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Mucus clearance as a primary innate defense mechanism for mammalian airways

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The conducting airways branch 20–25 times between the trachea and the alveoli as inhaled air passes from the relatively constricted nasal/tracheal passages to the large surface area of alveoli (70 m2), where gas exchange occurs. This branching anatomy leads to a surface area that expands greatly from proximal airways (e.g., third generation; ∼50 cm2) to distal airways (20th to 25th generation; ∼2 m2). The regional differences in airway surface area (or airway perimeters; ref. 1), which is often depicted by showing the airways as an inverted funnel (Figure (Figure1),1), pose interesting challenges for lung defense. Because many of the particles that settle on airway surfaces are infectious, airways have evolved innate defense mechanisms that constantly protect airways against bacterial and other types of infection. Figure 1 Pulmonary defense mechanisms preventing chronic bacterial infection. The lung is depicted as an inverted funnel, reflecting the relative surface area of distal versus proximal airways. The mechanical-clearance-of-mucus hypothesis is shown on the left. ... There is still little agreement on the nature of these innate airway defense mechanisms (2, 3) (Figure (Figure1).1). In the more traditional view, mechanical clearance of mucus is considered the primary innate airway defense mechanism (4–6). In this view, the role of the epithelia lining airway surfaces is to provide the integrated activities required for mucus transport, including ciliary activity and regulation of the proper quantity of salt and water on airway surfaces via transepithelial ion transport. More recently, a second view of innate airway defense has emerged as a result of studies of the pathogenesis of cystic fibrosis (CF) (7). This view emphasizes a role for a “chemical shield” in protecting the lung against inhaled bacteria (8). In this hypothesis, the two important functions for epithelia are the production of salt-sensitive defensins that are secreted into airway lumens, and the production of a low-salt (<50 mM NaCl) liquid on airway surfaces that renders defensins active (9). The predictions of each of these models and the relevant data have been extensively reviewed (2, 3, 10, 11). Here, we will focus on the role of mucus clearance in the lung as the more important innate defense mechanism in health and disease, including CF. We will attempt to fill in the gaps in our knowledge regarding important aspects of the mucus clearance system, and, where relevant, point out differences between the two views of innate airway defense.
Mucus layer and PCL transport by human airway epithelia. (a) Mucus transport in WD airway epithelial cultures was identified by the rotational movement of 1-µm fluorescent microspheres in the mucus layer. The image shown was acquired as a single 5-second exposure; the streaks represent the paths of individual microspheres (field diameter ∼2 mm). (b) Mucus transport rates were calculated as the linear velocities of fluorescent microspheres (a) and plotted against the distance from the center of the rotation (radial). (c) Transport of PCL revealed by photoactivation of caged fluorescein-conjugated dextrans . The ASL was labeled with caged fluorescein-conjugated dextran (10,000 Da) and the fate of photoreleased dextran fluorescence was determined. Conventional fluorescence microscopy at low power was used (bars = 0.2 mm). Left: Migration of the released fluorescent dextran during the 20-second period of observation. Right: After removing the mucus layer (+DTT), the migration of released fluorescent dextran was minimal. Both the mucus layer and the PCL are labeled by photoactivation. The absence of a " smear " during movement implies that both the mucus layer and the PCL move at the same velocity. The absence of PCL movement after mucus removal suggests that mucus movement is critical for PCL movement. (d) Models of ASL transport. Lateral fluid velocity profiles in ASL predicted for three different considerations , with the ordinate aligned to the diagram of the ciliary beat cycle at the left. Curve A approximates velocity profiles in the PCL predicted from theoretical considerations of ciliary propulsion of water (60, 61). Note nominally zero velocities at 70–75% of the ciliary length, below the level of the return stroke. Curve B depicts the velocity profile predicted for PCL flow driven solely by frictional interactions with the mucus layer. Curve C depicts the velocity profile for the PCL from the observations in this work that the flows of the PCL and mucus layer are essentially indistinguishable.
