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2007 35: 116 Toxicol Pathol
Alessandra Livraghi and Scott H. Randell
Cystic Fibrosis and Other Respiratory Diseases of Impaired Mucus Clearance
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Toxicologic Pathology, 35:116–129, 2007
Copyright C ?by the Society of Toxicologic Pathology
ISSN: 0192-6233 print / 1533-1601 online
Cystic Fibrosis and Other Respiratory Diseases of Impaired
ALESSANDRA LIVRAGHI1AND SCOTT H. RANDELL1,2
1Cystic Fibrosis/Pulmonary Research and Treatment Center, Department of Medicine, The University of North Carolina at Chapel Hill
2Department of Cell and Molecular Physiology, The University of North Carolina at Chapel Hill
regulated epithelial ion and water transport coordinated with mucin secretion, beating cilia, and cough results in continuous flow of fluid and mucus
over airway surfaces toward the larynx. This cleansing action is the initial and perhaps most quantitatively important innate defense mechanism.
Repeated lung infections and eventual respiratory insufficiency characteristic of human cystic fibrosis (CF) and primary ciliary dyskinesia (PCD)
illustrate the consequences of impaired mucus clearance. Altered mucus clearance likely contributes to the initiation, progression, and chronicity of
other airway diseases characterized by inflammation and mucous secretory cell hyper/metaplasia that afflict millions worldwide, including chronic
obstructive pulmonary disease (COPD). This review concisely discusses the pathophysiology of human diseases characterized by genetic defects that
impair mucus clearance. It then explores animal models in which components of the mucus clearance system have been disrupted. These models
firmly establish the importance of mucus clearance for respiratory health, and will help elucidate disease mechanisms and therapeutic strategies in
CF, PCD and COPD.
Chronic obstructive pulmonary disease; cilia; mucin; mucociliary clearance; cystic fibrosis; primary ciliary dyskinesia.
sult from complex interactions between the environment and
host genetics. Constantly exposed to the environment, the
airways and lungs have evolved to efficiently deliver inhaled
air to the alveoli, while protecting gas exchange structures
from potentially harmful airborne chemicals, particles, and
pathogens. A layer of fluid and mucus, propelled by cilia
beat and cough, continuously flows over conducting airway
surfaces towards the pharynx. Mucus clearance is a critical,
physiologically regulated, protective function of the airways
The genetic defects in cystic fibrosis (CF) and primary cil-
iary dyskinesia (PCD) impair mucus clearance, and study of
these diseases has provided key insights regarding the patho-
biology that occurs when constant protection of the respira-
tory tract by effective mucus clearance is lost. In this review,
PCD in humans. We will then summarize animal models in
which genes involved in the mucus clearance system have
been disrupted or modified to model the human diseases.
Room 4011 Thurston-Bowles Building, Chapel Hill, NC 27599; e-mail:
Abbreviations: CF, cystic fibrosis; PCD, primary ciliary dyskinesia;
COPD, chronic obstructive pulmonary disease; PCL, peri-ciliary layer;
ASL, airway surface liquid; CFTR, cystic fibrosis transmembrane con-
ductance regulator; ENaC, epithelial sodium channel; OMIM, Online
Mendelian Inheritance in Man; TEM, transmission electron microscopy;
PD, potential difference; Isc, short circuit current.
Respiratory Tract Physical Structure, Innate and Acquired
Integrated airway and lung defense is the product of a
complex network, acting at distinct levels of organization
(Figure 1). The nasal passages, nasopharynx and pharynx
constitute the upper airways and the trachea to ∼6th genera-
tion bronchi are the proximal, large lower airways. Portions
cipally of basal, intermediate, ciliated and mucous secretory
(goblet) cells. The proximal portion of the small, distal air-
ways (bronchioles) is also lined by a columnar pseudostrati-
fied epithelium, which transitions in more distal airways to a
replace mucous secretory cells.
I, and cuboidal type II alveolar epithelial cells. Thus, a con-
and circulatory system. Just as the relative abundance of cell
types changes along the respiratory tract axis, each segment
has specialized functions that are key to host defense. The
epithelial physical barrier, in combination with secreted an-
and natural killer cells constitutes the innate immune system
(Martin and Frevert, 2005; Zaas and Schwartz, 2005). This
system is constantly on guard and requires no previous ex-
posure to elicit a protective response.
inflammatory cells are induced when pathogen associated
patterns or other chemical motifs are recognized by innate
immune system receptors. The cell and cytokine/chemokine
Vol. 35, No. 1, 2007
FIGURE 1.—Schematic illustration of respiratory tract structure, including ion and mucus transport in normal (A) and CF (B) human airways. Ion channels in the
apical plasma membrane of airway epithelial cells regulate periciliary layer depth and mucus hydration. The epithelial Na+channel (ENaC) mediates Na+entry,
and Cl−and water follow through trans and/or paracellular pathways, decreasing airway surface hydration. In contrast, Cl−secretion through the cystic fibrosis
transmembrane conductance regulator (CFTR) and/or calcium activated Cl−channels (CaCC), increases surface hydration. All three channels are likely regulated by
ATP release and the action of ATP and its metabolites on cell surface receptors. P2Y2purinergic receptor activation by ATP leads to decreased PIP2, which inhibits
ENaC, and IP3-stimulated Ca2+release, which activates CaCC. Adenosine (ADO) derived from ATP activates the A2Badenosine receptor generating cAMP and in
turn activating CFTR. Active CFTR likely inhibits ENaC. In normal lungs, adequate hydration is maintained by balancing Na+absorption through ENaC and Cl−
secretion through CFTR and CaCC. In CF, absence of CFTR-mediated Cl−conductance and loss of ENaC down-regulation depletes the periciliary layer and causes
hyper-viscosity of mucus, impairing both mucociliary and cough clearance (black arrows). Opportunistic bacteria exploit static CF mucus, resulting in repeated
and chronic infection characterized by intense neutrophilic inflammation. It is still unclear whether a proportion of alveolar lining fluid egresses the distal lung via
mucociliary transport (grey arrow) or is maintained in a local circuit in the alveoli, or both.
