Clinical Science (1988)75, 113-120
113
Editorial Review
Secondary ciliary dysfunction
ROBERT WILSON
Host Defence Unit, Department of Thoracic Medicine, Cardiothoracic Institute, Brompton Hospital, London
INTRODUCTION
CILIARY DYSFUNCTION
The mucociliary system provides a primary defence
mechanism of the upper and lower respiratory tracts.
Ciliated epithelium lines the nasal passages, sinuses,
middle ear and eustachian tube, and the lower respiratory
tract from the trachea to the respiratory bronchioles. The
surface of each ciliated columnar cell contains approximately 200 cilia of uniform length and diameter [l, 21.
They beat in a two-layered system of fluid, consisting of a
water periciliary fluid phase which lies below a layer of
mucus. The distance of the mucus layer from the cell surface is approximately the perpendicular height of a vertical cilium. Inhaled particles adhere to the mucus surface,
and are moved towards the back of the throat by the coordinated movement of cilia: each individual cilium beating in concert with its neighbours, producing metachronal
waves of ciliary beating.
The potential consequences of impaired ciliary function
are portrayed by patients with primary ciliary dyskinesia
[ 10-121, who have absent or dyskinetic ciliary beating.
Children with this syndrome are physically well
developed and lie within normal growth curve limits for
age [ 121and may have a normal life expectancy [ 131.However, they suffer chronic infection of the upper and lower
respiratory tracts from an early age, subsequently
developing bronchiectasis. This outcome emphasizes the
contribution to lung defence made by mucociliary clearance. Alternative mechanisms of mucus clearance, such as
cough, and the other antibacterial defence mechanisms of
the bronchial tree are not sufficient to prevent chronic
respiratory infection in the absence of mucociliary clearance. More evidence is required to support the supposition that impaired mucociliary transport can result in
bacterial colonization in other circumstances, for example
after viral infection or in chronic bronchitis [ 14,151.
The term secondary ciliary dysfunction implies that
ciliary ultrastructure and performance were originally
normal. A number of measurements of ciliary dysfunction
have been made in vitro, and all have inherent difficulties
of interpretation. The absence of cilia or the presence of
morphologically abnormal cilia at sites of disease may be
important [ 16-19]. However, even when frequencies of an
abnormality are estimated, the sampling error may be
large. Slowed ciliary beating has been interpreted as being
deleterious to transport [20-221, but this may be fallacious if, in order to compensate, the power of the slowed
beating is increased. Perhaps the two most convincing
observations of secondary ciliary dysfunction are ciliary
dyskinesia and cessation of ciliary beating. Each cilium
has a stiff propulsive downstroke, and when the cilium is
erect small claws at its tip engage the mucus layer propelling it forward. Having completed the propulsive stroke
the cilium is withdrawn at right angles in a curved fashion
sweeping across the cell surface within the periciliary fluid
back to its starting point, so<avoidingfurther contact with
the mucus layer. Dyskinetic cilia may lose this pattern,
moving stiffly from side to side or losing co-ordination
with their neighbours: adjacent cilia moving in different
directions or in different planes.
Ideally, ciliary beating should be studied in vivo. The
technology to achieve this is unfortunately not available,
ABNORMAL MUCOCILIARY CLEARANCE
The system may malfunction in a number of ways, either
secondary to a disease process, or when a primary defect
occurs in one of its component parts. The ideal rheological properties of mucus for efficient transport are high
elasticity and low viscosity; these may change during
disease, for example becoming less elastic during viral
infection [3]or more viscous during bacterial infection [2,
41. It has been suggested that increase in the depth of the
mucus layer may also cause inefficient transport due to
uncoupling within it [2], so that the inner part is moved
forward by the beating cilia while the outer part, on which
particles stick, remains stationary. Changes in the depth of
the periciliary fluid may be important: a layer which is too
deep causing disconnection of the tips of the cilia from the
overlying mucus, and one which is too shallow causing
entanglement of cilia [5].In addition, loss of large areas of
ciliated epithelium, as may occur during viral [6, 71 and
bacterial [8, 91 infection, could cause stasis of the overlying mucus. Finally, the ciliary beating itself may become
either slow or disorganized (dyskinesia).It is these latter
two mechanisms on which this review concentrates.
