1 School of Optometry and
4 Department of Molecular and Cell
Biology, Fluid transport across cultures of bovine tracheal epithelium
was measured with a capacitance probe technique. Baseline fluid absorption (Jv)
across bovine cells of 3.2 µl · cm
bacteria; RpoN
IT HAS BEEN REPORTED THAT Pseudomonas
aeruginosa can cause pneumonia (3), pulmonary disease
in patients with autoimmune deficiency syndrome (4), and chronic airway
infections in patients with cystic fibrosis (10). The
combination of host and bacterial factors that contribute to infection
of airways by P. aeruginosa is
incompletely understood. Virulence factors expressed by
P. aeruginosa that may participate in
pathogenesis include 1) surface adhesins that mediate binding to respiratory epithelia or mucins (23),
2) the ability to invade airway
epithelial cells (7, 19), and 3) the
expression of toxins [e.g., proteases, exotoxin A, exoenzyme S,
exoenzyme U, rhamnolipids, and lipopolysaccharide (LPS)] that may
stimulate mucous secretion, cause ciliostasis, kill airway cells, and
activate host inflammatory and immune responses (5, 19, 25).
Mucociliary clearance, an important airway defense against infection,
is the mechanism by which bacteria are ensnared in mucus and cleared
from the lungs. This process requires ciliary beating, secretion of
respiratory mucus by submucosal glands, and regulation of water content
of the airway surface liquid (22, 29). The importance of
transepithelial fluid movements driven by active ion transport is
revealed by the genetic disease cystic fibrosis, in which changes in
chloride secretion (30) and active sodium absorption (2) by airway
epithelia are associated with inhibition of mucociliary clearance and
airway colonization by P. aeruginosa and other bacterial pathogens (10).
The relative hydration of the airway mucous secretions reflects a
balance between absorptive and secretory pathways. Liquid is added to
the airways by chloride secretion across gland acini (14). An important
mechanism for fluid removal is amiloride-sensitive sodium absorption
across the airway surface epithelium (13, 18). The purpose of this
study was to determine whether P. aeruginosa could affect fluid transport across airway
epithelia and, thereby, alter mucociliary clearance.
Bacteria.
Strains of P. aeruginosa were stored
frozen ( Cell culture.
Primary cell cultures of bovine airway surface tracheal epithelia (BTE)
were obtained as described previously (32). Briefly, strips of tracheal
epithelium were pulled away from underlying tissues, and epithelial
cells were isolated by overnight digestion (4°C) with 0.05%
(wt/vol) protease (type XIV, Sigma Chemical) in PBS. The next day,
epithelial cells were freed from the tissue strips by vigorous shaking
(30 s), and the action of protease was terminated by addition of FCS.
Cells were pelleted by centrifugation (200 g, 10 min) and resuspended in the
appropriate cell culture medium (32). Cells were plated at a density of
106
cells/cm2 onto 12-mm-diameter, 0.4 µm-pore-size Transwell filters (Costar, Cambridge, MA) coated with a
thin film of human placental collagen (20 µg/ml). Cells were grown at
an air-liquid interface (no medium added to the mucosal surface) and
fed every alternate day. Confluent sheets of airway surface epithelium
developed after ~7 days of culture at 37°C in an atmosphere of
5% CO2-95% air. For most
experiments, epithelial sheets were 8-17 days old. For BTE,
transepithelial resistance
(Rt) was 490 ± 250 Fluid transport.
Fluid transport
(Jv), TEP, and
Rt were measured
with a double-sided capacitance probe technique as previously described
(13). Briefly, a sheet of epithelium was mounted between two Lucite half-chambers. The chambers were perfused for 4 min with control Ringer
solution and then sealed. Capacitance probes were placed over the fluid
meniscuses in the reservoirs on either side of the epithelium to detect
changes in fluid height caused by transepithelial fluid transport. TEP
was recorded continuously, and
Rt was determined at 1-min intervals from the voltage deflections caused by
transepithelial current pulses of known magnitude.