118 LIVRAGHI AND RANDELL
involvement and development of the more specialized adap-
tive immune response; for example, specific memory and ef-
fector T and B cells. Mucous secretory and ciliated cells play
cough, provides mucus clearance, perhaps the quantitatively
most important airway innate defense mechanism (Knowles
and Boucher, 2002; Randell and Boucher, 2006).
Mucus, Mucous Secretory Cells, and Mucins
Mucus is the collective term for mucins, ions, water and
other substances normally present on mucosal surfaces. By
virtue of highly glycosylated secreted mucins, lipids, and
soluble proteins, mucus binds and entraps a broad array of
inhaled particles. Since increased airway secretions charac-
CF, particular attention has been paid to the regulation of
mucous secretory cell number, mucin gene expression and
diate mucin release (Thornton and Sheehan, 2004; Rose and
Voynow, 2006; Williams et al., 2006). Beyond obstruction of
airflow, excessive mucus that fails to clear may form plaques
and plugs that can serve as a nidus for infection by oppor-
tunistic pathogens, especially facultative anaerobic bacteria
such as P. aeruginosa, able to adapt to relatively hypoxic
Often, single cell, free-living bacteria (planktonic) evolve
into alginate-producing colonies or biofilms that are much
more resistant to attack from both neutrophils and antibi-
otics, thus becoming difficult to eradicate (Worlitzsch et al.,
remodeling of the airways perpetuating the vicious cycle ini-
tiated by mucus accumulation.
FIGURE 2.—Cilia structure in respiratory tract epithelial cells. (A) Air-liquid interface cultures of normal human tracheobronchial epithelial cells display mucous
secretory and ciliated cell differentiation similar to the in vivo morphology. The porous culture support is visible below the cells. Formalin-fixation and paraffin
section, H&E stain. (B) Low power TEM view across the apical plasma membrane of a ciliated cell demonstrates cilia axonemes and basal bodies (BB). (C and D)
Cross-section views of cilia axonemes at low and high power, respectively, reveal the 9 + 2 arrangement of microtubules and inner (IDA) and outer dynein arms
(ODA), and radial spoke apparatus (RSA) structures. B, C, and D were prepared using conventional TEM procedures.
in the human body (Afzelius, 2004). In the respiratory tract,
ciliated cells have ∼200 cilia per cell, each with a diameter
and large airways are denser and longer than in the bronchi-
oles. Each respiratory cilia axoneme has a typical 9 + 2 mi-
crotubule structure (9 outer doublets and 2 central single mi-
crotubules) with characteristic inner and outer dynein arms,
into the ciliated cell at a specialized structure that terminates
in a cytoplasmic basal body that resembles the centriole of
the mitotic apparatus (Figure 2). It is estimated that >200
unique proteins constitute the cilia and basal body structures
(Fliegauf and Omran, 2006). In normal lungs, cilia beat in
coordinated, directional, metachronal waves at a frequency
estimated to be ∼10–20 Hz, and mucus and the embedded
swallowed or expectorated, thereby promoting airway steril-
ity (Meeks and Bush, 2000).
The Peri-Ciliary Layer (PCL), Glands, and Cough.
gether with cilia and mucus, an adequate peri-ciliary layer
(PCL) is required for effective mucociliary clearance. The
PCL lines the airway surface and provides a low viscosity
milieu in which cilia can freely beat to propel overlying mu-
cus. The existence of the PCL has been demonstrated in vivo
by osmium/perfluorocarbon fixation techniques followed by
transmission electron microscopy (Sims et al., 1991) or in
vitro by confocal microscopy (Matsui et al., 1998; Tarran
and Boucher, 2002) (Figure 3). The ability of the airway ep-
layer required for cilia to beat and effectively clear mucus is
At least 8 categories of cilia, or cilia de-
Vol. 35, No. 1, 2007
FIGURE 3.—An adequate periciliary liquid (PCL) layer is essential for mucus
transport. (A) Perflurocarbon-OsO4fixation preserves the morphology of the
PCL and overlying mucus. The PCL is visible as a clear layer in a thin section
of a well-differentiatied air-liquid interface culture of human tracheobronchial
epithelial cells, Epon-embedding, Richardson’s stain. (B) TEM of an equivalent
area as depicted in the box in panel A. Dense mucus is visible above the clear
PCL. Two polystyrene beads, which were added to track mucus movement, are
visible at the mucus-PCL border (asterisks). (C) The PCL as viewed in a live
cell preparation by X-Z plane confocal microscopy. Texas red dextran-labeled
airway surface liquid (ASL) is on the apical surface (red) and the cells were
labeled with calcein (green).
critical for lung innate defense (Matsui et al., 1998; Tarran
et al., 2001, 2005).
As discussed in the legend of Figure 1, recent studies sug-
balancing of Cl−secretion and Na+absorption and is sensi-
cleotides and nucleosides) present in the PCL (Lazarowski
et al., 2004; Tarran, 2004, 2006a, 2006b). Upper and proxi-
airway secretions and amplify the ability of airway epithelial
cells to produce antimicrobial factors and mucus (Wine and
Joo, 2004; Inglis and Wilson, 2005). Finally, cough is an im-
portant innate defense mechanism that prevents pulmonary
aspiration, promotes ciliary activity and clears airway debris
(Chang, 2006; McCool, 2006). The combination of mucocil-
iary and cough clearance constitutes “mucus clearance.”
