Airway epithelial tight junctions and binding and cytotoxicity
of Pseudomonas aeruginosa
Alfred
Lee1,
Dar
Chow1,
Brian
Haus1,
Wanru
Tseng1,
David
Evans2,
Suzanne
Fleiszig2,
Grischa
Chandy1, and
Terry
Machen1
1 Department of Molecular and
Cell Biology, University of California, Berkeley 94720-3200; and
2 School of Optometry, University
of California, Berkeley, California 94720-2020
 |
ABSTRACT |
The role of tight junctions in the binding and
cytoxicity of Pseudomonas aeruginosa
to apical or basolateral membranes of lung airway epithelial cells was
tested with fluorescence microscopy on living cells. Binding of
noncytotoxic P. aeruginosa strain O1
was assessed with P. aeruginosa that
expressed green fluorescent protein. Binding of cytotoxic
P. aeruginosa strain 6206 was assessed with FITC-labeled P. aeruginosa;
cytotoxicity was determined from nuclear uptake of the impermeant dye
propidium iodide. The role of direct contact of P. aeruginosa to epithelial cells was tested with filters
with small (0.45-µm) or large (2.0-µm) pores. High transepithelial resistance
(Rt) Calu-3 and
cultured bovine tracheal monolayers
(Rt > 1,000
· cm2)
bound P. aeruginosa very infrequently
(<1 P. aeruginosa/100 cells) at the
apical membrane, but P. aeruginosa
bound frequently to cells near "free edges" at holes, wounds,
islands, and perimeters; cytotoxicity required direct interaction with
basolateral membranes. Wounded high
Rt epithelia
showed increased P. aeruginosa binding and cytotoxicity at the free edges because basolateral membranes were
accessible to P. aeruginosa, and dead
and living cells near the wound bound P. aeruginosa similarly. Compared with high
Rt epithelia, low
Rt CFT1
(Rt = 100-200
· cm2) and
EGTA-treated Calu-3 monolayers were 25 times more susceptible to
P. aeruginosa binding throughout the
monolayer. Cytotoxicity to CFT1 cells (throughout the confluent
monolayer, not only at the free edge) occurred after a shorter delay
(0.25 vs. 2.0 h) and then five times faster than to Calu-3 cells,
indicating that the time course of P. aeruginosa cytotoxicity may be limited by the rate of
gaining access through tight junctions and that this occurred faster in
low Rt than in
high Rt airway
epithelia. Cytotoxicity appeared to occur in a sequential process that
led first to a loss of fura 2 and a later uptake of propidium iodide.
P. aeruginosa bound three times more
frequently to regions between cells (tight junctions?) than to cell
membranes of low
Rt CFT1 cells.
epithelial cells; cystic fibrosis; cystic fibrosis transmembrane
conductance regulator; green fluorescence protein; Calu-3 cells; trachea; epithelial polarity
 |
INTRODUCTION |
ALTHOUGH IT IS COMMONLY ASSUMED that the critical first
steps in bacterial-induced pathogenesis in cystic fibrosis (CF) involve binding and subsequent direct and indirect cytotoxic effects of Pseudomonas aeruginosa to airway
cells, the respective roles of bacterial and host factors that
contribute to the pathogenesis of P. aeruginosa airway infection in CF remain controversial. One area of controversy has been the identity of P. aeruginosa receptors on the apical and/or basolateral
membranes of airway epithelial cells. Imundo et al. (19)
showed two- to threefold greater binding of P. aeruginosa to CF- versus CF transmembrane conductance
regulator (CFTR)-corrected epithelial cell lines (also see Refs. 30,
36). Because antibodies against
asialo-GM1 reduced P. aeruginosa binding and
asialo-GM1 appeared from
immunofluorescence to be on the apical membrane, it was proposed that
P. aeruginosa bound to this glycolipid
in the apical membranes of wild-type (WT) and especially CF airway
epithelial cells (19; also see Refs. 2, 9, 15, 22, 30, 32, 36).
Consistent with some aspects of these findings, De Bentzmann and
colleagues (2, 3) found that P. aeruginosa bound mainly to the dorsal, apparently apical, membranes of tracheal cells at the edges of wounds (also see
Refs. 10, 32, 37) and that binding was blocked by
anti-asialo-GM1 antibodies (2).
In contrast, Pier and colleagues (25-27) showed that
P. aeruginosa bound and were taken up
to a greater extent by cells expressing WT CFTR than cells expressing
either no CFTR or
F508 CFTR. They also showed that
P. aeruginosa invasion of epithelial
cells was increased during incubation of CF epithelial cells at
26°C (to increase expression of
F508 CFTR on the apical
membrane), whereas P. aeruginosa
invasion was blocked by an antibody against CFTR (26, 27) and electron
microscopy showed that P. aeruginosa bound to CFTR (25). To explain their data, Pier et al. (25) proposed
that P. aeruginosa bound to the
membranes of airway cells and that this binding of P. aeruginosa recruited CFTR to the membrane. CFTR then
served as an uptake mechanism for P. aeruginosa. Because CFTR is located at the apical
membrane of airway epithelial cells, it was reasonable to assume that
this binding and uptake of P. aeruginosa was similarly occurring at the apical membrane.
In contrast, other work has indicated that
P. aeruginosa bound and elicited
cytotoxicity by interacting with the basolateral membranes of
epithelial cells. Thus there was little binding when P. aeruginosa were added to the apical
sides of intact epithelial cell layers, and P. aeruginosa binding increased after tissue injury or
other treatment that allowed P. aeruginosa access to the basolateral membrane (e.g.,
Refs. 28, 32). Fleiszig et al. (10) recently found that
P. aeruginosa binding and/or
cytotoxicity increased 10- to 300-fold when basolateral membranes of
well-polarized tracheal, nasal, Madin-Darby canine kidney (MDCK), and
corneal cells were exposed by disrupting tight junctions (EGTA
treatment), by growing cells in a low-calcium concentration (prevents
tight junction formation), or by using subconfluent cells (10). In addition, scratch wounding of corneal epithelial cells leads to 10- to
100-fold increases in P. aeruginosa
adherence and rendered the cornea susceptible to infection (14, 28).
Apical addition of P. aeruginosa to
the apical surfaces of MDCK and corneal epithelial cells induced
cytotoxicity as expanding foci of dying cells, indicating that once
cytotoxicity was induced, cells adjacent to dying cells were affected
(1, 14). Also, direct contact of whole, viable P. aeruginosa was required for cytotoxicity to MDCK (1)
and corneal (14) epithelial cells, and P. aeruginosa were found beneath affected cells but not
under viable epithelial cells (1; also see Ref. 10). The recently
discovered protein ExoU (9; also see Refs. 12, 20), which is required
for the acute (within 3 h) cytotoxicity of P. aeruginosa, is likely to be secreted by a type III
mechanism (9, 34) that requires direct contact between
P. aeruginosa and epithelial cells.
Accordingly, it has been proposed that the maintenance of normal cell
polarity is a defense against infection (10).
