Am J Physiol Lung Cell Mol Physiol 301: L181–L186, 2011.
First published April 29, 2011; doi:10.1152/ajplung.00321.2010.
Mucociliary interactions and mucus dynamics in ciliated human bronchial
epithelial cell cultures
Patrick R. Sears,1 C. William Davis,1 Michael Chua,2 and John K. Sheehan1,3
1
Cystic Fibrosis Center, 2Department of Cell and Molecular Physiology, and 3Department of Biochemistry and Biophysics,
University of North Carolina, Chapel Hill, North Carolina
Submitted 10 September 2010; accepted in final form 27 April 2011
MUC5B; cilia; beads; adhesion; airways
that are deposited on the
mucus layer covering the epithelial surfaces of the lung. This
layer entraps these particles and is propelled toward the oropharynx under the action of ciliary beating. The current view of
the airway surface generally considers it to comprise a periciliary layer (PCL) and an overlying continuous mucus layer
(9, 13, 14, 18).
This view comes from images of fixed tissues and cultures.
Fixation of excised tissue and cultures generally shows mucus
as a variably thick internally heterogeneous layer (19, 20, 25).
Transmission electron micrographs of cultured cells show
mucus in a dense thin layer in some places and a thick layer in
others (13, 18). In electron and light micrographs of fixed
tracheas, the mucus layer had a much tighter mesh in
continuous contact with cilia (19). This tighter mesh has
also been seen in isolated mucus (16). The heterogeneity in
many of these images has been confirmed by bead-tracking
experiments (11).
The dynamics of mucus have also been investigated. Live
microscopy has been used in vivo, in excised tissue, and in
cultures (10, 12, 14, 25). These experiments investigated average mucus velocity over large areas. Only one study has
reported more detailed mucus velocity measurements by dif-
AIR WE BREATHE CONTAINS PARTICLES
Address for reprint requests and other correspondence: P. R. Sears, Cystic
Fibrosis Center, Univ. of North Carolina, 4021 Thurston-Bowles Bldg.,
CB7248, Chapel Hill, NC 27599 (e-mail: [email protected]).
http://www.ajplung.org
ferentiating between different radial positions in a circular
culture system with thick mucus (14). Possibly because of this
lack of data on mucus dynamics on the scale of cilia, models of
mucus flow have generally assumed that mucus flows freely
above cilia as a homogeneous layer (3, 22). Importantly, any
interaction of the cilia with this layer has been assumed to be
without an attachment.
However, the view of the mucus as a mostly passive continuous
layer that moves in bulk may not be appropriate for all areas of the
lung. Our hypothesis is that mucus may form a discontinuous
layer with dynamic attachments to the surface. We could find only
one example of mucus as a discontinuous layer: a scanning
electron micrograph of mucus on the surface of a trachea was
described as forming “rafts” and “strands” (17). Also, attachment
of mucus to the surface has been confined to reports from
pathogenic conditions such as cystic fibrosis (CF).
In early experiments to probe the PCL, we removed as much
of the mucus as possible from cultures and added albumincoated beads of various sizes. We expected these beads to
diffuse freely through the PCL, limited only by their size.
Instead, 20-nm beads were entrapped in mucus above the cilia
before they reached the PCL. Because this mucus was attached
to cilia, we developed the hypothesis that mucus binds to cilia
when there is little mucus present. This is in distinction to all
investigations of mucus to date, which have dealt with thick
mucus layers. As there is little mucus in the deep airways, we
supposed that a thin layer of mucus may be attached to the
surface and may only be released when contaminants reached
a critical level. In testing this hypothesis, we found that mucus
dynamics were more complex. We report these dynamics in the
present study.
MATERIALS AND METHODS
We aimed to determine how mucus behaves when there is little
mucus present. We observed well-washed live cultures by confocal
microscopy, and we recorded mucus dynamics in ⬃20-s videos with
a ⬃100-␮m field of view. We labeled the mucus nonspecifically with
albumin-coated fluorescent beads. One-micrometer beads labeled the
surface of the mucus, while 20-nm beads became entrapped inside
mucus. We also tested antibodies to specific mucins, and most did not
label mucins in the live culture. However, we successfully labeled the
gel-forming mucin MUC5B after its purification from saliva and, thus,
were able to add it back to the culture and observe its dynamics.
