CILIA AND MUCOCILIARY CLEARANCE

466 CILIA AND MUCOCILIARY CLEARANCE See also: Allergy: Allergic Reactions. Asthma: Overview; Extrinsic/Intrinsic. Cysteine Proteases, Cathepsins. Extracellular Matrix: Degradation by Proteases. Kinins and Neuropeptides: Neuropeptides and Neurotransmission; Tachykinins; Vasoactive Intestinal Peptide. Leukocytes: Mast Cells and Basophils. Lipid Mediators: Overview; Leukotrienes; Prostanoids. Serine Proteinases. Smooth Muscle Cells: Airway. Systemic Disease: Sarcoidosis.

Further Reading Cairns JA (2005) Inhibitors of mast cell tryptase beta as therapeutics for the treatment of asthma and inflammatory disorders. Pulmonary Pharmacology & Therapeutics 18: 55–66. Caughey GH (2001) New developments in the genetics and activation of mast cell proteases. Molecular Immunology 38: 1353– 1357.

Cocks TM and Moffatt JD (2001) Protease-activated receptor-2 (PAR2) in the airways. Pulmonary Pharmacology & Therapeutics 14: 183–191. Lan RS, Stewart GA, and Henry PJ (2002) Role of proteaseactivated receptors in airway function: a target for therapeutic intervention? Pharmacology & Therapeutics 95: 239– 257. Levi-Schaffer F and Piliponsky AM (2003) Tryptase, a novel link between allergic inflammation and fibrosis. Trends in Immunology 24: 158–161. Reed CE and Kita H (2004) The role of protease activation of inflammation in allergic respiratory diseases. Journal of Allergy and Clinical Immunology 114: 997–1008. Schmidlin F and Bunnett NW (2001) Protease-activated receptors: how proteases signal to cells. Current Opinion in Pharmacology 1: 575–582. Sommerhoff CP, Bode W, Matschiner G, Bergner A, and Fritz H (2000) The human mast cell tryptase tetramer: a fascinating riddle solved by structure. Biochimica et Biophysica Acta 1477: 75–89.

CILIA AND MUCOCILIARY CLEARANCE L E Ostrowski and W D Bennett, University of North Carolina, Chapel Hill, NC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Mucociliary clearance is an innate defense mechanism that protects the pulmonary system from the harmful consequences of inhaled agents, including those of biological, chemical, and physical nature. Ciliated cells, which line the surface epithelium of the airways, provide the force necessary for mucociliary clearance by the coordinated beating of their cilia. These highly specialized cells are therefore critical to the health and function of the pulmonary system. In this article, the basic biology of ciliated cells and the structure of the cilium are described. Their function and interaction with other components of the mucociliary system and the importance of mucociliary clearance to respiratory disease are discussed.

Introduction Mucociliary clearance is the process whereby the pulmonary system is cleared of inhaled foreign material by the continuous beating of ciliated cells to transport mucus and any entrapped material to the pharynx, where it is subsequently removed by swallowing. This well-coordinated system consists of mucous and serous cells in the submucosal glands and secretory goblet cells in the airway epithelium that secrete water, mucus, and other proteins to produce a fluid layer on the airway surface and also the ciliated cells that propel the fluid out of the lung toward the mouth (Figure 1). The fluid layer

consists of a low-viscosity sol phase near the cells’ apical surface and a more viscous gel (or mucus) phase near the tips of the cilia. Whether the two phases are entirely distinct or a viscosity gradient exists through the depth of the entire airway fluid is not clear. The efficiency of the mucociliary clearance (MCC) system at removing airway secretions and associated trapped substances depends on three primary factors: the beat frequency and coordination of the cilia, the quantity and rheology of airway secretions derived from surface goblet cells and submucosal glands, and the periciliary fluid depth that is modulated by ion transport of the airway epithelium. In health, this system is very effective at clearing mucus and associated bacteria and toxins from the lung. However, in a variety of airway diseases (e.g., chronic bronchitis, asthma, and cystic fibrosis (CF)) this apparatus becomes dysfunctional, leading to further exacerbation of airway inflammation and obstruction. Patients with primary ciliary dyskinesia (PCD), a genetic disease characterized by abnormal ciliary ultrastructure and function, have impaired MCC that results in chronic lung, sinus, and middle ear disease, demonstrating the critical importance of properly functioning cilia to the health of the pulmonary system.