… 
PCL is required for effective mucociliary and cough clearance. (a) Schema of microanatomy of normal ASL. Note mixing of bacteria in "turbulent" mucus. (b) Schema depicting hypothetical volume depletion of ASL covering CF airway epithelial surfaces. Note that the volume depletion is reflected in both the generation of a more concentrated mucus layer and the depletion of the PCL. PCL depletion allows interactions to occur between the tethered mucins of the glycocalyx and the mucus layer. Note motile bacteria penetrating into thickened, stationary mucus. (c) Evidence for ASL volume depletion in CF airway epithelia. ASL height was measured immediately, 12 hours, and 24 hours after deposition of PBS containing Texas red dextran on the epithelial surface of the cell (pseudocolored green). Left: Representative confocal microscopy images. Right: Mean data for normal (circles) and CF (squares) ASL heights. *CF ASL is significantly shallower than normal (P < 0.05; n = 6 per group). (d) Mucus (bead) rotational velocity 24 hours after administration of PBS containing fluorescent markers. At t = 0 hours, both normal and CF cultures exhibited rotational velocities of about 45 µm/s. (*P < 0.05, CF vs. normal; n = 6 per group). (e) Low-power electron micrograph of perfluorocarbon/osmium-fixed CF airway culture 24 hours after volume addition and with rotational mucus transport abolished. Note close apposition of mucus layer and the glycocalyx covering flattened cilia and the cell surface. (f) Light micrograph of freshly excised CF bronchus stained with Alcin blue periodic acid-Schiffs for mucus. As in the in vitro model, note close apposition (annealing) between secreted mucins and the cell surface (indicated by white arrow). NL, normal.
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The Journal of Clinical Investigation | March 2002 | Volume 109 | Number 5 571
PERSPECTIVE SERIES
Jeffrey A. Whitsett, Series Editor
Innate defenses in the lung
The conducting airways branch 20–25 times between
the trachea and the alveoli as inhaled air passes from
the relatively constricted nasal/tracheal passages to the
large surface area of alveoli (70 m
2
), where gas exchange
occurs. This branching anatomy leads to a surface area
that expands greatly from proximal airways (e.g., third
generation; 50 cm
2
) to distal airways (20th to 25th
generation; 2 m
2
). The regional differences in airway
surface area (or airway perimeters; ref. 1), which is often
depicted by showing the airways as an inverted funnel
(Figure 1), pose interesting challenges for lung defense.
Because many of the particles that settle on airway sur-
faces are infectious, airways have evolved innate defense
mechanisms that constantly protect airways against
bacterial and other types of infection.
There is still little agreement on the nature of these
innate airway defense mechanisms (2, 3) (Figure 1). In the
more traditional view, mechanical clearance of mucus is
considered the primary innate airway defense mechanism
(4–6). In this view, the role of the epithelia lining airway
surfaces is to provide the integrated activities required for
mucus transport, including ciliary activity and regulation
of the proper quantity of salt and water on airway sur-
faces via transepithelial ion transport. More recently, a
second view of innate airway defense has emerged as a
result of studies of the pathogenesis of cystic fibrosis (CF)
(7). This view emphasizes a role for a “chemical shield” in
protecting the lung against inhaled bacteria (8). In this
hypothesis, the two important functions for epithelia are
the production of salt-sensitive defensins that are secret-
ed into airway lumens, and the production of a low-salt
(<50 mM NaCl) liquid on airway surfaces that renders
defensins active (9).
The predictions of each of these models and the rele-
vant data have been extensively reviewed (2, 3, 10, 11).
Here, we will focus on the role of mucus clearance in
the lung as the more important innate defense mecha-
nism in health and disease, including CF. We will
attempt to fill in the gaps in our knowledge regarding
important aspects of the mucus clearance system, and,
where relevant, point out differences between the two
views of innate airway defense.
Microanatomy of the airway surface
With the advent of the capacity to fix airway surface
liquid (ASL) in vivo, using the perfluorocarbon/osmi-
um technique pioneered by Sims et al. (12), and the
development of well-differentiated (WD) human air-
way epithelial cultures that exhibit mucus transport in
vitro (13), it is now possible to investigate the
microanatomy of mucus transport on airway surfaces
at high resolution. Representative images depicting the
range of morphologic techniques that can be applied
to this culture system are shown in Figure 2.
Analysis of photomicrographs of this preparation,
combined with immunocytochemical studies, have
revealed several key features of the microanatomy of the
ASL compartment (13, 14). The ASL consists of at least
two layers, a mucus layer and a periciliary liquid layer
(PCL; Figure 2). The mucus layer consists of high–molec-
ular weight, heavily glycosylated macromolecules, prod-
ucts of at least two distinct genes (MUC5AC and
MUC5B), that behave as a tangled network of polymers
Mucus clearance as a primary innate defense mechanism
for mammalian airways
Michael R. Knowles and Richard C. Boucher
Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina, Chapel Hill, North Carolina, USA
Address correspondence to: Richard C. Boucher, Cystic Fibrosis/Pulmonary Research and Treatment Center,
7011 Thurston-Bowles Building, CB# 7248, The University of North Carolina, Chapel Hill, North Carolina 27599-7248, USA.
Phone: (919) 966-1077; Fax: (919) 966-7524; E-mail: rboucher@med.unc.edu.