Cystic Fibrosis (CF).
In the following section, we high-
light the seminal characteristics of CF. For a more a com-
prehensive treatise on the history, genetics, diagnosis, patho-
genesis, microbiology, treatment, or controversies in this in-
tensely studied disorder, the reader is directed to an excellent
recent textbook chapter (Boucher, 2005) and review articles
of the United States, respectively. It has been recognized as a
distinct disease entity since the 1930s. It is an autosomal re-
cessive, monogenic disorder resulting from mutations in the
gene, which was discovered in 1989 (Online Mendelian In-
heritance in Man (OMIM) # 602421, National Center for
The CFTR protein is a cAMP-activated transmembrane
anion channel expressed at a variety of epithelial surfaces,
where it is thought to principally conduct Cl−ions. How-
ever, CFTR also coordinately regulates the function of other
are over 1400 different recognized CFTR mutations but the
nicity, can be detected in a screen of 97 of the most common
3 base pairs resulting in the loss of phenylalanine at position
508, known as ?F508, is the most common CF mutation),
missense, frameshift, nonsense, and also mutations causing
alterations in RNA splicing.
Acting through a variety of molecular and cellular mech-
anisms, severe mutations result in an almost total absence of
resulting in partial CFTR function may produce abnormal-
ities in other organs but respiratory disease may be mild or
even absent. CF is a multi-system disorder with a childhood
onset. The defining feature is abnormal ion and water trans-
port across a variety of epithelial surfaces, which leads to
the characteristic salty sweat and elevated nasal and rectal
trans-epithelial electrical potential differences (PD) that can
be measured for diagnostic purposes. Relatively dehydrated,
thickened secretions and mucus accumulation/obstruction of
several tubular duct structures are characteristic of CF.
males due to in utero blockage, subsequent fibrotic oblitera-
vesicles. In the female, the uterine cervix can be inappropri-
principally associated with the gastrointestinal tract. Meco-
nium ileus in the newborn is highly suggestive of CF and
recurrent bouts of intestinal obstruction are common. Gas-
trointestinal changes and frequent coughing increases the
frequency of rectal prolapse. Biliary tract obstruction vari-
ably results in liver dysfunction and fibrosis. Pancreatic in-
which serves as the basis for the blood trypsinogen neonatal
Exocrine pancreatic insufficiency and duct blockage de-
crease digestive enzyme secretion into the intestine and can
result in significant nutritional deficiencies and steatorrhea
if not properly treated. Ultimately, postobstructive atrophy
results in complete replacement of the exocrine pancreas by
which can cause insulin dependent hyperglycemia. Progres-
sive pancreatic lesions are illustrated in Figure 4.
120 LIVRAGHI AND RANDELL
FIGURE 4.—Progressive obstructive pancreatic pathology in CF. Low (A) and higher power (B) views of the pancreas of a 9-year-old pancreatic insufficient CF
male that died of a non-CF related cause exhibits eosinophilic material in dilated ducts, residual exocrine pancreatic acini and numerous islets, whereas the pancreas
of a 47-year-old pancreatic insufficient CF female (C) contains mainly fat and islets with minimal fibrosis in this particular view. Routine formalin-fixed paraffin
sections, H&E stain.
Neonatal respiratory distress is not specifically associated
with CF and, except for dilated mucus gland ducts, the lungs
are apparently normal at birth. Postnatal respiratory tract le-
sions are, by far, the leading cause of morbidity and mor-
tality in CF. In the upper airways, rhinitis and sinusitis due
to infection are highly prevalent and development of nasal
polyposis is common. Recurrent bacterial bronchopneumo-
nia, often initially due to H. influenzae or S. aureus and then
P. aeruginosa, is frequent in newborns and young children
with CF. Evidence for lung infection with P. aeruginosa oc-
curs in ∼60% of CF individuals by age 3, which is likely to
be an underestimate (Burns et al., 2001; West et al., 2002).
Repeated and ultimately chronic infection with mucoid
P. aeruginosa, or other characteristic Gram-negative bacte-
ria, including Burkholderia sp. (previously known as Pseu-
domonas cepacia), S. maltophilia, A. xylosoxidans, as well
as variable involvement of Aspergillus and nontuberculous
mycobacteria, leads to progressive bronchiectasis, bronchi-
olectasis and respiratory insufficiency that ultimately causes
respiratory failure and death of >95% of CF patients,
currently with a median survival age of ∼37 years.
