBS plus 0.1% BSA.
The monolayers were loosened by the addition of 0.02% ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid
(EGTA), suspended by trituration, and fixed by the addition of 0.8% paraformaldehyde. Cell fluorescence was measured using a fluorescent-activated cell sorter star flow cytometer and monitoring at 540 nm. Gates were set and the
percentage of positive cells (n = 10,000) was calculated
and corrected by subtracting the signal from positive cells
in the secondary antibody control sample. A single bound
organism is sufficient to elicit a positive signal, as verified
by fluorescence microscopy. A minimum of three separate experiments was performed and representative experiments are presented. Statistical analysis was performed using a two-tailed t test or a Z test of proportions to compare
the percentages of positive cells in each group and test the
null hypothesis that there is no difference in the means of
two similar groups in which the distribution of results is
similar to a standard normal distribution for a large number of degrees of freedom.
Quantification of bacterial adherence.
Late log phase cultures of PAO1 were washed and resuspended in PBS, and
a 5 × 108 cfu/ml inoculum (or dilutions) was applied to the
monolayers for 60 min at 37°C. Unbound bacteria were removed by three washes with PBS. Bound organisms were
detected by incubating with rabbit antisera raised to PAO1
outer membrane proteins (OMPs) and bound antibody was
detected with a secondary antibody, goat antirabbit::FITC-labeled F(ab2'). Both the primary and secondary antibodies were incubated with the monolayers at 4°C for 30 min
in PBS plus 0.1% BSA. As described above, the monolayers were loosened from the plates with 0.02% EGTA and
fluorescence was measured by flow cytometry. Since the
adherent organisms are labeled by FITC-conjugated antisera after bacterial attachment has occurred, only organisms on the surface of the epithelial cells were detected.
Each data point was performed in triplicate and each experiment was performed at least three times to ensure reproducibility. Statistical analysis was performed using StatView software (Abacus Concepts, Berkeley, CA). A 
2 test
was used to compare these populations of > 10,000 cells analyzed by flow cytometry. The directly computed 2 value
was compared with appropriate critical values based on the large number of degrees of freedom in the samples,
and the probability of exceeding that value was calculated.
If P < 0.05, the null hypothesis (that there was no difference between the groups) could be properly rejected.
Fluorescence Microscopy
Samples were prepared as described above for flow cytometry and examined with a Nikon epifluorescent microscope. Fluorescent organisms were counted and expressed
as a fraction of the number of epithelial cells examined
over numerous representative fields. For each cell line, between 500 and 700 epithelial cells were examined. Experiments were repeated three times to ensure the reproducibility of the data. Statistical analysis was performed using
analysis of variance (ANOVA) calculations to compare
the ratios of adherent organisms with the three cell lines
expressing pCEP, pCEP-R, or F508 under each of the
two conditions tested (with and without anti-asialoGM1).
Quantification of IL-8 production.
Confluent monolayers of the 9HTEo cells expressing pCEP or pCEP-R were
plated at a density of 1 × 106 cells per well on vitrogen-coated
24-well plates in DMEM supplemented with 10% FBS, 2.5 mM L-glutamine, and 40 µg/ml hygromycin. At 24 h after
plating, the monolayers were fed with serum-free media and
incubated for another 18 h prior to stimulation. The monolayers were washed three times with Hanks' balanced salt
solution (HBSS; GIBCO BRL, Gaithersburg, MD) and stimulated with dilutions of PAO1 (1 × 109 cfu/ml) resuspended
in 0.5 ml HBSS. Aliquots of P. aeruginosa were incubated
with the monolayers for 60 min, then washed three times
with HBSS and sterilized with serum-free DMEM plus gentamicin (100 µg/ml). Wells incubated with IL-1 (20 ng/ml)
or tumor necrosis factor alpha (TNF-
) (100 ng/ml) served as
positive controls. Epithelial culture supernatants were harvested 18 h later and assayed for IL-8 content using an enzyme-linked immunosorbent assay (R&D Systems ELISA
kit, Minneapolis, MN). The epithelial monolayer was lysed
and protein concentration was determined by the bis-cinchonninic acid method of Pierce (Rockford, IL). Each data
point was performed in triplicate, and each experiment was performed three times. The data were analyzed for statistical significance using a two-tailed t test as described above.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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9HTEo/pCEP-R Cells Bind More P. aeruginosa PAO1
than Cells Expressing a Plasmid Vector Control
The effects of overexpression of the CFTR R domain on
the surface properties of the 9HTEo cells were tested
by comparing the relative avidity of the epithelial cells for
P. aeruginosa ligands in a bacterial binding assay. The
physiologic properties of the transfected 9HTEo cells
have been previously established; the pCEP vector is stable and does not integrate into the chromosome, and this
cell line continues to produce CFTR messenger RNA (27).
