State of the Art Clinical Aspects of Mucociliary Transport1 ADAM WANNER

Contents Introduction Normal Structure and Function Structure Function Cilia Mucus Measurement of Mucociliary Function Cilia Mucus Mucociliary Function Mucociliary Clearance Mucous Transport Physico-chemical Factors Humidity and Temperature Inorganic Salts and pH Ionized Air and Radiations Impairment by Inhalants Cigarette Smoke Short-Term Exposure Long-Term Exposure Effects of Filters Atmospheric Pollutants Sulfur Dioxide Nitrogen Dioxide Ozone Inorganic Sulfates and Nitrates Oxygen Hyperoxia Hypoxia Hypercapnia

Inhalation Anesthetics Miscellaneous Impairment in Disease States Chronic Bronchitis Cystic Fibrosis Bronchial Asthma Respiratory Infections Miscellaneous Endotracheal Intubation and Bronchoscopy Tracheal Resection and Lung Transplantation Postoperative Retention of Secretions Relationship of Impaired Mucociliary Transport to Carcinogenesis Depression by Pharmacologic Agents General Anesthetics Local Anesthetics Anticholinergic Agents Narcotics Stimulation by Pharmacologic Agents and Physical Therapy Adrenergic Agents Cholinergic Agents Biologically Active Amines Methyl-Xanthines Miscellaneous Pharmacologic Agents Antimicrobial Drugs Immunopharmacologic Agents Corticosteroids Mucolytic Agents Cardiac Glycosides Physical Therapy Summary

1

From the Division of Pulmonary Disease, Department of Medicine, Mount Sinai Medical Center, Miami Beach, Fla. 2 Some of the research reported herein was supported by National Institutes of Health Grant HL17816 and Contract 71-2205. 3 Requests for reprints should be addressed to Adam Wanner, M.D., Co-Chief, Division of Pulmonary Disease, Mount Sinai Medical Center, 4300 Alton Road, Miami Beach, Fla. 33140.

Introduction It has long been believed that the mucociliary a p p a r a t u s of the airways serves to remove inhaled particulate matter from the tracheobronchial mucosa, thereby c o n t r i b u t i n g to pulmonary host defense. Cilia were described by De H e i d e a n d L e u w e n h o u k in the seventeenth century (1), b u t mucous transport in the air-

AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 116, 1977

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ways and its modification by respiratory irritants has not received much attention until this century. The developments in this field have been extensively reviewed by Sleigh (1), Gray (2), Rivera (3), and Kinosita and Murakami (4). The clinical significance of the mucociliary transport system was the subject of a symposium in 1966 (5) and several individual reviews in the past decade (6-10). This review deals mainly with factors affecting mucociliary transport that may be of clinical importance.

Normal Structure and Function

Structure The respiratory epithelium from the proximal trachea to the terminal bronchioles is covered by cilia protruding from the luminal surface of columnar cells. The larynx contains over most of its surface a mucus-secreting squamous epithelium, and cilia are only present at the posterior commissure (11). Of the different cell types found in the respiratory epithelium, i.e., basal cells, intermediate cells, nonciliated columnar cells, ciliated columnar cells, and goblet cells (12, 13), only the latter 2 contribute directly to mucociliary function. Respiratory mucus is produced by submucosal glands and the goblet cells. Transmission electron microscopic and scanning electron microscopic studies provide further insight into the precise anatomic features of the respiratory epithelium (14-17). The nonciliated columnar cells either contain microvilli on their luminal surface or have large microvilli occupying the center of the cell apices (brush cell) (16, 18). Microvilli measure 0.3 fim in length and 0.1 /mi in diameter (16). They are covered by a cell membrane, and their filamentous cytoplasm is contiguous with the cytoplasm of the cell. These microvilli covering the nonciliated columnar cells may represent developing or degenerated cilia. The ratio of ciliated columnar cells to goblet cells is approximately 5 to 1 (17). The relative numbers of both goblet cells (12, 13) and ciliated columnar cells (19, 20) decrease from the trachea toward the peripheral airways. In the larger airways the major part of the epithelium is ciliated (21-23), with focal areas as large as 1 mm in diameter devoid of cilia (16). These areas are covered with nonciliated microvillous columnar cells, with the exception of one or 2 ciliated cells usually adjacent to the glandular openings. Small