The sequence of events predisposing to airway infection
in CF has been debated through the years, but clarified more
recently. Generalized immune deficiency and specific ab-
normalities in acquired immunity are highly unlikely, since
systemic infection is not characteristic of CF. In fact, sep-
sis due to P. aeruginosa, even after decades of lung infec-
tion, is rare, presumably due to effective humoral immu-
nity. More likely, the disease represents a failure of local
airway defense. It has been suggested that bacteria adhere
more readily to CF airway epithelial cells due to enhanced
expression of the cell surface ganglioside asialoGM1, pro-
moting infection (Saiman and Prince, 1993). Paradoxically,
it has also been proposed that CFTR serves as a bacterial re-
ceptor and that its absence leads to failure to internalize and
kill bacteria (Pier, 2000). Defects in anti-microbial activity
of airway fluid (Ganz, 2002) or in neutrophil phagocytosis
(Berger et al., 1989) likely occur in the inflamed CF airway
environment but are unlikely to be the primary defect. The
cells, even in the absence of infection, is debated (Machen,
2006). A consensus now exists that respiratory tract patho-
physiology in CF principally results from the inability to se-
crete Cl−and regulate Na+absorption, which causes relative
dehydration of the airway surface, depleting the PCL and
causing accumulation of hyper-viscous mucus that cannot
be cleared by mucociliary clearance or cough (Matsui et al.,
As discussed previously and as illustrated in Figure 5,
mucus plaques and plugs serve as a nidus for intra-luminal
infection. Interestingly, hypoxic microenvironments, which
are exploited by characteristic CF pathogens, develop in
macroscopic mucus accumulations, even within ventilated
airways (Worlitzsch et al., 2002). Bacteria resident within
the thickened luminal mucus may evade chemical antimi-
crobial factors and phagocytes (Matsui et al., 2005). Com-
plex bacterial evolution and host adaptation occurs in the
chronically infected airway, which is likely unique in CF
due to the constant and severe degree of mucus dehydration
and impaired mucus clearance. Bacterial colonies exhibiting
biofilm-like properties may develop, which are difficult or
impossible to eradicate. The continuous presence of bacte-
ria and the accompanying intense inflammation ultimately
remodel the airway wall, causing the ubiquitous mucous se-
cretory cell hyperplasia and metaplasia, submucosal gland
enlargement, hypertrophy of the bronchial circulation, ecta-
formation, sometimes progressing to cavitary disease, with
adjacent fibrosis and pleural involvement.
Primary Ciliary Dyskinesia (PCD).
importance of normally beating cilia to maintain healthy air-
ways. In recent years, there has been impressive progress
to elucidate the role of cilia in diverse biological processes.
It has been demonstrated that motile, rotating, 9 + 0 cilia
at the embryonic node function to break bilateral symmetry
PCD illustrates the
Vol. 35, No. 1, 2007
large, hilar airways were dissected away (not in picture) to reveal highly ectatic segmental bronchi exuding abundant mucopurulent secretions. 1 and 5 cc syringes
containing mucopurulent secretions extracted from the main stem and lobar bronchi are visible in the right lower corner. (B and C) Representative low and higher
power cross-sections of CF bronchus, respectively, routine formalin-fixed paraffin sections, H&E stain. Note the highly corrugated bronchial wall and clear patches
indicative of mucoid P. aeruginosa colonies within the luminal mucus. (D) Higher power view (100× objective) of mucoid P. aeruginosa colony illustrating single,
paired and small clumps of rod shaped bacteria (arrows), Alcian yellow-toluidine blue stain.
in the developing mammalian embryo and establish sided-
ness of the body plan (Hirokawa et al., 2006). A role for
immotile 9 + 0 cilia as sensing antennae and cell signal-
ing platforms in various cells has been suggested by recent
studies (Davenport and Yoder, 2005; Scholey and Anderson,
2006; Singla and Reiter, 2006). Forms of polycystic kid-
ney disease and retinitis pigmentosa are associated with
gene products expressed in renal 9 + 0 monocilia and in
the connecting cilium of retinal photoreceptor cells, respec-
tively (Eley et al., 2005). As in other aspects of cilia biology,
there have been recent strides in our understanding of PCD.
In the United States, the Genetic Diseases of Mucociliary
Clearance Consortium ?http://rarediseasesnetwork.epi.usf.
edu/gdmcc/index.htm?, a clinical research network spon-
sored by the National Institutes of Health Office of Rare
Diseases, includes PCD as a disease target. Furthermore, a
discuss the consequences of defects in motile 9 + 2 cilia, fo-
cusing on the respiratory tract, and refer the reader to excel-
and clinical information (Meeks and Bush, 2000; Afzelius,
2004; Geremek and Witt, 2004; Noone et al., 2004; Van’s
Gravesande and Omran, 2005; Badano et al., 2006; Stannard
and O’Callaghan, 2006).
PCD is less common than CF, with an estimated inci-
dence of 1 in 15,000 to 30,000 live births (OMIM #242650).
It is a genetically heterogeneous disorder that is usually
autosomal recessive. Due to multiple potential defective
genes, wide variability in symptoms, and similarity to ac-
quired abnormalities of cilia due to infection and inflam-
mation, the true incidence of PCD may be greater. PCD
is more frequent in certain isolated populations and in
consanguineous families. In the 1930s, 4 cases of indi-
viduals with the triad of situs inversus, chronic rhinos-
inusitis, and bronchiectasis were reported by Kartagener
(1936) and in the 1970s it was recognized that this syn-
drome was correlated with impaired motility of respiratory
122 LIVRAGHI AND RANDELL
tract cilia and spermatozoa in affected males (Afzelius,
We now know that Kartagener Syndrome is a subset
(∼50%) of PCD, because the abnormalities affecting res-
piratory cilia also impair the motile 9 + 0 cilia in the
embryonic node, resulting in the randomization of sidedness
during embryonic development. Randomization of laterality
is illustrated by the presence of situs solitus (normal) and
situs inversus totalis (mirror image of the thoracic and ab-
dominal organs) in monozygous twins, both of which have
PCD (Noone et al., 1999). Like CF, PCD usually presents
as an autosomal recessive disorder in affected families, but
X-linked cases of retinitis pigmentosa with respiratory tract
symptoms identical to PCD have been reported, and rarely,
other forms of inheritance.
The respiratory tract pathophysiolgy of PCD is attributed
to abnormal function of motile 9 + 2 cilia. The functional
changes can vary from subtle differences in beat frequency,
direction, strength or coordination (dysmotility) to complete
immotility and, rarely, complete absence of cilia (aplasia).