However, there have been no direct observations of
P. aeruginosa binding to
living airway epithelial cells. In addition, the question of polarized
cytotoxicity through direct contact of P. aeruginosa with either the apical or basolateral
membrane of airway epithelial cells has also not been definitively
answered; previous experiments (1) showed that 0.45-µm-pore filters
prevented cytotoxicity to MDCK cells, but it was not determined whether critical contact of P. aeruginosa was
with the apical or basolateral membrane. It is important to note in
this regard that MDCK cells are a low-resistance epithelium
[transepithelial resistance
(Rt) = 100
· cm2],
whereas most airway epithelia exhibit
Rt > 600
· cm2. It is
possible that these different
Rt values could
be crucial in the normal pathophysiological reactions occurring between
P. aeruginosa and epithelial cells.
For example, if cytotoxicity occurs from the basolateral surface, then
apically applied P. aeruginosa will
likely first have to traverse the tight junctions to gain access to the
basolateral membranes, and the tighter junctions of airway epithelia
could therefore lead to quite different pathophysiological circumstances compared with MDCK (renal proximal tubule) cells.
Given the contradictory findings regarding the interactions of
P. aeruginosa with different
epithelial cells and the potentially different pathophysiological
reactions that could occur in airway versus MDCK and other epithelial
cells, we made a direct determination (fluorescence and confocal
microscopy) of the binding and cytotoxicity of P. aeruginosa to two human, CFTR-expressing airway
epithelial cell lines, one with high
Rt (>1,000
· cm2) and
the other with low
Rt (<200
· cm2). We
also used primary cultures of bovine tracheal epithelial cell
monolayers for comparison.
 |
METHODS AND MATERIALS |
Cultured Calu-3 epithelial cells.
Calu-3 cells of human pulmonary adenocarcinoma origin (kindly provided
by Dr. Jonathan H. Widdicombe, Children's Hospital, Oakland, CA) were
used because they are a tracheal, serouslike cell line with high
resistance (1,000-2,000
· cm2)
that grows to confluence, and the cells express functional, cAMP- and
ATP-stimulated CFTR in their apical membranes in large amounts (17).
Thus these cells formed functional tight junctions, were well polarized
and physiologically responsive, and had CFTR in their apical membranes
(17, 31). The cells were maintained in Dulbecco's modified Eagle's
medium (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum
(University of California, San Francisco Cell Culture) in a humidifed
5% CO2-95% air atmosphere. Cells grown to 80% confluence were trypsinized with 0.25% trypsin-0.1% EGTA solution for 5-15 min. Cells were passaged at a 1:5 dilution, and the remaining cell suspension was seeded directly onto 25-mm cover
glasses or permeable filter supports (0.45- or 1.0-µm pore size;
Falcon, Becton Dickinson, Franklin Lakes, NJ) at a density of
106
cells/cm2. Cells grown on cover
glasses were then allowed to grow into small islands or to complete
confluence. Calu-3 cells grown as islands were used to investigate
binding to both "free edges" and confluent regions of the
monolayer. Cells grown on filters were monitored for
Rt with an
epithelial volt-ohmmeter (EVOM, World Precision Instruments), and
monolayers were used when
Rt was >1,000
· cm2. In
some cases, confluent monolayers on either cover glasses or filters
were mechanically wounded with a sterile filter tip, and the cells were
then placed back into the incubator for 2-24 h to allow the wound
to heal before experimentation.
Cultured bovine tracheal epithelial
cells. The method was based on the approaches of Wu et
al. (33) and Kondo et al. (21), and the cells were prepared in the
laboratory of J. Widdicombe. Briefly, bovine tracheae were obtained
from the slaughterhouse, and the cells were isolated from the
underlying muscle and connective tissue layers by enzyme treatment;
after centrifugation, the cells were plated on collagen-coated Falcon
filters (0.45-µm pore diameter) and then cultured with an air
interface on the apical side. These cell cultures are a mixed
population that come predominantly from surface-ciliated, nonciliated,
and mucus-secreting cells. After 7-10 days,
Rt, measured with
an epithelial volt-ohmmeter, reached 1,000-2,000
· cm2. Only
monolayers that had
Rt > 1,000
· cm2 were
used for these studies.
Cultured CFTR-corrected CF tracheal epithelial
cells. Immortalized CF tracheal epithelial cells that
express retrovirally mediated normal CFTR cDNA (clone CFT1-Exp1-C1)
were used (24). These cells are homozygous for the
F508 mutation and
also express WT CFTR. We refer to these cells as CFT1 cells. The CFT1
cells were cultured in hormone-supplemented Ham's F-12 medium (GIBCO BRL, Life Technologies) containing 100 U/ml of penicillin, 100 mg/ml of
streptomycin, and 4 mM glutamine (supplements: 5 µg/ml of insulin,
3.7 ng/ml of epidermal growth factor, 3 × 10
8 M triiodothyronine,
10
6 M hydrocortisone, and 5 µg/ml of transferrin). Cells were passaged at a 1:5 dilution, and the
remaining cells were plated onto 0.45-µm-pore filters (Falcon, Becton
Dickinson) at a density of 106
cells/cm2.
Rt was measured
with the volt-ohmmeter, and the monolayers used had
Rt = 100-200
· cm2. As
shown previously by Yankaskas et al. (35), CFT1 cells develop vectorial
ion transport, and Illek et al. (18) have previously shown
they exhibit apical anion conductance that is identical in properties
to CFTR. Thus CFT1 cells express physiological properties consistent
with typical tight junctions (albeit with low
Rt) and apical-basolateral polarity.
Solutions. Epithelial cells were
incubated in a Ringer solution containing (in mM) 135 NaCl, 1.2 MgSO4, 2 CaCl2, 2.4 K2HPO4, 0.6 KH2PO4,
20 HEPES, and 10 glucose (pH 7.4). Fluorescent dyes 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF)-AM, fura 2-AM, and fura red-AM were prepared as stock solutions
in DMSO plus the dispersing agent pluronic F-127. These
were added to the cells at a final DMSO concentration of
0.1-0.5%.
P. aeruginosa. P. aeruginosa strains 6206 and 103 (PA6206 and PA103,
respectively; both cytotoxic strains serogroup O11) and P. aeruginosa strain O1 (PAO1)
expressing green fluorescent protein (PAO1-GFP; invasive strain
serogroup O5; see Ref. 5) were maintained frozen in trypticase soy
broth with 10% (vol/vol) glycerol at
70°C. The day before
the experiments, P. aeruginosa were
grown on a trypticase soy agar surface at 37°C overnight and then
resuspended in sterile normal Ringer solution to a standardized density
of 108 colony-forming units
(cfu)/ml (0.1 absorbance, 650 nm). Dilutions of this stock
solution were then used for subsequent additions to the epithelial cells.
Bright-field and wide-field fluorescence and confocal
microscopy of airway epithelial cells and P. aeruginosa. Interactions between epithelial cells and
P. aeruginosa were observed with both
bright-field and fluorescence optics. PAO1-GFP cells were observed with
fluorescence. PA6206 cells were also observed with fluorescence after
treatment of the bacteria with 1 mg/ml of FITC for 1 h followed by
three to six centrifugations and washings to eliminate dye in the
solution (see Refs. 4, 6). Cellular patterns of FITC-labeled PA6206
binding were the same as those exhibited by PAO1, and rates of
cytotoxicity (see Figs. 9 and 10) induced by PA6206 were the same for
control and FITC-labeled bacteria, indicating that FITC labeling had no
deleterious effect on the bacteria.
Calu-3, CFT1, and bovine tracheal cells were observed with both
Nomarski and fluorescence methods. For fluorescence labeling of the
cytosol, cells were incubated with 2-10 µM acetoxymethyl ester
(membrane-permeant) version of either BCECF (green), fura 2 (blue), or
fura red (red) for 1 h followed by three washes with fresh solution.