Ciliated Cells and Cilia Ciliated cells, which provide the force necessary for MCC, are found throughout the trachea and large

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Ion channels

Mucin granules

Cilia

Mucus

Periciliary liquid Cl−

Na+

Ciliated cell

Goblet cell

Basal cell Basement membrane Figure 1 Schematic of airway epithelium illustrating the two major cell types, ciliated and goblet. The epithelium is covered by a fluid layer whose depth is regulated by ion transport across the cell membranes and within which mucins are secreted to trap foreign matter for mucociliary transport out of the lung.

airways and are present distally as far as the terminal bronchioles. Figure 2 shows a cross section of a normal human large airway illustrating the heavily ciliated surface. The percentage of ciliated cells in the human airway epithelium decreases from approximately 50% in the tracheal epithelium to o20% in the bronchiolar region. Ciliated cells are also present in areas of the upper respiratory tract, including the nasal sinuses and nasopharynx. On the apical surface of each ciliated cell is an organized array of up to 200 individual cilia; the adult lung has been estimated to contain approximately 3
1012 total cilia. A typical cilium from an adult tracheal cell is approximately 7 mm long and 0.1 mm in diameter. Ciliated cells in the more peripheral airways have fewer cilia, and the cilia are shorter as well. Each cilium demonstrates the highly conserved axonemal structure, consisting of a central pair of microtubules surrounded by a ring of nine microtubule doublets (Figure 3). Each of the outer doublets consists of a complete tubule (the A tubule, containing 13 tubulin subunits) and a partial tubule (the B tubule, containing 11 subunits). Attached to each complete tubule are two rows of large multiprotein complexes, the inner and outer dynein arms. Each dynein arm contains at least one catalytic dynein heavy chain that converts the energy of ATP hydrolysis into the bending motion of the ciliary axoneme by attaching to the adjacent microtubule and undergoing a conformational change, causing the microtubules to move relative to each other. The detailed structure of the human cilium is only beginning to be unraveled, but the realization that defects in ciliary function are

Figure 2 Ciliated cells of the human airway epithelia. Photomicrograph of a large airway showing the epithelial surface covered by numerous cilia extending from the tall, columnar ciliated cells. The paraffin-embedded large airway (bronchus) was stained with Richardson’s to visualize the cilia.

responsible for a much wider spectrum of disease than previously thought has stimulated studies on the assembly, structure, and function of this fascinating organelle.

468 CILIA AND MUCOCILIARY CLEARANCE

Radial spoke Radial spoke Inner dynein arm Central pair microtubule

Outer dynein arm

A microtubule B microtubule

Outer dynein arm

Inner dynein arm

Nexin link Figure 3 (Left) Schematic of a prototypical axoneme illustrating some of the structural features that are highly conserved among motile 9 þ 2 axonemes. (Right) Transmission electron micrograph showing a normal human cilium in cross section. Many of the conserved structures are easily visible.

Regulation of Ciliary Beat Frequency In the airway, cilia typically beat with a frequency of approximately 10–15 Hz, although reported values vary slightly depending on the conditions used. Analysis of ciliary waveform from nasal samples by digital high-speed video has revealed a mostly planar pattern, with the cilium being fully extended in the forward power stroke. During the recovery stroke, the cilium curls backwards in the same plane. Figure 4 shows an image from a video recording of an actively beating ciliated cell. During the recovery stroke, the cilium curls backwards in the same plane. The regulation of ciliary beat frequency (CBF) has been an area of research interest for many years, and many different agents have been reported to stimulate or inhibit CBF. For example, increases in the intracellular concentration of cAMP consistently stimulate CBF in airway epithelial cells. This increase is presumed to be due to the phosphorylation of axonemal proteins by the action of cAMP-dependent protein kinase (PKA). PKA has been demonstrated to be an integral component of the ciliary axoneme; however, the regulatory protein responsible for the increased CBF has yet to be identified. Agents that increase intracellular calcium also elevate CBF in mammalian airway cells, although, again, the detailed molecular pathway has not been elucidated. Other regulatory molecules, including cGMP, PKG, protein kinase C, calmodulin, phospholipase C, and nitric oxide, have all been reported to have effects on ciliary beating, although the different experimental systems used have sometimes produced conflicting results. It seems likely that the regulation of CBF occurs through the interaction of several pathways, and more research is required to determine the individual contributions of each to the overall regulation of CBF. For effective MCC to occur, ciliary beating must also be coordinated. Cilia in the airway