areas of squamous metaplasia may be found even in normal airways. The surface of each ciliated columnar cell contains approximately 200 cilia with an average length of 6 ^m and diameter of 0.2 /an. The cilia of human (17) and other mammalian (24) respiratory epithelia or cilia from lower animals are remarkably similar when compared by transmission and scanning electron microscopy (25-29). Each cilium contains longitudinal fibrils that appear to represent contractile elements. Two single fibrils form a central core, and 9 fibrils with a doublet structure are arranged in a circular fashion in the periphery of the cilium. The peripheral fibrils join to one structure toward the tip of the cilium. A basal body in the apex of the cell corresponds to each cilium. The fibrils, however, terminate at the level of the cell surface, and there is no direct connection between the fibrils contained in the ciiia and the basal body. A dense matrix surrounds the fibrils in the cilia, and this matrix continues down into the basal body. The fibrils consist of 10 to 15 substructures (filaments) of approximately 1-nm diameter. Lateral connections between doublets of fibrils have also been demonstrated. The structure, the distribution, and the histochemical characteristics of mucus-secreting glands (goblet cells, submucosal glands) have been reviewed by others (30, 31). They play only an indirect role in mucous transport, and will therefore not be further considered in this context. Submucosal glands are innervated predominantly by efferent postganglionic parasympathetic fibers (32). Sensory nerve endings seem to penetrate into the epithelium in the vicinity of goblet cells (17, 33). Although it is clear that respiratory mucus is secreted by goblet cells and submucosal glands, the role of materials released by Clara cells located in the bronchioles, where normally goblet cells and submucosal glands are absent, has not been clarified (34, 35). In 1934, Lucas and Douglas (36) developed a 2-layer concept of respiratory mucus. According to this concept, the cilia are surrounded by a periciliary fluid layer (sol), and are covered by a mucous layer (gel), which interacts with the tips of the cilia. In contrast to the periciliary fluid layer whose presence is difficult to prove experimentally, the existence of mucus (gel layer) has been clearly demonstrated (18, 21, 23, 37-39). Initially, it was believed that the mucous layer represented a contiguous blanket

CLINICAL ASPECTS OF MUCOCILIARY TRANSPORT

covering the surface of the tracheobronchial tree (8, 36, 40-42). It has subsequently been shown in mammals that the mucous layer, which is 5 to 10 /um deep, may be discontinuous, as demonstrated by electron microscopic examination showing foci consisting of mucous droplets and desquamated cells, macrophages, and cellular debris (18, 21, 22, 39). The ultrastructure of mucus reveals fine fibrillar formations that resemble the content of the secretory granules of goblet cells (18). A quantitative difference appears to exist between the concept of a mucous "blanket" interrupted by mucus-free islands, and the concept of foci of mucus floating like lily pads on the periciliary fluid (43). The distinction of these 2 concepts is especially important in the understanding of mucociliary function. Epithelial cells of the airways are continually being shed; they appear to regenerate from the basal cells (17). The life span of epithelial cells has been studied by autoradiography combined with electron microscopy. Such investigations have yielded turnover rates for the basal cells only (3 to 38 weeks), and precise life spans for the various cell types have not been established (35). It is not clear whether cilia can regenerate on a cell that has previously lost its cilia, or whether replacement of such epithelial cells by new columnar cells is required for restoration of cilia. Goblet cells seem to be capable of recovery after secretion with renewed formation of secretory granules for subsequent secretion. The mechanisms of epithelial regeneration and the time required for complete restoration after epithelial damage seem to be of more clinical relevance than turnover rates. Regeneration of ciliated respiratory epithelium injured by direct touch takes approximately 2 weeks in mammals (44). Complete restoration of the damaged epithelium requires the presence of vitamin A; in vitamin A-deficient animals, differentiation of the regenerating epithelium into the different cell types does not take place. After removal of more than 90 per cent of the epithelial cells in chicken trachea by a mechanical denudation process, restoration of a thin epithelium takes place within 2 to 4 days, but complete epithelial regeneration takes 29 days (45, 46). Thirty to 50 per cent of particle transport activity is present at a time when only 10 per cent of the epithelium is ciliated, and ciliary function is completely intact by 14 days, at a time when complete regeneration had not yet occurred, suggesting a great functional reserve of the muco-