J. Clin. Invest. 109:571–577 (2002). DOI:10.1172/JCI200215217.
Figure 1
Pulmonary defense mechanisms preventing chronic bacterial infection.
The lung is depicted as an inverted funnel, reflecting the relative surface
area of distal versus proximal airways. The mechanical-clearance-of-mucus
hypothesis is shown on the left. The schema depicts discrete mucus and
periciliary liquid layers and ascribes to the epithelium a volume-absorbing
function. The chemical shield hypothesis is shown on the right, with the
epithelium depicted as having a salt- but not a volume-absorbing function
to produce the hypotonic ASL required for defensin activity.
(15). The properties of the mucin gel are determined by
this tangled network, which reflects, in part, the water
content, monovalent and divalent ion concentrations,
and pH of the ASL. It appears that mucin macromole-
cules are well adapted to binding and trapping inhaled
particles for clearance from the lung, at least in part
because of the extraordinary diversity of their carbohy-
drate side chains. Because they provide, in effect, a com-
binatorial library of carbohydrate sequences, mucins can
bind to virtually all particles that land on airway epithe-
lia and can thus clear them from the lung (16). It is still
not known whether the mucus layer is continuous or
discontinuous in human airways in vivo, although it
appears likely at least that the layer is not homogeneous
but is deeper at some sites than at others.
The mucus-free zone at the cell surface, which approx-
imates the height of the outstretched cilia, has been pre-
viously described as the “sol” layer but is perhaps better
termed the “periciliary liquid layer” (PCL). The ability to
visualize this layer (Figure 2), both with perfluorocar-
bon/osmium/transmission electron microscopy fixa-
tion techniques and with confocal microscopy (Figure
3), has led to an appreciation of its vital importance in
lung defense. As discussed below, this liquid layer is cru-
cial both because it provides a low-viscosity solution in
which cilia can beat rapidly (about 8–15 Hz) and
because it shields the epithelial cell surface from the
overlying mucus layer.
One of the more important aspects of recent studies
of epithelial cell surfaces and their relationship to
mucus clearance has been the delineation of the size,
composition, and functional aspects of the “glycocalyx”
(Figure 2). A combination of more traditional freeze
substitution and ruthenium red staining with new anti-
bodies to cell surface–tethered mucins has revealed that
the apical surfaces of airway epithelial cells are lined by
highly glycosylated molecules that extend 500–1500 nm
into the lumen (17, 18). These data have set the stage for
studies that examine interactions in airway diseases
between the mobile, gel-forming mucus layer, consist-
ing of MUC5AC and MUC5B, and the cell surface–teth-
ered mucins, such as MUC1 and MUC4.
Physiology of mucus clearance from the lung
Macroscopic transport of ASL. A number of techniques
have been used to demonstrate that particles deposit-
ed on mammalian airway surfaces are cleared over rel-
atively short periods of time (4, 19). A useful approach
to quantitating basal mucus clearance in humans is to
measure the clearance of inhaled and deposited radio-
labeled particles from the human lung as a function of
time with external scintography (4, 20). The basal rate
of particle clearance depends on the interactions of cilia
with the overlying mucus. It is not yet clear what deter-
mines the basal rates of mucociliary clearance, which
vary within proximal and distal regions of the airways.
Certainly, the ciliary beat frequency and effectiveness
of cilia are primary determinants of the basal mucocil-
iary clearance rate, but the quantity and viscoelastic
properties of mucin, and possibly the viscous proper-
ties of the PCL, may also be important variables.