Treatment of Calu-3 cells with 10 µM probenecid during the loading
process was an effective way to reduce the loss of dye, but similar
results in terms of P. aeruginosa-Calu-3 cell interactions were obtained with
or without probenecid during loading. One to ten micromolar propidium
iodide (PI) was added to the Ringer solutions to identify dead
epithelial cells by staining the nuclei with the dye (red). In Calu-3
cells that had been loaded with either BCECF, fura 2, or fura red, cell
death was observed as a loss of cytosolic dye and uptake of PI into the
nucleus. Rates of PA6206-induced cytotoxicity were the same for control cells and for cells that had been loaded with fura 2 or BCECF (see
Figs. 9-11).
Two approaches were used for observing the interactions between
P. aeruginosa and epithelial cells. In
the first, cells grown on cover glasses were mounted in a chamber that
allowed perfusion to maintain the cells viable and could be mounted
either right side up or upside down. This chamber was mounted directly
on the stage of either an inverted microscope (Zeiss IM35 or Nikon
Diaphot) with standard wide-field fluorescence attachments and Nomarski optics or on an upright microscope (Nikon Optiphot) outfitted for
confocal microscopy (488-nm laser with filters appropriate for
fluorescein and Texas red; Bio-Rad MRC 600). Wide-field fluorescence microscopy was performed with a 75-W xenon light source and either 380 ± 5-, 490 ± 5-, or 530 ± 15-nm excitation and 410 long-pass, 520- to 550-nm band-pass, or 520 or 560 long-pass emission
filters. For observing P. aeruginosa-epithelial cell interactions on cover glasses, a Nikon oil-immersion lens (1.4 numerical aperture; Neofluar) was used.
Two methods were used for observations of epithelial cells grown on
filters. In the first, the filter cup was mounted directly into the
chamber, and the fluorescence of Calu-3 cells was observed with one of
the inverted microscopes through the filter with a long-working-distance (1.6-mm), water-immersion lens (0.75 numerical aperture; Zeiss). This approach had the advantage that the cells retained their normal polarized orientation, but it had the
disadvantage that the optics were compromised. In the second set of
experiments, filters were cut from the plastic cups and placed cell
side down in the chamber for observation with the inverted microscope.
This approach had the advantage of retaining somewhat better optics, although this required cutting the filter from the cup.
Images were recorded with two methods. In one set of experiments,
bright-field and wide-field fluorescence images were recorded photographically (35-mm Nikon), and the slides were scanned (Polaroid Sprint Scan 35) and manipulated with Adobe PhotoShop on a Macintosh computer. In another set of experiments, all images were recorded with
a Photometrics SenSys charge-coupled device. Digitized confocal images
were collected in 0.5-µm steps, stored on the hard disk of a Gateway
computer, reconstructed in 2.5-µm projection planes, and displayed
with Adobe PhotoShop.
To gain quantitative insights into the specific areas of epithelial
cells that bound bacteria, micrographs of monolayers and islands of
epithelial cells that showed PAO1-GFP still attached after 1 h of
incubation (with 107 cfu/ml) and
being washed were inspected. For Calu-3 cells, PAO1-GFP cells were
bound almost exclusively to cells that were within 70 µm of a free
edge. We measured (in both wide-field and confocal micrographs) the
linear distances that PAO1-GFP cells were found from random regions of
the free edges surrounding Calu-3 islands and adjacent to holes in
monolayers, wounded monolayers, and dead cells in wounded monolayers in
two to nine different samples. Averages of these 2-9 different
random regions were then averaged to obtain an average (±SD)
distance (also including range) that PAO1-GFP cells were found away
from the free edges of islands, holes in monolayers, wounded
monolayers, and dead cells in wounded monolayers for 3-15
different experiments; n refers to the
number of separate experiments. In addition, the punctate nature of
PAO1-GFP made it possible to count the number of PAO1-GFP cells bound
(after exposure of 106 to
107 cfu/ml for 1 h followed by
three washes to eliminate loosely adherent bacteria) to the apical and
basolateral surfaces of the epithelium with confocal images of the
apicalmost section and basalmost regions of both Calu-3 islands and
confluent monolayers that had been wounded and then allowed to heal.
Bound PAO1-GFP cells were defined as those bacteria that were found
within the boundaries of the cell.
For CFT1 cells, binding of P. aeruginosa occurred throughout the monolayer and
frequently to the tight junctional regions between adjacent cells. To
quantitate this aspect of binding, images of PAO1-GFP were overlaid on
bright-field images of the epithelial cells. P. aeruginosa were categorized as binding to either cells
or tight junctions (within 1 µm of distinct tight junctions). We did
not count P. aeruginosa that bound in
the few cases (<10%) where cells were inadequately distinct to tell
whether binding was to the cells or junctions.
 |
RESULTS |
PAO1-GFP cells bound heterogeneously to basolateral membranes of
high Rt Calu-3 cells near
free edges.
PAO1-GFP cells (106
to 108 cfu/ml) were
added to Calu-3 cells grown on either filters or cover glasses as
islands (and not subjected to any mechanical damage) for 1 h and then
washed to remove loosely adherent bacteria. Preliminary experiments
showed that there was little or no binding of PAO1-GFP cells to
confluent regions of Calu-3 cells grown on filters, although there was
binding to cells around the extreme perimeter of the filter. We
therefore adopted the following approach to test for binding to cells
in confluent regions versus at regions that had exposed lateral cell
borders. Monolayers were grown as described in
Cultured Calu-3 epithelial cells and wounded with a pipette tip. The
cells were then placed back in the incubator for 1-3 days to allow
the monolayer to repair so that there were no dead cells along the edge
of the wounded monolayer. This approach led to a filter covered with
confluent regions separated by several denuded regions. As shown in
Fig. 1, Aa
and Ab, PAO1-GFP cells did not bind to
confluent regions of Calu-3 cells grown on filters but only to cells
near the free edge. Nearly identical results were found for Calu-3
cells grown on cover glasses (Fig. 1,
Ba and
Bb), which had the experimental advantage of permitting bright-field observations of the epithelial cells: PAO1-GFP cells bound to both the glass and the cells with exposed free edges but not to cells that were more than two to three
cells from the free edge. These experiments indicated that patterns of
PAO1-GFP binding to Calu-3 cells were nearly identical whether the
cells were grown on filters or cover glasses.

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Fig. 1.
Pseudomonas aeruginosa (PA) strain O1
expressing green fluorescent protein (PAO1-GFP) bound to free edges but
not to confluent regions of Calu-3 cells grown either on filters
(A) to a transepithelial resistance
(Rt) > 1,000 · cm2 or on
cover glasses (B) nearly to
confluence, then wounded with a pipette tip, and allowed to heal for 3 days. PAO1-GFP cells [107
colony-forming units (cfu)/ml] were added to filter or cover
glass, and after 1 h, P. aeruginosa
were washed. In addition, 5 µM fura 2-AM was added to apical and
basolateral sides of filter so that cells could be visualized. Filter
or cover glass was then mounted in microscope chamber for observation
of cells. Aa: fluorescence image of
PAO1-GFP cells. Ab: merged overlay of
PAO1-GFP cells (bright white) on filter-grown, fura 2-loaded Calu-3
cells. PAO1-GFP cells bound to free edges of Calu-3 cells that had not
completely covered the filter. Ba:
fluorescence image of PAO1-GFP cells that were bound to cover
glass-grown Calu-3 cells. Bb:
fluorescence image of PAO1-GFP was inverted (i.e., to black) to improve
contrast and overlaid on bright-field Nomarski image of cover
glass-grown Calu-3 monolayers to create merged image.