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ciliary transport system. The regeneration time of human tracheobronchial ciliated epithelium after damage is not exactly known; it appears that after mechanical injury, regeneration does not begin until after 2 to 3 days (47). Function Cilia. Early reviews of the physiologic features of ciliary movement were published in the late 1920s and early 1930s (2, 48). With the advent of electron microscopy, new information about the fine structure of cilia has contributed considerably to a better understanding of ciliary function (1, 3). In contrast to flagella, which are characterized by undulating motion, cilia beat in one plane with a fast effective stroke (power stroke) and a slow recovery stroke. This pattern of ciliary motion, which is remarkably uniform among different species, has been investigated with high-speed cinematography of isolated cilia or isolated cells (49). The effective stroke is 2 to 3 times faster than the recovery stroke. An increase in the beating frequency shortens both the effective and recovery phases. Cilia treated with glycerine, which destroys the ciliary membrane but preserves the filament doublets, resume motility on addition of adenosine triphosphate (ATP) to the medium, thereby identifying these filament doublets as contractile elements. The following theory of ciliary bending has been proposed (50). One of the 2 subfibers of the 9 peripheral filament doublets is shorter than the other, and bending of the cilium results from sliding between the 2 subfibers. The sliding is accomplished by molecules (dynein arms) bridging the space between the subfibers. The force of the bending filament doublets is transferred to the ciliary membrane by radial fibers. It seems that the primary function of the basal body is to provide skeletal support for the ciliary bending (51). ATP has been identified as the energy source for ciliary motion, as demonstrated in isolated cilia (52, 53), cilia of lower animals (54), or in ciliated epithelia (55, 56). An ATPase has been found in the cytoplasm of the cell and the cilia (54), and in isolated cilia, ciliary motion can be enhanced by adding ATP and other nucleoside triphosphates to the medium (53). The intensity of ciliary motion is directly related to the level of cellular energy production (54, 57). It has been long known from isolated cell or ciliated epithelium preparations that the motion of adjacent cilia on an individual cell or

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the motion among cilia of adjacent cells is coordinated (58). This interciliary pattern of motion by which adjacent cilia beat one after another to generate a wave of ciliary motion is called metachronism (58, 59). This metachronal coordination creates the least interference between the beating of adjacent cilia. The velocity of the metachronal wave propagation appears to be a function of the recovery stroke velocity (49). The directional relationship of the ciliary effective stroke to the metachronal wave propagation has been described and classified by Knight-Jones (60). The metachronal wave pattern is "symplectic" when the wave direction is in the direction of the effective stroke, "antiplectic" when the wave direction is opposite to the direction of the effective stroke, and "diaplectic" when the wave direction is at right angles to the direction of the effective stroke. In most preparations, metachronal wave patterns seem to be antiplectic (1, 49). In the airways, the effective stroke of the ciliary motion is in the cephalad direction. The mechanisms responsible for the coordination between adjacent cilia resulting in the metachronal wave pattern have not been established; both mechanical and humoral controls have been suggested (4, 57, 59). Brokaw (61) and Iravani (62) proposed that the coordination among adjacent cilia depends on the presence of mucus, which interacts mechanically with the tips of cilia, and that the efficiency of this coordination is influenced by the rheologic properties of mucus. The neurohumoral control of ciliary coordination has been based on observations that 5-hydroxy-tryptamine serves as a pacemaker in molluscs (63, 64), and changes in ciliary activity have been demonstrated by electrical manipulation in similar preparations (4, 57, 65-69). With the exception of the frog palate, which was suggested to be under nervous control as early as 1930 (70) and in which sympathetic nerve stimulation results in increased ciliary activity and parasympathetic nerve stimulation, in decreased ciliary activity (71), nervous control of ciliary coordination has not been demonstrated in vertebrates. Although early workers strongly suggested a nervous control of ciliary beating in the mammalian trachea based on circumstantial evidence (72), more recent studies have failed to substantiate this (57, 62, 73). Mucus. The natural history of respiratory secretions in the tracheobronchial tree has been

