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Originally published In Press as doi:10.1074/jbc.M208826200 on October 3, 2002
J. Biol. Chem., Vol. 277, Issue 52, 50710-50715, December 27, 2002
Absent Secretion to Vasoactive Intestinal Peptide
in Cystic Fibrosis Airway Glands*
Nam Soo
Joo ,
Toshiya
Irokawa ,
Jin V.
Wu ,
Robert C.
Robbins§,
Richard I.
Whyte§, and
Jeffrey J.
Wine ¶
From the Cystic Fibrosis Research Laboratory,
Stanford University, Stanford, California 94305-2130 and the
§ Cardiothoracic Surgery and School of Medicine, Stanford
University, Stanford, California 94305-5407
Received for publication, August 29, 2002
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ABSTRACT |
We are testing the hypothesis that the
malfunctioning of airway gland serous cells is a component of cystic
fibrosis (CF) airway disease. CF is caused by mutations that disrupt CF
transmembrane conductance regulator, an anion channel essential
for proper fluid secretion in some epithelia. Submucosal glands
supply most of the mucus in upper airways, and gland serous cells are
the primary site of CF transmembrane conductance regulator expression
in airways. We have discovered a major defect in CF glands by in
situ optical monitoring of secretions from single human airway
glands. CF glands did not secrete to agents that elevated
[cAMP]i (0 responses/450 glands, 8 subjects), whereas glands
were responsive in all donor tracheas (605/827 glands, 15 subjects) and
in bronchi from subjects who were transplanted because of other lung
diseases (148/166 glands, n = 10). CF glands secreted
to cholinergic stimulation, and serous cells were abundant in glands
from all CF subjects. The complete absence of secretion to agents that
elevate [cAMP]i suggests that altered secretion of gland
mucus could contribute to CF lung disease.
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INTRODUCTION |
Cystic fibrosis is characterized by widespread dysfunction of
exocrine organs (1). Organs that secrete mucus or macromolecules, including the sinuses, vas deferens, pancreas, and intestine, become
partially or completely filled with inspissated secretions, often
before or shortly after birth, leading in some cases to complete
blockage and degeneration (2-4). Inadequate hydration of epithelial
fluids underlies much of this pathology. The proposed role of salt and
water was made even before the discovery that CF1 is caused by mutations in
CFTR, an anion channel and channel regulator essential for proper salt
and water movement across some epithelia (5). There is increasing
evidence that this general dysfunction also plays an important role in
CF airway disease (6).
The most devastating clinical consequence of CF is chronic infection of
airways with normally innocuous bacteria and fungi. There is as yet no
consensus on how this is related to altered epithelial salt and water
transport, but the earliest and most persistent hypothesis has been
that some defect in airway mucus is responsible. The role of mucous
clearance as a primary innate defense mechanism of the airways has
recently received renewed attention (6). In CF airways, infecting
organisms are confined to the mucus (6-9) (for discussion, see Ref.
10), but they are neither killed nor cleared, and their diffusible
products provoke an intense but ineffective neutrophilic inflammatory
response that further degrades the airways, eventually leading to death from pulmonary failure (11).
Within airways, CFTR is most highly expressed in serous cells of
submucosal glands (12). Submucosal glands, which are estimated to
supply >95% of upper airway mucus (13), occur with a frequency of
~1/mm2 in human trachea (14). Each gland comprises
multiple tubules that feed into a collecting duct, which then narrows
into a ciliated duct that is continuous with the airway surface (Fig.
1 and Ref. 15). Tubules are lined with
mucous cells in their proximal regions and with serous cells in the
distal acini (15). Normal glands are ~60% serous and 40% mucous
cells by volume, and the abundant serous cells secrete water,
electrolytes, and a rich mixture of antimicrobial, anti-inflammatory
and antioxidant substances, whereas mucous cells provide most of the
mucin component (16, 17). Because of their key role in fighting mucosal
infections, serous cells have been described as "immobilized
neutrophils" (16).

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Fig. 1.
Living human submucosal
gland. A brightfield image at high contrast of a human submucosal
gland following stimulation with carbachol is shown. The gland is
viewed through an oil layer, the surface epithelium, and the lamina
propria, but all deeper tissue was dissected away. The bubble of mucus
secreted by the gland is visible in the oil layer (open
arrow). The presumptive collecting duct (C.D.), mucous
tubules (M.T.), and serous acini (S.A.) are
labeled by reference to the gland reconstruction by Meyrick et
al. (15).