P. aeruginosa bound to Calu-3 cells at
free edges of monolayer but not in confluent region of Calu-3
monolayer.
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Similar results were obtained for Calu-3 cells grown as islands on
cover glasses with no mechanical perturbation (Fig.
2A). PAO1-GFP cells were found an average of 11 µm from the free edges of
the islands but no farther than 40 µm from the edges of islands (Table 1). In some
experiments, there were small holes in the otherwise confluent
monolayer, and PAO1-GFP cells adhered only to cells surrounding these
holes (data not shown) an average of 17 µm from the free edges of the
holes and no farther than 35 µm (Table 1). The pattern of PAO1-GFP
binding to Calu-3 cells was heterogeneous, with some cells binding a
few or many bacteria, whereas their neighbors bound none. PAO1-GFP
cells often bound in larger numbers to dead Calu-3 cells and to cells
near the dead cells (Fig. 3), but there was
no preferential binding solely to dead cells. Inspection of regions
adjacent to dead cells (within 10 µm) in wounded monolayers showed
that PAO1-GFP bound an average of 26 µm from the edges of the wounded
monolayers and no farther than 70 µm from the free edge (Table 1).
Inspection of the same wounded monolayers showed PAO1-GFP bound an
average of 29 µm from the edges of intact cells and no farther than
70 µm from the free edge (Table 1).

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Fig. 2.
PAO1-GFP cells bound to perimeters of Calu-3 islands and around holes
in monolayers grown on cover glass. PAO1-GFP cells
(107 cfu/ml) were added to Calu-3
cells grown as islands on cover glasses, and after 1 h,
P. aeruginosa were washed
off to eliminate loosely bound bacteria. Propidium iodide (PI; 1 µM)
was added to solution to identify dead Calu-3 cells. Nomarski images of
epithelial cells were merged with fluorescence images of bacteria.
A: PAO1-GFP cells (bright white spots)
fluorescence image. B: bacterial
fluorescence was inverted (i.e., black) and merged with Nomarski image
of Calu-3 island to show heterogeneous binding of P. aeruginosa to cells along free edge.
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Table 1.
PAO1-GFP binding along free edges of islands and wounds and to
apical and basal membranes of Calu-3 cells
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Fig. 3.
PAO1-GFP cells bound to both dead and live cells at free edge of Calu-3
monolayer grown on cover glass. PAO1-GFP cells
(107 cfu/ml) was added to Calu-3
cells grown to confluence on cover glasses, and after 1 h,
P. aeruginosa were washed
off to eliminate loosely bound bacteria. PI (1 µM) was added to
solution to identify dead Calu-3 cells.
A: fluorescence image showing both
PAO1-GFP- and PI-stained nucleus (d) of dead Calu-3 cell.
B: Nomarski image of Calu-3 cells was
merged with inverted fluorescence image of bacteria (i.e., black to
increase contrast) and PI-stained nucleus (d) from dead Calu-3 cell.
PAO1-GFP bound both along free edge of otherwise confluent monolayer
and to cells near dead cell. Because this preparation had not been
mechanically damaged, this dead Calu-3 cell was present
spontaneously.
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Similar experiments were also performed on fura red-loaded confluent
islands grown on cover glasses with confocal microscopy. A typical
series of 2.5-µm sections is shown in Fig.
4,
Aa-Ad, where it can be seen that PAO1-GFP cells bound to the glass and to the
basal and lateral regions of the cells adjacent to the free edge (Fig.
4, Ac and
Ad). Even in sections in which a few PAO1-GFP cells were present in the topmost confocal sections, the
bacteria appeared to bind to the lateral regions of cells that had
exposed free edges (Fig. 4, Aa and
Ab). There was little or no binding
of PAO1-GFP cells to the apical membranes of Calu-3 cells (Fig.
4Ad). Individual PAO1-GFP cells were
counted in confocal images from the apicalmost and basalmost regions of
islands like those shown in Fig. 4. We detected an average of only two
PAO1-GFP cells at the apicalmost aspects of the islands (Table 1). In contrast, there was an average of 43 PAO1-GFP cells in the basalmost sections of the same islands (Table 1), all within 40 µm of a free
edge (Table 1).

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Fig. 4.
Confocal sections of Calu-3 cells exposed to PAO1-GFP.
A: PAO1-GFP cells bound to basal and
lateral sides of cells near free edges of Calu-3 island. Calu-3 cells
grown as islands on a cover glass were loaded with fura red, and
5 × 107 cfu/ml
were added for 1 h. Cells were then washed with bacteria-free solution
to remove loosely adherent P. aeruginosa, mounted in chamber, and examined with
confocal microscope. Sections (0.5 µm) were stored and then displayed
in 2.5-µm composite sections. Aa:
section through topmost portion of island.
Ab: section from middle portion of
cell. Ac: section from bottom of cell.
Note that majority of PAO1-GFP binding occurred in this bottommost
section. Ad: 4 Z-sections were
reconstructed from regions of monolayer shown by dark bars that connect
to bottommost 2.5-µm section. These Z-sections emphasized binding of
PAO1-GFP to basal and lateral surfaces of Calu-3 cells adjacent to free
edge of island and lack of binding to apical membranes except at free
edge of monolayer. B: confluent Calu-3
cells that had been mechanically wounded had PAO1-GFP cells bound only
to basal and lateral sides of cells near free edges of wound. Calu-3
cells grown to confluence on a cover glass were loaded with fura red,
mechanically wounded with a needle, and then allowed to heal for 1 h.
PAO1-GFP cells (5 × 107
cfu/ml) were added to cells for 1 h. Cells were then washed with
bacteria-free solution containing 1 µM PI (to identify dead cells)
and then mounted in chamber and examined with confocal microscope.
Sections (0.5 µm) were stored and then displayed in 2.5-µm
composite sections similar to those in Fig.
2A. Topmost
(Ba) and middle
(Bb) sections showed little or no
binding of PAO1-GFP. Bottommost section
(Bc) showed extensive binding of
PAO1-GFP, and PAO1-GFP cells were found farther from free edge in these
wounded monolayers compared with island in Fig.
2A. Z-sections
(Bd) were reconstructed from regions
of monolayer shown by dark bars that connect to bottommost 2.5-µm
section. Z-sections showed binding of PAO1-GFP to basal and lateral
surfaces of Calu-3 cells near free edge of wound, but there was little
PAO1-GFP binding to apical membranes.