extensively studied and reviewed by Reid (74, 75). Respiratory secretions consist of mucus produced by mucous and serous submucosal glands and goblet cells, and tissue fluid. The total volume of mucus-producing structures has been estimated to be approximately 4 ml in human lungs; submucosal glands comprise most of this volume. The submucosal glands and goblet cells produce either acid or neutral glycoproteins. Although the volumetric, rheologic, and clinical examination of "normal" respiratory secretions is difficult because of contamination with saliva, some useful information has been obtained from tracheostomized subjects. The daily volume of respiratory secretions eliminated through the trachea has been estimated to be 10 to 100 ml (74, 76). The submucosal glands are under parasympathetic nervous control (73, 74, 77), whereas goblet cells seem to secrete on direct irritation. "Normal" human respiratory secretions (mucus and tissue fluid) contain approximately 95 per cent water (78). The rest consists of micromolecules (electrolytes and amino acids) and macromolecules (lipids, carbohydrates, nucleic acids, mucins, immunoglobulins, enzymes, and albumin). Mucociliary interaction depends primarily on the rheologic properties of respiratory secretions, but the biochemical characteristics of respiratory secretions are of interest in that they determine their rheologic properties (disulfide cross linking and hydrogen bonding between glycoprotein molecules and water) (79). In situ, the respiratory secretions might take the form of 2 layers, i.e., periciliary fluid (sol phase), and mucus (gel phase). Whereas mucus has been clearly shown to be the product of submucosal glands and goblet cells, the origin of the periciliary fluid has not been firmly established. This periciliary fluid could be the product of as yet unidentified epithelial cells, could result from a liquefying effect of ciliary beating on mucus (80), could represent alveolar or bronchiolar secretions (81), or could result from active water and solute transport across the respiratory epithelium. The latter concept has gained support by the demonstration of epithelial protein transport (82) and of active sodium, chloride, and water transport across the respiratory epithelium to the luminal surface (83). This fluid might also modify the viscoelastic properties of mucus by decreasing its viscosity (84). Early observations of mucociliary transport in the mammalian trachea were made by Sharpey

CLINICAL ASPECTS OF MUCOCILIARY TRANSPORT

in 1830 (85) and Lommel in 1908 (86). The efficiency of this apparatus has been subsequently measured by a great number of investigators in various animal species and in human subjects. Of the different contributions, some are of special interest because they are of help in more critically assessing past and ongoing techniques of mucociliary transport measurement. Stewart (87) investigated the weight-carrying capacity of ciliated epithelium. He found that up to a weight of 20 mg per mm 2 there was no decrease in test particle transport, and that no acceleration of particle transport occurred with lighter weights. If these findings are extrapolated to the human trachea, a mucous layer with a depth in excess of 20 mm would be required to impair ciliary activity, a situation that is hardly compatible with life. Despite the presence of normal ciliary function, however, an extremely deep mucous layer could, theoretically, decrease the velocity of surface particles due to uncoupling within the mucous layer. The remarkable force of mucociliary transport has also been suggested by Hilding (88), who examined the transport of larger mucous plugs occluding the whole lumen of excised hen tracheas that were clamped distally. Transport of the mucous plug toward the proximal end of the trachea created a negative pressure of approximately 60 cm H 2 0 , which was attributed to mucociliary transport rather than air absorption. Although the general direction of mucous transport is cephalad, specific mucociliary streaming patterns have been described in mammalian airways. Hilding (89) described whirlpool formations and areas of stasis at the sites of airway divisions, and Iravani (90) observed focal derangement of metachronal coordination, ciliary inactivity, reversal of transport direction, and an abnormal pattern of mucous production in normal rat bronchi. Many investigators reported spiral mucociliary pathways in mammalian tracheas (including human subjects) (39, 91-93, and Wood, R. E.: Personal communication). The direction of transport appears to be clockwise when viewed from the larynx, at least in the northern hemisphere. Between such pathways, areas of stasis have been clearly demonstrated in intact normal tracheas. This should be taken into consideration when using single-particle techniques for mucociliary transport determination, because the results obtained may vary considerably depending on where the marker has been deposited. The work of Camner and associates (94, 95) suggests a

