Opinion
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Vol.13 No.6
Cystic fibrosis: a disease of
vulnerability to airway surface
dehydration
Richard C. Boucher
Cystic Fibrosis Pulmonary Research and Treatment Center and the UNC Virtual Lung Group, The University
of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Cystic fibrosis (CF) lung disease involves chronic
bacterial infection of retained airway secretions (mucus).
Recent data suggest that CF lung disease pathogenesis
reflects the vulnerability of airway surfaces to dehydration and collapse of mucus clearance. This predisposition is caused by mutations in the CF transmembrane
conductance regulator (CFTR) gene, resulting in (i) the
absence of CFTR-mediated ClS secretion and regulation
of epithelial Na+ channel (ENaC) function; and (ii) the sole
dependence on extracellular ATP to rebalance these ion
transport processes through P2 purinoceptor signaling.
Recent clinical studies indicate that inhalation of hypertonic saline osmotically draws sufficient water onto CF
airway surfaces to provide clinical benefit.
Difficulties in translating mutations in CFTR into
pathogenesis in CF lung disease
Research into the molecular pathogenesis of the syndrome
of cystic fibrosis (CF) has evolved rapidly over the past
15 years. The identification and cloning of the gene encoding
the cystic fibrosis transmembrane conductance regulator
(CFTR) (see Glossary) protein was a seminal event in this
evolution [1,2]. Subsequently, the molecular and cellular
pathogenesis of the most common CFTR mutation, DF508
CFTR, and other common severe CFTR mutations, was
demonstrated to result in a failure of the mutated CFTR
to fold properly and progress through maturational processes within the cell [3–5]. Hence, the mutated CFTR
protein is degraded prior to insertion into the plasma membrane.
However, despite these great strides, there has been
less insight into how mutations in the CFTR protein
produce disease in CF-affected organs. Perhaps most perplexing has been the relationship between mutant CFTR
function and the disease phenotype of CF in the lung,
which is manifest as a failure of innate airways defense
against inhaled bacteria that produces chronic bacterial
infection of CF airway lumens and, ultimately, airway
obstruction, bronchiectasis and death [6] (Box 1).
CF lung pathogenesis has been proposed to reflect
problems in cell biology. One hypothesis has focused on a
role for mutated CFTR to generate an epithelial inflammatory syndrome [7]. However, such a hypothesis fails to
Corresponding author: Boucher, R.C. ([email protected]).
Available online 23 May 2007.
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account for the predisposition to infection, and studies of
primary cultures of airway epithelia have failed to detect
differences in inflammatory phenotypes between CF and
normal airway epithelia [8,9]. A second cell biological hypothesis has focused on the role of wild-type CFTR to function as a receptor to promote epithelial clearance and killing
of bacteria, and a failure of CF airway epithelia to do so in the
context of a mutant CFTR [10]. However, there is little
evidence for epithelial killing of bacteria in the normal lung,
and no good explanation for why mutant CFTR proteins that
appear in the apical membrane (e.g. G551D) produce a CF
phenotype similar to that of mutations with no protein in the
apical membrane (e.g. DF508 CFTR) (see earlier).
There have been three attempts to link mutations in
CFTR to failures of lung defense through the ion transport
functions of CFTR. In the first, a role for CFTR in controlling airway surface liquid (ASL) salt composition was
proposed. In this hypothesis, it was suggested that the
absence of CFTR led to a failure to absorb Cl (and Na+),
producing a relatively hypertonic ASL, and degradation of
defensin-mediated antimicrobial activity [11,12]. However, it seems implausible to ‘trust’ the defense of the lung
to antimicrobial activities because, for example, problems
of resistance would probably soon emerge. Furthermore,
most recent studies have failed to detect differences in
tonicity of ASL between normal and CF airways [13,14]. In
the second hypothesis, the role of CFTR in airway submucosal gland secretion was emphasized [15–20]. Evidence suggests that CF submucosal glands are defective
in secreting liquid in response to cAMP-regulated agonists
(e.g. vasoactive intestinal peptide) but not acetylcholine
receptor agonists. It is likely that defective gland function
Glossary
Airway surface liquid (ASL): the water contained in the periciliary and mucus
layers.
Cystic fibrosis transmembrane conductance regulator (CFTR): protein product
of the CFTR gene that exhibits, as its name implies, Cl channel and ENaC
regulatory properties.
Epithelial Na+ channel (ENaC): heteromultimeric Na+ channel composed of
products of three or four separate genes, including a, b, g and, possibly,
1 ENaC.
Mucus layer: layer composed of unrestrained secreted mucins in tangled
networks interacting with globular protein ‘stickers’.
Periciliary liquid layer (PCL): a domain surrounding the cilia and cell surface
that is a grafted polyelectrolyte gel, composed, in part, of tethered mucins; it is
not a liquid layer.
1471-4914/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2007.05.001
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Box 1. Outstanding questions
Is the superficial epithelium entirely responsible for ASL dehydration, or do the submucosal glands contribute significantly?
Is the early inflammation in CF due to failure to clear mucus,
undiagnosed infection, or an intrinsic defect in epithelial regulation of inflammation?
Is hypertonic saline exerting its beneficial action by hydrating
airway surfaces or by inducing cough?
Will early eradication of selected bacteria treat CF lung disease
effectively in the absence of restoring hydration and clearance?
contributes to disease pathogenesis in the large airways by
reducing their ability to respond to insults with secretion
of antimicrobial agents and a modest amount of liquid.
However, submucosal gland dysfunction is unlikely to
contribute to disease pathogenesis in the CF small airways, the initial and major site of disease [6,21]. The third
hypothesis proposed that CF airways disease results from
a failure of the mechanical clearance system that constitutes the primary innate defense against bacterial infections – that is, mucus clearance [22]. This failure reflects
the inability of CF superficial airway epithelia to maintain
the hydration of CF airway surfaces required for efficient
mucus clearance due to abnormal regulation of Na+ and
Cl transport (see later) [23]. Based on mounting evidence
from in vitro cell culture data, mouse models and human
clinical trials with hydration agents, reviewed later, this
hypothesis is preferred by the author, and constitutes the
basis for this review.