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We also examined PAO1-GFP binding to Calu-3 monolayers that had been
mechanically damaged, allowed to heal for 2-24 h, and then exposed
to 106 to
108 cfu/ml of PAO1-GFP for 1 h
(rather than the 1-3 days as in other experiments) before unbound
P. aeruginosa was washed off and
polarity of P. aeruginosa adherence
was monitored. Dead cells (PI-positive nuclei) were found only along
some wounds (presumably because dead cells had been sloughed from the
monolayer during the healing time), whereas other regions had one or
several dead epithelial cells lining the wound. PAO1-GFP cells always
bound to dead cells and their undamaged nearest neighbors but more
randomly to undamaged cells along the wound, similar to the binding of
PAO1-GFP cells along wounded then healed monolayers that exhibited no
dead cells. Confocal images showed that PAO1-GFP cells bound to basal
and lateral membranes of Calu-3 cells that were adjacent to the wound (Fig. 4, Bc and
Bd), but there was little or no
binding to the apical membranes (Fig. 4,
Ba and
Bb). We detected an average of 3 PAO1-GFP cells at the apicalmost aspects of the monolayers. In
contrast, there was an average of 54 PAO1-GFP cells in the basalmost
sections of the same epithelial monolayers (Table 1), all within 70 µm of the free edges (Table 1).
There was somewhat more extensive PAO1-GFP binding along wounds or
along free edges containing dead cells than at the free edges of
unwounded islands: comparisons of monolayers that had been wounded with
those of unwounded islands showed that PAO1-GFP cells were found
farther from the free edge in the wounded epithelia than in the
unwounded islands. As mentioned above, PAO1-GFP was found an average of
11 µm from the free edge of the island (Table 1). Similar
measurements on monolayers that had been wounded and then allowed to
heal showed that PAO1-GFP cells were found an average of 29 µm from
the edges of cell islands (Table 1), and there were no PAO1-GFP cells
bound to regions farther than 70 µm from the free edges of the
monolayers. PAO1-GFP cells were found an average of 26 µm from dead
cells at the edges of monolayers and no farther than 70 µm from the
free edge (Table 1). Therefore, PAO1-GFP cells were found closer to a
free edge of either undamaged islands or holes (within 11-17 µm)
and farther away from free edges of mechanically damaged monolayers
(within 26-29 µm; Table 1).
An implication of the data to this point was that binding of
P. aeruginosa to the basolateral
membranes of cultured Calu-3 cells in confluent areas was prevented
because P. aeruginosa could not
migrate past the tight junctions. We tested whether confluent monolayers of primary, cultured bovine tracheal epithelial cells behaved similarly. Cells were grown to confluence on filters with an
air-water interface and
Rt > 1,000
· cm2. We
then added 200 µl of Ringer solution containing 1-5 × 107 cfu/ml of PAO1-GFP and 10 µM
fura 2-AM to the apical side for 1 h and washed the surface three
times. The filters were then cut from the plastic support and placed in
the chamber. As shown in Fig. 5, the
confluent monolayers bound few bacteria. In 18 different randomly
selected fields (3 different filters), we observed an average of 4 ± 3 P. aeruginosa bound/field of
300-500 epithelial cells.

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Fig. 5.
Few PAO1-GFP cells bound to confluent primary cultured bovine tracheal
epithelial cell monolayers. PAO1-GFP cells (2 × 107 cfu/ml) and fura 2-AM (10 µM) were added to apical side of monolayers grown to confluence, with
Rt > 1,000 · cm2.
Bacteria and dye were washed out, and filter was cut from filter cup
and mounted on stage of microscope. Fluorescence image of the 2 bacteria was inverted and overlaid on fura 2 fluorescence of cells
(~500 cells) to show cellular binding of P. aeruginosa (small black dots).
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PAO1-GFP binding to low
Rt monolayers: Calu-3
monolayers in Ca-free solution and CFT1 monolayers.
We tested whether tight junctions were limiting access of
P. aeruginosa to critical basolateral
binding sites using two approaches. First, Calu-3 cells were treated
with a 1 mM EGTA-containing, Ca-free solution for 30 min (to open tight
junctions) and then switched to a normal Ca-containing solution
containing 107 cfu/ml of PAO1-GFP
for 1 h. This treatment of Calu-3 cells grown on filters caused
Rt to decrease
from 1,000-2,500 to 0
· cm2,
consistent with the idea that the tight junctions had been disrupted. Cells were finally washed three times with normal Ringer solution to
remove loosely adherent bacteria. After this treatment, PAO1-GFP cells
bound to many, but not all, regions of the confluent monolayer that
would normally have excluded P. aeruginosa (Fig. 6). The "patchy" binding of PAO1-GFP likely indicated the regions where EGTA was disrupting the tight junctions. The regions of the Calu-3 monolayers that bound PAO1-GFP cells often correlated with regions of
the Calu-3 monolayers that appeared to have lifted off the cover glass
but were still confluent.

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Fig. 6.
Ca-free treatment of confluent monolayers of Calu-3 cells led to
PAO1-GFP binding to cells that previously excluded bacteria. Calu-3
cells were grown to confluence on cover glasses and then treated for 30 min with Ca-free EGTA (1 mM) solution. Cells were returned to
Ca-containing solution containing PAO1-GFP cells
(107 cfu/ml) for 60 min and then
washed with fresh Ca-containing Ringer solution. Bacterial fluorescence
(A) was overlaid on Nomarski
micrograph of epithelial cells to generate merged image
(B) showing PAO1-GFP cells (inverted
to black to improve contrast) bound to many cells within confluent
region.
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The second general approach was to perform binding experiments on
confluent CFT1 cell monolayers, a low
Rt (100-200
· cm2),
CFTR-expressing, tracheal-derived cell line. Ringer solution containing
107 cfu/ml of PAO1-GFP was placed
on the apical surface, and after 1 h, the monolayers were washed three
times to remove loosely adherent bacteria. The filter was then cut from
the plastic cup and mounted in the chamber. In sharp contrast to the
results obtained with Calu-3 cells, many PAO1-GFP cells bound to
confluent regions of the CFT1 monolayer, particularly to the junctional
regions between adjacent cells. Similar results were obtained when CFT1 cells were grown on cover glasses where it was possible to compare PAO1-GFP binding to cellular morphology. A typical example is shown in
Fig. 7. These experiments showed that,
unlike Calu-3 and bovine tracheal epithelial cells, PAO1-GFP cells
bound similarly to the confluent regions of CFT1 cells that had been
grown on either filters or cover glasses and also that there was
apparently preferential binding of bacteria to the junctions compared
with that to the cells.

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Fig. 7.
PAO1-GFP bound throughout confluent regions of low
Rt CFT1
monolayers. CFT1 cells were grown to confluence on cover glasses.
PAO1-GFP cells (107 cfu/ml) were added to CFT1 cells for 1 h, and bacteria were then washed. Fluorescence image of PAO1-GFP cells
(A) was inverted (black) and
overlaid on Nomarski image to yield merged image
(B) showing that P. aeruginosa bound prominently to junctional regions
between adjacent cells as well as to cells in confluent regions of
monolayer.
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We quantitated cellular and junctional binding sites by moving the
microscope stage to random regions of the monolayer where individual
epithelial cells could be easily observed. Then we collected a
fluorescence image of PAO1-GFP cells that were bound in the same
region. Fluorescence and bright-field images were overlaid, and
PAO1-GFP cells that had bound to cells versus tight junctions were
counted. In some cases (<10%), it was impossible to determine
whether bacteria were binding cellular or junctional regions, and these
bacteria were ignored. On average, when
107 cfu/ml of PAO1-GFP were used
for binding, there were 20 ± 8 bacteria bound to ~200 cells in
each field (89 images from 9 monolayers, 5 different cultures). Thus
there was more binding of PAO1-GFP to CFT1 cells compared with that to
bovine monolayers and Calu-3 cell monolayers. In the same experiments,
PAO1-GFP appeared to bind preferentially to the regions between
adjoining cells: an average of 14 ± 7 bacteria bound to this
intercellular (junctional) region, whereas 4 ± 2 bacteria bound to
cells in any one field (~200 cells/field of view). Thus, in CFT1
cells, there was a >3-fold preference for binding to regions just
between adjacent cells compared with that in other portions of the
cell. Similar results were obtained with JME monolayers, a low
Rt (10-100
· cm2)
F508 CFTR nasal epithelium.