Materials. Cell culture medium was purchased from GIBCO BRL
(Gaithersburg, MD) and its supplements from Collaborative Research
(Bedford, MA). DMEM-Ham’s F-12 nutrient mixture (DMEM-F12)
was also purchased from GIBCO BRL. Transwell-Col (T-Col) and
Transwell-Clear (T-Clear) membrane supports were obtained from
Costar. Beads were obtained from Invitrogen (Carlsbad, CA). Antibodies were obtained from Sigma-Aldrich (St. Louis, MO).
Cell culture. Human bronchial epithelial (HBE) cells were obtained
from normal human bronchi, as previously described (4). Briefly,
1040-0605/11 Copyright © 2011 the American Physiological Society
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Sears PR, Davis CW, Chua M, Sheehan JK. Mucociliary interactions
and mucus dynamics in ciliated human bronchial epithelial cell cultures. Am
J Physiol Lung Cell Mol Physiol 301: L181–L186, 2011. First published
April 29, 2011; doi:10.1152/ajplung.00321.2010.—The airway epithelial
surface liquid is generally considered to be composed of two layers,
a periciliary layer and a continuous thick mucus layer moving in bulk.
This view may not be appropriate for all areas of the lung. Our
hypothesis, that mucus may form a discontinuous layer with dynamic
attachments to the surface, is investigated using a culture system. We
used live-cell confocal microscopy to investigate thin mucus layers
and fluorescent beads and exogenous MUC5B to visualize mucus
dynamics on ciliated human bronchial cultures. A continuous mucus
layer was not observed. In sparsely ciliated cultures, mucus attached
to ciliated cells; however, in highly ciliated cultures, mucus formed
strands several hundred micrometers long. As with increases in
ciliation, increases in bead concentration caused the appearance of
mucus strands. We confirmed the involvement of mucins in the
binding of mucus to cilia by adding labeled purified MUC5B to the
cultures. These data suggest that mucins may have an intrinsic ability
to form attachments to cilia. The significance of these findings is that
aberrant modulation of such an intrinsic property may explain the
initiation of highly adherent mucus in cystic fibrosis lung disease.
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Labeling of cultures with low concentrations of 20-nm beads. For
experiments involving the addition of low concentrations of 20-nm
beads, a working solution of beads was diluted to 1:10,000 from their
stock concentration of 2% solids. While the culture on the microscope
was monitored, solution changes were made by addition of 50 ␮l of
bead solution to a 200-␮l volume on the culture surface followed by
removal of 50 ␮l to maintain constant surface volume. A total of five
additions were made. Several regions of the culture surface were
videotaped between each solution change.
Two-color sequential bead addition. The sequential addition of
20-nm beads of two different colors was done, such that the addition
of the second color (green) occurred after all the beads of the first
color (red) were entrapped in mucus. Addition of red-fluorescent
20-nm beads was followed by four solution changes using buffer with
no beads. The solution changes changed half the surface fluid volume
and were done every 10 min. Then addition of green-fluorescent
20-nm beads was followed by four more solution changes. With four
solution changes, the final bead concentration would be 1/16th of the
original bead concentration, if there was no mucus to entrap beads.
With mucus present, the free red bead concentration was negligible at
the time of green bead addition. This allowed new mucus to be labeled
exclusively with green beads.
Microscopy. Imaging was done in the Michael Hooker Microscopy
Facility at the University of North Carolina at Chapel Hill. For the
fluorescence measurements, a Zeiss 510 Meta laser scanning confocal
microscope with a ⫻40/1.2 NA C-Apochromat water immersion
objective was used. For determination of the level and heterogeneity
of ciliation in cultures, a Nikon Eclipse TE-2000 microscope equipped
with a ⫻40 objective was used. A MegaPlus ES-310 T camera
(Redlake, Tucson, AZ) was used to record differential interference
contrast images of ciliary activity at 125 frames per second, under the
control of the Sisson-Ammons video analysis system (Ammons Engineering, Mt. Morris, MI) (21).
RESULTS
To easily refer to mucus structures we observed, we introduce the following three terms. We will use “plumes” for
⬃10-␮m-wide mucus structures that taper as they extend
directly away from the culture for ⬃30 ␮m, “strands” for
structures that are much longer than wide and lie horizontally
on the culture, and “clumps” for other structures that do not fit
well into the other two categories.