Mechanical (mucus) clearance as the primary innate
defense mechanism for airways
As shown in Figure 1, the effective clearance of particles
deposited on airway surfaces requires the coordinated
activities of a two-phase gel system on the airway surface:
(i) the periciliary layer that extends from the cell surface to
the height of the extended cilium; and (ii) the mucus layer
that is positioned atop the cilia [24]. The layer that surrounds the cilia was originally thought to be a liquid but is
more likely to be a grafted polyanionic gel [25]. Intriguingly, the properties of this type of gel provide an ideal lowfriction environment for effective ciliary beat, and also
provide a lubricant activity that prevents adhesion of
the mucus layer to the cell surface [26]. The mucus layer
is composed of extremely long, highly glycosylated polymers known as the secreted mucins. A combination of the
mucins MUC5AC and MUC5B, secreted predominantly in
health, by goblet cells and submucosal glands, respectively, constitute this gel layer [27]. Biophysical studies
suggest that the mucins are organized as a mesh, with a
pore size optimal for trapping and retaining virtually any
inhaled particle [28].
Recent evidence, primarily from mouse models, suggests
that hydration of the two-phase ASL compartment is the
primary determinant of the efficacy of mucus transport [29].
As shown in Figure 1, the hydration of this compartment is
determined by the net activities of active ion transport
systems. Normal airway epithelia have the capacity both
to absorb and to secrete salt, with water moving osmotically
in response to the generated salt gradients [30,31]. Under
normal conditions, active Na+ transport is the dominant ion
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transport pathway. The dominance of this process reflects
the large volume of ASL funneling up airways with converging surfaces that must be absorbed to maintain a thin film
of ASL on airway surfaces. Active Na+ transport is mediated
by a rate-limiting channel in the apical membrane, the
epithelial Na+ channel (ENaC), and Na+ is extruded from
the cell through the basolaterally located Na+/K+-ATPase.
Under conditions when there might be too little ASL on the
airway surface, the ENaC is inhibited, electrochemical driving forces for Cl secretion are generated, and Cl is secreted
through two apical membrane Cl channels. It seems that
the Cl channel that mediates the basal Cl channel activity
of the airway epithelium is the CFTR Cl channel, whereas
a second Cl channel, the calcium-activated Cl channel
(CaCC), responds to stresses [32]. It should be noted that
airway epithelia are relatively ‘leaky’, so the counter-ion for
absorption (Cl) or secretion (Na+) moves through the paracellular path.
The CFTR protein exerts a major influence on the
capacity of the airways to maintain normal hydration, with
CFTR functioning both as a Cl channel and as an inhibitor of the ENaC. Hence, as shown in Figure 1b, it is
predicted that in CF airway epithelia, with an absence
of either molecular or functional CFTR in the apical membrane, there will be unregulated Na+ absorption and a
decreased capacity to secrete Cl. Both defects combine to
produce dehydration of the airway surface, with a collapse
of the periciliary layer (PCL), concentration of mucins
within the mucus layer, and adhesion of mucus to the
airway surface [22]. It is predicted that the adhesion of
mucus to airway surfaces initiates the airflow obstruction,
inflammation and chronic infection that are features of CF.
Thus, the simplest form of the ASL volume depletion and
dehydration hypothesis involves unremitting and inappropriate absorption of ASL.
Evidence for ASL volume depletion in CF
Evidence emanating from three systems favoring the low
volume and dehydration hypothesis can be summarized as
follows. First, observations from air–liquid interface cultures maintained under static conditions have demonstrated that normal airway epithelia maintain adequate
ASL volume on airway surfaces (defined as liquid height
being equal to the extended cilium – i.e. 7 mm) for
extended periods of time, whereas CF cultures do not
[22,32] (Figure 1c,d). Bioelectric studies revealed that
the normal airway epithelia adjust the rates of Na+ and
Cl secretion to maintain adequate salt, and hence water,
on airway surfaces for normal hydration, whereas CF airway epithelial cells are locked into an unregulated Na+absorptive mode, with little capacity to secrete Cl ions.
These studies demonstrated that the failure to maintain
adequate hydration on CF airway cell culture surfaces led
to a complete failure of mucus transport and, ultimately,
adhesion of mucus to cell surfaces [22,41].
Second, mouse models have provided important in vivo
data. The normal mouse lung expresses little CFTR and,
hence, mice with targeted disruptions of the murine CFTR
gene (i.e. ‘CF mice’) exhibit little or no pulmonary disease
phenotype [33]. Consequently, to test the importance of
ASL dehydration in the pathogenesis of airways disease,
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Figure 1. Normal mucus clearance mechanisms and failure of CF mucus clearance due to airway surface dehydration. (a) Normal airways coordinate rates of Na+
absorption and Cl secretion to hydrate airway surfaces and promote mucus clearance. Under basal conditions, with ASL funneling up converging airway surfaces, Na+
transport is dominant (accompanied by paracellular Cl absorption; not shown). To fine-tune ASL volume, ASL can be secreted if the ENaC is inhibited and electrochemical
driving forces favoring Cl secretion generated (accompanied by paracellular Na+ secretion; not shown). Water moves in response to the net transport (absorption or
secretion) of NaCl. (b) In CF, the absence of CFTR function in the apical membrane leads to unregulated Na+ (Cl follows passively through the paracellular path; not
shown), a failure to secrete Cl and, hence, inappropriate water absorption. The deficit of water on CF airway surfaces (i.e. dehydration) depletes the PCL of volume,
concentrates mucus and produces mucus adhesion to CF airway surfaces, and, hence, a failure of both ciliary and cough-dependent mucus clearance. (c) Confocal images
of ASL covering normal and CF airway surfaces. At t = 0 hours, the water component of ASL is labeled with a green fluorescent dextran, and the mucus layer with red
fluorescent microspheres. Note that the beads at t = 0 hours are excluded from the PCL in both normal and CF cultures. At t = 24 hours, the normal cultures have absorbed a
component of ASL but maintain a distinct PCL. By contrast, all available ASL has been absorbed from CF culture surfaces and the microsphere-labeled mucus layer has
invaded the PCL. (d) Perfluorocarbon–osmium-fixed cultures at t = 24 hours. In the normal cultures, a distinct PCL is visualized, with cilia fully extended. In CF cultures, the
cilia are flattened, and the mucus layer has invaded the PCL space and adhered to ciliary surfaces. Parts (c,d) reproduced, with permission, from Ref. [22].