Cytotoxic effects of PA6206 on high
Rt Calu-3 monolayers
required direct interaction between bacteria and basolateral membranes
of epithelial cells.
Cellular patterns of binding and cytotoxicity of PA6206 (response to
108 cfu/ml) on Calu-3 cells were
investigated in cells grown both on filters and cover glasses. When
106 to
108 cfu/ml of PA6206 were added to
the apical surfaces of these monolayers, Rt remained
stable and then decreased rapidly (Fig. 8).
Cytotoxicity (i.e., PI uptake) occurred only at the extreme periphery
of these confluent monolayers, in regions adjacent to the edge of the
plastic (data not shown). This effect was difficult to quantitate
because it was impossible to determine which cells were growing on the filter and which were growing up the plastic sides. We therefore performed experiments similar to those used to investigate PAO1-GFP binding. Calu-3 cells were grown to confluence and then wounded and
allowed to heal for 3 days. FITC-labeled PA6206 cells
(107 cfu/ml) were added to the
apical surface for 1 h and then washed three times to remove loosely
adherent bacteria. The cells were left in this configuration for an
additional 2 h, which control experiments showed allowed sufficient
time for cytotoxic reactions just to have begun. One micromolar PI was
added to the solution to stain the nuclei of dead cells. As shown in
Fig. 9, PA6206 cells, like PAO1-GFP cells,
bound at the free edge of the monolayer, and cytotoxicity was initiated
at the free edge where bacterial binding was occurring, not in
confluent regions of the monolayer.

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Fig. 8.
Effects of apically added PA strain 6206 (PA6206) on
Rt of confluent
Calu-3 monolayers. PA6206 (106,
107, and
108 cfu/ml) were added to apical
surface of confluent Calu-3 monolayers, and
Rt was measured
at times shown.
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Fig. 9.
PA6206 binding and initiation of cytotoxicity at free edge of Calu-3
monolayer grown on filter. PA6206 cells (5 × 107 cfu/ml) that had been FITC
labeled plus 5 µM fura 2-AM were added to a Calu-3 monolayer that had
been grown to confluence, wounded, and then allowed to heal for 3 days.
After 1 h, cells were washed, and 1 µM PI was added to monolayer,
which was left for an additional 1 h. Cover glass was then washed again
and mounted in chamber for observation.
A: fluorescence from PA6206 was
inverted (i.e., black) and overlaid along with PI-stained nuclei (d) of
dead cells on Nomarski bright-field image to show cellular pattern of
bacterial binding along free edge of monolayer.
B: fluorescence from PA6206 (shown as
bright white to improve contrast) was overlaid on fluorescence of fura
2-stained cells (dimmer white monolayer) and PI-stained nuclei (d) to
show relationships among live and dead Calu-3 cells and bound PA6206.
Note that P. aeruginosa were bound to
both epithelial cells that had taken up PI and lost their fura 2 and
cells that retained fura 2 and excluded PI.
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Longer-term experiments on random regions of Calu-3 cells with exposed
free edges on either cover glasses or filters showed that cytotoxicity
was dose dependent (Fig. 10) and occurred
after ~2 h, first at the free edge of the epithelium, and was
followed by further killing of epithelial cells inward from the free
edge toward the confluent region of the monolayer (Fig.
11). Unlike the results obtained on low
Rt MDCK cells
(17), cytotoxicity was never initiated in confluent portions of the
monolayers. Similar results were obtained with PA103 (data not shown).
Thus cytotoxicity by both PA6206 and PA103 appeared to require that
P. aeruginosa have access to the
basolateral membranes of Calu-3 cells.

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Fig. 10.
Dose dependence of PA6206-induced killing of Calu-3 cells vs. time.
Calu-3 cells were grown as islands, and
106,
107, or
108 cfu/ml were added in presence
of 1-10 µM PI. At times shown, PI-positive nuclei were counted
in 10 random edges (where cytotoxicity occurred) of islands. Values are
averages from these 10 random edges. Increasing doses of PA6206 caused
increasing amount of cytotoxicity. Results are typical of 3 other
experiments.
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Fig. 11.
PA6206 required direct contact with Calu-3 cells to elicit
cytotoxicity. Calu-3 cells were grown to confluence on 0.45-µm-pore
(A,
C, and
E) or 1.0-µm-pore
(B,
D, and
F) filters and then left in a
confluent state or mechanically damaged and allowed to heal for 24 h.
Cells were loaded with
2',7'-bis(2carboxyethyl)-5(6)-carboxyfluorescein
(BCECF; green fluorescence), and then
108 cfu/ml were added to apical
(A and
B) or basolateral
(C-F)
surfaces of filters. PI (1 µM) was added to solutions to stain nuclei
of dead cells (red). When PA6206 cells were added to apical side of
cells grown on either 0.45-µm-pore
(A) or 1.0-µm-pore
(B) filters, cytotoxicity (as shown
by red-orange PI staining of nuclei) occurred only along free edges of
wounds after 3.5 h. There was similar but more extensive cytotoxicity
after 4.5 and 5.5 h along free edges of monolayers (data not shown).
When PA6206 cells were added to basal sides of monolayers
(C-F),
there was no cytotoxicity to cells grown on 0.45-µm-pore filters
after 3.5 h (C) but widespread and
random (i.e., no preference for wound edge) cytotoxicity to cells grown
on 1.0-µm-pore filters (D). Note
in D that PI-stained nuclei were
intermingled with BCECF-loaded cells that did not take up PI, showing
random nature of cytotoxicity. Similarly, addition of PA6206 for 4.5 h
to basal surface of confluent monolayers elicited no cytotoxicity if
cells were grown on 0.45-µm-pore filters
(E) but extensive, random
cytotoxicity to cells grown on 1.0-µm-pore filters
(F).
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Experiments to test directly whether PA6206-induced cytotoxicity
occurred only when P. aeruginosa
actually contacted the basolateral surfaces of Calu-3 cells were
performed by adding PA6206 to either the apical or basolateral surface
of Calu-3 cells grown to confluence (Rt > 1,000
· cm2) on
filters with 0.45-µm (too small to permit PA6206 access) or 1.0-µm
(large enough to permit PA6206 access) pores. When
108 cfu/ml of PA6206 were added to
the apical surfaces of cells grown on either 0.45- or 1.0-µm-pore
filters, cytotoxicity occurred only along the free edges of the
monolayers (Fig. 11, A and
B). There was no cytotoxicity in
confluent regions. Thus the addition of PA6206 to the
apical side of Calu-3 cells grown on 0.45- or 1.0-µm-pore filters elicited the same pattern of cytotoxicity as that
exhibited by Calu-3 cells grown on cover glasses. These results showed
that apical addition of bacteria elicited cytotoxicity only along the
free edges of Calu-3 islands.