Correspondence: Dr R. Wilson, Host Defence Unit, Department of Thoracic Medicine, Cardiothoracic Institute, Brompton
Hospital, Fulham Road, London SW3 6HP.
114
R. Wilson
although mucociliary wave frequency has been measured
in the maxillary sinus through an operating microscope
[23]. This technique utilized the variation that occurs in
light reflected from the illuminated surface of the mucus
blanket, and it is probably an index of ciliary beating. The
mucociliary wave frequency of human sinus epithelium
measured after surgical excision was unchanged from that
measured in vivo before the excision, suggesting that
removal of tissue from the host did not disturb ciliary
beating 1231. Many studies have compromised and
examined either cilia taken from sites of disease, or the
effect of mediators or drugs on normal cilia in vitro.
Unfortunately, because of their easy availability, animal
cilia have often been used and sometimes conflicting
results have been obtained when the experiments have
been repeated using human tissue [24]. For instance, a
factor was described in the serum of patients with cystic
fibrosis, and in the concentrated euglobulin fraction of the
serum from obligate heterozygotes, but not from most
normal controls, which disorganized the beating of cilia in
rabbit tracheal explants. This finding led to work to
characterize the factor and to investigate its activity in
inhibiting other ciliary systems. Subsequently, cystic fibrosis serum was found not to inhibit human ciliary beating in
vitro [24]. Whereas this test could possibly prove useful in
the diagnosis of cystic fibrosis, it does not suggest a
mechanism applicable to the pathogenesis of delayed
mucociliary clearance in these patients.
Direct measurement of mucociliary clearance can be
achieved in vivo in a number of ways [25-271. In certain
disease states mucociliary clearance is delayed and the
contribution to this delay made by secondary ciliary dysfunction has been investigated. Abnormal or slow ciliary
beating may be due to factors directly affecting the cilium
itself, or may reflect damage to the epithelial cell. The
rheological properties of mucus may influence ciliary
beating [ 281, although the interpretation of such results is
difficult, because the mucus under investigation may also
contain factors toxic to ciliated cells (see below).
Several techniques have been used to measure ciliary
function [29, 301, and in general they employ methodology in which the ciliated epithelium is relatively mucus
depleted. These conditions are not physiological, and it is
possible that the ciliary beat frequency (CBF) and its
response to toxins will differ because of this alone. For
example, betamethasone and betamethasone with neomycin nose drops were found to be ciliotoxic when
human ciliated epithelium was exposed to them in vitro,
due to the effects of the preservatives benzalkonium
chloride and thiomersal. However, after topical application of the drops in vivo in healthy subjects, nasal mucociliary clearance and CBF of biopsied nasal ciliated
epithelium in vitro were not adversely affected. In addition, treatment with the drops for 4 weeks did not affect
these indices in two groups of patients with rhinitis [31].
Thus, despite being ciliotoxic to epithelium in vitro, the
drops did not adversely affect ciliary beating measured
after topical application in the nose. One explanation of
these results is that protection is afforded to the cilia by
the mucus blanket in vivo.
Secondary ciliary dysfunction has been investigated in
the following conditions.
Chronic bronchitis
Mucociliary clearance is probably delayed in patients
with chronic bronchitis [32, 331, although other reports
have produced variable results mainly because of difficulties in controlling for cough and airways obstruction
[2]. Chronic bronchitis is a disease in which there are
changes in the respiratory mucosa which is thicker and
shows more frequent metaplasia [34]. There is hypertrophy of the submucosal glands and an increase in the
goblet cell numbers, leading to increased mucus volume.