Direct microscopic examination of cilia beating patterns in
nasal and/or bronchial brushing or curettage specimens is
a useful clinical test for PCD, although some PCD patients
to infection, allergic inflammation, pollution or smoking,
of cilia in routine histological sections can be suggestive but
is not usually diagnostic (Figure 6).
Ultrastructural abnormalities can be confirmed by cross-
the cilia axoneme, but again, acquired abnormalities may re-
sult in false positives. The structural changes identifiable by
TEM fall into distinct categories, including absence of inner
or outer dynein arms, radial spoke or microtubule defects, or
other rare alterations. However, dysmotile cilia from patients
with PCD may appear normal by TEM. Extended in vitro
FIGURE 6.—Subtle changes in PCD cilia visible in routine formalin-paraffin sections. (A) Normally aligned and extended cilia are visible in bronchial sections
from an individual with idiopathic pulmonary fibrosis (IPF) and end-stage lung disease. In this view of a CF specimen (B), cilia generally appear flattened and
of variable height, and often pointing in different directions.
culturing of respiratory epithelial cells at an air-liquid inter-
face, enabling maintenance of the ciliated cell phenotype,
removes confounding in vivo influences, thereby eliminating
secondary abnormalities and likely improving the specificity
of the functional and TEM tests (Jorissen et al., 2000).
For unknown reasons, nasal nitric oxide (NO) production
is typically very low in PCD patients (Noone et al., 2004).
and there may be overlap in nasal NO levels between PCD
and CF or other allergic or infectious rhinosinusal diseases.
Due to the wide range of symptom severity and the potential
non-specificity of clinical tests, the diagnosis of PCD can be
As noted before, it is estimated that >200 unique proteins
constitute the cilia and basal body apparatus (Fliegauf and
Omran, 2006) and mutations in any of these genes, or even
in non-cilia genes responsible for cilia assembly or mainte-
nance may cause PCD. The model di-flagellated unicellular
manipulation and analysis of structural-functional correla-
tion, and is a very useful tool to study homologous genes and
proteins present in mammalian cilia (Silflow and Lefebvre,
Genome-wide linkage studies in human PCD have not
been highly useful to elucidate specific mutations, but in-
stead confirm genetic heterogeneity, identifying several po-
characterized by absent or reduced outer dynein arms and
the study of C. reinhardtii has identified mutations caus-
ing similar structural and functional defects. This obser-
vation pointed towards human orthologs of C. reinhardtii
genes as candidate genes for PCD. Mutations in DNAI1 and
DNAH5, the human orthologs of the C. reinhardtii IC78 and
dynein γ-heavy chain genes, respectively, have been identi-
Fliegauf et al., 2005). It has also been reported that the gene
DNAH11, which is homologous to the gene mutated in the
Vol. 35, No. 1, 2007
FIGURE 7.—PCD lung pathology. Representative routine formalin paraffin sections of lung tissue removed from a 60-year-old male PCD patient during lung
transplantation. (A) Accumulated mucus is visible in the lumen of an ectatic, highly corrugated bronchiole that is surrounded by an enlarged and inflamed fibrotic
sheath, H&E stain. (B) Higher power view of an Alcian blue-PAS stained section demonstrates mucous secretory cells in the epithelium and luminal mucins. (C)
Neutrophils are abundant in the luminal mucus, H&E stain.
Ird mouse model of situs inversus (Supp et al., 1999), is mu-
(Bartoloni et al., 2002), but this patient is also homozygous
for ?F508 CFTR, and it is unclear whether the individual
truly had PCD or had acquired cilia defects due to CF and a
tation caused situs inversus but not PCD (Van’s Gravesande
and Omran, 2005).
Recent more detailed studies of PCD families have better
defined the spectrum and frequency of DNAI1 and DNAH5
gene mutations (Hornef et al., 2006; Zariwala et al., 2006).
These studies are important because they suggest the future
applicability of novel clinical tests for PCD. First, antibody
detectable absence or abnormal localization of mutant cilia
proteins may enable novel tests on nasal or bronchial cells
(Fliegauf and Omran, 2006). Second, the combination of
DNAI1 and DNAH5 mutations likely define ∼40% of all
known cases of PCD, potentially ushering in an era of ge-
netic testing for this underappreciated and potentially diffi-
cult to diagnose disease (Bush and Ferkol, 2006). Accurate
and timely diagnosis is key, since aggressive and appropriate
therapy may delay the ultimate development of severe lung
The pathogenesis of respiratory tract disease in PCD has
been generally thought of as being similar to CF, but some-
what milder in general, and with unique features. There is an
with PCD but not CF, apparently due to a failure to rapidly
not well understood but may involve a role for cilia action in
is prominent in both PCD and CF, but childhood otitis media
is much more common in PCD, and patients frequently seek
initial treatment at the otolaryngology clinic.
Incidence of lung infection, offending organisms, devel-
opment of bronchiectasis and longitudinal declines in lung
function are similar to CF but appear to be delayed, and seri-
patients is generally modeled on CF treatment paradigms. A
factor possibly explaining the “PCD vs. CF delay” is that
ion transport mechanisms regulating airway surface hydra-
tion are preserved in PCD. Thus, airway secretions are less
dehydrated than in CF, and the PCL does not become as
Although the progression of airway disease may be delayed,
the physiologic impairment due to progressive mucus accu-
mulation, infection and lung pathology in PCD can become
severe and similar to end stage CF (Figure 7), and require
lung transplantation as a final therapeutic option.