In contrast, addition of PA6206 to the basal surface of cells grown on
0.45-µm-pore filters elicited no cytotoxicity along the wound edge
(Fig. 11C). This showed that
small-pore filters prevented PA6206 from eliciting cytotoxicity. In
contrast, when PA6206 cells were added to the basal side of Calu-3
monolayers grown on 1.0-µm-pore filters, there was extensive, random
damage throughout the monolayer of Calu-3 cells (Fig.
11D). Addition of PA6206 to the
basal surface of confluent monolayers had no effect on either
Rt (data not
shown) or cytotoxicity when cells were grown on 0.45-µm-pore filters
(Fig. 11E), whereas at the same
time, P. aeruginosa on the basal side
of confluent monolayers of Calu-3 cells grown on 1.0-µm-pore filters
elicited random and extensive epithelial cell killing (Fig.
11F).
Role of junctional tightness on P. aeruginosa-induced cytotoxicity: effects of PA6206 on low
Rt CFT1 cell
monolayers.
CFT1 cells were used to test the apparent role of tight junctions on
the pattern of PA6206-induced cytotoxicity. Because previous experiments showed that P. aeruginosa
binding to CFT1 cells was similar whether they were grown on filters or
coverslips, we utilized cover glass-grown CFT1 cells to facilitate
microscopic observations. CFT1 grown on cover glasses were exposed to a
solution containing 5 µM fura 2-AM and
107 cfu/ml of FITC-labeled PA6206.
After 1 h, the cells were washed three times to remove the dye and
loosely adherent bacteria. The cells were left for an additional
0.5-1 h, which control experiments showed was sufficient time just
to begin expression of cytotoxicity. One micromolar PI was added to the
cells, which were then examined. Typical images are shown in Fig.
12. Several aspects should be noted.
PA6206 cells, like PAO1-GFP cells, bound frequently to regions between
adjacent epithelial cells throughout the confluent monolayer. Also,
nuclei throughout the confluent CFT1 monolayer were stained with PI,
not just at the free edges of the monolayer as in Calu-3 cells. In
addition, many PA6206-treated epithelial cells throughout the monolayer
lost fura 2 even though they had not yet become permeable to PI.
Control experiments showed that CFT1 cells that had not been exposed to
PA6206 were stained uniformly by fura 2 and did not take up PI. Thus
PA6206 bound and elicited cytotoxicity to CFT1 cells throughout the
monolayer, and there appeared to be preferential binding of PA6206 to
junctional regions between cells. Time-course experiments showed that,
compared with the free-edge type of killing exhibited by PA6206 on
Calu-3 cells, the apparently random PA6206-induced killing of CFT1
cells occurred after a shorter delay (15-20 vs. 120 min) and once
cytotoxicity occurred more quickly (18 Calu-3 vs. 120 CFT1 cells/h with
108 cfu/ml of PA6206).

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Fig. 12.
Cellular pattern of PA6206 binding and cytotoxicity to CFT1 cells. CFT1
were grown on cover glass, and 107
cfu/ml of FITC-labeled PA6206 and 5 µM fura 2-AM were added and then
washed off after 1 h. After an additional 1 h, 1 µM PI was added, and
monolayer was examined. A:
fluorescence of FITC-labeled PA6206 (shown as black dots to improve
contrast) and PI-stained nuclei (d) of dead cells were overlaid on
Nomarski image. B: fluorescence of
FITC-labeled PA6206 (white to improve contrast) and PI-stained nuclei
(d) of dead cells were overlaid on fura 2 fluorescence image to show
patterns of PA6206 binding and cytotoxicity.
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 |
DISCUSSION |
Tight junctions of high
Rt tracheal epithelial
cells prevent airway disease by restricting access of bacteria to
basolateral membranes.
Fleiszig et al. (10) previously noted that there was an
inverse correlation between
Rt of different
epithelial cell types and susceptibility to P. aeruginosa binding and cytotoxicity and also that
all epithelial cell types were more susceptible to the bacteria once
tight junctions had been disrupted. The present results are consistent
with these findings and have extended them in a number of ways.
P. aeruginosa both bound and exerted
cytotoxicity heterogeneously, but selectively, to high
Rt Calu-3 cells
by interacting with basolateral membranes of cells that were positioned
near a free edge (within a few cell diameters).
P. aeruginosa were always
located within 2-70 µm of the free edges of Calu-3 monolayers, and cytotoxicity was initiated from these free edges where
P. aeruginosa had access to the
basolateral membranes. There was little or no detectable binding of
apically added P. aeruginosa (either PAO1 or PA6206) in confluent Calu-3 or bovine tracheal monolayers in regions that were >70 µm from a free edge, and
cytotoxicity was initiated in confluent regions only when holes or
other epithelial discontinuities were present. Confocal microscopy
showed that there were ~25 times as many PAO1 cells bound to the
basal surfaces of the same Calu-3 cells that bound only very small
numbers of P. aeruginosa at the apical
surface. PAO1 cells were detected farther from free edges of wounded
monolayers (26-29 µm) compared with Calu-3 islands that had not
been wounded (11-17 µm).
It might be argued that the binding studies with PAO1-GFP were
artifactual because PAO1-GFP bound to abundant basolateral sites with
low affinity and that the methods were insufficiently sensitive to
observe PAO1-GFP binding to more critical high-affinity but less
abundant sites on the apical surface. However, cytotoxicity to high
Rt Calu-3 cells
at all doses of PA6206 (and PA103) began only when bacteria had access
to exposed basolateral membranes, i.e., cells with a free edge on the
periphery of islands or adjacent to small holes or healing areas of
mechanically damaged epithelium. This implied that, similar to the
binding of PAO1, the critical receptors involved in the susceptibility
to cytotoxicity were at the basolateral, not at the apical, sides of
Calu-3 cells. Also, apically added P. aeruginosa bound to Calu-3 cells in confluent monolayers in which the basolateral membranes had become exposed to the
luminal solution by EGTA treatment to open tight junctions.
PA6206-induced cytotoxicity required direct contact of
P. aeruginosa with the basolateral membranes of Calu-3
cells. Unlike low
Rt CFT1 and MDCK
cells (1), P. aeruginosa did
not induce cytotoxicity when added to the apical side of high
Rt Calu-3 cells unless P. aeruginosa had access to the
basolateral membrane at free edges of wounds or holes in the monolayer.
P. aeruginosa did not elicit
cytotoxicity from the basolateral side if the filters had pores that
were too small to allow access of P. aeruginosa to the Calu-3 cells, indicating that
secreted factors were insufficient in themselves to elicit
cytotoxicity. Instead, P. aeruginosa
exerted cytotoxicity to Calu-3 cells only when they had access through the large-pore filters to the basolateral membrane. Our results therefore showed for the first time that P. aeruginosa-induced cytotoxicity required direct contact
of bacteria with basolateral membranes of airway epithelial cells, a
finding consistent with recent experiments (9) showing that ExoU, which
is likely secreted by a type III secretion mechanism, is critical for
P. aeruginosa-induced cytotoxicity.
The use of large-pore filters will provide a useful experimental
approach for testing the specificity of basolateral P. aeruginosa-epithelial cell interactions and
cytotoxicity in future experiments.
Low Rt CFT1 monolayers
bound more P. aeruginosa and allowed random
cytotoxicity throughout the confluent region of the monolayer compared
with high Rt Calu-3 cells.