It is likely that there is an absolute reduction in ciliated
cell numbers. Epithelial changes have been described at
necropsy in the peripheral airways of young smokers
dying of non-respiratory causes, without a history of
chronic airways obstruction or pathological changes of
emphysema [35].
The pathogenesis of chronic bronchitis is unknown but
is associated with infection and inhalation of a variety of
pollutants especially tobacco smoke. An increased proportion of cilia with abnormal ultrastructure has been
reported [36]. In two studies of patients with chronic
bronchitis, the CBF of samples of bronchial epithelium in
vitro did not differ from normal [37,38]. However, in a rat
model of bronchitis [39-411 there was a small decrease in
overall CBF compared with normal rats, and areas of
static and reversed ciliary beating were noted in the bronchial tree. These latter changes could halt mucus transport in vivo, and would easily be missed if biopsy samples
alone were examined.
Small quantities of whole cigarette smoke or its
aqueous extract cause cessation of ciliary beating on
human respiratory epithelium in vitro [42, 431. This may
be due to the effects of hydrogen cyanide, acrolein, formaldehyde, ammonia and phenols, which are toxic to
mammalian cilia in vitro [44]. Although patients with
chronic bronchitis related to smoking have delayed mucociliary clearance [45], clearance studies in healthy
smokers and non-smokers have been much less conclusive [ S , 46-48]. Acute exposure to cigarette smoke in vivo
has given even more conflicting results, causing slowing
[49], acceleration [50],no effect [5, 511 or variable results
[52]. CBF measured in vitro by studying nasal biopsies
did not differ between smokers and non-smokers, and did
not change when nasal biopsies were examined before
and immediately after smoking two cigarettes, and exhaling through the nose [5].The slowing of CBF by cigarette
smoke in vitro is reversible, providing exposure is stopped
before ciliostasis occurs [42], which could explain the disparity between the results in vitro and some of those
obtained after exposure to cigarette smoke in vivo. Alternatively, the mucus layer may again act as a protective
barrier [311.
Sulphur dioxide is a common pollutant whose detrimental effects on pulmonary function have been studied
[2]. In the rat it may cause reduction of mucus transport
and in some cases reduction of ciliary beating [53].
Secondary ciliary dysfunction
Viral infection
Mucociliary clearance is delayed during viral infections
[6, 7, 54, 551, although not in patients with subclinical
infection, and not before the onset of overt symptoms [7].
Loss of ciliated epithelium may be largely responsible for
this effect as demonstrated in nasal epithelial biopsies
taken during colds [6, 71. During influenza infection the
bronchial epithelium shows degeneration and desquamation of cells [56]. Acquired defects of the ciliary microtubules were observed in nasal epithelial biopsies of
children with viral infections [19]. These defects were
associated with cytopathic changes in epithelial cells and
progressive loss of ciliated cells from the epithelium.
Ciliated cells have been shown in the blown secretions of
patients with colds [57]. In organ cultures of ciliated
respiratory epithelium a number of viruses cause loss of
ciliary activity and degeneration of ciliated cells [58,591.
There does, however, seem to be a variation in toxicity
after infection with different viruses [59-6 13.
In one study a small reduction in CBF was observed in
the ciliated cells remaining during overt infection [6],
although this was not confirmed in a subsequent study [7].
It would not, however, be surprising if CBF was slower
during the cytopathic process before shedding of the cells,
although small changes in CBF are likely to be less
important than loss of large areas of ciliated epithelium.
An increase in mucus volume and a change in mucus
rheology to a more watery secretion are also likely to
adversely affect mucociliary clearance [31.
Bacterial infection
Mucociliary clearance of the upper and lower respiratory tracts is delayed in conditions where bacterial infection is present and purulent secretions are produced.