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Remarkably, despite their possible relevance for CF lung disease, the
behavior of intact, CF submucosal glands has never been directly
studied. However, ion and fluid secretion is reduced in cultures of CF
gland cells (18, 19) and in glands pharmacologically treated with CFTR
inhibitors (20). This is consistent with patch clamp studies of primary
cultures of serous cells (21) and the Calu-3 serous cell model (22),
which indicate that CFTR is the only physiologically relevant apical
anion channel in such cells. Ussing chamber studies with permeabilized
cell sheets of Calu-3 cells confirm those studies and further indicate
that functional CFTR is required for secretion to calcium-elevating
agonists (23, 24). Thus, it is predicted that fluid secretion from
serous cells in CF glands should be deficient to all mediators.
However, because glands also contain mucous cells that do not appear to contain CFTR (12), the expected serous cell defect should be most
easily detected if an agonist could be found that preferentially activated serous cell secretion.
Vasoactive intestinal peptide (VIP) stimulates macromolecular secretion
from ferret submucosal glands by elevating [cAMP]i (25, 26)
and degranulates ferret gland serous cells (26). In isolated submucosal
glands from cats, VIP stimulates glycoconjugate release without
stimulating contraction of myoepithelial cells (27). Binding sites for
VIP are detected on human submucosal glands (28), and the Calu-3
human serous cell model has functional VPAC1 (VIP/PACAP-II)
receptors (29). In addition to stimulating macromolecular secretion, we
recently showed that VIP stimulates sustained fluid secretion from pig
submucosal glands (30). Therefore, VIP seemed to be a good choice to
test the hypothesis that serous cell secretion is defective in CF
glands (24); forskolin was also used to circumvent variations in VIP
receptor density (28).
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MATERIALS AND METHODS |
Airway Preparations--
Human tracheal and bronchial tissues
were obtained following lung transplants, or in one CF case, from a
necropsy specimen obtained 4 h post-mortem. These studies were
approved by the Institutional Review Boards of Stanford
University. Usable data were obtained from 33 subjects. Subject
characteristics are given in Table I.
Other diseases consisted of patients diagnosed with 1
antitrypsin deficiency (n = 4), chronic obstructive
pulmonary disease (n = 3), and bronchiolitis obliterans
pulmonary emphysema, and cardiomyopathy with pulmonary fibrosis plus
IgA deficiency (n = 1 each). No obvious differences in
gland secretion were observed among the non-CF disease conditions, and
so they were grouped for analysis.
Tissues were transported to the laboratory in cold
PhysiosolTM solution (Abbott) and were then transferred to
ice-cold Krebs-Ringer bicarbonate buffer bubbled with 95%
O2, 5% CO2, where they were maintained until
use. The Krebs-Ringer bicarbonate buffer composition was: 115 mM NaCl, 2.4 mM K2HPO4,
0.4 mM KH2PO4, 25 mM
NaHCO3, 1.2 mM MgCl2, 1.2 mM CaCl2, 10 mM glucose, and
1.0 µM indomethacin. pH was 7.4, and osmolarity was
adjusted to ~290 mosM. A piece of ventral trachea or
bronchus of ~1.5 cm2 was pinned mucosal-side-up, and the
mucosa with underlying glands was dissected from the cartilage and
mounted in a 35-mm diameter, Sylgard-lined plastic Petri dish with the
serosa in the bath (~2 ml volume) and the mucosa in air. The tissue
surface was cleaned and blotted dry with cotton swabs and further dried
with a stream of gas, after which 30-70 µl of water-saturated
mineral oil was placed on the surface. The tissue was warmed to
37 °C at a rate of ~1.5 °C min 1 and continuously
superfused with warmed, humidified 95% O2, 5% CO2. Pharmacological agents were diluted to final
concentration with warmed, gassed bath solution and were added to the
serosal side by complete bath replacement.
Two experimental paradigms were used in an attempt to detect minor
levels of VIP/forskolin-mediated secretion in CF tissues. In one, we
waited until basal secretion (if present) was stopped or stable and
then added 1 µM VIP or 10 µM forskolin
serially or together. Forskolin was used to control for the possibility that VIP receptors are decreased in CF tissues. After intervals of 40 min to 1 h, 10 µM carbachol was then added to test
for gland viability. In the second procedure, we first stimulated
transiently with a 2.5 µM solution of carbachol until
small bubbles of mucus formed over some gland ducts and then
repeatedly replaced the bath with fresh Krebs-Ringer bicarbonate buffer
until secretion either stopped or returned to basal values. VIP + forskolin was then applied, and the secreted droplets of mucus were
followed for at least 40 min to detect any slight increase in the rate of secretion. A second application of 10 µM carbachol was
then applied.