Mall et al. [29] engineered murine airways to accelerate
Na+ absorption. Transgenic overexpression of a single
ENaC subunit, the bENaC subunit, produced an approximate 2.5-fold increase in Na+ absorption; a depletion of
ASL, as measured morphometrically in the PCL and
physicochemically in the mucus layer (percentage solids
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content of the mucus layer); and mucus obstruction.
Indeed, the mucus obstruction was so severe that 50%
of the transgenic mice died owing to asphyxia within
the first 30 days of life. An unexpected but important
result was that the failure to clear mucus was associated
with a sterile, neutrophilic inflammation mediated by the
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production of proinflammatory cytokines. Finally, these
mice had difficulty in clearing bacteria, and recent studies
have shown that they exhibited a robust inflammatory
response to respiratory viruses. Thus, these studies have
strongly suggested that ASL depletion can produce major
features of CF lung disease in vivo.
Third, evidence for ASL volume depletion and
dehydration emanates from several types of human clinical
studies. Perhaps the first data indicating the importance of
dehydration and mucus plugging were reported by Zuelzer
and Newton [21] in the 1940s, in infants who had died from
meconium ileus. These pathologists demonstrated that the
earliest lesions in the CF lung, as early as 2–4 days after
birth, were ‘bland’ mucus plugs in the bronchioles. Subsequent experiments in the late 1960s from Matthew et al.
[34] and Potter et al. [35,36] revealed that the water
content of CF secretions, expressed as percentage solids,
was reduced as compared with disease controls and normal
subjects. Although the subject of much debate, studies from
the 1990s indicated that this dehydration is an isotonic
dehydration – that is, CF and normal airway secretions
both have a concentration of NaCl that is approximately
isotonic [13,14,37].
The collective data indicating that CF airways might be
dehydrated led to therapeutic trials with aerosolized
hypertonic saline (HS) [38,39]. As discussed in detail later,
the concept tested was that the inhalation of solutions
containing high concentrations of salt (7%) would produce transient hypertonicity on airway surfaces (note:
plasma and interstitial salt concentrations are 0.9%) to
‘rehydrate’ these surfaces osmotically. In brief, short-term
(two-week) inhalation of HS four times daily increased
mucus transport both acutely and chronically, and this
response correlated with improvements in lung function
and quality of life [38]. In parallel, long-term studies of HS
in Australia revealed that there was a sustained improvement in lung function in CF patients on HS therapy (7%,
twice daily) as compared with vehicle, and, importantly,
there was a major reduction in infectious exacerbations in
HS-treated subjects [39]. These data suggest that rehydration of CF airway surfaces is indeed beneficial, consistent
with the in vitro culture and mouse model data supporting
the low volume and dehydration hypothesis.
‘Key caveats’ to the notion of Na+ hyperabsorption
and failed ClS secretion in the pathogenesis of CF
airways dehydration
Recent studies have suggested that the notion of simple,
persistent Na+ hyperabsorption, coupled with failure to
secrete Cl, is an oversimplified version of what might
happen in the CF patient [32]. Indeed, these studies were
stimulated by predictions from in vitro studies (see earlier)
that CF airways should rapidly and homogeneously fill
with a mucus gel soon after birth, in contrast to the clinical
data that demonstrated that CF airways disease becomes
manifest many months to years after birth [6,22,40]. Study
of this apparent incongruity led to two major modifications
of the simple volume hyperabsorption hypothesis.
First, it seems that CF airways disease reflects a
vulnerability to collapse of ASL volume in response to
exogenous stresses (e.g. viral infections or aspiration).
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The data that led to this concept emerged from studies
in which the normal shear stresses that occur during tidal
breathing in vivo were applied to the cell culture preparations described earlier [32,41]. These studies revealed
the importance of signaling molecules within the ASL
itself in controlling ASL volume [42]. Although this signaling system is not understood in its totality, it seems
that two extracellular signaling molecules, ATP and adenosine (ADO), are vital for determining ASL volume and
hydration in a state optimal for normal host defense. As
shown in Figure 2, normal airway epithelia release ATP
into the luminal compartment by both a constitutive (probably vesicular mediated) and a shear stress-regulated
process. ATP released into the luminal compartment has
two important fates. (i) ATP interacts with luminal P2Y2
purinoceptors that mediate inhibition of the ENaC and
activation of both the CFTR and CaCC [43]. (ii) ATP is
metabolized by cell surface ectoenzymes to produce ADO
[44]. ADO interacts with luminally positioned A2b receptors that, through cAMP-dependent mechanisms, activate
CFTR, and, by an as- yet-unknown mechanism, induce
CFTR to inhibit the ENaC [45]. The volume of ASL seems
to be regulated by the magnitude of shear stress-induced
ATP release, and subsequent studies have shown that both
ATP and ADO are important for supporting the physiological volume of ASL on normal airway surfaces.
By contrast, CF airway epithelia fail to express a CFTR
protein, whether functional or otherwise, at the apical
membrane (Figure 2b). CF airway epithelia release ‘normal’
amounts of ATP in response to shear stress, and the released
ATP effectively regulates the ENaC and the CaCC through
P2Y2 receptor signaling [41,42,46,47]. However, in contrast
to normal airway epithelia, luminally formed ADO does not
activate Cl secretion and inhibit the ENaC because of the
absence of the target of A2b signaling (i.e. CFTR). Thus, CF
airways epithelia are vulnerable in two respects from an
ASL volume and hydration perspective: (i) they are predicted to have an ASL volume that is less than normal but
probably sufficient for mucus transport in times of health;
and (ii) they depend solely on ATP for the production of
sufficient ASL to maintain mucus transport.