Tracheobronchial clearance of radioaerosol is delayed in
bronchiectasis [62, 631 and cystic fibrosis [64]. Tracheal
mucus velocity ( W )
of Teflon discs observed
bronchoscopically has also been shown to be slow in
patients with cystic fibrosis [65]. In this study there was
slow movement in several patients with minimal lung
disease, while there was normal movement in one patient
with advanced lung disease, suggesting no clear association with disease severity. However, mucociliary tracheal
transport of radiolabelled albumin microspheres is also
delayed in cystic fibrosis, and slow transport correlated
with severity of disease as measured by the Schwachman
score [66]. This latter result suggests a relationship
between mucociliary transport and disease severity, perhaps because of the less invasive nature of the study and
assessment of a greater area of the bronchial tree.
Mucociliary clearance in the upper respiratory tract is
also delayed in chronic sinusitis [20, 22, 671. In addition,
patients with serologically verified infection with Mycoplasma pneumoniae have delayed tracheobronchial clearance compared with the results obtained when the study
was repeated in the same patients 3 weeks later [68]. On
balance, the results suggest that infection is associated
with a generalized reduction in mucociliary clearance.
115
Histological evidence of loss of cdiated cells [9, 371,
structural abnormalities of the epithelium [34] and an
increased frequency of cilia with abnormal ultrastructure
[ 161 indicates the damage which occurs in the bronchial
tree during chronic infection. Several studies have found
slowed CBF in biopsies taken from patients with these
diseases [20, 22, 691. The CBF was reduced in upper
respiratory tract biopsies taken from patients with
mucopurulent sinusitis compared with healthy controls,
and in patients with bronchiectasis and mucopurulent
sinusitis compared with patients with bronchiectasis alone
[20]. Such studies may underestimate the slowing of CBF
in vivo, as cilia are removed from a hostile environment
and placed in cell culture fluid, so diluting any toxic
factors present [20].In a study of epithelial biopsies taken
from sites of purulent infection, one patient from whom
Pseudomonas aeruginosa was cultured had ciliary beating
which appeared dyskinetic, but this had returned to
normal when the biopsy was repeated after eradication of
the organism by antibiotic treatment [20]. In two studies
[20, 691 improvement in CBF has been demonstrated
from biopsies taken after a course of effective antibiotic
treatment.
The reason for ciliary slowing during infection may be
multifactorial. Certain bacteria produce factors which
slow ciliary beating and damage epithelium: P. aeruginosa
[70-721, M. pneumoniae [73,74], Haemophilus influenzae
[75-771, Bordetella pertussis [78],some strains of Staphylococcus aureus [76, 791, Streptococcus pneumoniae [80],
Neisseria meningitidis [8 11and Neisseria gonorrhoeae [82].
These factors may play an important role in the pathogenesis of respiratory infection. After inhalation the bacterium adheres to the mucus layer and would normally be
removed by mucociliary clearance. In order to colonize
the epithelium it would be an advantage for the bacterium
to delay clearance, and penetrate the mucociliary barrier
to reach the epithelial surface where disruption of the
epithelium would facilitate colonization or permit
systemic invasion. Characterization of the factors produced by micro-organisms which are responsible for
changes in ciliary function in vitro will allow a study of
their relevance in vivo to be made. For example, P. aemginosa produces a number of small hydrophobic molecules which affect ciliated epithelium [70, 711.
1-Hydroxyphenazine causes immediate ciliary slowing
and dyskinesia [70], while pyocyanin [70] and rhamnolipid [71] slow CBF and disrupt the integrity of the
epithelial surface. All three of these molecules can be
found in sputum [83, 841, and both 1-hydroxyphenazine
and pyocyanin delay mucociliary clearance in vivo in an
animal model [85].