Optical Measurements--
Bubbles of mucus within the oil layer
were visualized by oblique illumination, and digital images were
captured with a CCD sensor mounted on a microscope (small field) or
were obtained directly with the macro lens of a Nikon digital camera
(large field). For macro images, each image contained an internal
reference grid to compensate for any minor adjustments in magnification made during the experiment. Stored images were analyzed either by
direct measurement or with Scion Image software (Scion Corp., Frederick, MD). Mucous volumes were determined from the size of the
spherical bubbles; bubbles that were not spherical were omitted from
secretion rate analyses. Details of these methods are given in Refs. 30
and 31.
Reagents--
Compounds (Sigma) were made fresh or maintained at
20 °C in the following solvents: carbachol and VIP in distilled
water, indomethacin in ethanol, and forskolin in
Me2SO. All were diluted 1:1,000 with bath solution (except
indomethacin, which was diluted 1:10,000) immediately before use at the
concentrations indicated.
Statistics--
Data are means ± S.E., and Student's
t test for unpaired data was used to compare the means of
different treatment groups unless otherwise indicated. The difference
between the two means was considered to be significant when
p < 0.05.
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RESULTS |
Pieces of human tracheal or bronchial epithelia were prepared so
that spherical bubbles of uncontaminated mucus formed within an oil
layer on the surface (Fig. 2). The
bubbles of mucus, which remained attached to the gland ducts, were
optically monitored, and their volumes were estimated by assuming that
they were spheres (31). Glands sometimes began secreting at room
temperature or started secreting as the bath was warmed (Fig.
2a). This basal secretion was variable, usually diminished
or stopped within 30 min after the tissue reached 37 °C, and in
general was less pronounced than in sheep and pigs (30, 31).

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Fig. 2.
Mucus secretion from individual submucosal
glands in a donor tracheal epithelium. a and b,
two successive images of the same field after 30 min of basal secretion
(a) and after 30 min of forskolin-stimulated secretion
(b).
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Stimulation of Donor Tracheal Glands and Non-CF Bronchial Glands
with VIP/Forskolin--
In many glands, including pig bronchial
submucosal glands (30, 32), agents that elevate [cAMP]i
produce secretion. In the pancreas and in the Calu-3 serous cell model
(33, 34), fluid secretion produced by the elevation of
[cAMP]i is mediated by HCO
secretion and is CFTR-dependent. Prior studies of
[cAMP]i-mediated, serous cell protein secretion emphasized
pathways through -adrenergic receptors (16), but isoproterenol
produces minimal mucous secretion in sheep (31), pigs (30, 32),
and humans,2 in contrast with
VIP (30) and forskolin (32), so these latter agents were used.
Treatment of control tissues with VIP or forskolin resulted in gland
mucous secretion that reached a maximal value at about 10 min and was
then sustained (Figs. 2b and
3). Secretion in response to VIP or
forskolin was observed in each of 15 donor tracheas and in bronchial
glands from each of 10 subjects with non-CF diseases (100%).

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Fig. 3.
Control glands secrete in response to VIP or
forskolin. a, responses to VIP and then carbachol of
two glands from a donor trachea, monitored with the small field method.
b, responses to 1 µM VIP + 10 µM
forskolin of 20 glands from a disease control broncheal preparation,
monitored with the large field method. Note the long latency of
response, variations in response profiles, and 7-8-fold range of
volumes secreted.
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Secretion Rates--
Secretion rates and profiles in response to
VIP/forskolin were quantified for a subset of glands in donors and
other diseases (Fig. 3). For a subset of 68 glands from donor tracheas,
secretion rates varied from 0 to a maximal rate of 4.50 nl·min 1 gland 1 with a mean rate of
1.04 ± 0.23 nl·min 1 gland 1. In a
subset of 48 glands from non-CF diseases, secretion rates ranged from 0 to 5.02 nl·min 1 gland 1 with a mean rate
of 1.14 ± 0.35 nl·min 1 gland 1. As
with responses to carbachol in sheep (31) and pigs (30), we observed
wide variations in secretion rates to VIP/forskolin among glands within
subjects (Figs. 2 and 3).