77

genetic determination of mucociliary clearance in human subjects. They reported highly comparable clearance patterns in monozygotic twins, less congruence in heterozygotic twins, and least similarity among normal unrelated nonsmokers. Two subjects with Kartagener's syndrome had almost no mucociliary clearance (95). Although the latter had chronic bronchitis, which by itself can impair mucociliary clearance mechanisms, the deposition patterns of aerosols differed considerably from those observed in patients with chronic bronchitis who did not have Kartagener's syndrome. Camner and associates (95) suggested a genetic defect of ciliary motion, which was also supported by the observation that spermatozoa from these 2 patients with Kartagener's syndrome lacked tail motility. In our laboratory, Goodman and co-workers (96) recently investigated the effect of aging in normal nonsmokers. Mean tracheal mucous velocity (5.7 mm per min) was significantly slower in the elderly subjects 56 to 70 years of age than in young healthy subjects 19 to 28 years of age (9.7 mm per min). All particles in the young subjects showed cephalad motion, whereas 10 per cent of deposited particles in the elderly subjects failed to show any movement at all. Because most of the elderly subjects had lived the major part of their lives in a northeastern industrial environment, it cannot be determined with certainty from these observations whether the age-related decrease in tracheal mucous velocity was an aging phenomenon or stemmed from long-term, low-level exposure to atmospheric pollutants. The exact mechanisms by which the cilia interact with mucus are still incompletely understood, although recent work has provided important new information. Particle transport has been shown to fail in the absence of mucus (38), but can be restored by placing autologous or heterologous mucus on the ciliated epithelium (38). The interaction between ciliary activity and mucus seems to be a function of the rheologic properties rather than the biochemical characteristics of mucus (97). Also, the thickness of the mucous layer appears to influence the propagation of mucus, i.e., surface particles move faster at a critical mucous thickness, with slower motions when the mucous layer is thinner or thicker (98). By placing mucus or sputum of different rheologic properties on ciliated epithelia, it has been shown that mucous transport is directly proportional to elastic recoil and inversely proportional to the viscosity of the sam-

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ADAM WANNER

pie (81, 99-101). Mucus with high elasticity and low viscosity is transported at the fastest rate, and the influence of elasticity appears to be more important than that of viscosity. It has been suggested that the ideal viscoelastic properties of mucus ensure the fastest mucociliary transport rate by optimal mechanical coordination of adjacent cilia (102). The results of these experiments have been supported by mechanical and mathematical models of mucociliary interaction (102, 103). The efficiency of the system appears to be decreased by thinning of the sol layer, increased viscosity of the sol layer, and decreased elasticity of the gel layer. Viscosity of the gel layer appears to be of secondary importance only, and gravitational and inertial forces seem to be negligible except under extreme conditions. In summary, these investigations indicate that (1) the direction of mucous transport in the tracheobronchial tree is basically cephalad, whereas the direction and rate of periciliary fluid transport are unknown; (2) the presence of mucus is required for surface particle transport; (3) mucous transport rates are influenced by the rheologic properties of mucus; and (4) the thickness of the mucus and possibly the periciliary fluid layer has an influence on mucous transport. A decrease of mucous transport rates from central to peripheral airways has been clearly demonstrated (19, 65, 91, 104), and may relate to the following, all of which decrease with increase in airway generation: ciliary beating frequency (48, 104), ciliary length, and number of ciliated cells (20). Despite this increase in mucous transport velocity toward the larger airways, the concept of a continuous mucous layer of 5-/>tm thickness throughout the tracheobronchial tree is difficult to accept because of the convergence of mucus toward the central airways, unless active resorption of mucus takes place in the airways, a mechanism that has not yet been demonstrated. The concept of focal distribution of mucus in the airways is more consistent with most experimental data than a continuous mucous blanket (105). Measurement of Mucociliary Function

The techniques that have been described for the determination of mucociliary function can be grouped into those which assess ciliary activity, those which measure the volume and physical properties of mucus and periciliary fluid, and finally, those which determine the efficiency of mucociliary interaction. The terms "mucous