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
2 · h
1
was inhibited by ~78% after 1 h of exposure to suspensions of Pseudomonas aeruginosa, with a
concomitant decrease in transepithelial potential (TEP) and increase in
transepithelial resistance
(Rt). Effects
of P. aeruginosa were blocked by
amiloride, which decreased Jv by 112% from
baseline of 2.35 ± 1.25 µl · cm
2 · h
1,
increased Rt by
101% from baseline of 610 ± 257
· cm2, and
decreased TEP by 91% from baseline of
55 ± 18.5 mV.
Microelectrode studies suggested that effects of P. aeruginosa on amiloride-sensitive Na absorption were
due in part to a block of basolateral membrane K channels. In the
presence of Cl transport inhibitors
[5-nitro-2-(3-phenylpropylamino)-benzoic acid,
H2-DIDS, and bumetanide],
P. aeruginosa induced a fluid secretion of ~2.5 ± 0.4 µl · cm
2 · h
1
and decreased Rt
without changing TEP. However, these changes were abolished when the
transport inhibitors were used in a medium in which Cl was replaced by
an impermeant organic anion. Filtrates of P. aeruginosa suspensions had no effect on
Jv, TEP, or
Rt. Mutants
lacking exotoxin A or rhamnolipids or with defective lipopolysaccharide still inhibited fluid absorption and altered bioelectrical properties. By contrast, mutations in the rpoN gene encoding
a
factor of RNA polymerase abolished actions of P. aeruginosa. In vivo, changes in transepithelial salt
and water transport induced by P. aeruginosa may alter viscosity and ionic composition of
airway secretions so as to foster further bacterial colonization.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C) in trypticase soy broth containing 10%
(vol/vol) glycerol. They were grown on a trypticase soy agar surface at
37°C overnight, then resuspended into prewarmed (37°C) sterile
normal Ringer solution to a concentration of ~4 × 107 colony-forming units (cfu)/ml.
Filtrates of bacterial cell suspensions were prepared by passage
through a 0.2-µm membrane filter (Millipore). Unless otherwise
stated, experiments were performed with P. aeruginosa strain PA0l.
· cm2 and
transepithelial potential (TEP) was
66 ± 18 mV (means ± SD, n = 8 tracheae).
Microelectrodes.
As described previously (16), calomel electrodes in series with
Ringer-agar bridges were used to measure TEP, and the signals from
intracellular microelectrodes were referenced to the apical or basal
bath to measure the apical and basolateral membrane potentials (VA and
VB, where TEP = VB VA).
Microelectrodes were made from fiber-filled borosilicate glass tubing
of 0.5 mm ID and 1 mm OD (Sutter Instrument, Novato, CA). They were
backfilled with 150 mM KCl and had resistances of 100-170 M
.
Silver-silver chloride wire was used to make the electrical connection
between the microelectrode and the electrode amplifier (Axoprobe 1A,
Axon Instruments). The Rt and the
apparent ratio of the apical to basolateral membrane resistance
(RA/RB)
were obtained by passing bipolar 4-µA current pulses across the
tissue for 3 s every 30 s and measuring the resulting changes in TEP,
VA, and
VB
(RA/RB
VA/VB).
Solutions. Bovine Ringer solution (BRS) contained (in mM) 120 NaCl, 23 NaHCO3, 10 glucose, 5 KCl, 1.8 CaCl2, and 1 MgCl2 (osmolarity = 290 ± 5 mosmol). In chloride-free BRS, NaCl was replaced by CH3SO3Na; all other chloride was replaced by gluconate. Osmolarity was adjusted to that of BSR using mannitol. Bicarbonate- and chloride-free BRS was the same as chloride-free BRS, except NaHCO3 was replaced by sodium HEPES. Solutions were equilibrated with 5% CO2-10% O2-85% N2 to pH 7.4, except for bicarbonate- and chloride-free BRS, which was equilibrated with 10% O2-90% N2 (pH 7.4). Addition of P. aeruginosa to Ringer solutions had no detectable effect on osmolarity.