Sparsely ciliated cultures. In sparsely ciliated cultures
(⬍60% of the surface), 20-nm beads became entrapped in
mucus structures on ciliated cells (Fig. 1). These structures
extended upward between 10 and 30 ␮m and waved as the cilia
beat, so we named them plumes. The 1-␮m beads bound to
these plumes but did not enter them. We do not know if the
plumes were present before addition of the beads, but, once
formed, they were only removed by vigorous washing, after
which they reformed within minutes.
Fig. 1. Sparsely ciliated cultures in 2 views.
A: confocal x,y scan at the level of cilia. Redfluorescent beads were 1 ␮m diameter; greenfluorescent beads were 20 nm diameter. B: x,z
scan for fluorescence and differential interference
contrast (DIC) in which only 20-nm greenfluorescent beads were present. One-micrometer
beads (arrow 1) remained outside the structures
labeled by 20-nm beads (arrow 2). Structures
often extended for ⬎10 ␮m above the cell surface (arrow 3). See Supplemental Material for 1
movie for each view (Movies 1A and 1B). Scale
bars, 10 ␮m.
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HBE cells were isolated and grown on plastic culture dishes in
bronchial epithelial cell growth medium (BEGM) and passaged at
⬃70% confluence, and first-passage cells were seeded onto collagencoated, 12-mm T-Col or T-Clear permeable supports at 250,000 cells
per support. After confluence, the cells were maintained under airliquid interface (ALI) conditions in ALI culture medium [BEGM
modified as described elsewhere (4)], which was changed at the
basolateral surface three times a week. HBE cell cultures were used
for experiments 4 –10 wk after confluence, when the columnar cells
are well differentiated.
Bead and MUC5B preparation. Beads were 20-nm or 1-␮mdiameter and were coated with albumin to decrease bead clumping at
physiological salt concentrations. Beads were diluted from their stock
solutions into a solution containing 1 mg/ml albumin in PBS. A
microscope was used to check the final solutions for clumping. The
1-␮m beads were centrifuged, and the precipitate was brought up in
the experimental working buffer. The 20-nm beads were always used
at a dilution that reduced any sodium azide levels, having no effect on
ciliary beat frequency. MUC5B was purified from saliva by density
gradient centrifugation and gel filtration chromatography, as previously described (15, 24). MUC5B was labeled with a rabbit primary
antibody to MUC5B and then with a FITC-labeled anti-rabbit secondary antibody (F-4890, Sigma). After gel filtration, several size
fractions were pooled and used within 2 days.
Handling of cultures before and during microscopy. The apical
surfaces of the cultures were soaked in PBS for 30 min in an incubator
at every feeding. In addition, 4 days before the experiment, the
cultures were washed with PBS containing 1 mM DTT to remove as
much surface-attached mucus as possible. During the 3 days preceding the experiment and on the morning of the experiment, cultures
were washed without DTT. Before they were placed on the microscope stage, the cultures were altered to be used with a highnumerical-aperture objective and to control evaporation. First, any
protrusions were removed from the underside of the culture. Then a
small circular plastic ridge surrounding the underside of the culture
was carefully trimmed off with a scalpel. Side openings were plugged
with putty. Finally, the culture was placed on a #1 cover glass on the
microscope stage, buffer was added to the apical surface, and a large
#2 cover glass was placed on top. The volume of added buffer ensured
that there was ⱖ200 ␮m of fluid above the cells in the center of the
culture, with one exception (see Fig. 1). Once the cultures were on the
microscope stage, the top cover glass was lifted, and the solution was
gently pipetted down and up several times, so that the new solution
was diluted into the apical fluid without approaching the ALI to the
culture surface. Unless otherwise mentioned, the test solution containing the beads, antibody, or exogenous MUC5B was added at the
start of the experiment, then the solution was changed at a rate of
one-half volume every 20 min.
Preparation of cultures for addition of exogenous MUC5B. For
experiments involving the addition of exogenous MUC5B, the cultures were washed and allowed to stand in PBS with 1 mM DTT and
100 ␮M ATP prior to the microscopy. ATP depleted the mucin stores,
while DTT helped remove as much of the mucus as possible and also
temporarily interfered with new mucin synthesis.
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Fig. 2. Mucus behavior on fully ciliated cultures
shown as fluorescence on the culture surface from
red-fluorescent 1-␮m beads. A: fluorescence just
after bead addition (also see Supplemental Movie
2A). B: fluorescence a few minutes later, when thick
strands were moving across the field of view (also
see Supplemental Movie 2B). Inset: 20-nm (green)
and 1-␮m (red) beads much later, when strands have
stopped flowing. Scale bars, 30 ␮m.
attachment was delicate enough that vigorous flow or larger
flowing mucus structures (strands) could clean the surface.