transport" and "mucociliary clearance" are most frequently used to describe the latter function, although "mucous transport" should be reserved for techniques that measure particle transport in an anatomically defined airway. "Mucociliary clearance," as a rule, is used to describe the elimination of inhaled or insufflated aerosols from the tracheobronchial tree. Some of these methods are quantitative (ciliary beat frequency, angular velocity of cilia, particle transport rate, mucociliary clearance, and mucous rheology), whereas others are more descriptive and provide predominantly qualitative information (ciliary coordination, mucous streaming patterns, and nonmorphometric histologic studies). The anatomic features of the mucus-producing structures in the airway wall and the morphologic features of mucus in the airway lumen have been examined histologically, whereas the biochemistry and the viscoelastic properties of respiratory secretions usually require removal from the airway for examination. A great number of studies have been reported in which ciliary beat frequency and mucous transport rates have been determined separately in the same preparation; however, the direct effects of irritants or pharmacologic agents on ciliary activity cannot be determined in such preparations because of interaction between cilia and mucus, both of which might be primarily altered by the intervention. Therefore, modification of ciliary function should be assessed in preparations of cilia that are free of respiratory secretions. Cilia Ciliary function has been measured by using preparations of isolated cilia (106-109). The experimental techniques for separation, isolation, and reactivation of cilia have been reviewed by Goldstein (109). In vitro investigations on the physiologic and pharmacologic characteristics of cilia have been carried out in protozoa and metazoa (see 110 and 111 for reviews). Corssen and Allen (112) developed an in vitro technique for indirect assessment of ciliary function, and their method was subsequently used by other investigators (113, 114). In an organ culture, explants of ciliated human respiratory epithelium tend to curl up, so that the cilia cover the outside of a globe. Because of ciliary activity, these globes rotate, and the number of revolutions per unit time can be measured using a microscope. The direct effect of

CLINICAL ASPECTS OF MUCOCILIARY TRANSPORT

pharmacologic agents on ciliary activity is determined by introduction of the drug into the perfusion chamber. Isolated ciliated cells can be obtained from the respiratory tract by brushing (115) or other means (49) for examination under the microscope. Such preparations, however, may not be devoid of mucus. Finally, ciliary activity has been determined in mucus-free epithelial preparations (116). Measurement of ciliary beating frequency in preparations of respiratory epithelium has been accomplished by both in vitro and in vivo techniques (117, 118). In contrast to isolated cells or preparations of cilia in which a light beam can be transmitted through the preparation chamber, thus allowing observation of ciliary beating under the microscope, an incident light technique is required for the study of epithelial preparations. When a parallel light beam is directed at the epithelium along the visual axis, the flickering light reflection can be observed through the microscope, because the light is reflected either directly by the cilia or by the mucus surrounding the cilia. The flickering frequency can be measured by high-speed cinematography (119), the use of stroboscopy (114), a photoelectric cell (120), or the subjective synchronization of a variable acoustic signal with the ciliary flicker by the observer (115). The latter technique is probably inaccurate for frequencies greater than 600 to 900 beats per min. "Normal" ciliary beat frequencies vary considerably with temperature, humidity, and duration of experiment; values as low as a few beats per min up to values of approximately 1,600 beats per min have been reported. In mammals, the average "normal" frequency seems to be approximately 1,000 beats per min. Most in vivo investigations have used the tracheal window technique described by Dalhamn (121). In tracheas of normal cats, a mean ciliary beat frequency of 876 beats per min was associated with a mean particle transport rate of 10.5 mm per min (122). Mucus Interest in the rheologic properties of nonNewtonian fluids such as respiratory secretions has resulted in the development of many procedures and instruments capable of describing viscoelastic characteristics. Davis (123) and Aiache and Molina (124) have reviewed the various rheologic techniques for respiratory secretions. Although some of the methods are highly

79

sophisticated and permit the determination of elasticity and viscosity, there are many problems associated with such analyses. Inconsistencies in results have been related to inhomogeneity of the sample, decomposition of respiratory secretions after removal from the airways, dependence of rheologic properties on humidity and temperature, the inability to analyze frozen samples because of changes in consistency, day-to-day intrasubject variability, and the current lack of methods for obtaining normal respiratory secretions. Most investigators have used expectorated sputum, which probably is not representative of lower airway secretions because of contamination with saliva. Various modifications of the original techniques have been suggested to obtain more accurate results (125, 126), some of which require only small samples measuring 1 ml or less (127, 128); however, major advances in the examination of rheologic properties must come from improved collection of secretions and from methods capable of measuring viscoelastic properties in situ or in vivo. Wardell and associates (129), using a tracheal pouch method in dogs, found considerable interand intraindividual variability in the rheologic properties of respiratory secretions, which Lutz and associates (130) attributed to spontaneous variation in pH and ionic strength causing alteration of glycoprotein structure. Another technique for the collection of normal tracheal mucus uses a glass fiber screen placed within the trachea of normal dogs (131, 132) and gives secretion rates of approximately 1 mg of mucus per kg per min. The measurement of rheologic properties in situ has been described by Gilboa and Silberberg (133), who introduced a small glass probe into the mucous layer of an epithelial preparation at various depths. They calculated viscosity at different shear rates by displacing the preparation and measuring the transmitted force to the glass probe with appropriate transducers. Although their method measured the rheologic properties of mucus in situ, it cannot be applied in intact animals or human subjects. Recently, Michaelson and co-workers (134) described an in vivo method for rheologic measurements of respiratory mucus. A small 2-capillary viscometer, similar to that developed by Philippoff and associates (135) and Barnett and Dulfano (136) was connected to a polyethylene tube, which in turn was connected to a withdrawal pump allowing variable shear rates. This