Stimulation of CF Tracheal and Bronchial Glands with
VIP/ Forskolin--
In marked contrast with the gland secretory
responses seen in all non-CF subjects, submucosal glands from CF
subjects were completely refractory to stimulation with VIP or
forskolin or to combined treatment (Figs.
4 and 5). We observed no gland secretion to either agent, alone or in combination,
in any of eight CF subjects followed for periods of 40-60 min
(p < 0.001 for CF versus either control
group, Chi square). All of the glands counted as refractory to
forskolin were otherwise functional because they secreted in response
to the [Ca2+]i-elevating agonist carbachol (Figs.
4 and 5) (35). The percentage of carbachol-responsive glands that also
responded to forskolin was 73% for donor trachea (605/827 glands),
89% for bronchi from diseases other than CF (148/166 glands), and 0%
for CF (0/450 glands) (Fig. 6).

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Fig. 4.
CF glands do not secrete to VIP + forskolin. Left column, whole field; right
column, an enlargement of the approximate field shown by the
box in image a. Bronchial epithelium from a CF
subject was transiently stimulated with 2.5 µM carbachol
to produce the mucus secretion shown in a. Just after
image a, the tissue was stimulated with 1 µM
VIP + 10 µM forskolin. Image b, taken 64 min
later, shows zero increase in mucus volume during that period. Just
after image b, the tissue was again stimulated with 10 µM carbachol, and image c was taken 30 min
later. Scale of grid = 0.5 mm.
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Fig. 5.
Forskolin failed to stimulate mucus secretion
from CF glands. Initial secretion was produced by stimulating with
2.5 µM carbachol. After a thorough wash, two glands were
followed for 45 min during which a low rate of secretion continued
(0.15 nl·min 1 gland 1). Stimulation for 70 min with 10 µM forskolin caused no increase in the
secretion rate, but glands were still viable because they subsequently
responded vigorously to 10 µM carbachol.
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Fig. 6.
Summary of airway
submucosal gland responses to VIP/forskolin. a,
subjects tested (open bar) and responding (closed
bar). b, glands tested (open bar) and
responding (closed bar). The asterisk indicates
zero responses of all CF glands in all subjects.
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Time-dependent Changes in Gland Responsiveness--
In
our animal studies of carbachol-mediated secretion, we observed no
diminution in responsiveness for tissues up to 24 h after harvest.
In human donor tissues, responses to carbachol also appeared to be
undiminished for at least 24 h after harvest, but responses to
VIP/forskolin were less robust and declined in responsiveness after
~10 h (Fig. 7), although some control
glands were observed responding to forskolin for up to 40 h after
harvest. The mean ages of tissues at the time of experiments for
donors, other diseases, and CF were 15 ± 11, 13 ± 8, and
11 ± 9 h, respectively. Thus, CF tissues were tested on
average several hours earlier than controls, eliminating a
time-dependent change in responsiveness as a basis for the
absence of responses in the CF tissues.

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Fig. 7.
Time-dependent changes in gland
responses to VIP/forskolin. Eleven large format experiments with
donor tissues (solid diamonds) and disease controls
(open circles) were conducted at varying intervals after
harvest. The ordinate shows the percentage of carbachol-responsive
glands that also responded to VIP/forskolin. Each point is based on
17-208 responding glands.
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Serous Cells Are Abundant in CF Airway Glands--
One possible
explanation for the complete lack of cAMP-mediated secretion from CF
glands is that such secretion is proposed to originate from serous
cells, and conversion of serous to mucous cells has been observed in
chronic bronchitis (13, 36). However, such conversion has not been
claimed for CF glands, and histological examination of glands from the
CF and non-CF subjects we studied showed abundant serous cells in the
CF glands (Fig. 8). As reported by others
(37-39), simple inspection revealed CF glands to be much larger than
glands in the non-CF groups. Although we have not yet quantified the
extent of CF gland hypertrophy in our samples, the volume of serous
cells in the CF airways we studied is clearly greater than in control
tissues, further highlighting the remarkable absence of responsiveness
to VIP or forskolin.

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Fig. 8.