Studies of patients with airway diseases and analyses
of the daily variability of clearance rates in normal
human subjects reveal a wide range of mucociliary
clearance rates, implying endogenous regulation of this
process (4). It is not clear what mechanisms regulate
mucociliary clearance rates in response to airway stress-
es. There is no cholinergic efferent innervation of the
superficial airway epithelium (21), and it is likely that
tachykinin-mediated neural systems are present pre-
dominantly in the upper airways, making it unlikely
that neural mechanisms regulate lung mucus transport
(21). Similarly, systemic hormonal responses, for exam-
ple, to epinephrine, have time constants that are too
long for this function. There has been interest in
autocrine and paracrine airway epithelial signals that
may control basal and stimulated rates of mucus clear-
ance in the airways. The recognition that 5 nucleotides
applied to airway surfaces regulate all components of
the mucus clearance system, including epithelial ion
transport, ciliary beat frequency, and mucus secretion,
has suggested a role for these compounds in this func-
tion (22–26). These speculations have been buttressed
by recent data suggesting that shear stress induces
572 The Journal of Clinical Investigation | March 2002 | Volume 109 | Number 5
Figure 2
Microanatomy of human ASL. (a) WD human airway epithelial culture
exhibiting rotational mucus transport (see Figure 3), fixed with perfluo-
rocarbon/osmium. Note a distinct mucus layer atop a distinct PCL. (b)
Visualization of glycocalyx on WD human airway epithelia by the freeze
substitution technique. Note the high degree of organization of this bar-
rier. (c) Left: X-Z confocal image of living WD human airway epithelial
culture. The cells were stained with calcein, AM, (green), and the ASL was
visualized with Texas red dextran. Scale bar = 10 µm. Top right: Fluores-
cent “dissection” of mucus layer and PCL in living WD airway epithelia
by confocal microscopy. The mucus layer is visualized as green fluores-
cent beads and the PCL as the “bead-free zone” interposed between the
mucus layer and cell surface (black). Scale bar = 10 µm. Bottom right:
Detection of glycocalyx by fluorescence/confocal microscopy. The ker-
atan sulfate component of the glycocalyx is visualized by Texas
red–labeled anti–keratan sulfate (anti-KS) antibodies. Scale bar = 5 µm.
nucleotide release in airway epithelia and that the air-
ways express a large number of extracellular nucleoti-
dases that regulate extracellular nucleotide concentra-
tions (27–30). Collectively, the data suggest that
nucleotide release, perhaps in response to ambient and
cough-induced shear stress, regulates mucus clearance
rates in response to luminal stresses.
It should be emphasized that the lungs possess an
additional mechanism to clear mucus from the lung,
namely cough clearance. By definition, cough clearance
is independent of the actions of cilia, but cough effi-
ciency is heavily dependent on several variables perti-
nent to ASL (31). The overall height and volume of liq-
uid on the airway surface are directly related to the
efficiency of cough clearance, whereas the viscosity of
the luminal material is inversely related to the efficien-
cy. As highlighted previously by Zahm and colleagues,
the PCL appears to be an extraordinarily important con-
tributor to the efficiency of cough clearance (32). Specif-
ically, the lubricating function of the PCL facilitates
mucus movement along airway surfaces in response to
coughing. As discussed below, the absence of the PCL
allows adhesive interactions between mobile mucins in
the mucus layer and tethered cell-surface mucins, great-
ly reducing the efficiency of cough clearance.
Microscopic physiology of ASL/mucus transport. The devel-
opment of cell culture methods in which WD lung
epithelia expressing rotational mucus transport are
examined using confocal microscopy to study the move-
ment of the individual components of the mucus layer
and PCL has provided great insight into the physiology
of ASL clearance (Figure 3). Recent studies have demon-
strated complex interactions among cilia, the mobile
mucins in the mucus layer, and the underlying PCL (13,
14). For example, it appears that both the mucus layer
and the PCL are moved unidirectionally along airway
surfaces via ciliary actions from the small airways to the
proximal airways and larynx (13). Interestingly, howev-
er, recent data have suggested that ciliary actions also
impart vertical movements within the mucus layer (33).
Thus, particles deposited on the surface of the mucus
layer are rapidly mixed within the mucus layer. This fea-
ture of the mucus layer, i.e., entrapment, may constitute
a second mechanism that ensures clearance of virtually
any particle that deposits on airway surfaces. These stud-
ies also showed for the first time that the PCL moves
with the mucus layer, elucidating a mechanism that
clears the lung of hydrophilic noxious solutes dissolved
in the PCL, along with particles trapped in mucus.
From a mechanistic point of view, these studies sug-
gested that the movement of ASL on airway surfaces
involved two steps. First, the ciliary power stroke acts
on the undersurface of mucus to move the mucus layer
unidirectionally on the airway surface. Second, the fric-
tional interaction of the mucus layer with the PCL
allows this underlying layer to travel at velocities simi-
lar to those of the overlying mucus layer (a form of
“secondary” transport). These data are consistent with
the fact that mammalian airway cilia are too short, and
their recovery stroke not sufficiently close to the cell
surface, to move the PCL directly.