There are unique non-pulmonary manifestations of PCD
that are uncommon in CF. Male infertility is common to both
CF and PCD. However, men with PCD are not azoosper-
mic, but exhibit sperm tail dysmotility. As noted above, si-
tus inversus totalis, namely a mirror image of the thoracic
and abdominal organs, affects ∼50% of PCD patients and
is not seen in CF. A significant percentage of PCD patients,
on the order of 7%, appear to have heterotaxy, also known
as situs ambiguous, which represents a range of abnormali-
ties between situs solitus (normal) and situs inversus totalis
that may include isolated or paired reversals, midline organ
distribution, aspleenia or polyspleenia. About half of the in-
defects (Personal communication, Dr. Michael R. Knowles,
The University of North Carolina). If substantiated, it will be
informative to explore the mechanistic links between genes
causing both PCD and congenital heart disease.
Hydrocephalus is a rare finding, inconsistent even within
affected families, but is definitely associated with PCD. Hy-
drocephalus frequently occurs in mouse models of PCD (see
below). The exact mechanism is unknown but ependymal
cells with motile 9 + 2 cilia lining the ventricles may partici-
which may become damaged, obstructed and ultimately fi-
brotic in the absence of effective cilia beat.
Role of Impaired Mucus Clearance in COPD?
a leading cause of disability and death worldwide. Like CF
124 LIVRAGHI AND RANDELL
and PCD, submucosal gland enlargement, mucous secretory
cell hyperplasia in the large airways and metaplasia in the
small airways and sputum production are common features
of COPD (Szilasi et al., 2006). Thickening of the bronchi-
olar wall due to fibrosis, an increased volume of epithelial
cells and the presence of luminal inflammatory mucus exu-
dates are correlated with the severity of COPD (Hogg et al.,
2004). Toxic particles and gases, the main cause of COPD,
inhibit cilia function and Cl−secretion (Sisson et al., 1994;
Kreindler et al., 2005; Cantin et al., 2006), and emphysema
and loss of small airways tethers may distort and/or promote
collapse of the airway wall, all of which would decrease
the efficiency of mucociliary and cough clearance. Bacte-
rial infection and exacerbation by viral illness are prominent
features of COPD, and this sequence is also important in
CF and PCD. Thus, reduced efficiency of mucus clearance
likely plays an important role in the pathogenesis of COPD.
A more detailed comparison of mucus clearance in CF and
COPD is available in a recent review (Randell and Boucher,
Animal Models of CF.
gene pointed the CF research community towards generation
or mutated, to enable study of the pathophysiology and po-
tential treatments. To date, both knockout mice and those
harboring specific mutations in murine Cftr, notably ?F508,
have been created. A brief summary of CF mouse genotype
and phenotype, adapted from the European working group
on CFTR expression web site ?http://pen2.igc.gulbenkian.pt/
cftr/vr/f/scholte mouse models table.pdf?, is given in Ta-
ble 1. Detailed reviews of the pathophysiological features
of CF mice have been published (Grubb and Boucher, 1999;
Guilbault et al., 2006). Briefly, a consistent phenotype is ex-
hibited in the gastrointestinal tract of CF mice. Untreated
mice typically die shortly after birth due to meconium ileus
or after weaning from intestinal obstruction/rupture caused
by impaired luminal fluid secretion, which correlates with
the absence of functional CFTR protein in the gut epithelium
and resembles the human CF intestinal phenotype. The inci-
dence of death due to intestinal obstruction can be reduced
by a liquid diet or by supplementing the drinking water with
a laxative (Colyte).
An unexpected finding was the lack of an overt pul-
monary phenotype. All of the CF mice generated exhib-
ited inconsistent or minor pathological alterations in the
lungs. In Cftrtm1Unc(knockout) mice, the nasal septal ep-
ithelium exhibited hyperplastic and hypertrophic mucous se-
cretory cells (Snouwaert et al., 1992) in conjunction with
reduced PCL volume, in comparison to wild-type littermates
(Tarran et al., 2001). When Cftrtm1Uncmice were backcrossed
onto the C57BL/6 strain, patchy alveolar distension, intersti-
tial thickening, early mild neutrophilic infiltrate at day 30, an
(Kent et al., 1997; Durie et al., 2004;). G551D CF mice ex-
hibited inspissated eosinophilic material in the lumen of the
pharyngeal submucosal glands (Delaney et al., 1996).
A number of hypotheses have been formulated to explain
minimal pulmonary pathology in CF mice. Except for the
proximal trachea, murine airways are devoid of submucosal
glands (Pack et al., 1981), which may be an important dif-
ference from humans, but is not consistent with the presence
The cloning of the human CFTR
of disease in gland-free human distal bronchioles. As dis-
cussed previously, human CF is characterized by both the
and Na+hyperabsorption, due to unregulated ENaC activity
in the upper and lower airways. Short circuit current (Isc), as
across the epithelium.
In excised mouse nasal epithelium virtually all of the
basal Iscis inhibitable by amiloride (ENaC-mediated) and
in Cftrtm1Uncmice amiloride-sensitive Isc is elevated indi-
cating sodium hyperabsorption (Clarke et al., 1992). This
mimics the higher nasal PD found in CF humans, which
is also thought to be due to up-regulation of ENaC activ-
ity. However, in the mouse tracheal and bronchial epithe-
lium, the amiloride-sensitive Isc accounts for only part of
the basal Isc. Tracheas from both wild-type and Cftrtm1Unc
mice respond with significant Cl−secretion when stimulated
with forskolin, typically used to activate adenyl cyclase, in-
excised mouse tracheas, forskolin also elevates intracellu-
lar Ca2+, activating an alternative (non-CFTR,) Cl−channel
(Grubb et al., 1994).
