Mucociliary clearance - Wiley Online Library

Clin Physiol Funct Imaging (2014) 34, pp171–177

doi: 10.1111/cpf.12085

INVITED REVIEW

Mucociliary clearance: pathophysiological aspects Mathias Munkholm1 and Jann Mortensen1,2 1

Department of Clinical Physiology, Nuclear Medicine & PET, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark and 2Department of Medicine, The Faroese National Hospital, Torshavn, Faroe Islands

Summary Correspondence Jann Mortensen, Department of Clinical Physiology, Nuclear Medicine & PET, Rigshospitalet, Copenhagen University Hospital, DK-2100 Copenhagen Ø, Denmark E-mail: [email protected]

Accepted for publication Received 05 April 2013; accepted 21 August 2013

Key words cough clearance; diagnostic test; mucociliary clearance; overview

Mucociliary clearance has long been known to be a significant innate defence mechanism against inhaled microbes and irritants. Important knowledge has been gathered regarding the anatomy and physiology of this system, and in recent years, extensive studies of the pathophysiology related to lung diseases characterized by defective mucus clearance have resulted in a variety of therapies, which might be able to enhance clearance from the lungs. In addition, ways to study in vivo mucociliary clearance in humans have been developed. This can be used as a means to assess the effect of different pharmacological interventions on clearance rate, to study the importance of defective mucus clearance in different lung diseases or as a diagnostic tool in the work-up of patients with recurrent airway diseases. The aim of this review is to provide an overview of the anatomy, physiology, pathophysiology, and clinical aspects of mucociliary clearance and to present a clinically applicable test that can be used for in vivo assessment of mucociliary clearance in patients. In addition, the reader will be presented with a protocol for this test, which has been validated and used as a diagnostic routine tool in the work-up of patients suspected for primary ciliary dyskinesia at Rigshospitalet, Denmark for over a decade.

Introduction When breathing, inhaled particles such as dust and bacteria inevitably reach the conducting airways. As a respond to this constant threat of inflammation and infection the airways have evolved different innate defence mechanisms (Wanner et al., 1996). Mucociliary clearance is known to be of particular importance in this first line of defence, which becomes clearly evident in patients with defects related to this system such as patients with cystic fibrosis or primary ciliary dyskinesia as these patients typically present themselves with recurrent infections of the airways (Robinson et al., 2000; Knowles & Boucher, 2002; Sagel et al., 2011). In recent years, our understanding of the composition and function of this important defence mechanism has grown, and possible ways to evaluate as well as enhance mucociliary clearance are currently being investigated. (Yoo & Koh, 2004; Marthin et al., 2007; Amirav et al., 2009). The aim of this review is to give an overview of the anatomy, physiology, pathophysiology, and clinical aspects related to mucociliary clearance and to describe a method that can be

used in the assessment and diagnosis of patients with defects of the mucociliary system.

Anatomy and physiology related to mucociliary clearance The cilia In humans, cilia are found lining the respiratory tract including the middle ear and the sinuses, the ductuli efferentes of males, the Fallopian tubes of females, and the ependyma of the brain (Meeks & Bush, 2000). Each cilium is about 6 lm long and has a diameter of 250 nm. The amount of cilia in the airways is at the level of 109 cilia per cm2 usually being longer and denser packed in the larger respiratory airways than in the bronchioles (Livraghi & Randell, 2007). The inner cytoskeletal structure of the cilia called the axoneme has a distinctive 9 + 2 microtubule structure as well as some important microtubular-associated proteins some of which can be seen with electron microscopy (Fig. 1). They consist of outer and inner dynein arms, the so-called radial spokes that link the

© 2013 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd 34, 3, 171–177

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172 Mucociliary clearance: pathophysiological aspects, M. Munkholm and J. Mortensen

Figure 1 Structure of a normal cilium as seen under an electronic microscope.