Figure 4 Image of an actively beating human ciliated cell. Ciliated cells isolated from human bronchial tissue were visualized using differential interference contrast optics and high-speed video images were recorded.

beat in a metachronal wave, and, again, the mechanisms that initiate and maintain the proper orientation and coordination between the ciliated cells are unknown.

Measurement of Mucociliary Clearance MCC rates can be measured in vivo by assuming that a nonpermeating, inhaled marker depositing on the airway surface moves out of the lung at the same rate as the airway secretions in which it is immersed. The most common technique is to use inhaled, radiolabeled particles, aqueous or dry, that upon deposition in the lung can be followed by a gamma camera or scintillation detectors to determine their rate of egress from the lung. Using these techniques, scientists have been able to study MCC in healthy subjects and in patients with airways disease and to assess

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the effects of various therapies on MCC. The rate of tracheal mucus velocity in healthy controls has been estimated to be 4 or 5 mm min  1 using this technique, with slower rates reported in the more distal airways. Increases in CBF have been shown to translate into increased rates of clearance, and it has been reported that a 16% increase in CBF resulted in a 450% increase in tracheal mucus velocity. Thus, agents that increase CBF increase MCC and can provide benefits to patients with a range of airway diseases.

Mucociliary Clearance in Pulmonary Disease A number of short-acting b-adrenergic agonists (salbutamol, isoproterenol, and albuterol) intended to ease bronchoconstriction in patients with airways disease have also been shown to enhance the rate of MCC from the lung. Figure 5 illustrates this effect in a healthy subject. Figures 5(a) and 5(c) show the

gamma camera images of a healthy person’s lungs immediately after inhalation of such radiolabeled particles. Figure 5(b) shows the gamma camera image 30 min after depositing the radiolabeled particles and no intervention (i.e., a control measure of MCC), illustrating a mild loss of image intensity during the 30-min period (i.e., in comparison to Figure 5(a)). On the other hand, Figure 5(d) illustrates a 30-min posttreatment gamma camera image in the same individual after inhaled albuterol treatment, showing more rapid movement of radiolabeled particles out of the lung compared to the control condition (compare Figures 5(b) and 5(d)). This enhancement in MCC is likely due to increases in CBF induced by the b-adrenergic agonist and consequent increases in intracellular cAMP discussed previously, although it is clear that these agents have pleiotropic effects. Table 1 provides a list of other pharmacological agents that can stimulate or depress in vivo MCC. Although the mucociliary apparatus in the normal lung responds quite well to pharmacological stimulation, it is less responsive in patients

Figure 5 Stimulation of mucociliary clearance by a b-adrenergic in a healthy subject. Top panels illustrate clearance associated with no intervention. (a) A gamma camera image of the lungs immediately after inhalation of radiolabeled particles (i.e., particle deposition image). (b) Thirty-minute postdeposition image. Bottom panels illustrate clearance in the same subject associated with inhaled albuterol treatment. (c) Particle deposition image that was immediately followed by inhaled albuterol treatment. (d) Thirty-minute postdeposition (and treatment) image showing increased clearance of the radiolabeled particles.

470 CILIA AND MUCOCILIARY CLEARANCE Table 1 Factors affecting mucociliary clearance

Conclusions

Factor

MCC is a remarkably effective mechanism for maintaining pulmonary health. Impaired MCC is a common feature of many airway diseases, and agents that improve MCC are beneficial in the treatment of these diseases. Many of these agents have a direct effect on ciliated cells and increase CBF. However, further research is needed to understand the complex interactions between cilia, airway surface liquid, mucus, and MCC so that more effective treatments may be developed.