pCEP-R expresses the R domain of CFTR under the control of the CMV promoter. By SPQ assay the 9HTEo/
pCEP-R cells lack the cAMP-dependent increase in chloride efflux shown by normal cells and by 9HTEo cells
containing the empty pCEP vector. Basal Cl efflux in the
pCEP-F508 cells is similar to the pCEP control cells, but
there is no response to isoproterenol-induced elevation of
cAMP levels compared with the cells that express the
pCEP vector alone (D. Kube, manuscript in preparation).
We postulated that the cell lines with overexpression of
the R domain would bind increased numbers of P. aeruginosa (Figure 1A). As increasing inocula of PAO1 were incubated with the 9HTEo cells, the available receptor sites
were saturated on the cells expressing the pCEP vector at
a lower inoculum of organisms (
5 × 107 cfu/ml) than the
pCEP-R cells with the overexpressed R domain (> 2 × 108 cfu/ml). At bacterial concentrations of 2 × 108 cfu/ml
the number of epithelial cells with adherent organisms was
significantly greater for the pCEP-R cells (38% of the epithelial cell population studied) compared with 10% of the
pCEP vector control cells (2 analysis, P < 0.005).
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Increased Bacterial Adherence to the pCEP-R Cells Is Dependent on the Expression of Pili
P. aeruginosa adherence to epithelial cells involves the expression of several adhesins and receptors. One of the major bacterial ligands is pilin, which specifically recognizes
the GalNAc1-4Gal moiety exposed in certain asialylated
glycolipids, such as asialoGM1. If the increased bacterial
adherence to the cells expressing pCEP-R is due to recognition of asialylated receptors for pilin, binding by a pil
mutant, isogenic for other properties of P. aeruginosa, should be equivalent in the cells expressing pCEP or
pCEP-R. Adherence of the pil mutant PAO/NP to the epithelial cells expressing pCEP was identical to the adherence to pCEP-R cells over the range of inocula tested
(Figure 1B), suggesting that the increased attachment of
PAO1 to the pCEP-R cells (Figure 1A) is pilin-related and
dependent upon the recognition of asialylated receptors.
Note that the y-axis in Figure 1B is almost an order of
magnitude less than that in Figure 1A, which reflects the
diminished binding of the pil mutant.
AsialoGM1 Is More Abundant on the Surface of Epithelial Cells Overexpressing the R Domain
Flow cytometry was used to estimate the relative abundance of asialoGM1 on the surface of the 9HTEo cells expressing pCEP-R compared with those expressing pCEP. For each of the cell lines, the fluorescence associated with
bound anti-asialoGM1 was determined (Figure 2A). At a
saturating concentration of polyclonal anti-asialoGM1 antibody, the mean fluorescence of cells expressing pCEP-R
was significantly greater than that of the cells expressing
pCEP (unpaired t test, t = 5.34, P < 0.0005; Figure 2B). At
greater than saturating amounts of anti-asialoGM1 there
is a decrease in fluorescence attributed to squelching. This
occurred with both cell lines when sufficient anti-asialoGM1 was added. Using FITC-labeled CTB subunit as a marker
for cell-surface GM1, virtually all of the epithelial cells expressing either pCEP or pCEP-R were labeled (data not
shown), as might be expected for a major epithelial membrane component. The relative increase in the amount of
asialoGM1 on the surface of the pCEP-R cells suggests a
specific defect in terminal sialylation.
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Bacterial Adherence to pCEP and pCEP-R Epithelial Cells Correlates with the Availability of AsialoGM1 Receptors
The number of epithelial cells that bound P. aeruginosa
was quantified by flow cytometry. We identified the percentage of epithelial cells with associated FITC-labeled
organisms from a background of epithelial cells with no associated bacteria. In these experiments a 9HTEo/pCEP-F508 cell line was included to compare directly the adherence phenotype of the cells overexpressing the R domain
with cells known to have CFTR dysfunction on the basis of
deletion of F508 in the nucleotide binding domain as expressed in the 9HTEo cells. The available asialoGM1 residues on these cells was quantified by tagging with anti-asialoGM1 antibody. Representative flow cytometry data
shows a population of epithelial cells shifted to the right, representing the fraction of the total with adherent organisms, which is larger in the pCEP-R and F508 cell lines
than in the pCEP group (Figure 3A). Under saturating conditions (1 × 108 cfu/ml inoculum), 38% of the cells expressing pCEP had adherent organisms, compared with 53% of
the pCEP-R cells (P < 0.005; unpaired, two-tailed t test),
or 55% of the cell line expressing pCEP-F508 (P < 0.005;
Figure 3B). The percentage of epithelial cells with associated bacteria was roughly proportional to the distribution of asialoGM1 as a marker for the GalNAc1-4Gal receptor;
10% of the pCEP cells, versus 33% and 35% of the pCEP-R
and F508 cells, respectively, had accessible asialoGM1 residues under these experimental conditions.