inner microtubules with the outer ones, and the nexin links linking the outer microtubules to one another (Meeks & Bush, 2000). Especially the dynein arms have been extensively studied and are known to be essential for normal ciliary movement generated by ATP hydrolyses (Porter & Johnson, 1989). The function of the cilia in the respiratory tract is to beat in a synchronized manner thereby propelling mucus as well as substances trapped within the mucus to the pharynx where they will be swallowed (Wanner et al., 1996). In health, this works effectively. However, in a variety of airway diseases, it will be insufficient, and to a great extent these patients then have to rely on cough clearance, which means the ability to shift mucus towards the pharynx by means of coughing. Both of these clearing mechanisms can be disrupted in different ways thus leaving the patient vulnerable to infection (Meeks & Bush, 2000; Randell & Boucher, 2006; Bennett et al., 2010). The mucus layer Apart from the numerous ciliated cells, the epithelial lining of the intrapulmonary airways consists mainly of secretory cells. These cells release different antimicrobial molecules (defensins, lysozyme and IgA), immunomodulatory molecules (e.g. cytokines) and large glycoproteins called mucins that bind considerable amounts of water whereby the deformable gel known as mucus is generated (Evans et al., 2010). The mucus is lifted away from the cilia by the periciliary liquid layer, which has two main functions. Because of its low viscosity, it allows the cilia to beat rapidly, and it prevents the glycoproteins of the mucus layer from adhering to the glycocalyx of the epithelial apical membrane (Knowles & Boucher, 2002). In healthy individuals, mucus from the airways contains 97% water and only 3% solids of which mucins constitute around 30% (the rest is non-mucin proteins, lipids, salts and cellular debris). With this composition, the mucus will have a consistency resembling egg white and can easily be cleared from the airways by the ciliary beating. However, this balance of hydration can be disrupted either by mucin hypersecretion or dysregulation of the volume of surface liquid resulting in thicker and more elastic mucus, which will be harder to clear from the airways (Fahy & Dickey, 2010). Because of the large amount of water contained in the mucus layer, it serves as a buffer for the periciliary liquid

layer so that this layer can maintain its volume during physiological variations of airway hydration (Tarran et al., 2001). Recently, Button et al. (2012) proposed a more detailed explanation of the composition of the periciliary liquid layer formerly thought to consist mainly of water. According to this new theory, the periciliary liquid layer is occupied by large amounts of mucins and large glycoproteins both of which are tethered to the cilia thereby creating a fine mesh. This prevents larger molecules such as inhaled particles and mucins of the mucus layer from entering the periciliary liquid layer. Thus, as opposed to the former so-called gel-on-water model, this new theory (gel-on-brush model) can explain why the mucus layer and the periciliary liquid layer can exist in such close proximity without getting mixed up. In addition, this theory is better at explaining the changes seen in the two layers in relation to dehydration or excess hydration of the airway surface. For a more detailed explanation of the gelon-brush model, the reader is encouraged to study the article by Button et al. (2012).

Pathophysiological and clinical aspects of mucociliary clearance There are two main reasons why mucociliary clearance could be hampered. Either the movements of the cilia can be hindered directly, for example by genetic defects in central proteins of the axoneme or by temporary dysfunction caused by infection or environmental influences (Reimer et al., 1980; Afzelius, 2004; Escudier et al., 2009; Wang et al., 2012); or the mucus layer can constitute the main problem when dehydration of the mucus leads to increased viscosity whereby the ciliary clearance becomes ineffective. Moreover, such dehydration can cause the periciliary liquid layer to shrink causing the cilia to become squeezed underneath the mucus layer impeding their movement. If the periciliary liquid layer gets increasingly thin, the mucin glycoproteins of the mucus will bind to the epithelial glycocalyx much like Velcro impeding both ciliary and cough clearance radically (Knowles & Boucher, 2002; Boucher, 2007; Fahy & Dickey, 2010). Aetiology and classification Conditions directly affecting the movement of the cilia of the respiratory tract are traditionally divided into primary and

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secondary ciliary dyskinesia (PCD and SCD). PCD is usually an autosomal recessive disorder, but occasionally X-linked or autosomal-dominant transmission has been reported as well. It is both phenotypically and genetically a heterogeneous condition presumably reflecting the molecular complexity of the axoneme whereby mutations in many different genes can cause defects of the ciliary movement. To this date, only a few known mutations have been confirmed to cause PCD; and as these mutations are causative only in around 25% of the diagnosed patients, genetic testing is still far from universally applicable (Chodhari et al., 2004; Bush et al., 2007). Today, the diagnosis primarily rests on clinical presentation of the patient (recurring sinopulmonary infections from birth), nasal NO measurements (low nasal NO points towards PCD), electron microscopy of ciliary ultrastructure, and microscopy of ciliary beat pattern and frequency (Bush et al., 2007). In some centres, these tests are supplemented by other tests, for example pulmonary radioaerosol mucociliary clearance, which is described in detail later in this review. Secondary ciliary dyskinesia on the other hand includes a variety of temporary, acquired defects of ciliary movement, which can be caused by viral or bacterial infection or by air pollutants such as ozone, aldehydes or cigarette smoke. In some instances, the condition can be hard to differentiate from PCD due to overlapping findings in the tests meant for diagnosing PCD (Carson et al., 1980; Reimer et al., 1980; Rautiainen et al., 1992; Calderon-Garciduenas et al., 2001; Randell & Boucher, 2006; Livraghi & Randell, 2007; Wang et al., 2012). Apart from these conditions directly affecting the cilia, many other diseases have abnormal mucociliary clearance as a central part of their pathogenesis. For example, this applies to cystic fibrosis, which is caused by mutations in the gene Table 1