Therefore, CFTR seems to have a small role in the mouse
ternative, Ca2+-activated Cl−channels. Thus, minimal spon-
taneous lung pathology in CF mice is most likely due to re-
gional, species-specific differences in ion transport between
mice and humans. There is an active research effort to cre-
ate CF in larger animals that may be physiologically more
similar to humans, including ferrets, pigs and sheep (Scholte
et al., 2004; Li et al., 2006).
Scnn1b Transgenic Mice.
hydration of the airway surface represents the balance be-
tween Cl−secretion and Na+absorption, mice that hyper-
absorb Na+in the lower airways were created by overex-
pressing the β subunit of ENaC (encoded by the Scnn1b
gene) using the airway-specific Clara cell secretory protein
(CCSP) promoter (Mall et al., 2004). The tracheas of neona-
mates. Accordingly, tracheal and bronchial PCL height was
significantly reduced, the percent solids content of lower air-
way mucus was significantly increased and mucus transport,
measured in vivo, was significantly reduced in comparison
to wild-type littermates.
Scnn1b overexpression did not have adverse effects on fe-
tal survival, but Scnn1b mice showed a significant postnatal
mortality that began in the first days of life and continued up
to 4 weeks, with an overall mortality of ∼50%. Scnn1b mice
exhibited postnatal mucus accumulation and obstruction in
the large and small airways associated with goblet cell meta-
plasia and neutrophil infiltration (Figure 8). The neutrophil-
attracting chemokines macrophage inflammatory protein-2
(MIP-2) and KC (the two murine analogues of human IL-8)
were significantly increased. Lungs were free of cultureable
bacteria, indicating that the neutrophilic inflammation oc-
curred in the absence of active infection. Interestingly, the
upregulation of MIP-2 and KC was not a direct effect of
Scnn1b overexpression in the epithelial cells, since their se-
cretion was not increased in epithelial cultures derived from
Based on the hypothesis that
Vol. 35, No. 1, 2007
TABLE 1.—Mouse models of CF.
background MouseMutation SurvivalPhenotypeReference
KO, Inframe stop
UndetectableC57Bl/6 × 129/Sv
BALB/C × 129/Sv
<5% Severe intestinal disease,
10–50% weight reduction.
Damaged gallbladder. No
changes in pancreas and liver.
Atrophy of nasal serous
glands, dilation of gland ducts.
No defect in reproductive
organs. Disruption of serous
acini in submaxillary glands.
Mild intestinal disease- colon
dilation with mucus
accumulation, vas deferens
occlusion. No lung or pancreas
pathology. Residual CFTR
activity (Dorin et al., 1994)
Severe intestinal disease. 50%
weight reduction Meconium
ileus, blockage of pancreatic
ducts Lacrimal gland
pathology No lung pathology
Severe intestinal disease. Survival
affected by genetic background
(CD1, C57BL/6 and BALB/cJ
strains have better survival).
Severe intestinal disease. 70%
weight reduction. Dilation of
acini in sublingual glands.
Mild inflammation in
pancreatic duct. No pathology
in lungs, trachea, liver, or
Severe intestinal disease. No lung
Severe intestinal disease. ?F508
Severe intestinal disease. 50%
weight reduction. No
pathology in lung, pancreas,
gallbladder, male reproductive
tract, lacrimal glands and
Mild pathology. 20% weight
reduction. Focal hypertrophy
of intestine goblet cells. No
pathology in lung, pancreas,
liver, bile ducts, vas deferens
and salivary glands. Residual
Milder intestinal disease- 30-50%
weight reduction. Focal biliary
cirrhosis. Abnormal salivary
glands. No pathology in
pancreas, lung, and
reproductive tract. Residual
CFTR activity in caecum.
No severe intestinal disease. No
weight loss, mild intestinal
goblet cell hypertrophy. CFTR
activity present in caecum, but
absent in the nose
Severe intestinal disease. 40%
weight reduction vs. 20% in
mixed strain. Reported
spontaneous, progressive lung
fibrosis. Bronchioli layered
with mucus-like material.
Liver, pancreas, salivary
glands, vas deferens develop
CF-like pathology by 1–2 years
(Snouwaert et al.,
10% of wild-typeMF1 × 129/Sv
C57Bl/6 × MF1?
(Dorin et al., 1992)
UndetectableMF1 × 129 C57Bl/6
<5% (Ratcliff et al., 1993)
Undetectable CD1 × 129/Sv
129/Sv × DBA/2J
129/Sv × C57BL/6
129/Sv × BALB/cJ
C57Bl/6 × 129/Sv
25% (Rozmahel et al.,
<2% of wild-type40%
(O’Neal et al., 1993)
KO, Inframe stop
?F508 by exon 10
Undetectable C57Bl/6 × 129/Sv
C57Bl/6 × 129/Sv
40% (Hasty et al., 1995)
30% of wt
(Colledge et al., 1995)
?F508 by exon 10
low in intestine
(Zeiher et al., 1995)
?F508 by exon 10
“Hit and Run”
Mutant mRNA at
FVB × 129/Sv100%
(van Doorninck et al.,
G551D by exon
53% of normal
CD1 × 12970%
(Delaney et al., 1996)
G480C by exon 10
“Hit and Run”
Mutant mRNA at
C57Bl/6 × 129/Sv 100%(Dickinson et al.,
UndetectableC57Bl/66.4% vs 15.5%
on a liquid
(Durie et al., 2004;
Kent et al., 1997)
126LIVRAGHI AND RANDELL
FIGURE 8.—Lung pathology in Scnn1b (βENaC-overexpressing) mice. (A and B) Low power view of lung tissue sections from an adult Scnn1b mouse, H&E and
Alcian blue-PAS stain, respectively. (C–H) Higher magnification views of boxed areas in panels A and B, H&E (C–E) and Alcian blue-PAS stain (F–H). Note the
variable degree of highly adherent, accumulated mucus in different airways and the presence of neutrophils in luminal mucus (inset in panel C).