Furthermore, highly ciliated areas generated the strands needed
to clean the surface.
Effect of bead concentration on mucus dynamics. To determine
how 20-nm beads changed mucus dynamics, we slowly increased
the bead concentration in highly ciliated cultures. Figure 3 shows
a culture at bead concentrations much lower than those that
caused the development of strands (see Supplemental Movies
3AB, 3CD, and 3EF). When fluorescence from beads first became
visible, it appeared as a thin layer of small clumps attached to cilia
(Fig. 3, C and D, arrowhead in C; compare with Fig. 3, A and B,
with no beads). These small clumps were attached to cilia and
aligned with the flow. As the bead concentration increased (Fig. 3,
E and F; see Supplemental Movie 3EF), the attached clumps
extended lengthwise, and occasional detachment and flow were
followed by reattachment. So, in summary, low concentrations of
20-nm beads labeled highly ciliated areas, and this thin fluorescence only started flowing as the bead concentration increased.
Fig. 3. Effect of bead concentration on mucus dynamics shown in 3 stages during a slow increase in
concentration of 20-nm beads. Each row of panels
and each movie in Supplemental Material (Supplemental Movies 3AB, 3CD, and 3EF) show a different bead concentration. Each pair of images shows
fluorescence (green on black) and merged fluorescence-DIC (green on gray). Areas imaged in the 3
rows are not identical but are highly ciliated. A, C,
and E: movie frame in which no fluorescence was
moving across the field of view. Frame represents
attached mucus. B, D, and F: frame for which the
most fluorescence was moving. A and B (Supplemental Movie 3AB): culture before addition of
beads. The same movie frame was used for A and B.
Some autofluorescence can be seen. C and D (Supplemental Movie 3CD): culture after second addition of beads, at which point an increase in fluorescence was first seen. E and F (Supplemental Movie
3EF): culture after last addition of beads. Some
attached clumps are marked with arrowheads (C and
E), and some larger flowing clumps are shown with
arrows (D and F). Fluorescent images of E and F
were taken using a slightly lower gain. All images
were processed in exactly the same way to enhance
contrast. Scale bars, 40 ␮m.
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Mucus behavior on fully ciliated cultures. Upon further
investigation of mucus plumes, we found that they did not form
on fully ciliated cultures. Instead, small clumps of mucus
visible on cilia immediately after addition of beads (Fig. 2A;
see Supplemental Movie 2A, available online at the Journal
website) coalesced into long structures that we named strands
(Fig. 2B; see Supplemental Movie 2B). The 20-nm and 1-␮m
beads had this effect, although the mass concentration of 1-␮m
beads required was higher, since they interacted much less with
the mucus. These strands extended for several hundred micrometers and flowed, remaining in contact with the culture
surface. While these strands flowed (⬃1 h), most of the culture
remained free of fluorescence, and plumes were never seen.
Occasionally, small clumps of fluorescence were seen on cilia,
but these were quickly removed by the passage of strands.
Eventually, the strands developed strong attachments to some
spots on the culture surface and stopped flowing (Fig. 2B,
inset). So it appeared that, in the presence of beads, mucus
attached to cilia (small clumps and plumes) but that this
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Because the labeling of highly ciliated areas at the lowest concentration was so thin, it may only represent the labeling of
tethered mucins, but the longer structures and intermittent flow
that developed later suggest the involvement of gel-forming mucins, even at bead concentrations only slightly above our limit of
detection.
Two-color sequential addition of beads. We showed the
attachment of mucus to cilia but not the source of this mucus.
As reported previously (7), mucins undergo a maturation
process upon secretion. So, mucus that we labeled may not be
representative of newly secreted mucus. We were able to show
that the beads did label newly secreted mucus by first adding
red 20-nm beads and then green 20-nm beads [Fig. 4 (profile
view); see Supplemental Movie 4 (top-down view)]. As usual,
the mucus formed strands that flowed over the culture. Most
importantly, there were distinct red- and green-labeled regions
with little combined labeling, suggesting that the green-labeled
mucus was newly secreted.