A mechanism by which an exogenous insult can lead to
collapse of CF mucus transport defenses in response to a
reduction in ATP levels in the ASL compartment has
recently been described [32]. As shown in Figure 3, an
early manifestation of respiratory syncytial virus (RSV)
airways infection is an upregulation of a cell surface
ectoenzyme that metabolizes ATP and reduces ASL ATP
concentrations. Lesser P2Y2 receptor activation reduces
ENaC inhibition and stimulation of Cl secretion, the net
effect being ASL volume depletion and collapse of mucus
transport. Thus, the ASL low volume hypothesis predicts a
sensitivity to viral respiratory infections, consistent with
the observations that CF patients have a more severe
clinical response to RSV infections than do normal individuals [48].
Second, it is important to emphasize that CF lung
disease is heterogeneous – that is, the CF phenotype does
not appear and progress homogeneously throughout the
lung. Perhaps the first evidence of this notion emanated
from pathology studies that showed that the upper lobes of
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Figure 2. Regulation of ASL volume homeostasis by ATP release. (a) Airway epithelial cells release ATP onto the airway surface by basal (vesicle)-mediated and shear
stress-regulated pathways. ATP is metabolized by cell surface ectoenzymes to produce ADO. (b) Normal airway epithelia exhibit ATP-mediated P2Y2 receptor regulation of
both the ENaC (inhibition) and CFTR and CaCC (activation), and ADO-mediated A2b receptor regulation of the CFTR and, ultimately, ENaC (not shown). (c) Normal airway
epithelia balance Na+ absorption against Cl secretion to maintain ASL homeostasis, through regulatory effects of both ATP and ADO. Note that counter-ions for both
transcellular Na+ absorption and Cl secretion permeate the epithelium through this paracellular path (not depicted). (d) CF airway epithelia do not express functional CFTR
protein in the apical membrane. However, P2Y2 receptor signaling regulates the ENaC and CaCC normally. By contrast, the absence of the CFTR renders A2b signaling
ineffective for Cl secretion, and, indeed, paradoxically activates the ENaC. (e) CF airway epithelia have a tendency to absorb more salt and water than normal because of
the absence of CFTR inhibition of ENaC and CFTR Cl secretion. This tendency is held in check during basal intervals by P2Y2 receptor signaling to the ENaC and CaCC. Not
depicted is the likely dominance of the paracellular path for absorption of Cl as the counter-ion for transcellular Na+ absorption in CF.
CF subjects are typically first affected, whereas the lower
lobes could be normal [49]. More recently, CT scanning
studies of young infants have also shown a predilection for
early bronchiectasis in the upper lobes of the lung, with
relatively normal structures in the lower lobes [50]. Clinically, it has long been known that CF exacerbations are
often associated with ‘new clinical findings’ in circumscribed areas of the lung, suggesting that progression is
not uniform but heterogeneous. Finally, the recent mucociliary clearance data from Donaldson et al. [38] support
the notion that mucus clearance in the CF lung is also
heterogeneous (Figure 4).
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There are two interesting speculations with regard to
the heterogeneity of CF lung disease. First, there is the
simple question of why this happens, given the fact that
the CF lung is homogeneously deficient in CFTR function.
One speculation is that with normal tidal breathing, shear
stresses are greater in the lower lobes, where, by virtue of
gravitational forces, more than two-thirds of the normal
tidal ventilation occurs [32]. Thus, it is predicted that there
is more shear stress in the lower lobes and hence more
ATP release, producing more P2Y2-mediated inhibition of
Na+ absorption and induction of CaCC-mediated Cl
secretion, and a relative increase in ASL volume. The
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Figure 3. Effect of RSV infection on CF ASL volume homeostasis. (a) RSV induces upregulation of an extracellular ATPase (eNPP2) that greatly reduces ASL ATP
concentrations. (b) Due to reduced ASL ATP concentrations, P2Y2 receptor signaling to inhibit the ENaC and activate CaCC is reduced or abolished (indicated by broken lines
– compare with Figure 2b). (c) With reduced P2Y2 receptor signaling, CF airway epithelia revert to an unregulated mode of salt and water absorption that produces ASL
depletion and mucus stasis.
second speculation is that it might be possible to arrest
the progression of CF lung disease if the stimuli that drive
the progression are identified and if effective therapies can
be appropriately delivered to these regions (see later).
Thus, it seems that the inclusion of compensatory
mechanisms to regulate the balance of Na+ absorption
and Cl secretion – that is, extracellular ATP signaling
(Figures 2,3) – provides a more accurate description of the
pathogenesis of CF lung disease than the simpler model
depicted in Figure 1.
Events ‘downstream’ of ASL volume depletion in CF
airways pathogenesis
What is unique about the CF bacterial bronchitis
phenotype is the persistence of the bacterial infection. For
example, CF patients can be infected virtually life-long with
Figure 4. Schema describing regional heterogeneity of mucus clearance in a CF patient with mild–moderate lung disease (forced expiratory volume in one second 75%
predicted). Shown is the absence of mucociliary clearance (##MCC) in bronchiectatic apices (red), reduced mucus clearance in bronchioles (yellow) and relatively normal
mucus clearance in central, lower zones (green). Measurements of radioparticle clearance from the lungs of CF patients with mild–moderate disease is shown on the right.
The 24-hour clearance is an index of bronchiolar clearance and is reduced in CF. Central airway mucus clearance reflects large airways, and it is not decreased in the lower
lobes of CF patients with this level of disease severity. Asterisk denotes significant difference (P<0.05) between normal and CF. Data shown are from Ref. [38].