Confluent, low Rt
tracheal epithelial CFT1 monolayers bound about 25 times more
P. aeruginosa than did confluent
regions of the high
Rt Calu-3 or
primary bovine tracheal cell monolayers. CFT1 (and also low
Rt JME)
epithelial cells bound P. aeruginosa selectively in the regions between adjacent cells, indicating that
binding sites may be localized to these regions (tight junctions?) of
the cells. P. aeruginosa also bound,
although three times less frequently, to cell membranes of CFT1 cells.
Our methods did not provide enough resolution to determine whether
P. aeruginosa were bound to the apical
surfaces of junctions in the shallow furrows somewhat below the apical
cell membranes that protruded slightly above the level of the
junctions. It was also possible that the bacteria gained access in some
way to the lateral intercellular space and were bound there. In any
case, these experiments showed that P. aeruginosa binding to the low
Rt CFT1 cells was
very different from that exhibited by the high
Rt Calu-3 cells.
We acknowledge that although the
Rt of CFT1
monolayers was generally 100-200
· cm2 when
grown on filters and results with filter- and cover glass-grown cells
were similar, it was impossible to determine whether all the cells in
the monolayers were completely polarized. However, our studies clearly
showed that apically added P. aeruginosa bound very differently to the apical
membranes of high and low
Rt airway epithelial cells: P. aeruginosa bound
only at the perimeters, near the edges of the filters, in confluent
high Rt Calu-3
monolayers, whereas P. aeruginosa
bound throughout the confluent monolayer of low
Rt CFT1 cells. A
corollary of our results is that comparisons of P. aeruginosa binding to epithelial cells will require
using epithelia that are closely matched in terms of
Rt. Further
microscopy-based studies to determine whether different physiological
states (e.g., different hormonal treatments) are associated with
differences in P. aeruginosa binding
to cell versus junctional regions of airway epithelial cells could
provide insights into the interactions between P. aeruginosa and airway epithelial cells in both control and disease states.
In addition to differences in binding, P. aeruginosa-induced cytotoxicity occurred after a much
shorter delay and faster and randomly throughout confluent CFT1
monolayers rather than only at the free edges of the Calu-3 monolayers.
We observed many images of monolayers in which some cells had lost fura
2 and taken up PI, whereas other cells had only lost cytosolic fura 2 but had not yet taken up PI and still others had retained fura 2 and
were impermeant to PI. One possible explanation was that cells were damaged in a time-dependent, graded fashion, first losing fura 2 and
later becoming freely permeable to PI. In any case, the faster time
course of cytotoxicity in low versus high
Rt epithelia indicated that the delay between the time of P. aeruginosa addition and the initiation of cytotoxicity
likely reflected in part the time required for P. aeruginosa to gain access to the critical sites, and
these sites were more accessible in low versus high Rt airway
epithelia. After binding, cytotoxicity seems to involve tyrosine
kinase-coupled reactions (7), and the cells then appear to become leaky
such that fura 2 is lost from the cells followed by the uptake of PI.
Further microscopy-based time-course studies of P. aeruginosa-induced cytotoxicity may yield insights into the cellular reactions involved.
We observed similar patterns and amounts of P. aeruginosa binding and cytotoxicity to both CFT1 cells
(which express CFTR) and JME cells (which do not), indicating that
these processes did not depend on CFTR. Similar comments pertain to
binding of P. aeruginosa to
asialo-GM1 (32). Our studies
instead emphasize the importance of junctional tightness in
P. aeruginosa binding and cytotoxicity.
Binding of P. aeruginosa to Calu-3 cells was enhanced
in mechanically damaged regions of the monolayer. PAO1
cells were found at larger distances from the free edges of wounded
versus control Calu-3 islands and holes. However, PAO1 bound equally
well to injured and healthy Calu-3 cells along wounds. This indicated that injured or dead Calu-3 cells did not specifically bind more P. aeruginosa than control, nondamaged
cells in the same mechanically disturbed region of the monolayer, i.e.,
cells did not necessarily have to be injured to be susceptible to
P. aeruginosa binding and sequelae.
Thus mechanical disruption of the Calu-3 monolayer increased
P. aeruginosa binding because bacteria
had increased access to the basolateral membranes, not because dead
cells bound more P. aeruginosa than
live cells. It therefore seems likely that mechanical damage, likely
including the "loosening" of cell attachments to the underlying
support and/or matrix near the wound, was responsible for the fact that
PAO1 cells bound and/or migrated farther away from the free edge toward
the intact, unwounded epithelium in mechanically damaged compared with
control epithelial sheets. Thus our findings with Calu-3 cells
suggested that the reason cell injury promotes adherence to and
infection of the trachea (28, 29) is that the damage exposes the
basolateral surfaces of the cells and that this, not cellular injury,
increased susceptibility to P. aeruginosa binding of wounded tissues as proposed
previously by Finck-Barbançon et al. (9).
Identity of P. aeruginosa binding sites on epithelial
cells remains unknown. Although the specific binding
sites for P. aeruginosa on epithelial
cells remain unknown, several possibilities have emerged from these
studies. Many PAO1 cells bound near the basal aspect of Calu-3 cells,
so it seems possible that there is a role for the extracellular matrix
or epithelial junctional proteins in binding. Based on the fact that
P. aeruginosa bound preferentially to
regions between adjacent CFT1 (and JME) cells and that there were
images in which P. aeruginosa also
bound to regions between adjacent Calu-3 cells, it seems possible that
a junctional protein may also have been involved. Mistargeting of
basolateral proteins to the apical sides of epithelial cells may also
play a role in P. aeruginosa-induced
pathogenesis (4, 11). In any case, the binding sites appear to be
localized near regions between adjacent cells (at intercellular
junctions?) in low
Rt epithelial cells. In addition, for airway epithelia with
Rt > 1,000
· cm2,
apical P. aeruginosa did not bind or
exert cytotoxicity. In contrast, the addition of PAO1 to the apical
sides of intermediate Rt (200-500
· cm2)
tracheal epithelial cells led to high levels of P. aeruginosa binding (up to 1 P. aeruginosa/epithelial cell) and dramatic changes in ion
and fluid transport properties of the tissue (8), and we found that
apical addition of P. aeruginosa to
low Rt CFT1 and
JME cells led to high-level binding and rapid cytotoxicity. The
implication is that P. aeruginosa
bound and exerted effects from the apical surface of low (20-100
· cm2) and
intermediate (200-500
· cm2)
Rt but not of
high Rt (>1,000
· cm2)
airway epithelial cells.
 |
ACKNOWLEDGEMENTS |
We thank G. O'Toole and G. B. Pier (Harvard University Medical
School, Boston, MA) for supplying Pseudomonas
aeruginosa strain O1 expressing green fluorescent protein.
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FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-51799 (to T. Machen); National Eye
Institute Grant EY-11221 (to S. M. J. Fleiszig); and a grant from the
American Lung Association of California (to D. J. Evans).
A portion of the work presented in this paper has been published
previously in abstract form (W. Tseng, A. Lee, B. Haus, D. Evans, G. Chandy, D. Chow, and T. Machen. Pediatr. Pulmon.
Suppl. 14: 286, 1997).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. Machen, Dept.
of Molecular and Cell Biology, 231 LSA, Univ. of California, Berkeley,
CA 94720-3200 (E-mail: machen{at}socrates.berkeley.edu).
Received 8 June 1998; accepted in final form 19 March 1999.
 |
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