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instrument could be passed through the inner channel of a bronchofiberscope and placed directly into the respiratory secretions within the airways. By knowing the geometry of the small distal capillary, both elasticity and viscosity could be calculated from the obtained pressure curve in the system. In addition to the possibility of measuring rheologic properties in vivo, this method requires only a small sample size (approximately 0.01 ml), and has the capability of estimating yield stress and surface tension. Perhaps this and other in vivo techniques will make it possible to describe more precisely the direct action of irritants and pharmacologic agents on the rheologic properties of mucus in normal subjects and patients with lung disease. Mucociliary Function Mucociliary activity, or the over-all function of the mucociliary apparatus, can be assessed by 2 basically different methods: airway clearance of inhaled particles (mucociliary clearance) and the transport of markers placed on the mucosa (mucous transport). The former has been widely used in intact animals and human subjects, whereas the latter is the usual in vitro technique but has also been widely used in vivo. A comparison of the results obtained by the 2 different methods is difficult, largely because of inherent methodologic variations. Most markers used to measure mucous transport are relatively large, do not penetrate into the mucous layer, and might reflect surface mucous velocity, whereas inhaled aerosols consist of small particles that might penetrate into the deeper layers of the mucus or periciliary fluid. The superficial and deep markers might not be transported at the same rate, but this has not been proved. Another major difference between the 2 techniques is that mucous transport is measured in anatomically defined airways in contrast to most aerosol techniques, which assess over-all regional mucociliary clearance in the lung. Mucociliary clearance. Morrow and associates (137) and a number of other investigators (138— 142) have examined the deposition and clearance patterns of inhaled aerosols. The differences in results among various investigations might be related to particle size and to the use of monodispersed or heterodispersed particles and the type, number, and location of scintillation detectors. Mucociliary clearance of inhaled particles depends on the deposition pattern because central zones normally have faster clear-

ance rates than distal zones (137). The deposition pattern itself has been found to depend on respiratory rate and tidal volume, and more central deposition of inhaled particles occurs in patients with obstructive lung disease. Because of the relatively long observation period of several hours required for these methods, particle clearance by coughing must be taken into consideration. In normal subjects, Morrow and associates (137) observed mean biologic half-lives of 2.7 min in the trachea, approximately 28 min in the intermediate zones, and 100 min to 2 months in the distal zones (depending on the depth of penetration), with comparable values for 1- and 6.5-fim particles. These biologic half-lives for central airways compared well with those reported by other investigators (139, 143). The overall biologic half-life of inhaled particles of intermediate size (6 fim) in the lung seems to be approximately 60 min (141). The slowest clearance rates (days) probably represent nonmucociliary clearance mechanisms (144). Yeates and co-workers (145) described a technique that combines the characteristics of both the mucociliary clearance technique and the mucous transport technique. Boli of albumin microspheres labeled with technetium-99m ( 99m Tc) in aqueous aerosol are inhaled by the subject so that the deposition occurs at different sites in central airways. The cephalad motion of individual boli is monitored with scintillation detectors. Tracheal mucous transport rates measured by this technique are considerably lower than those obtained with the surface particle transport methods. Mucociliary clearance has also been examined by insufflating tantalum powder into the airways and estimating its clearance semiquantitatively in selected airways (146). Although this radiographic method has the advantage of assessing anatomic rather than regional clearance, it is more invasive than the labeled aerosol techniques because it requires placement of a catheter in the trachea for insufflation of tantalum. Mucous transport. The techniques for in vivo and in vitro measurement of mucous transport rates in the airways were reviewed by Giordano and Morrow in 1972 (147) and Asmundsson and Kilburn (10) in 1973. In this review, attention will be directed to techniques that assess the effects of irritants and pharmacologic agents on mucous transport, or evaluate mucous transport in disease states (table 1). The principle of this method is based on the placement of one or more