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To demonstrate that the increased bacterial binding associated with the pCEP-R or the pCEP-F508 cells was, in
fact, due to a difference in asialylated glycolipids, the number of bacteria associated with the epithelial cells was quantified after incubating the monolayers with anti-asialoGM1
to compete for the pilin-specific receptors (Figure 4). Under these experimental conditions the number of organisms
per epithelial cell was equivalent in the three cell lines examined, whereas the expected increase in bacterial adherence in the pCEP-R and F508 cell lines was confirmed under control conditions in the absence of anti-asialoGM1.
Using ANOVA to compare the means from each of the
experimental conditions, we found a significant difference
in the binding for each cell line in the presence of antibody
to asialoGM1 compared with control conditions (F-value = 12.17; P < 0.0001). There were no statistically significant
differences between the binding of PAO1 to each of the
lines in the presence of anti-asialoGM1 (F-value = 2.11, P > 0.14). There was no effect of antibody to cadherin on the
adherence of the organisms to the various epithelial cell lines
(data not shown). Thus, pretreatment of the epithelial cells
with antibody to the asialoGM1 receptor, specific for pilin,
abrogates any differences in the amounts of bacterial attachment to the different cell lines.
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Cells that Express pCEP-R Have Increased IL-8 Production in Response to P. aeruginosa
We compared the amount of IL-8 production in epithelial
cells expressing either pCEP or pCEP-R as stimulated by
adherent P. aeruginosa PAO1 (Figure 5). The endogenous
IL-8 expression was somewhat higher in the pCEP-R cells
even without exogenous stimulation, but there was no significant difference in the response of the cell lines to TNF-,
a positive control. At an inoculum of 108 cfu/ml, less than
saturating under these conditions, the amount of IL-8 expressed was equivalent in the pCEP and pCEP-R cells. However, at an inoculum of 109 cfu/ml PAO1, there was a
statistically significant increase in the amount of IL-8 expressed by the pCEP-R cells compared with those transfected with the empty vector (P = 0.042).
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Discussion |
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Introduction
Materials and Methods
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Discussion
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The molecular basis for the unusual association between
P. aeruginosa and the CF airway epithelial cell has been
difficult to establish despite a great deal of information regarding the biochemistry and function of CFTR. The experiments described here demonstrate a relationship between CFTR Cl channel dysfunction, the availability of
asialoGM1 on the cell surface as a marker representative
of undersialylated gangliosides, and the number of receptor sites for P. aeruginosa binding. Moreover, the increased
affinity of P. aeruginosa for the epithelial cells with abnormal
CFTR function was limited to organisms which expressed pili, the ligand with specificity for asialoGM1. For these
studies we used P. aeruginosa PAO1, a genetically well-characterized strain that is prototypic for the environmental organisms that initiate infection. The alginate-producing,
mucoid organisms associated with CF pulmonary disease
are associated with chronic infection and represent the mutants that are selected by conditions found in the CF lung.
Because we are interested in the earliest stages of lung infection, these studies focused on strains typical of the environmental types of P. aeruginosa. PAO1 is motile and piliated and expresses the exoproducts required to initiate
pulmonary infection (30), properties that are important in
pathogenesis because they are required both for adherence and to stimulate a polymorphonuclear leukocyte inflammatory response (10).
The 9HTEo cell lines used in these studies have been
extensively characterized. In contrast to the parent 9HTEo
cell line or cells transfected with the empty vector, the
HTEo/pCEP-R cells and the HTEo/pCEP-F508 cells
fail to respond to isoproterenol with an increased rate of
chloride efflux as measured by SPQ fluorescence (27; D. Kube, unpublished results). Thus, they assume a CF-like
phenotype. In addition, cells expressing either pCEP-R or
pCEP-F508 have decreased levels of 2,6 sialic acid on
their surface as measured by binding of elderberry bark
lectin (21). Although CFTR cannot be identified on the
cell surface of either the pCEP or pCEP-R cells using currently available antibodies to CFTR, overexpression of the
R domain did not have an appreciable effect on the level of
endogenous CFTR message as determined by semiquantitative reverse transcription-polymerase chain reaction (27).