Serous cells are abundant in CF submucosal
glands. A 5-µm paraffin section through formalin-fixed bronchial
tissue stained with Hematoxylin and Eosin. a
(inset), gland at lower magnification, with a portion of the
duct opening onto the airway surface at top. Tissue had been stimulated
with both forskolin and carbachol. The surface epithelium is missing,
and the cartilage, which would normally have occupied the semicircular
areas, was micro-dissected away to allow mounting of the flat
epithelial sheet required for optimal optical measurements.
b, higher magnification of the boxed area of the
gland, showing serous (S) and mucous (M)
tubules.
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DISCUSSION |
We found that glands from cystic fibrosis airways completely lack
secretion stimulated by VIP or forskolin. Our experiments with intact,
individually monitored airway glands are the first to demonstrate a
defect in CF airway gland volume secretion, but they were preceded by
highly informative experiments with primary cultures of
serous-like cells derived from CF airway glands, which showed
defects in stimulus-evoked short circuit current (18) and fluid
secretion (19, 40). Together with localization of CFTR to gland serous
cells (12), the present and previous studies establish serous cell
dysfunction as a consistent feature of CF submucosal glands.
In our study, serous cell dysfunction was not caused by general
debility, inactivity, or inflammation because every non-CF transplant
patient responded to VIP/forskolin stimulation. Because forskolin was
also ineffective, a deficiency in VIP receptors cannot explain the
results, nor can serous-mucous cell conversion because direct
inspection revealed abundant serous cells within CF submucosal glands.
The absence of [cAMP]i-mediated serous cell fluid secretion
in CF glands generates a series of questions about gland secretion,
which are discussed with reference to the model of gland secretion
shown in Fig. 9. In that model, gland
mucus is the joint product of serous cells and mucous cells, which
normally secrete together. In the following discussion, we distinguish between fluid secretion, which we use as shorthand for
electrolyte-driven water transport, and macromolecular secretion, which
refers to the secretion of everything else, including mucins and
non-mucin proteins. Normal mucus is ~98% water.

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Fig. 9.
Schematic model of submucosal gland.
Four functional compartments have been proposed based on anatomical and
immunohistochemical data with CFTR located primarily in serous cells.
Pathways that elevate [Ca2+]i, such as ACh, are
hypothesized to activate fluid and macromolecular secretion from both
serous and mucous cells. Pathways that elevate [cAMP]i, such
as VIP, are hypothesized to stimulate serous cells and mucin but not
fluid secretion from mucous cells. The CFTR-dependent
fluid-secreting pathway is deleted in cystic fibrosis glands.
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What Is the Mechanism of Serous Cell Fluid Secretion?--
Our
results are consistent with the serous cell model developed by studies
of Calu-3 cells (22) and primary cultures of serous cells (18, 19), in
which CFTR is required for anion secretion to all mediators (see
Introduction). Secretion in response to agents that elevate
[Ca2+]i is effected either because CFTR is
normally open or because stimulation releases ATP and activates CFTR
via an apical autocrine pathway (41).
What Is the Mechanism of Mucous Cell Fluid Secretion?--
Ballard
and colleagues (32) showed that mucus secretion by pig airways is
maximally stimulated by ACh, that forskolin produces about 60% of that
volume, and that the two agonists are not additive. CFTR expression,
studied with a well characterized polyclonal antibody and with in
situ hybridization, was observed in serous but not mucous tubules
(12), although others have reported more extensive expression (42). If
CFTR is localized to serous cells, then the absence of
forskolin-stimulated secretion in CF glands suggests that normal mucous
cells do not secrete fluid in response to forskolin, whereas the
continued ability of CF glands to secrete to ACh suggests that fluid
secretion by mucous cells does not require CFTR.
How Is Macromolecular Secretion Linked to Fluid
Secretion?--
Most prior studies of secretion by cultured
gland cells or explants have studied either the release of labeled
macromolecules or short circuit current, but these two features of
secretion may be uncoupled, especially in glands expressing a genetic
defect. Prior studies with explants of whole bronchial segments (43) or
primary submucosal gland cell cultures (44) from CF subjects found
defective stimulus-evoked secretion of macromolecules. We have not
addressed that possibility in the present experiments.
Is Secretion of CF Glands to Cholinergic Agents Normal?--
Given
the total loss of secretion to VIP and forskolin in CF glands, is the
secretion that remains to carbachol indistinguishable from normal
secretion? That is, can this Ca2+-elevating agent induce CF
serous cells to secrete? The model of gland function shown in Fig. 9
predicts that it cannot, resulting in gland fluid secretion to
cholinergic agents that will be mediated only by fluid secretion from
mucous cells. It is not a simple matter to determine whether such
secretion is deficient. As documented by others, the CF glands in our
study appeared much larger than normal, but we have not yet established
that point with morphometry. Given the expected hypertrophy of CF
submucosal glands (a 4-fold increase was observed in a recent study
(39)), a meaningful comparison of secretion rates requires that the
rates be expressed relative to gland volumes. Such studies are now underway.