Fig. 5. Exogenous MUC5B. FITC-labeled exogenous
MUC5B was added to a culture. Left: fluorescence;
right: merged fluorescence and DIC. A: highly ciliated
area a few minutes after MUC5B addition. Arrow 1,
thin MUC5B strand. B: images obtained 2 h after
MUC5B addition showing an area that is highly ciliated, except for a band running from bottom left (circled 3) diagonally up and to the right (circled 4). In this
band, MUC5B (green) could be seen to temporarily
attach to ciliated cells (gray DIC motion in Supplemental Movie 5B). Arrow 2, one of the thicker clumps
that showed temporary attachment to a ciliated cell.
Scale bars, 20 ␮m.
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Fig. 4. Two-color sequential addition of beads. Redfollowed by green-fluorescent 20-nm beads were
added to a culture seen here in profile. From top to
bottom: red fluorescence, green fluorescence,
merged red and green, and merged fluorescence and
DIC. At 3 min after the final wash (A), green and red
fluorescence was seen in separate areas, with some
green fluorescence closer to the culture surface (arrow 1). At 20 min (B), green fluorescence was often
seen circumscribing areas of red fluorescence (arrow 2). At 36 min (C), some areas of fluorescence
appeared thin, as if stretched (arrow 3), and other
areas were starting to show combined red and green
fluorescence (arrow 4). Scale bars, 23 ␮m.
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cultures, the mucus is composed of 5–10 times more MUC5B
than MUC5AC (2, 6, 23). The investigation of the mucociliary
interactions when exogenous MUC5AC, instead of MUC5B, is
present may explain the need for two gel-forming mucins in the
airways. Finally, surfactant may also modulate these interactions, since it has been observed between cilia and the mucus
layer (5).
Significance of mucociliary interactions. Our model presumes the appropriate modulation of the mucociliary interactions. Pathological mucus may be permanently shifted
toward stronger interactions. This would explain highly
adherent mucus as an early sign of disease in the CF lung.
It may also explain why CF lung disease originates in the
deepest airways (1), where there are fewer ciliated cells,
since we observed the strongest interactions on sparsely
ciliated surfaces.
ACKNOWLEDGMENTS
The authors thank the members of the Cystic Fibrosis Center Tissue Culture
Core at University of North Carolina at Chapel Hill.
DISCUSSION
We have shown that mucus can form discontinuous layers
with temporary attachments to the surface. More specifically,
we have shown that beads on cultures label specific structures
that we have called plumes and strands and that the formation
of these structures depends on cilia. We do not know the extent
to which beads cause mucus to form these structures. Most
importantly, the mucus interacts with cilia, as opposed to
nonciliated surfaces, and newly secreted mucus, as well as the
gel-forming mucin MUC5B, does the same. Attachments are
dynamic, in the sense that they are short-lived when the culture
is well ciliated.
Model of mucociliary interactions. These observations
lead us to the following model of mucociliary interactions.
Gel-forming mucins are secreted into the mucus layer,
where they are responsible for its weak interactions with
cilia (the labeling of the surface in Fig. 3E is thin). Upon the
deposition of contaminants on this layer, the mucus collapses around these contaminants. The interactions with the
cilia (Fig. 1) are strengthened by this collapse, but this leads
to the bundling of the collapsed mucus into strands by the
cilia (Fig. 2) and its transport in a packaged form. The
increased mucociliary interactions resulting from the collapse of the mucus cause further mucus secretion (Fig. 4),
which will help bundle the contaminants and regenerate the
layer of mucus in its less sticky form.
Full model of the airway liquid layers. We believe that the
two layers of the airway liquid work together to modulate the
mucociliary interactions according to the level of contamination. The last layer of defense would be the tethered mucins,
MUC1, MUC4, and MUC16, which are known to be present in
our cultures (6). We were able to detect MUC1 (data not
shown) at the level of the microvilli and shed into the layer
above the cilia, where it has been found in exosomes (8). The
larger MUC4 and MUC16 mucins have been identified in the
mucus layer and on cilia by conventional staining methods. On
cilia, they provide a barrier that may also carry a small amount
of beads (Fig. 3C). So a full model would include the shedding
of these mucins from the PCL into the mucus layer as cilia
bundle contaminants. The gel-forming mucin MUC5AC will
also be important in mucociliary interactions, although in our
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GRANTS
The project was supported by National Heart, Lung, and Blood Institute
Grant HL-084934-04.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the
authors.
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