encoding the Cl channel known as CFTR. This defect leads to dehydration of the mucus layer and shrinkage of the periciliary liquid layer, thereby impeding mucus clearance by both ciliary beating and coughing as described earlier (Fahy & Dickey, 2010). Impaired mucociliary clearance is also seen in patients with asthma and chronic obstructive pulmonary disease. An important part in their pathogenesis is thought to be the hypersecretion of mucin leading to excessive amounts of mucus with an increased viscosity. This rubbery mucus is hard to clear from the airways and in severe cases can end up forming mucus plugs whereby infection or localized atelectasis is likely to follow (Randell & Boucher, 2006; Fahy & Dickey, 2010). An overview of different conditions affecting mucociliary clearance is presented in Table 1. Clinical aspects The primary symptoms of impaired mucociliary clearance are productive cough and dyspnoea. Dyspnoea is a result of mucus obstructing airflow in numerous airways, which in cases of total obstruction can lead to atelectasis (Hogg, 2004; Bosse et al., 2010; Fahy & Dickey, 2010). As described earlier, coughing can to some extent substitute for impeded ciliary clearance. This may help explain why diseases in which only the cilia are involved (such as PCD) tend to be less severe than those primarily caused by dehydration of the mucus (e.g. cystic fibrosis) as this will impede both ciliary and cough clearance (Knowles & Boucher, 2002; Livraghi & Randell, 2007; Fahy & Dickey, 2010). Recurring sinopulmonary infections starting in early childhood are almost universal in patients with substantial and

Characteristics of different conditions affecting mucociliary clearance. Primary ciliary dyskinesia

Secondary ciliary dyskinesia

Aetiology

Genetic defect in gene related to movement of the cilia

Temporary dysfunction of the cilia caused by viral or bacterial infection or by air pollutants

Genetic defect in gene encoding the Cl channel known as CFTR

Multifactorial disease Smoking is the most common cause

Multifactorial disease Atopi or bronchial hyperresponsiveness plays an important part

Primary causes of defective mucociliary clearance

Immotile or dysmotile cilia

Temporarily immotile or dysmotile cilia

Dehydration of the airway mucus leading to increased mucus viscosity Shrinkage of the periciliary liquid layer impeding ciliary movement

Goblet cell metaplasia and hyperplasia and submucosal gland enlargement leading to hypersecretion of mucin. Thereby mucus viscosity and amount is increased

Goblet cell metaplasia leading to hypersecretion of mucin. Thereby mucus viscosity is increased Narrowing of airways combined with rubbery mucus can form mucus plugs obstructing mucociliary clearance

Cystic fibrosis

Chronic obstructive pulmonary disease

Asthma

(Afzelius, 2004; Soler-Cataluna et al., 2005; Kondo et al., 2006; Randell & Boucher, 2006; Livraghi & Randell, 2007; Mall, 2008; Fahy & Dickey, 2010; Lommatzsch, 2012). © 2013 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd 34, 3, 171–177