The Journal of Clinical Investigation | March 2002 | Volume 109 | Number 5 573
Figure 3
Mucus layer and PCL transport by human airway epithelia. (a) Mucus
transport in WD airway epithelial cultures was identified by the rota-
tional movement of 1-µm fluorescent microspheres in the mucus
layer. The image shown was acquired as a single 5-second exposure;
the streaks represent the paths of individual microspheres (field diam-
eter 2 mm). (b) Mucus transport rates were calculated as the linear
velocities of fluorescent microspheres (a) and plotted against the dis-
tance from the center of the rotation (radial). (c) Transport of PCL
revealed by photoactivation of caged fluorescein-conjugated dex-
trans. The ASL was labeled with caged fluorescein-conjugated dextran
(10,000 Da) and the fate of photoreleased dextran fluorescence was
determined. Conventional fluorescence microscopy at low power was
used (bars = 0.2 mm). Left: Migration of the released fluorescent dex-
tran during the 20-second period of observation. Right: After remov-
ing the mucus layer (+DTT), the migration of released fluorescent dex-
tran was minimal. Both the mucus layer and the PCL are labeled by
photoactivation. The absence of a “smear” during movement implies
that both the mucus layer and the PCL move at the same velocity. The
absence of PCL movement after mucus removal suggests that mucus
movement is critical for PCL movement. (d) Models of ASL transport.
Lateral fluid velocity profiles in ASL predicted for three different con-
siderations, with the ordinate aligned to the diagram of the ciliary
beat cycle at the left. Curve A approximates velocity profiles in the
PCL predicted from theoretical considerations of ciliary propulsion
of water (60, 61). Note nominally zero velocities at 70–75% of the cil-
iary length, below the level of the return stroke. Curve B depicts the
velocity profile predicted for PCL flow driven solely by frictional inter-
actions with the mucus layer. Curve C depicts the velocity profile for
the PCL from the observations in this work that the flows of the PCL
and mucus layer are essentially indistinguishable.
Although there have been a number of model sys-
tems designed to simulate cough clearance in artifi-
cial tubes, there have been no studies to evaluate the
relationship between the mucus and the periciliary
liquid layers, using cell culture models and confocal
microscopy, in response to intraluminal air flow, as
occurs during coughing.
ASL antimicrobial factors and the kinetics of mucus clear-
ance. Clearance of bacteria from peripheral airways by
mucus transport may require up to 6 hours. Since the
number of bacteria can (under optimal conditions)
double every 20 minutes, large increases in bacterial
number on airway surfaces could occur during the
6-hour clearance period. However, it appears likely
that antimicrobial substances (see Ganz, this Perspec-
tive series, ref. 34) in ASL suppress bacterial growth
during mucus clearance. For example, the studies of
Cole et al. have revealed that bacteria added to undi-
luted nasal surface liquid are initially “killed” for 3–6
hours by endogenous antimicrobial factors (35). These
studies identified lactoferrin, lysozyme, and secretory
leukoproteinase inhibitor as the major antimicrobial
substances in ASLs; in contrast, defensins are present
only in trace quantities. Cole et al. (35) also showed
that the quantities of endogenous antimicrobial sub-
stances in ASL are sufficient to be “salt-insensitive”;
that is, they were fully active in the salt concentrations
(isotonic) measured in nasal ASL in these experiments.
However, residual bacteria in these liquids acquired
resistance to the ambient antimicrobial factors, and
rapid bacterial growth recurred by 24 hours. This find-
ing demonstrates an important kinetic interaction
between antimicrobial substances in ASL and mucus
clearance rates. Specifically, the antimicrobial sub-
stances can suppress bacterial growth for the short
period (about 2–6 hours) that is normally required to
clear inhaled bacteria from the airways by mucus
transport. The prediction is that a failure to clear
mucus would ultimately reveal the inability of chemi-
cal shields (antimicrobial substances) to chronically
suppress bacterial growth in the lung.
Genetic diseases associated with abnormal
mucus clearance
Primary ciliary dyskinesia: the importance of cough clearance.
Patients with primary ciliary dyskinesia (PCD) exhibit
recurrent middle ear infections, chronic airway infec-
tion, predominantly lower-lobe bronchiectasis, male
sterility, and sometimes situs inversus. Patients with
PCD exhibit a milder airway disease than do those with
CF and typically live at least to middle age. This syn-
drome reflects defective ciliary function, with abnor-
malities in beat, stroke, or coordination, associated
with genetically determined abnormalities of the struc-
tural/functional components of the ciliary shaft. The
first of the genes associated with PCD has recently been
cloned; it encodes an intermediate chain dynein (IC78)
that appears important in assembling and coordinat-
ing the activities of several proteins, including the
heavy chain dyneins of the ciliary shaft (36, 37).