The epithelial damage seen in chronic infection is not
mediated by bacterial products alone. Polymorphonuclear
leucocytes move to the lung in response to infection. This
can be monitored by labelling patient's granulocytes with
"lIn and following their movement by gamma s c h g
[86].In bronchiectasis, the labelled granulocytes appear in
affected areas of the bronchial tree within 24 h. Enzymes,
for example elastase, collagenase and cathepsin G, may
escape from the neutrophil during the host cell-bacterial
116
R. Wilson
eosinophils, lymphocytes and plasma cells. There is
destruction and detachment of epithelial cells, the number
of ciliated cells are reduced and the goblet cells increased.
Mucus impaction is observed in small-and medium-sized
airways at post mortem in patients dying from an acute
asthmatic attack [96, 971. Even in remission mucus plugging occurs, as does eosinophil infiltration, loss of ciliated
cells and ultrastructural abnormalities of the cilia themselves [98].
The sol phase from some asthmatic sputum samples
has been shown to cause a reduction in the CBF of human
bronchial biopsies, and in some cases this progressed to
complete stasis [98]. The character of the sputum was
important, the effect being associated with ‘slurry’ sputum
which was watery, contained few plugs and was slightly
turbid and foamy. Mucoid sputum produced the effect
much less frequently. The factor responsible was shown
to have a molecular mass of about 8000 daltons. The disappearance of the sputum toxicity seemed to correlate
with clinical improvement, but the degree of cilio-inhibition did not correlate with sputum eosinophilia.
Recent evidence, however, would suggest that the
eosinophil has a potential role in causing damage to the
respiratory epithelium in asthma [99]. The eosinophil
granules contain a number of cationic proteins that have
been characterized, including major basic protein, eosinophi1 cationic protein and eosinophil-derived neurotoxin
[ 1001. Major basic protein (100 pg/ml) caused exfoliation
of epithelial cells from human bronchial ring cultures, so
that after 19 h no ciliary beating was visible 11011. Alteration of ciliary beating was observed before complete stasis
but was not quantified. Electron microscopy demonstrated exfoliation of the surface epithelium leaving
behind only basal cells, and the exfoliated cells showed
obvious signs of degeneration [ 1011. Major basic protein
( 100 pg/ml) also reduced the area of observed ciliary
beating on rabbit tracheal rings, but greater concentrations were required to cause a significant fall in CBF
interaction. These enzymes may overwhelm the capacity
of inhibitors (such as a ,-antiproteinase)in the pulmonary
secretions to neutralize them. Thus free elastolytic activity
has been demonstrated in purulent sputum [87,88].
Human leucocyte elastase and neutral proteinase arrest
ciliary activity and subsequently cause superficial epithelial destruction when they are incubated with rabbit
tracheal ring preparations [89].When purulent sputum sol
phase, with free elastolytic activity, was added to human
nasal epithelium in vitro it caused a gradual slowing of
CBF [211. This response was abrogated by the prior addition of sufficient a,-antiproteinase to the sol to neutralize
all elastolytic activity, suggesting that the active factor
causing ciliary slowing was a serine proteinase, possibly
neutrophil elastase. Sputum sol obtained after a course of
antibiotic treatment no longer had elastase activity and
had little effect on CBF. In a similar study, it has been
shown that the ciliary slowing properties of some samples
of purulent sputum sol are not affected by prior exposure
to a,-antiproteinase, and may be due to bacterial
products [90]. It seems likely that, in the presence of infection, both host- and bacteria-derived products can
adversely affect ciliary function (Fig. 1).
Asthma and allergic rhinitis
Mucociliary transport is delayed in patients with
asthma [91-931. A reduction in the TMV of asthmatic
patients follows allergen challenge [9 11 independently of
the degree of bronchospasm produced. Similarly, transport is delayed in the upper respiratory tract of patients
with allergic rhinitis [67], but after nasal antigenic challenge conflicting results have been obtained [94, 951, and
the transport time may differ depending upon the mode of
antigen administration [95].