174 Mucociliary clearance: pathophysiological aspects, M. Munkholm and J. Mortensen

permanent defects of mucociliary clearance (Sagel et al., 2011). At least partially, this is thought to be a result of mucus plugs blocking smaller airways thereby creating a local environment that promotes bacterial growth (Worlitzsch et al., 2002; Livraghi & Randell, 2007). Such long-term localized infection leads to mucus gland hypertrophy and epithelial damage further impairing mucociliary clearance from that area. Eventually, the inflammation can lead to bronchiectasis characterized by permanent dilation of the airway and thickening of the bronchial wall. Because of these changes, areas like this can function as a seedbed for future infections (Bilton, 2008; Ilowite et al., 2008; Javidan-Nejad & Bhalla, 2009; King, 2009; Goeminne & Dupont, 2010). Recurring infections are thought to be the main reason why, in time, these patients develop a decrease in lung function. However, the rate of lung function decline of the individual patients can be difficult to foresee, but tend to appear earlier and to be more profound in patients with cystic fibrosis compared with patients with PCD (Livraghi & Randell, 2007). Nevertheless, all severe chronic diseases involving defective mucociliary clearance result in substantial morbidity in terms of dyspnoea, recurring sinopulmonary infections, and frequent and productive coughs. The amount, frequency and appearance of the sputum will vary according to the disease, but generally with increased infection, the amount increases and it gets more viscous and purulent as often seen in patients with cystic fibrosis. The close relationship with infection is also evident in patients with acute infectious exacerbation of chronic obstructive pulmonary disease where large amounts of dark/ green and viscous sputum are produced (Rubin, 2009; Fahy & Dickey, 2010; Miravitlles et al., 2010; Stenbit & Flume, 2011). In severe cases of chronic defective mucociliary clearance, the decline of lung function can lead to the need for lung transplantation or early mortality (Livraghi & Randell, 2007; Marthin et al., 2010).

infections and/or bronchiectasis, a universal and very slow mucociliary clearance can reflect an underlying PCD, while this is excluded if mucociliary clearance is normal or only regionally abnormal. In this case, the radioaerosol is cleared normally from the lung except at a few sites corresponding to the bronchiectasis (Marthin et al., 2007). There are only about ten centres in the world that regularly perform pulmonary radioaerosol mucociliary clearance studies, and only few of these use it as a diagnostic tool on a routine basis. There is considerable variation in the specific techniques applied considering choice of radiocolloid (e.g. albumen/sulphur colloids), aerosol generator and particle size, inhalation technique (slow/fast, depth), gamma camera acquisition period (0–2, 0–6, 0–24 h) and type (posterior/anterior, static/ dynamic/SPECT), characterization of initial deposition (central/peripheral ratio, penetration index), etc. The variation in the applied techniques reflects the lack of consensus upon the ‘best’ method and that the choice of ‘best’ method depends upon the specific aim of the test. For example, assessment of mucociliary clearance from the small and large airways needs different deposition patterns and timing of acquisition (Bennett et al., 2010). The simplified pulmonary radioaerosol mucociliary clearance technique described in Table 2 has been used as a diagnostic routine tool in the work-up of patients suspected for PCD in 30–40 patients yearly for more than a decade at Rigshospitalet, Denmark. This radioaerosol clearance technique was originally validated in three sequentially performed studies. First, it was used in a cross-sectional study in patients with established PCD. Second, it was applied in a prospective blinded trial of patients referred for suspicion of PCD. Third, it was implemented in a trial using the method in PCD workup. The results were compared with nasal ciliary motility studies, electron microscopy of cilia and the final clinical diagnosis (Marthin et al., 2007).

Can mucociliary clearance be enhanced? Measurement of mucociliary clearance Radioaerosols can be used to study the mucociliary transport from the ciliated airways. Insoluble 99mTc labelled colloids are inhaled and deposited, and their subsequent clearance can be assessed by scintigraphy. If the person does not cough during the study period, the radioaerosol clearance reflects mucociliary clearance. However, if the person does cough during measurement, the measured clearance will reflect a combination of mucociliary clearance and cough clearance. Hence, pulmonary radioaerosol clearance studies can be used for assessment of mucociliary clearance, cough clearance or the combination of both. Most pulmonary radioaerosol mucociliary clearance studies are performed to investigate a possible link between mucociliary dysfunction and pathophysiology of lung diseases or pharmacological challenges to the mucociliary apparatus. However, as reference values are available, the technique can also be used as a diagnostic tool to assess if a person’s mucociliary clearance is within or outside reference limits. In a patient with recurrent

As described, mucociliary clearance is crucial in the pathogenesis of many different diseases, which has led to the development of a wide range of therapies seeking to augment the clearance of mucus from the airways. Roughly, these therapies can be divided into two categories as seen below. Physiotherapeutic regimens These include postural drainage, positive expiratory pressure, forced expiration techniques, voluntary coughing and regular exercise. They are primarily thought to augment clearance mechanisms largely independent of ciliary function. Thus, even though these therapies have shown convincing positive results in a variety of diseases with dysfunctional mucociliary clearance, this is not thought to be a result of an increase in ciliary clearance; more it is a result of finding ways to circumvent this defect (Mortensen et al., 1991a; Pryor, 1999; Lester & Flume, 2009).