Increase

Decrease

Pharmacological Adrenergic agonists Cholinergic agonists Histamine ATP Amiloride Hyperosmolar solutions

Anesthetics Atropine Antigen

Pathological

Cystic fibrosis Primary ciliary dyskinesia Chronic bronchitis Asthma Respiratory infections

Environmental

Pseudohypoaldosteronism Chronic cough

Acute exposure to Sulfur dioxide Cigarette smoke Ozone

Chronic exposure to Sulfur dioxide Cigarette smoke Ozone NO2

with airways disease. This may be due in part to the loss of ciliated epithelium associated with chronic inflammation (e.g., patients with chronic obstructive airway disease who have smoked cigarettes most of their lives). Other pathological conditions for which MCC is known to be depressed are also listed in Table 1. In support of the need to hydrate mucus for effective MCC, patients with pseudohypoaldosteronism, a rare inherited disease characterized by poor Na þ absorption from the airway surface, have MCC rates three or four times higher than normal. The increased rate of clearance in these patients may be due to enhanced ciliary beating in response to an increased low-viscosity load on the cilia. In contrast, the airway surfaces of CF patients are poorly hydrated, leading to decreased MCC and promoting the formation of adherent mucus plaques on CF airway surfaces, ultimately causing airflow obstruction and promoting chronic infection. Finally, a number of environmental pollutants can influence the rate of MCC, either acutely or chronically, and are also listed in Table 1. For example, although sulfur dioxide may acutely stimulate MCC, chronic exposure to it causes a depression in MCC. This is likely due to a remodeling of the airway epithelium (i.e., a loss of ciliated cells and increase in mucus-producing cells) similar to that seen with chronic cigarette exposure.

Acknowledgments The authors thank R Wonsetler, Dr P Spears, Dr M Chua, and the Hooker Microscopy Facility for preparing the ciliated cell video; K Burns and the histology core for excellent technical services; L Brown for assistance in preparing the figures; Dr S H Randell and the Cell Culture Facility for samples; and the individuals who donated tissue specimens. See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Cystic Fibrosis: Overview. Environmental Pollutants: Overview. Mucins. Mucus. Primary Ciliary Dyskinesia.

Further Reading Bennett WD (2002) Effect of b-adrenergic agonists on mucociliary clearance. Journal of Allergy and Clinical Immunology 110: S291–S297. Bennett WD, Noone PG, Knowles MR, and RC Boucher (2001) Regulation of mucociliary clearance by purinergic receptors. In: Salathe M, et al. (eds.) Cilia and Mucus: From Development to Respiratory Defense. New York: Dekker. Brody SL (2004) Genetic regulation of cilia assembly and the relationship to human disease. American Journal of Respiratory Cell and Molecular Biology 30(4): 435–437. Knowles MR and Boucher RC (2002) Mucus clearance as a primary innate defense mechanism for mammalian airways. Journal of Clinical Investigation 109(5): 571–577. Noone PG, Leigh MW, Sannuti A, et al. (2004) Primary ciliary dyskinesia: diagnostic and phenotypic features. American Journal of Respiratory and Critical Care Medicine 169(4): 459–467. Ostrowski LE, Blackburn K, Radde KM, et al. (2002) A proteomic analysis of human cilia: identification of novel components. Molecular & Cellular Proteomics 1(6): 451–465. Robinson M and Bye PT (2002) Mucociliary clearance in cystic fibrosis. Pediatric Pulmonology 33(4): 293–306. Snell WJ, Pan J, and Wang Q (2004) Cilia and flagella revealed: from flagellar assembly in chlamydomonas to human obesity disorders. Cell 117(6): 693–697. Wanner A, Salathe M, and O’Riordan TG (1996) Mucociliary clearance in the airways (state of the art). American Journal of Respiratory and Critical Care Medicine 154: 1868–1902.