Does the Composition of CF Gland Mucus Differ from
Normal?--
Surprisingly, secretions of pig glands in response to
forskolin or carbachol had equivalent pH values (30), and again
surprisingly, [Na+] and pH values for
carbachol-stimulated gland secretions were equivalent in CF and control
subjects (35). Whether the loss of CFTR will alter protein secretion
from serous cells in situ, as occurred for cultured CF gland
cells (44), is uncertain. It remains possible that the only component
missing from CF mucus is CFTR-dependent salt and water flux.
Does VIP Stimulation of Glands Play an Anti-inflammatory
Role?--
VIP is one of the most abundant peptides in the lungs, and
a plethora of studies have implicated VIP pathways in the suppression of inflammation and cell damage (45). Exactly how this occurs is
unknown. If selective VIP activation of serous cells occurs naturally,
it is possible that it could contribute to the anti-inflammatory role
of VIP in the airways, and its loss in CF may contribute to the
heightened inflammatory state that is a hallmark of CF airway disease
(46, 47).
Does the Gland Defect Help Explain the Initiation of Infections in
CF Airways?--
People with cystic fibrosis die primarily because
their lungs become chronically infected with bacteria and fungi that
are easily cleared from normal lungs. Importantly, the pathogens in CF
airways are trapped within the mucus (8), but the ability of even
normal mucus to inhibit the growth of pathogens is incomplete and wanes
over time, emphasizing the importance of mucociliary clearance and
cough in limiting the residence time of pathogens in the airways (48).
The loss of serous cell secretion may dispose the airways to infections
via multiple mechanisms, including slower transport of mucus, absolute
stasis of mucus caused by tethering to glands (49), reduced secretion
of antimicrobials and anti-inflammatory agents, and reduced
bioavailability of these agents because of inadequate dispersal (50).
All of these features will be exacerbated by increased fluid absorption
and decreased fluid secretion by the surface epithelium (6, 51)
Summary--
Based on the above results and reasoning, our working
hypothesis is that secretion by CF submucosal glands lacks the
electrolyte-driven fluid component normally supplied by serous cells.
Macromolecular secretion by both serous and mucous cells, as well as
electrolyte-driven fluid secretion by mucous cells, are hypothesized to
be intact. Because the rheological properties of mucous depend
critically on the concentration of macromolecules during initial
formation of the gel and are resistant to subsequent changes, we
hypothesize that a deficiency in electrolyte-driven water transport
deep within the gland tubules will increase the concentration of gland
mucus, adversely affecting mucus clearance from the glands and
contributing to impaired mucociliary and cough clearance.
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ACKNOWLEDGEMENTS |
We thank B. A. Reitz, N. R. Henig,
J. Theodore, G. J. Berry, and the staff of the Stanford Transplant
team for aid in obtaining post-transplant tissue specimens and T. E. Robinson, N. R. Henig, and H. Furthmayr for help in obtaining
an autopsy specimen. We are grateful to W. Finkbeiner, M. E. Krouse, P. Quinton, and J. H. Widdicombe for criticisms of an
earlier manuscript, to R. Dhillon C. Tseng, and T. Hsu for data
analysis, and to M. F. Wine for help in obtaining informed
consents. We are especially grateful to the families of organ donors,
whose generosity has allowed both life-saving transplants and research
into the root causes of lung diseases.
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FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants DK-51817 and HL-60288 and by the Cystic Fibrosis Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Cystic Fibrosis Research Laboratory, Rm. 450, Bldg.
420, Main Quad, Stanford University, Stanford, CA 94305-2130. Tel.:
650-725-2462; Fax: 650-725-5699; E-mail: wine@stanford.edu.
Published, JBC Papers in Press, October 3, 2002, DOI 10.1074/jbc.M208826200
2
N. S. Joo and J. J. Wine, unpublished observations.
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ABBREVIATIONS |
The abbreviations used are:
CF, cystic fibrosis;
CFTR, CF transmembrane conductance regulator;
VIP, vasoactive
intestinal peptide;
PACAP, pituitary adenylate cyclase activating
peptide.
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