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Table 2

Example of a pulmonary radioaerosol mucociliary clearance protocol.

Preliminary procedure 20 inhalations (slow inspiration/forced expiration) of 99mTc-albumin colloid aerosol (Venticoll, Solco) Gamma camera measurements of pulmonary radioactivity Repeated dynamic and static acquisitions for 2 h A static acquisition after 24 h A 81mKr ventilation acquisition of the ventilated lung area Reference values Lung retention at 1 and 2 h is compared with predicted values calculated from penetration index, age and sex using own reference equations (Mortensen et al., 1994) Interpretation Interpretation is based on visual analysis and quantitative analysis of the radioactivity retention based on the following three measurements 1) Is the bolus transport in the trachea normal? 2) Is lung retention at 1 and 2 h within the predicted values? 3) Is there no focal retention in the airways after 24 h? (Focal retention indicates regional/general impaired mucociliary clearance) Conclusion of a test Test results can typically be divided in the following 4 main groupings Normal mucociliary clearance It is the case, if all three measurement parameters are normal. Thereby, both PCD and secondary mucociliary defects can be excluded Abnormal mucociliary clearance It is the case, if all three measurement parameters are abnormal. This is compatible both with PCD and SCD. Subsequently, nasal ciliary function testing and electron microscopy can be used to discriminate between these entities Regional abnormal mucociliary clearance It is the case, if 1) and 2) are normal while 3) is abnormal. It is compatible with focal SCD, for example bronchiectasis, but excludes PCD Inconclusive test It can be due to coughing, too peripheral initial distribution or inconsistency between the three measurement parameters

Pharmacological therapies In recent years, great interest in finding ways to directly stimulate mucociliary clearance has led to many different new therapies. Some focus on changing the viscosity of the airway mucus thereby making it easier to clear either by ciliary movement or by coughing. This includes inhalation of aerosols of hypertonic saline or a dry powder formulation of mannitol, which promotes the flux of water across the lung surface. Other approaches for changing mucus viscosity have been to directly modulate chloride or sodium channels of the lung in order to increase hydration of the mucus. Yet another approach has been to degrade extracellular DNA found in the mucus by inhalation of rhDNAse, thereby reducing the mucus viscosity (Yoo & Koh, 2004; Rogers, 2005; Amirav et al., 2009). Others have tried to directly stimulate the beating of cilia by b-adrenergic agents. This has long been known to increase in vitro ciliary beat frequency, and numerous studies have also shown enhancement of in vivo mucociliary clearance in patients with various airway diseases although this improvement is generally more pronounced in the normal lung (Mortensen et al., 1991b; Bennett, 2002; Yoo & Koh, 2004). In addition, some interesting new therapies, primarily explored in relation to cystic fibrosis, focus on assisting or repairing the production of certain defective proteins in epithelial cells of the airways. This kind of treatment is still only on an experimental basis, but shows exciting potential for future enhancement of chronic defects of mucociliary clearance (Amirav et al., 2009). In conclusion, several studies have provided preliminary data supporting the ability of different types of agents to augment mucociliary clearance. However, further investigation is

needed in order to determine the potential usefulness of each therapy in the broad array of different diseases.

Conclusion Mucociliary clearance is an important airway defence mechanism. Unfortunately, it can be affected by exogenous challenges such as smoke, dust and infections, and reduced mucociliary transport is an important part of the pathophysiology of many lung diseases. Today, many different mechanisms have been elucidated whereby mucociliary clearance can be hampered, and this has led to a wide variety of approaches to augment the impeded clearance. However, further investigation is needed before the usefulness of each therapy in the broad array of different diseases can be determined. In addition, still many questions remain unanswered especially regarding the specific gene defects underlying conditions such as PCD. Further knowledge within this field might facilitate the development of therapies able to correct chronic defects of mucociliary clearance. Today, scintigraphic clearance of inhaled insoluble radioaerosols can be used in the study of mucociliary clearance to assess the pathophysiology of lung diseases and the importance of mucociliary dysfunction in this regard. This means that the procedure can be used as a clinical diagnostic tool in work-up of patients with recurrent airway infections. Furthermore, effects of physical or pharmacological interventions on mucociliary transport can be studied by way of this technique, which may be useful in the clinical testing of novel therapies aiming to augment mucociliary clearance.

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Conflict of interest The authors declare no conflict of interest.

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