Bacterial adherence. As described previously (7), tissue culture medium was removed, and confluent cell sheets were washed once with 500 µl of sterile BRS. P. aeruginosa suspension (200 µl containing ~4 × 107 bacteria) was added to the apical surface and incubated for 1 h at 37°C. Bacteria were removed, and the epithelial surface was washed gently three times with BRS (500 µl). The tissue was excised from the Transwell filter, washed once more, then homogenized for 15 min at room temperature in 1 ml of 0.25% Triton X-100 in trypticase soy broth. This lysed the epithelial cells and released bacteria into the medium without affecting bacterial viability. Counts of the viable bacteria in the lysate were performed. In each experiment, three tissue sheets were used to assess the binding for each bacterial strain. Comparisons between strains were made on the same day to control for variability in binding that occurs on cells of different age and from different sources. Representative experiments are presented in RESULTS. Experiments were repeated up to three times with similar results.
Ion transport inhibitors. Amiloride and bumetanide were obtained from Sigma Chemical, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) from Research Biochemicals International (Natick, MA), and H2-DIDS from Molecular Probes (Eugene, OR). Drugs were dissolved directly into Ringer solutions ~30 min before use, except NPPB, which was dissolved in DMSO before it was diluted 1:100 into BRS.
Data analysis. Values are means ± SD unless otherwise stated. Statistical assessments of differences between means were performed with Student's t-test, with P < 0.05 considered significant.
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RESULTS |
---|
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---|
Effects of P. aeruginosa on Jv.
The data shown in Fig. 1
illustrate the effects of P. aeruginosa on
Jv,
Rt, and TEP
across a sheet of BTE.
Jv decreased from ~5 to ~2
µl · cm2 · h
1,
Rt increased from
390 to 630
· cm2, and
TEP decreased from
50 to
31 mV. In six similar
experiments the bacteria-induced decrease in
Jv was 2.5 ± 1.4 µl · cm
2 · h
1
from a baseline absorption of 3.2 ± 1.3 µl · cm
2 · h
1,
the increase in
Rt was 302 ± 164
· cm2
from a baseline of 628 ± 215
· cm2, and
the decrease in TEP was 19 ± 9 mV from a baseline of
45 ± 19 mV.
|
Blockade by amiloride.
Figure 2 shows an experiment in which
apical amiloride (20 µM) decreased
Jv from 4.4 to
0.8 µl · cm2 · h
1,
increased Rt from
520 to 2,000
· cm2, and
decreased TEP from
77 to
7 mV. Subsequent addition of P. aeruginosa caused no further change
in steady-state
Jv,
Rt, or TEP. Two
other experiments produced practically the same result. In other
experiments (not shown), removal of amiloride resulted in recovery of
tissue responses to P. aeruginosa
similar to those shown in Fig. 1. With 50 µM amiloride
(n = 3) the results were indistinguishable from those with 20 µM. For all six experiments, amiloride reduced TEP from
55 ± 7.6 to
4.8 ± 1.7 mV, increased Rt
from 610 ± 105 to 1,226 ± 248
· cm2, and
decreased Jv from
2.35 ± 0.5 to
0.28 ± 0.6 µl · cm
2 · h
1
(means ± SE). Changes in
Jv,
Rt, and TEP
induced in BTE by P. aeruginosa in the presence of amiloride, 0.1 ± 0.3 µl · cm
2 · h
1,
44 ± 38
· cm2, and
2 ± 1.5 mV (means ± SE), respectively, were not
significantly different from zero and were significantly less than the
values obtained in the absence of amiloride (see above).
|
|
Role of chloride.
The experiment shown in Fig. 4 is
representative of three similar experiments in which we used NPPB (100 µM, mucosal), H2-DIDS (500 µM,
mucosal), and bumetanide (500 µM, basolateral) to inhibit known anion
transport pathways (27). In the example shown, these blockers decreased
Jv from 4.5 to
0.5 µl · cm2 · h
1,
increased Rt from
310 to 419
· cm2, and
decreased TEP from
70 to
14 mV. Subsequent addition of P. aeruginosa to the tissue in the
presence of the chloride transport inhibitor cocktail caused little
change in TEP but induced a significant fluid secretion of
2.8
µl · cm
2 · h
1
(Fig. 4). In three experiments the mean fluid secretion induced by
P. aeruginosa was
2.5 ± 0.4 µl · cm
2 · h
1.