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the original Pseudomonas organism that colonizes and
infects their lungs [51]. The acquisition of this persistent
infection and the responses to it are complex but many
aspects seem to be congruent with the dehydrated, adherent
mucus plaques predicted by the low volume hypothesis.
One reason for CF airways infections being so persistent
is that bacteria grow in biofilm-type physiologies and
morphologies [52,53]. Bacteria growing as biofilms are
difficult to eradicate by secondary host defense and by
antimicrobial agents [54]. Recent studies suggest that
Pseudomonas interacts with the dehydrated mucus of
CF airways in a manner that favors biofilm formation
[28]. A normally hydrated mucus layer exhibits a mucindependent mesh size of 0.5–10.0 mm in diameter. Bacteria deposited on a normally hydrated mucus gel (2%
solids) can ‘swim’ through the gel and remain in a planktonic state within it. By contrast, with depletion of salt and
water, the mucin-dependent meshwork in a CF mucus gel
becomes ‘tight’ (mesh size of <100 nm). The small mesh
size restricts the mobility of Pseudomonas that deposit on
the surface of CF mucus, resulting in the bacteria replicating at the site of deposition faster than they migrate from
it. This phenomenon leads to accumulation of bacteria and
a high local production of autoinducers that produce
biofilm formation [55,56]. This motility restriction, coupled
with a reduced permeation of autoinducers from the site of
their production through concentrated mucus, triggers
biofilm formation in CF, but not normal, mucus.
Airways have secondary defense mechanisms against
bacteria that are not normally cleared by mucus clearance.
These defenses include soluble antimicrobial agents (e.g.
lactoferrin and lysozyme) and neutrophils that can migrate
into the airway lumen and capture and kill bacteria.
Recent studies have shown that the concentration of mucus
that occurs in CF airways reduces the ability of lactoferrin
and lysozyme to permeate this environment to inhibit
bacterial growth [28]. Similarly, the reduction in the mesh
size of the mucin network in concentrated CF mucus limits
the ability of neutrophils to penetrate mucus and subsequently capture and kill bacteria [57].
An interesting and important consequence of persistent
intraluminal bacterial infection is the host inflammatory
response. Again, the unusual characteristic of CF is the
persistence of this bacterial stimulus, with patients having
>106 microorganisms/ml of intraluminal mucopurulent
material in some airways for the majority of their lives.
Recent data suggest that the persistent intraluminal bacterial infection upregulates the normal host response to
luminal bacterial pathogens [9,58]. One response is to
increase the production of proteins that have antimicrobial
and anti-inflammatory properties, in addition to proteins
involved in the repair of bacterial- and inflammationinduced cell damage. This increased protein synthetic
response initiates a form of the ‘unfolded protein response’,
which is associated with expansion of the endoplasmic
reticulum (ER) compartment and increased capacity to fold
and synthesize proteins [59–61]. One consequence of ER
expansion is to increase the inositol trisphosphate-releasable Ca2+ stores. Thus, Ribeiro et al. [58] have reported that
freshly excised CF cells, or indeed normal cells chronically
exposed to CF airway luminal mucopurulent material,
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exhibit increased Ca2+-dependent interleukin-8 release in
response to inflammatory mediators that signal through
Ca2+-dependent processes (e.g. bradykinin). At one level,
this amplified inflammatory response might be beneficial to
the host, particularly if the host has the capacity to clear the
offending agent. However, in CF, where the bacteria persist
within the immobilized mucopurulent material and mucus
plaques, the amplified inflammatory response might
damage airway walls, producing the bronchiectasis typical
of CF. Unresolved are questions of whether there are other
mechanisms that produce hyperinflammatory responses,
and, indeed, if CF airway epithelia are intrinsically hyperinflammatory consequent to CFTR defects [62–64].
Thus, it seems that many of the heretofore perplexing
problems of infection and inflammation in CF pathogenesis
can be explained by interactions of infectious agents and
host inflammatory processes with the abnormally thick,
adherent mucus that characterizes the CF airways.
Therapies directed at airway surface rehydration
in CF
Mammalian airway surfaces are relatively permeable to
water [65]. Thus, rehydration therapies require the
addition of salt to airway surfaces, to draw water onto
airway surfaces osmotically. In general, there are two ways
to ‘add salt’ to CF airway surfaces. The first is for patients
to inhale HS (see earlier). The second is to administer
agents to the CF airway surface that will redirect ion
transport towards the secretory direction.
As described earlier, HS seems to have a therapeutic
benefit over the short term (pulmonary function) and over
the long term (reduction in exacerbations) in CF patients
[39,66–68]. Interestingly, HS might be more therapeutically beneficial in CF than in normal airways (Figure 5). In
normal subjects, when HS is deposited on airway surfaces,
a transepithelial chemical gradient for Na+ and Cl is
generated that can be dissipated through movement of
Na+ through the ENaC and Cl through the CFTR channel. Indeed, there is a ‘kinetic horse race’ between NaCl
absorption and water transport in the direction of the
osmotic (salt) gradient – that is, towards the lumen. In
the normal airway, NaCl is absorbed sufficiently rapidly
that osmotically driven water transport to the airway
surface is modest. By contrast, when HS is deposited on
CF airway surfaces, Na+ can theoretically permeate the
epithelium through the open ENaC; however, Cl cannot
permeate in parallel because the CFTR Cl channel is not
present or active. The remaining pathways for Cl movement across the CF epithelium down the imposed chemical
gradient are more minor – for example, CaCC and the
paracellular pathway, so that Na+ and Cl are absorbed
more slowly from the CF airway surface. This phenomenon
enables water to move efficiently to the airway surface in
response to the persistent Na+ and Cl gradient, producing
greater volume expansion on the CF airway surface.