It is unlikely that the availability of superficial CFTR in the
pCEP and pCEP-R cells is affected by overexpression of
the R domain. Single-channel studies in the planar lipid bilayer show that unphosphorylated exogenous R-domain
protein blocks the wild-type CFTR channel (24). Thus, the
9HTEo cells, transfected with pCEP-R vector, express a
CF-like phenotype with respect to chloride transport in the
face of continued expression of wild-type CFTR.
The pCEP-F508 cells, which also had reduced cAMP-stimulated chloride transport, had increased PAO1 adherence and availability of asialoGM1 as well. Although it is
not apparent why expression of F508-CFTR results in reduced chloride transport, the results obtained with this cell
line further demonstrate that surface properties of airway
epithelial cells are entrained in part by reduced function of
CFTR, by whatever mechanism functional disruption of
CFTR is achieved.
Overexpression of the R domain had a number of interesting effects on the superficial properties of the 9HTEo
cells. Most relevant to the issue of bacterial attachment,
the amount of asialoGM1 on the cell surface was significantly
increased. Biologic consequences of increased P. aeruginosa
adherence were suggested in these studies because in response to the same bacterial stimulus, a greater amount of
the proinflammatory cytokine IL-8 was produced by the
pCEP-R cells than by cells expressing the empty vector.
CF cells with increased numbers of pilin receptors have
been shown to produce more IL-8 in response to an identical inoculum of bacteria than a "corrected" CF cell line
transfected with a wild-type copy of CFTR (10). Consistent
with those observations, expression of IL-8 is increased in
response to PAO1 in pCEP-R cells. Although the pCEP-R
cells had a somewhat (but not significantly) greater amount
of endogenous IL-8 expression, the IL-8 response to PAO1
was still excessive even after correcting for the endogenous amount of IL-8 expressed by each of the cell lines.
Both cell lines responded equivalently to a TNF- signal,
suggesting that the increased response to P. aeruginosa is a
consequence of the greater stimulation provided by the
occupation of more receptors on the pCEP-R cells. It has
been suggested that "cell stress" due to the accumulation
of mutant protein in the ER, as might occur with mistrafficked mutant CFTR, can cause activation of NF-
B and
perhaps the stimulation of proinflammatory cytokine expression (30). It remains to be established whether the excess R-domain protein expressed in the pCEP-R cells causes
activation of a similar pathway. The "excessive" cytokine
response to bacteria associated with cells with CF physiology is consistent with clinical data demonstrating increased
amounts of proinflammatory cytokines in bronchoalveolar
lavage fluid from CF patients compared with control subjects (13, 14). These in vitro studies suggest a direct correlation between the increased receptor availability and IL-8
production by cells with abnormal CFTR function.
Additional CFTR-dependent abnormalities in epithelial cells have been proposed recently to explain the predilection of P. aeruginosa for the CF airway. One theory attributes the pathogenesis of infection to failure of local
defense mechanisms in the milieu of the CF airway surface fluid (4). This may account for increased numbers of
P. aeruginosa that persist within the CF airway, which could
then initiate an inflammatory response. It has also been suggested that decreased internalization of Pseudomonas
by mutant CFTR residues accounts for increased numbers
of luminal organisms in CF (31). The methods used in the
studies reported here detected bacteria that were adherent
to, but not internalized by, the epithelial cells. Organisms
were tagged with FITC::conjugated antibodies following
adherence; thus, ingested organisms were not labeled. The
9HTEo cell lines should have similar amounts of apical
CFTR and identical CFTR primary structures. Differences
in bacterial association with these cells are more likely attributable to a functional consequence of CFTR dysregulation by the R domain. Our previously published studies
showed that 35S-labeled organisms, which are detectable
inside and outside the epithelial cell, associated with CF
nasal polyp cells in primary culture and with CF epithelial
cell lines (5) to a significantly greater extent than they associated with wild-type nasal polyp cells or "corrected" CF
epithelial cell lines (5, 8, 9). The current study similarly
supports the hypothesis that CFTR-dependent sialylation
of surface glycolipids is responsible for the relative distribution of asialylated receptors that mediate pilin-associated, superficial P. aeruginosa attachment.