This bacteria-induced fluid secretion was accompanied by a 27% decrease in Rt
from a baseline of 327 ± 101
· cm2.
Chloride channel blockers in combination with replacement of chloride
by methylsulfate (n = 3) or
replacement of chloride and bicarbonate
(n = 3) completely blocked the
induction of secretion and resistance changes induced by
P. aeruginosa (data not shown).
|
Bacterial factors.
A mutant strain of P. aeruginosa
(PG201 rhlR) that is deficient in the
production of rhamnolipid (and elastase) toxins (20) decreased
Jv from 8.0 to
3.9 µl · cm2 · h
1,
increased Rt from
300 to 440
· cm2, and
decreased TEP from
82 to
59 mV. An exotoxin A mutant
(PA103 tox::
) decreased
Jv from 3.7 to
0.1 µl · cm
2 · h
1,
increased Rt from
780 to 850
· cm2, and
decreased TEP from
56 to
49 mV. Similar results were
obtained in a second experiment with each mutant. Thus rhamnolipids,
elastase, and exotoxin A may not be involved in the actions of
P. aeruginosa on
Jv,
Rt, and TEP.
Filtrate of a P. aeruginosa suspension
had little or no effect on
Jv,
Rt, or TEP,
causing a small increase in
Jv of 0.7 ± 0.6 µl · cm
2 · h
1
from a baseline absorption of 3.9 ± 2 µl · cm
2 · h
1,
a decrease in Rt
of 10 ± 26
· cm2 from a
baseline of 440 ± 317
· cm2, and a
decrease in TEP of 4 ± 3 mV from a baseline of
57 ± 22 mV (n = 3).
|
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DISCUSSION |
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Airway epithelia exhibit baseline absorption of fluid. The inhibition
of absorption by amiloride implicates active sodium transport as a
driving force (13, 32). Here, we show that this fluid absorption is
inhibited by P. aeruginosa, which also raises Rt and
decreases TEP. These effects were achieved by adding bacteria to apical
and basolateral cell surfaces. This was done for two reasons:
1) to avoid artifacts in recorded
Jv changes and
2) because exposure of basolateral
cell surfaces to bacteria may occur frequently in vivo because of
inflammation and tissue injury. Recently, it was shown that interaction
with basolateral cell surfaces may be significant in the pathogenesis
of P. aeruginosa infections (6).
Amiloride blocked all the effects of P. aeruginosa without altering bacterial binding,
suggesting that this pathogen acts by inhibiting one or more components
of the amiloride-sensitive sodium transport pathway. These findings are
consistent with previous studies showing that P. aeruginosa decreased short-circuit current and reduced
net sodium absorption across short-circuited airway epithelial cells
(9, 26). Bacterial cells were required to alter
Jv,
Rt, or TEP;
filtrates of P. aeruginosa suspensions
were ineffective. Loss of production of rhamnolipids, LPS O antigen, and exotoxin A did not affect these actions of P. aeruginosa. By contrast, a mutant unable to express
adhesins under transcriptional control of the factor RpoN not only
showed markedly reduced binding to the airway epithelial cells but also
had no effect on fluid absorption. Thus direct interaction of bacterial
cells with the airway epithelium may be needed for P. aeruginosa to inhibit fluid absorption.