It was proposed that inflammation occurs because en-
vironmental inflammatory stimuli accumulate in the lungs
of Scnn1b mice due to poor mucus clearance. Finally, H.
In summary, the Scnn1b mice exhibit airway Na+hyper-
absorption, a hallmark of human CF, and recapitulate many
features of the human disease, including mucus accumula-
tion and neutrophilic airway inflammation. This mouse is
thus a useful in vivo tool for investigating pathophysiologic
mechanisms relevant to CF and other diseases.
curs naturally in both dogs and pigs, which appears identical
to that seen in humans, except that hydrocephalus is a promi-
nent feature (Edwards et al., 1989; Roperto et al., 1993). The
genetic basis of canine and porcine PCD-like airway disease
remains unknown. Numerous mouse models with left-right
symmetry defects have been identified and are the subject of
classified as PCD models (Geremek and Witt, 2004). Table
2 summarizes the mouse models with known genetic defects
affecting 9 + 2 motile cilia function.
Mouse axonemal dynein heavy chain 5, encoded by the
deficient mice exhibit early mortality (∼4–5 weeks), hydro-
cephalus, respiratory cilia immotility due to the complete
lack of outer dynein arms, randomization of sidedness, and a
opment of chronic rhinitis (Ibanez-Tallon et al., 2002). Tar-
geted mutation of the mouse homologue of human DNAI1,
Mdhc7, which encodes for an inner dynein arm heavy chain
results in reduced sperm motility and male infertility. These
mice show a 50% reduction of the respiratory ciliary beat
frequency but apparently this does not cause airway disease
(Neesen et al., 2001; Vernon et al. ,2005; Woolley et al.,
2005). Other candidate genes for PCD include transcription
factors implicated in ciliated cell differentiation. Hfh4 (also
ithelial cell cilia and may be a model for cilia aplasia. These
mice exhibit hydrocephalus and randomization of sidedness,
but still have monocilia in the embryonic nodal pole (Chen
et al., 1998; Brody et al., 2000). However, mutations in the
FOXJ1 gene have not been found in PCD patients, generally
excluding it as a candidate gene for PCD (Maiti et al., 2000).
It was reported that deletion of DNA polymerase λ gene
(Poll) in mice produced inner dynein arm defects in ependy-
mal and respiratory cilia and a phenotype characteristic of
PCD, including hydrocephalus, situs inversus, chronic sup-
purative sinusitis, and male sterility (Kobayashi et al., 2002).
Vol. 35, No. 1, 2007
TABLE 2.—Mouse models of PCD.
function MouseGene MutationPhenotype Reference
Insertional KO Axonemal
yesPerinatal mortality, growth
in nasal sinuses, middle ear
infection, situs inversus,
in outer dynein arms
Reduced sperm motility,
reduced ciliary beat
frequency, no airway
Perinatal mortality, growth
failure situs inversus,
absence of cilia in the
airways, partial penetrance
of hydrocephalus, nodal
cilia are present
inversus, chronic sinusitis,
infertility. Defective inner
dynein arms of ependimal
and respiratory cilia
et al., 2002)
no (Neesen et al.,
Insertional KO Transcription
(Brody et al.,
et al,. 1998)
Poll Dpcd POLL and DPCD
Insertional KOUnknown no
(Kobayashi et al,.
et al., 2004a)
However, it was difficult to reconcile a mutation in a DNA
polymerase with an axonemal defect and there was evidence
that mice with deletion of the sequence encoding for the cat-
alytic domain of Poll were viable and fertile (Bertocci et al.,
mice was examined in further detail.
The region of chromosome 19 that was deleted to generate
the Poll−/− mouse also included the first exon and the start
codon of a newly identified gene, Dpcd, which is transcribed
from the opposite strand relative to Poll. Dpcd is predicted
to encode for a 23 KDa protein of unknown function. Since
Dpcd mRNA is expressed in airway epithelial cells and its
gested that mutation in Dpcd can cause PCD, although thus
far no mutations in the human homolog of Dpcd have been
found in 51 unrelated PCD patients (Zariwala et al., 2004).
Collectively, the existing animal models of PCD are char-
acterized by a high incidence of hydrocephalus and perina-
tal mortality. These pathologic features hamper the estab-
lishment of models suitable for long-term studies of chronic
infection characteristic of the human pulmonary PCD phe-
notype. Nevertheless, mouse models where a homolog of
a candidate human gene has been targeted can confirm the
likely role of that gene in PCD, and novel animal models
may also reveal important differences in the physiology of
ment of such mice may avoid hydrocephalus, enabling more
in-depth study of the lower respiratory tract.
SUMMARY AND CONCLUSION
Mucus clearance, the continuous flow of fluid and mucus
over airway surfaces towards the larynx, is a vital protective,
innate defense mechanism. Failure of this system, due to ge-
netic defects in specific components, occurs in CF and PCD
and results in repeated lung infections and eventual respira-
derlying efficient mucus clearance and the pathophysiologic
consequences of its impairment. Altered mucus clearance
likely plays a role in several common acquired respiratory
tract diseases, including COPD. A greater understanding of
the basic science of mucus clearance and new animal models
will promote advances in the diagnosis and treatment of this
important class of diseases.
The authors thank Dr. Michael R. Knowles for critical as-
sessment of the manuscript.
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