P. aeruginosa expresses a number of surface ligands that can recognize epithelial receptors. The experiments performed with pil mutants demonstrated that organisms that do not express this ligand for asialoGM1 fail to discriminate between epithelial cells with normal versus mutant CFTR function. This pilin-dependent adherence of P. aeruginosa appears to be distinct from the process of epithelial ingestion of bacteria, which is postulated by Pier and colleagues to be mediated by lipopolysaccharide (31). The increased association of P. aeruginosa with the pCEP-R cells was demonstrated only in experiments using organisms with functional pili. The pil mutant PAO/NP did not demonstrate any increased affinity for the pCEP-R cells even when saturating inocula of organisms were used, suggesting that pilin is the major ligand responsible for the excess adherence to epithelial receptors in CF-phenotype cells.
The experiments described indicate a correlation between abnormalities in CFTR regulation and function, increased amounts of asialoGM1, and the adherence of piliated P. aeruginosa. Different CFTR mutations, with their
varying effects on CFTR function (22), may differentially
impact upon levels of sialylation and the amount of proinflammatory cytokine expression elicited in response to bacteria; this may help to explain the wide range of phenotypes observed with respect to susceptibility and severity
of P. aeruginosa infection. If Pseudomonas binding and its
consequences are important in disease pathogenesis in CF,
preventing it may be an important consideration as therapeutic modalities to normalize CFTR function are developed and tested. It is unclear how much "normal CFTR" may be necessary to prevent P. aeruginosa infection and
how this relates to the amount of functional CFTR that is
required to normalize cAMP-dependent Cl transport. Decrease in asialoGM1 level may provide a convenient marker
of CFTR function in cells treated with agents to increase the insertion of functional CFTR to the apical surface of
the epithelium, or may provide a quantitative measure of
the efficacy of gene replacement strategies.
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Footnotes |
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Address correspondence to: Alice Prince, M.D., Dept. of Pediatrics, College of Physicians and Surgeons, Columbia University, 650 W. 168th St., New York, NY 10032. E-mail: asp7{at}columbia.edu
(Received in original form January 6, 1997 and in revised form November 10, 1997).
Acknowledgments: The authors thank Daniel Ng and Junta Liu of the Flow Cytometry Facility, Columbia University Irving Cancer Center, for expert assistance. This work was supported by the Cystic Fibrosis Foundation (R.B., A. Prince, P.D., A. Perez, D.K.) and National Institutes of Health grants DK36963 (A. Prince), HL/DK 49003 (P.D., A. Perez, D.K.), and DK27651 (P.D., A. Perez, D.K.).
Abbreviations
BSA, bovine serum albumin;
cAMP, cyclic adenosine monophosphate;
CF, cystic fibrosis;
CFTR, cystic fibrosis transmembrane conductance regulator;
FITC, fluorescein isothiocyanate;
IL, interleukin;
PBS, phosphate-buffered saline;
R domain, regulatory domain;
TNF-, tumor necrosis factor alpha.
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References |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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1. Hudson, V. L., C. L. Wielinski, and W. E. Regelman. 1993. Prognostic implications of initial oropharyngeal bacterial flora in patients with cystic fibrosis diagnosed before the age of two years. J. Pediatr 122: 854-860 [Medline].
2. Kerem, E., M. Corey, R. Gold, and H. Levison. 1990. Pulmonary function and clinical course in patients with cystic fibrosis after pulmonary colonization with Pseudomonas aeruginosa. J. Pediatr 116: 714-719 [Medline].
3. Konstan, M., and M. Berger. 1993. Infection and inflammation in the lung in cystic fibrosis. In Cystic Fibrosis. P. Davis, editor. Marcel Dekker, New York. 221-276.
4. Smith, J. J., S. M. Travis, E. P. Greenberg, and M. J. Welsh. 1996. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85: 229-236 [Medline].
5. Saiman, L., and A. S. Prince. 1993. Pseudomonas aeruginosa pili bind to asialoGM1 which is increased on the surface of cystic fibrosis epithelial cells. J. Clin. Invest. 92: 1875-1880 .
6.
Sheth, H. B.,
K. K. Lee,
W. Y. Wong,
G. Srivastava,
O. Hindsgual,
R. S. Hodges,
W. Paranchych, and
R. T. Irvin.
1994.
The pili of Pseudomonas
aeruginosa strains PAO and PAK bind specifically to the carbohydrate
sequence GalNAc(1-4)Gal found in glycolipids asialoGM1 and asialoGM2.
Mol. Microbiol
11:
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