The syndrome of PCD is informative with respect to
many aspects of mucus clearance in lung defense. For
example, measurements of mucociliary clearance,
using external radionuclide tracers, have revealed vir-
tually no basal, cilium-dependent mucus clearance in
patients with PCD (38–40). However, studies of mucus
clearance with inhaled radionuclide tracers show that
cough-dependent mucus clearance is well preserved in
PCD patients (39, 40). Thus, PCD patients may have a
nearly normal rate of mucus clearance over time that
is mediated solely by repetitive coughing. It appears
likely that the retention of coughing as a backup
574 The Journal of Clinical Investigation | March 2002 | Volume 109 | Number 5
Figure 4
PCL is required for effective mucociliary and cough clearance. (a) Schema
of microanatomy of normal ASL. Note mixing of bacteria in “turbulent”
mucus. (b) Schema depicting hypothetical volume depletion of ASL cov-
ering CF airway epithelial surfaces. Note that the volume depletion is
reflected in both the generation of a more concentrated mucus layer and
the depletion of the PCL. PCL depletion allows interactions to occur
between the tethered mucins of the glycocalyx and the mucus layer. Note
motile bacteria penetrating into thickened, stationary mucus. (c) Evi-
dence for ASL volume depletion in CF airway epithelia. ASL height was
measured immediately, 12 hours, and 24 hours after deposition of PBS
containing Texas red dextran on the epithelial surface of the cell (pseudo-
colored green). Left: Representative confocal microscopy images. Right:
Mean data for normal (circles) and CF (squares) ASL heights. *CF ASL is
significantly shallower than normal (P < 0.05; n = 6 per group). (d) Mucus
(bead) rotational velocity 24 hours after administration of PBS contain-
ing fluorescent markers. At t = 0 hours, both normal and CF cultures
exhibited rotational velocities of about 45 µm/s. (*P < 0.05, CF vs. nor-
mal; n = 6 per group). (e) Low-power electron micrograph of perfluoro-
carbon/osmium–fixed CF airway culture 24 hours after volume addition
and with rotational mucus transport abolished. Note close apposition of
mucus layer and the glycocalyx covering flattened cilia and the cell sur-
face. (f) Light micrograph of freshly excised CF bronchus stained with
Alcin blue periodic acid-Schiffs for mucus. As in the in vitro model, note
close apposition (annealing) between secreted mucins and the cell sur-
face (indicated by white arrow). NL, normal.
mechanism for mucus clearance in PCD explains the
milder pulmonary phenotype of PCD relative to CF.
The presence of effective cough-dependent mucus
clearance in PCD patients suggests that the volume of
liquid on airway surfaces is normal or increased. Indeed,
the few reports quantifying ion transport in airway tis-
sues freshly excised from PCD patients or in nasal
epithelia studied in vivo have revealed no abnormalities
that might adversely affect the volume of ASL (41). Thus,
the apparently normal ASL volume in PCD patients may
both preserve the lubricant function of the PCL and pro-
vide sufficient hydration and height of mucus for effec-
tive cough-dependent clearance. Interestingly, agents
such as UTP that increase the volume of liquid on airway
surfaces can acutely increase the efficiency of cough
clearance in PCD patients (39). Long-term studies, how-
ever, will be required to test the clinical benefit of such
therapeutic strategies in these patients.
Cystic fibrosis: the importance of the periciliary liquid and
mucus hypoxia. Although controversy still exists, it now
seems likely that CF lung disease reflects chronic deple-
tion of PCL volume rather than high salt concentra-
tions in the CF ASL. This assertion rests upon evidence
that ASL is isotonic both in the normal state (in
humans and other large mammals) and in CF (5, 6,
42–44). Further, it is congruent with the findings that,
under physiologic conditions, the rates of net epithe-
lial Na
+
transport and isotonic volume absorption are
higher in CF than in normal airway epithelia (6, 14, 45,
46). Consistent with accelerated volume absorption are
reports of an increased number of Na
+
-K
+
-ATPase bind-
ing sites and activity, increased amiloride-sensitive and
ouabain-sensitive O
2
consumption in CF airway epithe-
lia, and the early pathology of the non-infected CF lung
(33, 41, 47–49). Finally, recent data have directly con-
nected reduced ASL volume, but not ion composition,
with morphologic evidence of spontaneous airway dis-
ease in CF mice (50).