Multiple different types of ion channel modulators have
been explored in the pharmacotherapy of CF. Several
agents described as ‘alternative’ Cl channel (ClC-2,
Ca2+-activated) activators are in current clinical testing
– for example, SPI-8811 and Moli 1901 [69]. Although
these novel agents show promise, it is possible that
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Figure 5. Effects of inhalation of HS (1000 mM NaCl, 7%) on ASL volume in normal and CF airway epithelia. (a) Deposition of droplets containing 7% HS on a normal airway
(mucus) surface that is isotonic (150 mM NaCl). Note that the interstitium is, by definition, isotonic (0.9%). (b–d) Normal airway epithelia. (b) The NaCl chemical gradient
generated by deposition of 7% HS is rapidly dissipated by transcellular absorption through the basally activated ENaC and CFTR channels. Water moves towards the airway
surface from the interstitium in response to an osmotic gradient generated by HS deposition. (c) Modest expansion of the ASL volume that is ‘stored’ in the expanded
mucus layer reflects rapid absorption of NaCl and, hence, a relatively smaller gradient for H2O permeation. (d) The added ASL is gradually reabsorbed by active Na+
transport. (e)–(g) CF airway epithelium. (e) The NaCl chemical gradient generated by HS deposition is not dissipated quickly because Cl cannot permeate rapidly through
the relatively Cl-impermeable CF cell. Hence, the HS- generated osmotic (NaCl) gradient persists for a larger interval than on normal airway surfaces, and, hence, a larger
amount of water permeates to the apical surface. (f) The large water flow to the airway surface produces a larger increase in ASL volume on CF than on normal airway
surfaces; compare this with (c). (g) The added ASL is absorbed by active Na+ transport but the greater initial volume expansion produces a larger ASL volume expansion at
t = 60 than normal [compare height of mucus layer in (d) with that in (g)].
activating Cl channels in the absence of inhibiting the
ENaC might paradoxically increase absorption rather than
induce secretion. Alternatively, there are agents that selectively block the ENaC to conserve liquid on airway surfaces
and perhaps generate driving forces for CaCC-mediated
Cl secretion. These agents include small-molecule open
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channel ENaC blockers (e.g. PS552) and agents that
inhibit the cell surface proteases that activate the ENaC
when it appears on the cell surface (e.g. Aerovance 152) [70].
Finally, there are agents that both inhibit ENaC and initiate
Cl secretion. The most advanced of these compounds is
INS37217, a metabolically stabilized UTP derivative that
Opinion
TRENDS in Molecular Medicine
interacts with the P2Y2 receptor to inhibit Na+ absorption
and initiate CaCC-mediated Cl secretion [71]. Moli 1901,
A152 and PS552 are currently in Phase II testing, and
INS37217 is currently in Phase III testing. Of note, it might
be most rational to combine molecules that have ENaCblocking activity with those that have Cl-secretory activity
or HS, to hydrate airway surfaces maximally.
Concluding remarks
Data derived from multiple experimental systems suggest
that CF airways are vulnerable to dehydration-induced
loss of mechanical (mucus) clearance of airway surfaces.
The dehydration that characterizes CF airway surfaces
reflects the inability to regulate Na+ and Cl transport
coordinately owing to the absence of the CFTR function in
the apical membrane of airway epithelia. New directions of
research to understand this proximate component of CF
airways disease pathogenesis should involve gaining a
physiochemical understanding of the competition for water
between the grafted polyanionic gel constituting the PCL,
and the unrestrained mucin gel constituting the mucus
layer. A corollary is to elucidate the mechanisms by which
mucus adheres to airway surfaces when water is limited –
for example, in CF. New research is also required to
translate these concepts into new therapies. A major hurdle is to develop sensitive and reproducible assays that
measure the hydration of airway surfaces. Technologies
using exhaled breath condensates, measuring the ratio of
protein biomarkers to urea, might be feasible. Finally,
although hypertonic saline seems to be surprisingly effective at treating CF lung disease, its effectiveness in treating small airways is at best modest, and it produces only a
40–60% reduction in exacerbation frequencies. Thus,
strategies must be developed to improve the hydrating
capacity of inhaled therapies and increase their durability.
In parallel, it will be useful to identify approaches to detach
mucus from airway surfaces (e.g. through inhaled detergents) to rescue lung function, in addition to protecting it.
The next few years will see intensive efforts to optimize
rehydration therapies to develop the most effective and
convenient forms of what might be chronic but potentially
‘curative’ therapies for CF lung disease.
References
1 Riordan, J.R. et al. (1989) Identification of the cystic fibrosis gene:
cloning and characterization of complementary DNA. Science 245,
1066–1073
2 Kerem, B. et al. (1989) Identification of the cystic fibrosis gene: genetic
analysis. Science 245, 1073–1080
3 Cheng, S.H. et al. (1990) Defective intracellular transport and
processing of CFTR is the molecular basis of most cystic fibrosis.
Cell 63, 827–834
4 Riordan, J.R. (2005) Assembly of functional CFTR chloride channels.
Annu. Rev. Physiol. 67, 701–718
5 Guggino, W.B. and Stanton, B.A. (2006) New insights into cystic
fibrosis: molecular switches that regulate CFTR. Nat. Rev. Mol. Cell
Biol. 7, 426–436
6 Davis, P.B. (2006) Cystic fibrosis since 1938. Am. J. Respir. Crit. Care
Med. 173, 475–482
7 Perez, A. et al. (2007) CFTR inhibition mimics the cystic fibrosis
inflammatory profile. Am. J. Physiol. Lung Cell. Mol. Physiol. 292,
L383–L395
8 Becker, M.N. et al. (2004) Cytokine secretion by cystic fibrosis airway
epithelial cells. Am. J. Respir. Crit. Care Med. 169, 645–653
www.sciencedirect.com
Vol.13 No.6
239
9 Ribeiro, C.M.P. et al. (2005) Chronic airway infection/inflammation
induces a Ca2+i-dependent hyperinflammatory response in human
cystic fibrosis airway epithelia. J. Biol. Chem. 280, 17798–17806
10 Pier, G.B. et al. (1996) Role of mutant CFTR in hypersusceptibility of
cystic fibrosis patients to lung infections. Science 271, 64–67
11 Smith, J.J. et al. (1996) Cystic fibrosis airway epithelia fail to kill
bacteria because of abnormal airway surface fluid. Cell 85, 229–236
12 Zabner, J. et al. (1998) Loss of CFTR chloride channels alters salt
absorption by cystic fibrosis airway epithelia in vitro. Mol. Cell 2, 397–
403
13 Knowles, M.R. et al. (1997) Ion composition of airway surface liquid of
patients with cystic fibrosis as compared to normal and disease-control
subjects. J. Clin. Invest. 100, 2588–2595
14 Kotaru, C. et al. (2003) Desiccation and hypertonicity of the airway
surface fluid and thermally induced asthma. J. Appl. Physiol. 94, 227–
233
15 Verkman, A.S. et al. (2003) Role of airway surface liquid and
submucosal glands in cystic fibrosis lung disease. Am. J. Physiol.