Possible mechanisms. Net transepithelial fluid movement across airway surface epithelium reflects the balance of active secretory and absorptive solute transport. Thus inhibition of fluid absorption across airway epithelium by P. aeruginosa could be due to a reduction of active solute absorption and/or an increase in secretion. In airway epithelia, major solute transport processes are amiloride-sensitive active sodium absorption and active secretion of chloride (27), the former inhibited by amiloride and the latter by bumetanide, NPPB, and DIDS. As in our previous studies on human tracheal cells (13), pretreatment of BTE with amiloride brought Jv to approximately zero while increasing Rt and decreasing TEP, consistent with its known inhibitory action on apical membrane sodium channels (1). In Fig. 3B the hyperpolarization of VA and the large increase in RA/RB in the presence of an increase in Rt point to amiloride's predominant action being on apical membrane sodium channels. In the presence of amiloride, P. aeruginosa had no effect on Jv. Taken together these results indicate that this pathogen acts predominantly by inhibiting active sodium absorption rather than by stimulating a secretory process.
Microelectrode studies were used to help determine the site(s) of action of P. aeruginosa on active sodium absorption. In the continuous record of Fig. 3A, Rt did not change significantly. However, we believe that the lower-than-normal Rt and TEP of this tissue were due to edge damage. If this is so, the resulting high shunt conductance would have obscured the effects of changes in RA and RB on Rt. Pooled results from all tissues showed that P. aeruginosa increased Rt from ~500 to ~800Bacterial factors. P. aeruginosa may induce changes in ion-coupled fluid transport through a variety of mechanisms, including toxin secretion or binding to an epithelial cell surface receptor. Our data show that if a toxin is involved, it is not secreted into the surrounding environment before airway cell contact, since the filtrate of a bacterial suspension did not affect ion or fluid transport. This does not rule out toxin involvement, however. Some toxins are only secreted by bacteria on host cell contact (type III secretion), a form of delivery identified in P. aeruginosa and other gram-negative bacteria (31).
A glycolipid hemolysin of P. aeruginosa has been shown to reduce sodium absorption and unidirectional chloride fluxes across human bronchial epithelium (26). Low concentrations of purified P. aeruginosa rhamnolipids decrease amiloride-sensitive short-circuit current across sheep tracheal epithelium (9). Finally, P. aeruginosa exotoxin A has been shown to increase fluid absorption across distal airways in vivo (21). In the present study a rhamnolipid-deficient (and elastase-deficient) mutant of P. aeruginosa and an exotoxin A-deficient mutant inhibited fluid absorption and altered the bioelectrical properties of BTE. Thus, although we cannot preclude a role for these toxins in mediating fluid transport changes in vivo, our data indicate that P. aeruginosa can alter ion-coupled fluid transport in airway cells without these toxins. Inhibition of fluid absorption by P. aeruginosa requires RpoN, aClinical implications. The P. aeruginosa-induced alterations in salt and water transport across airway epithelium could affect further bacterial colonization in one of two ways. First, decreased fluid absorption would dilute the mucous secretions, thereby reducing clearance of secretions and entrapped P. aeruginosa (17). The resulting buildup of mucus, albeit dilute, could also encourage colonization by increasing the number of binding sites for P. aeruginosa (23). Second, P. aeruginosa could promote further colonization by altering the salt content of airway surface liquid. Joris et al. (15) reported that cystic fibrosis airway surface liquid has higher-than-normal NaCl levels (120 vs. 80 mM). More recently, it was demonstrated that the killing ability of antimicrobials secreted by the airway epithelium was inhibited at higher salt levels (8, 24). Bacterial inhibition of active sodium absorption could increase bacterial colonization by elevating NaCl concentrations in the airway surface liquid.
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ACKNOWLEDGEMENTS |
---|
We thank Kefu Yu for expert technical assistance and Dr. Suzanne Fleiszig for helpful discussions and advice. We also acknowledge and thank Drs. Barbara Iglewski, Stephen Lory, and Urs Ochsner for generating P. aeruginosa mutant strains.
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FOOTNOTES |
---|
This work was supported by a grant from Cystic Fibrosis Research (Palo Alto, CA), a grant from the American Lung Association of California to D. J. Evans, and National Heart, Lung, and Blood Institute Grant HL-42368 to J. H. Widdicombe.
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: D. J. Evans, School of Optometry, 387A Minor Hall, University of California, Berkeley, CA 94720-2020.
Received 17 March 1998; accepted in final form 25 June 1998.
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