What is new in the area of CF pathogenesis is the
detailed understanding of the relationships between
volume depletion on airway surfaces and mechanisms
for mucus transport. Volume depletion on airway sur-
faces could be associated with selective removal of liq-
uid from the mucus layer, or could reflect depletion of
both the mucus layer and the PCL. In vitro studies have
revealed that volume depletion normally occurs sequen-
tially, first from the mucus layer, and then from the
PCL. Specifically, early volume depletion occurs from
the mucus layer, until about 50% of volume is removed,
after which the volume is removed from the PCL (51).
Thus, the mucus layer can normally act as a reservoir for
liquid, but in CF, where volume regulation on airway
surfaces is severely perturbed, the capacity of the mucus
layer to buffer the PCL volume may be exceeded.
Another important insight from these studies has
been identification of the relative importance of deple-
tion of the PCL in the pathogenesis of CF. Depletion of
the PCL predicts adverse interactions between the
mucus layer and the airway epithelial cell surface (Fig-
ure 4). Indeed, in vitro data indicate that depletion of the
PCL allows the gel-forming mucins in the mucus layer,
such as MUC5AC and MUC5B, to come in contact with
the tethered mucins MUC1 and MUC4 on the cell sur-
face (Figure 4). Similar findings have been made in
analyses of frozen sections of freshly excised CF airways.
Thus, it appears likely that the annealing that occurs
between the carbohydrate side chains of the mobile
mucins and cell surface mucins, which effectively glues
mucins in mucus to cell surfaces (the “Velcro effect”),
will abolish cough clearance. Hence, excessive ASL vol-
ume absorption constitutes a double hit on mucus
clearance mechanisms. The loss of volume depletes the
PCL, which removes the liquid in which cilia can extend
and beat, thus inhibiting mucociliary clearance. It simul-
taneously compromises the PCL’s lubricant function,
allowing the mucus layer to adhere to cell surfaces and
inhibiting cough clearance. This dual effect may
account for the severe phenotype of CF lung disease, rel-
ative to PCD. Interestingly, the importance of tethered
mucins in the pathogenesis of organ obstruction has
been better documented in the gut, where the meconi-
um ileus syndrome associated with the CF mouse intes-
tine has been ameliorated by genetic ablation of the
MUC1 mucin, which serves as the cell surface adhesive
ligand for meconium in the intestine (52).
Recently, a link between mucus stasis and a predilec-
tion for Pseudomonas aeruginosa infection in CF has been
revealed (33). In brief, despite the failure of mucus clear-
ance, goblet cells continue to secrete, generating thick
mucus plaques and plugs on airway surfaces. CF airway
epithelia exhibit high rates of cellular O
2
consumption
to fuel raised Na
+
transport, which creates hypoxic zones
in adherent mucus plaques near the cell surface. P. aerug-
inosa inhaled and deposited on the surfaces of mucus
plaques “swim” into the mucus plaques and adapt to the
hypoxic zones with alginate production and biofilm for-
mation, setting the stage for chronic infection.
The initiating event in CF, ASL volume hyperabsorp-
tion, reflects two CF-specific defects in airway epithe-
lial ion transport. Recent in vitro studies suggest that
active Na
+
absorption by normal airway epithelia medi-
ates ASL volume absorption under basal conditions
and that normal airway epithelia can slow Na
+
absorp-
tion and induce Cl
secretion, when ASL volumes are
depleted (51). In CF, there appear to be defects in both
the Na
+
-absorptive and Cl
-secretory mechanisms for
regulating ASL volume. Specifically, CF airway epithe-
lia have an accelerated basal rate of Na
+
(and volume)
absorption that reflects the absence of the tonic
inhibitory effect of CFTR on the epithelial Na
+
channel
(ENaC) activity (53, 54). However, CF airway epithelia
are also missing the capacity to add liquid back to air-
way surfaces when ASL volumes are depleted, due to
the absence of CFTR functioning as a Cl
channel (55,
56). Thus, therapies directed both at slowing the abnor-
mally raised volume absorption, e.g., Na
+
channel
blockers, and at initiating Cl
channel activity, e.g.,
UTP-dependent activation of Ca
2+
-regulated Cl
chan-
nels, provide routes for the treatment of the primary
defect of the CF airway epithelium (20, 57).