Cell Physiol. 284, C2–C15
16 Song, Y. et al. (2006) Hyperacidity of secreted fluid from submucosal
glands in early cystic fibrosis. Am. J. Physiol. Cell Physiol. 290, C741–
C749
17 Wu, J.V. et al. (2007) Acinar origin of CFTR-dependent airway
submucosal gland fluid secretion. Am J Physiol Lung Cell Mol.
Physiol 292, L304–L311
18 Joo, N.S. et al. (2006) Hyposecretion, not hyperabsorption, is the basic
defect of cystic fibrosis airway glands. J. Biol. Chem. 281, 7392–7398
19 Wine, J.J. and Joo, N.S. (2004) Submucosal glands and airway defense.
Proc. Am. Thorac. Soc. 1, 47–53
20 Inglis, S.K. and Wilson, S.M. (2005) Cystic fibrosis and airway
submucosal glands. Pediatr. Pulmonol. 40, 279–284
21 Zuelzer, W.W. and Newton, W.A., Jr (1949) The pathogenesis of
fibrocystic disease of the pancreas. A study of 36 cases with special
reference to the pulmonary lesions. Pediatrics 4, 53–69
22 Matsui, H. et al. (1998) Evidence for periciliary liquid layer depletion,
not abnormal ion composition, in the pathogenesis of cystic fibrosis
airways disease. Cell 95, 1005–1015
23 Tarran, R. et al. (2001) The CF salt controversy: in vivo observations
and therapeutic approaches. Mol. Cell 8, 149–158
24 Knowles, M.R. and Boucher, R.C. (2002) Mucus clearance as a primary
innate defense mechanism for mammalian airways (‘Perspective’).
J. Clin. Invest. 109, 571–577
25 Randell, S.H. et al. (2006) Effective mucus clearance is essential for
respiratory health. Am. J. Respir. Cell Mol. Biol. 35, 20–28
26 Raviv, U. et al. (2003) Lubrication by charged polymers. Nature 425,
163–165
27 Rose, M.C. and Voynow, J.A. (2006) Respiratory tract mucin genes and
mucin glycoproteins in health and disease. Physiol. Rev. 86, 245–278
28 Matsui, H. et al. (2006) A physical linkage between CF airway surface
dehydration and P. aeruginosa biofilms. Proc. Natl. Acad. Sci. U. S. A.
103, 18131–18136
29 Mall, M. et al. (2004) Increased airway epithelial Na+ absorption
produces cystic fibrosis-like lung disease in mice. Nat. Med. 10, 487–
493
30 Tarran, R. et al. (2001) The relative roles of passive surface forces and
active ion transport in the modulation of airway surface liquid volume
and composition. J. Gen. Physiol. 118, 223–236
31 Tarran, R. et al. (2006) Soluble mediators, not cilia, determine airway
surface liquid volume in normal and cystic fibrosis superficial airway
epithelia. J. Gen. Physiol. 127, 591–604
32 Tarran, R. et al. (2005) Normal and cystic fibrosis airway surface liquid
homeostasis: the effects of phasic shear stress and viral infections.
J. Biol. Chem. 280, 35751–35759
33 Grubb, B.R. and Boucher, R.C. (1999) Pathophysiology of genetargeted mouse models for cystic fibrosis. Physiol. Rev. 79, S193–S214
34 Matthews, L.W. et al. (1963) Studies on pulmonary secretions. I. The
over-all chemical composition of pulmonary secretions from patients
with cystic fibrosis, bronchiectasis, and laryngectomy. Am. Rev. Respir.
Dis. 88, 199–204
35 Potter, J.L. et al. (1967) Studies of pulmonary secretions. II. Osmolarity
and the ionic environment of pulmonary secretions from patients with
cystic fibrosis, bronchiectasis and laryngectomy. Am. Rev. Respir. Dis.
96, 83–87
240
Opinion
TRENDS in Molecular Medicine
36 Potter, J.L. et al. (1969) Studies on pulmonary secretions. III. The
nucleic acids in whole pulmonary secretions from patients with cystic
fibrosis, bronchiectasis, and laryngectomy. Am. Rev. Respir. Dis. 99,
909–916
37 Caldwell, R.A. et al. (2002) In vivo airway surface liquid Cl analysis
with solid-state electrodes. J. Gen. Physiol. 119, 3–14
38 Donaldson, S.H. et al. (2006) Mucus clearance and lung function in
cystic fibrosis with hypertonic saline. N. Engl. J. Med. 354, 241–250
39 Elkins, M.R. et al. (2006) A controlled trial of long-term inhaled
hypertonic saline in patients with cystic fibrosis. N. Engl. J. Med.
354, 229–240
40 Rosenfeld, M. et al. (2001) Early pulmonary infection, inflammation,
and clinical outcomes in infants with cystic fibrosis. Pediatr. Pulmonol.
32, 356–366
41 Button, B. et al. (2007) Differential effects of cyclic and constant stress
on ATP release and mucociliary transport by human airway epithelia.