In status asthmaticus the respiratory mucosa is oedematous, the capillaries in the submucosal tissues are
dilated. and there is a cellular infiltration which includes
-
Patient before
antibiotic treatment
t
t
Purulent sputum
containing:
J
Free
elastolytic
activity
t
Gradual onset
of CBF slowing
and epithelial
disruption
J.
Effect neutralized
by a,-antiproteinase
Patient after
antibiotic treatment
Mucoid sputum
containing:
t
\
Bacterial
products
No elastolytic activity
and reduced
bacterial products
4
No effect on CBF
Immediate CBF
slowing, followed
later by epithelial
disruption
.i
+
Effect neutralized
by chloroform
extraction
Fig. 1. Schematic representation of the effect of sputum sol phase on human CBF in vitro. Purulent sputum contains bacterial products and host cell-derived proteolytic activity, both of which
adversely affect ciliary function in vitro [ 21,76,90].
Secondary ciliary dysfunction
[ 1021. Major basic protein is present at sites of damage to
the bronchial epithelium in asthma [ 1031, and the amount
in sputum is increased [loll in asthmatic patients (up to
9 3 pg/ml). The molecular mass of major basic protein is
similar to that of the cilio-inhibitory factor described in
asthmatic sputum [98], but it was not found in sputum
samples with known cilio-inhibitory activity, and did not
slow ciliary beating in this short study which only
measured CBF for 2 h [98].Thus the role of this protein
remains uncertain.
The effect of other eosinophil basic proteins on
epithelium needs to be evaluated. Eosinophil cationic protein causes rapid and sustained cell membrane depolarization due to the production of transmembrane pores
[104] which may be a mechanism of target cell damage.
Thus mediators, some of which are released from the
eosinophil, damage the respiratory epithelium of
asthmatic patients. Further work is required to fully
characterize the mediators involved and their respective
roles.
In young non-smokers with allergic asthma the fall in
TMV produced by a specific antigen challenge was prevented by prior treatment with disodium cromoglycate
[91]. In another study, the reduction in TMV produced by
inhalation of ragweed antigen by susceptible patients, was
prevented by immediate treatment with FPL-55712, an
antagonist of slow-reacting substance of anaphylaxis
[ 1051. Such studies suggest that reduction of TMV may be
related to mediators of anaphylaxis. However, two studies
using sheep ciliated cells suggested that these mediators
do not cause ciliary dysfunction. First, ciliated cells
obtained from sensitized animals after exposure to
specific antigen showed a small increase in CBF at a time
when TMV was decreased [ 1061. Secondly, several mediators of anaphylaxis, including prostaglandin E, and
leukotriene C4, either had no effect or again produced an
increase in CBF without loss of co-ordination [107]. It
may be that these mediators exert their deleterious effect
on mucociliary function by other mechanisms. Several
mediators, including the leukotrienes C4 and D,, increase
the release of mucus from human airways in vitro [108,
1091, and various changes occur in the composition of
asthmatic mucus which may alter its rheological properties [ 110, 1111and hence, clearance.
Other causes of secondary ciliary dysfunction
In addition to cigarette smoke, other environmental
pollutants [2] and certain drugs may slow mucociliary
clearance. There is some evidence for secondary ciliary
dysfunction in both cases. However, interspecies variation
[24] and concentration in vitro of the compound studied,
compared with that likely to be achieved in vivo in man,
needs further consideration.
CONCLUSION
Mucociliary clearance is impaired in a number of
diseases. Slowing, cessation or disorganization of ciliary
117
beating may contribute to this delay. Loss of ciliated
epithelium, and changes in the quantity or composition of
mucus and periciliary fluid, are also likely to be very
important factors. Abnormal ciliary. beating may reflect
more generalized changes occurring to the ciliated cell,
rather than being confined just to cilia abnormalities
themselves. The characterization of molecules responsible for damage to the respiratory epithelium in vifro
should lead to an assessment of their involvement in the
morphological and physiological manifestations in these
diseases in vivo.
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