Pseudohypoaldosteronism: a surprise with respect to mucus
clearance. Pseudohypoaldosteronism (PHA) is a clinical
The Journal of Clinical Investigation | March 2002 | Volume 109 | Number 5 575
syndrome that reflects, at the genetic level, loss-of-func-
tion mutations in ENaC subunits (58). Characterization
of the pulmonary phenotype of this syndrome became
pertinent because of the contrasting predictions of the
compositional (chemical shield) and volume (mucus
clearance) hypotheses with respect to the effects of the
loss of Na
+
channel function on ASL composition ver-
sus volume. Specifically, the compositional theory pre-
dicted that PHA patients would have raised ASL NaCl
concentrations and inactivation of defensins, with
resultant chronic airway infections. In contrast, the vol-
ume hypothesis predicted that PHA patients would
have an excess of isotonic volume on airway surfaces,
perhaps with a syndrome of airway obstruction.
Studies of PHA subjects showed that Na
+
transport is
absent, at least as measured by the transepithelial poten-
tial difference technique (59). Analyses of both the
upper (nasal) and lower (bronchial) airways revealed
that the ASL is isotonic in PHA patients, as it is in
healthy subjects, but that the volume of their ASL is
greatly increased. These patients suffer intermittent air-
way obstruction and infection as young children, but
after the age of 6 years, their lung function is normal,
and they are free of chronic bacterial airway infections
or bronchiectasis, consistent with normal lung defense.
Studies of mucus clearance in these subjects with the
external radiolabeled tracer technique (59) show that
basal mucus clearance occurs at an astonishing rate,
equaling or exceeding rates in normal subjects after
acute exposures to β-agonists or purinergic agents. No
comprehensive explanation for this observation is avail-
able, but, given the reservoir function of the mucus
layer, extra liquid added to this mucus layer could
account for a modest increase in mucus clearance rates.
Indeed, recent in vitro studies have confirmed that liq-
uid added to the ASL preferentially partitions into and
swells the mucus layer, accelerating mucus transport
(51). Whereas liquid addition probably improves mucus
viscoelasticity, it likely also changes the characteristics
of the PCL, perhaps decreasing its viscosity, which could
also accelerate mucus transport.
Therefore, the mucus transport data from the PHA
subjects have highlighted two principles. First, as long
as mucus clearance is maintained, chronic airway infec-
tions do not occur, despite any postulated adverse
effects of isotonic (high salt) ASL on antimicrobial
activity. Second, expansion of ASL volume triggers a
series of events in the ASL compartment that acceler-
ates the rate of mucus transport.
Future directions
It has long been speculated that mucus clearance is
important for airway defense, but only recently have
important details of this system become available. For
example, it has only been recently recognized that the
nonspecific binding capacity of the carbohydrate side
chains of mucins, coupled with the turbulent behav-
ior of the mucus layer, mediates clearance of virtually
all inhaled substances from the airways. Similarly, we
have only recently recognized that the PCL not only
promotes effective ciliary beat by providing a low vis-
cosity solution but also serves an important role as a
lubricant between the mobile and cell surface mucins.
Finally, it has been primarily through the analysis of
various genetic diseases of mucus transport that the
importance of both the basal mucociliary clearance
system and the back-up, cough-dependent system has
become appreciated.
Many aspects of the airway defense system, however,
remain to be elucidated. First, it is not yet clear at the
physiologic level how this noninnervated epithelial sys-
tem can regulate mucus transport rates over a more
than threefold range. The preliminary observations that
nucleotides are released by airway epithelia and interact
with luminal purinoceptors provide the most tantaliz-
ing clues about the local regulation of mucus transport
rates. Second, it is not yet clear how airway epithelia
sense and regulate the volume of liquid on their sur-
faces. Although recent data indicate that regulation of
ASL by airway epithelia involves the reciprocal regula-
tion of active Na
+
absorption and Cl
secretion (51), it is
not known whether the signals to the epithelium
emanate from the ASL, or how such signals are trans-
duced by the epithelium. Finally, whereas β-agonists
and other agents that increase mucociliary clearance via
regulation of ciliary beat frequency have become clini-
cal mainstays, it now appears that other classes of drugs
that affect the volume of ASL may be equally, or more,
effective in many conditions and could be uniquely
effective in specific diseases, such as CF. Thus, studies
of airway function in health and disease have provided
unique insights into how “wet-surface” epithelia can
achieve a functional form of innate defense by unique
adaptation of mechanical clearance mechanisms.
Acknowledgments
This work was supported by NIH grants HL34332,
HL60280, R000046, and Cystic Fibrosis Foundation
grant R026. The authors wish to thank their col-
leagues W. Bennett, C.W. Davis, H. Matsui, P.G.
Noone, R. Pickles, S.H. Randell, M.J. Stutts, and R.
Tarran, who have contributed to the knowledge
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The Journal of Clinical Investigation | March 2002 | Volume 109 | Number 5 577
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