J. Physiol. 580, 577–592
42 Lazarowski, E.R. et al. (2004) Nucleotide release provides a mechanism
for airway surface liquid homeostasis. J. Biol. Chem. 279, 36855–36864
43 Lazarowski, E.R. and Boucher, R.C. (2000) UTP as an extracellular
signaling molecule. News Physiol. Sci. 16, 1–5
44 Picher, M. et al. (2004) Metabolism of P2 receptor agonists in human
airways: implications for mucociliary clearance and cystic fibrosis.
J. Biol. Chem. 279, 20234–20241
45 Stutts, M.J. et al. (1995) CFTR as a cAMP-dependent regulator of
sodium channels. Science 269, 847–850
46 Watt, W.C. et al. (1998) Cystic fibrosis transmembrane regulatorindependent release of ATP. Its implications for the regulation of
P2Y2 receptors in airway epithelia. J. Biol. Chem. 273, 14053–14058
47 Okada, S.F. et al. (2006) Physiological regulation of ATP release at the
apical surface of human airway epithelia. J. Biol. Chem. 281, 22992–
23002
48 Abman, S.H. et al. (1988) Role of respiratory syncytial virus in early
hospitalizations for respiratory distress of young infants with cystic
fibrosis. J. Pediatr. 113, 826–830
49 Tomashefski, J.F., Jr et al. (1985) Pulmonary air cysts in cystic fibrosis:
relation of pathologic features to radiologic findings and history of
pneumothorax. Hum. Pathol. 16, 253–261
50 Long, F.R. et al. (2004) Structural airway abnormalities in infants and
young children with cystic fibrosis. J. Pediatr. 144, 154–161
51 Smith, E.E. et al. (2006) Genetic adaptation by Pseudomonas
aeruginosa to the airways of cystic fibrosis patients. Proc. Natl.
Acad. Sci. U. S. A. 103, 8487–8492
52 Baltimore, R.S. et al. (1989) Immunohistopathologic localization of
Pseudomonas aeruginosa in lungs from patients with cystic fibrosis.
Implications for the pathogenesis of progressive lung deterioration.
Am. Rev. Respir. Dis. 140, 1650–1661
53 Sriramulu, D.D. et al. (2005) Microcolony formation: a novel biofilm
model of Pseudomonas aeruginosa for the cystic fibrosis lung. J. Med.
Microbiol. 54, 667–676
Vol.13 No.6
54 Moskowitz, S.M. et al. (2005) Use of Pseudomonas biofilm
susceptibilities to assign simulated antibiotic regimens for cystic
fibrosis airway infection. J. Antimicrob. Chemother. 56, 879–886
55 Costerton, J.W. et al. (1999) Bacterial biofilms: a common cause of
persistent infections. Science 284, 1318–1322
56 Davies, D.G. et al. (1998) The involvement of cell-to-cell signals in the
development of a bacterial biofilm. Science 280, 295–298
57 Matsui, H. et al. (2005) Reduced 3-dimensional motility in dehydrated
airway mucus prevents neutrophil capture and killing bacteria on
airway epithelial surfaces. J. Immunol. 175, 1090–1099
58 Ribeiro, C.M.P. et al. (2005) Cystic fibrosis airway epithelial Ca2+i
signalling. The mechanism for the larger agonist-mediated Ca2+i
signals in human cystic fibrosis airway epithelia. J. Biol. Chem. 280,
10202–10209
59 Mori, K. (2000) Tripartite management of unfolded proteins in the
endoplasmic reticulum. Cell 101, 451–454
60 Patil, C. and Walter, P. (2001) Intracellular signaling from the
endoplasmic reticulum to the nucleus: the unfolded protein response
in yeast and mammals. Curr. Opin. Cell Biol. 13, 349–355
61 Kaufman, R.J. et al. (2002) The unfolded protein response in nutrient
sensing and differentiation. Nat. Rev. Mol. Cell Biol. 3, 411–421
62 Muhlebach, M.S. et al. (1999) Quantitation of inflammatory responses
to bacteria in young cystic fibrosis and control patients. Am. J. Respir.
Crit. Care Med. 160, 186–191
63 van Heeckeren, A. et al. (2006) Response to acute lung infection with
mucoid Pseudomonas aeruginosa in cystic fibrosis mice. Am. J. Respir.
Crit. Care Med. 173, 288–296
64 Srivastava, M. et al. (2004) Digitoxin mimics gene therapy with CFTR
and suppresses hypersecretion of IL-8 from cystic fibrosis lung
epithelial cells. Proc. Natl. Acad. Sci. U. S. A. 101, 7693–7698
65 Matsui, H. et al. (2000) Osmotic water permeabilities of cultured, welldifferentiated normal and cystic fibrosis airway epithelia. J. Clin.
Invest. 105, 1419–1427
66 Chen, E.Y. et al. (2004) The DF508 mutation disrupts packing of the
transmembrane segments of the cystic fibrosis transmembrane
conductance regulator. J. Biol. Chem. 279, 39620–39627
67 Eng, P.A. et al. (1996) Short-term efficacy of ultrasonically nebulized
hypertonic saline in cystic fibrosis. Pediatr. Pulmonol. 21, 77–83
68 Wark, P.A. et al. (2005) Nebulised hypertonic saline for cystic fibrosis
(Cochrane Review). Cochrane Database Syst. Rev. CD001506
69 Zeitlin, P.L. et al. (2004) A phase I trial of intranasal Moli1901 for cystic
fibrosis. Chest 125, 143–149
70 Hirsh, A.J. et al. (2006) Design, synthesis, and structure–activity
relationships of novel 2-substituted pyrazinoylguanidine epithelial
sodium channel blockers: drugs for cystic fibrosis and chronic
bronchitis. J Med Chem. 49, 4098–4115
71 Yerxa, B.R. et al. (2002) Pharmacology of INS37217 [P1-(uridine 50 )-P4(20 deoxycytidine 50 ) tetraphosphate, tetrasodium salt], a next
generation P2Y2 receptor agonist for the treatment of cystic fibrosis.
J. Pharmacol. Exp. Ther. 302, 871–880
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