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CLINICAL ASPECTS OF MUCOCILIARY TRANSPORT

TABLE 1 NORMAL IN VIVO TRACHEAL MUCOUS TRANSPORT RATES IN MAMMALS*

Reference

Mean Transport Rate (mm/min)

Species

Anesthesia

Berke and Roslinski (148)

Rat

General

Radiographic

Barium sulfate suspension

Dalhamn (121)

Rat

General

Microscopy (incised trachea)

Debris

13.5

Iravani (149)

Rat

General

Microscopy (exposed intact airway)

Debris and mucus

11

Cat

General

Translumination (microscope)

Carbon-lycopodium

13

Laurenzi et al. (151)

Cat

General

Translumination (microscope)

Carbon-lycopodium

20

Hilding (89) Giordano and Holsclaw (152)

Dog Dog

None General

Bronchoscopy Scintillation detector

Sakakura and Proctor (153)

Dog

General

Scintillation detector

"mTc-labeled resin beads

Marin and Morrow (154)

Dog

General

Scintillation detector

"

Wanner et al. (155)

Dog

General

Cinebronchofiberscopy

Teflon discs

13.5

Landa et al. (16

Sheep

None

Cinebronchofiberscopy

Teflon discs

17.5

Friedman et al. (157)

Man

Local*

Radiography

Radiopaque teflon diiscs

10

Yeates et al. (145)

Man

None

Scintillation detector

" m T c - l a b e l e d albumin microspheres

Goldhamer eta/.

(150)

(156)

Technique

Marker

India ink [ 9 9 m T c ] pertechnetate solution

m

Tc0

4

solution

6-10

14-15 10 10.5 16

3.5

Representative references. * Topical anesthesia in upper airway, not in trachea.

markers on the respiratory epithelium and the measurement of the distance traveled per unit time, from which mucous transport rate or mucous velocity can be calculated. Because of the easy accessibility and the relative simplicity of the preparation, most in vitro studies have been carried out using frog palates and esophagi, or mammalian tracheas (158-161). Some ciliated epithelial preparations require careful temperature control; in addition, in preparations exposed to air, adequate humidity must be ensured. India ink (91), lamp black (162), carbon-lycopodium (163), and cellular debris (121) have been used as markers, and their motion has been observed either by the naked eye or through a microscope. Dalhamn (121) described a method by which tracheal mucous transport can be observed through an incision or window in the rat trachea; this technique was later also adopted for cats (164). Translumination of the exposed intact cat trachea for the measurement of par-

ticle transport was described by Goldhamer and co-workers (150) and was extensively used by Laurenzi and associates (151, 165). Another interesting in vivo technique uses the temporary exteriorization of the mobile chicken trachea for the direct observation of particle transport (166). Iravani (167) described a method by which bronchi of a lobe are isolated so that both ciliary beat frequency and the transport of mucus and debris could be observed in rats and hamsters. As recognized by Iravani himself, artefacts might have been introduced into the measurement of mucociliary function because of the high inflation pressures (approximately 70 cm H 2 0 ) required to keep the airways open. Injection of barium sulfate (148, 168) or solutions of radioisotopes (153, 169) into the airway has alio been used to measure mucous transport rates either radiographically or by using 2 scintillation detectors (collimators). In contrast to the particle transport techniques, which allow averaging of individual particle transport

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rates, these methods determine the frontal movement of a solution or suspension that is faster than the average transport rate. A direct observation of particle transport or of mucociliary clearance in the nose is relatively easy to determine, but nasal transport or clearance rates may not correlate with transport and clearance rates in the lower airways (43, 170172). In our laboratory, we developed a cinebronchofiberscopic technique for the measurement of particle transport in the trachea of intact animals and human subjects (173). Standardized teflon discs are blown through the inner channel of the bronchofiberscope onto the tracheal mucosa and their cephalad motion is filmed. Because of considerable variability in individual particle transport rates due to the anatomic and functional characteristics of the respiratory mucosa, we analyze 10 to 20 particles distributed circumferentially around the airway lumen. As the discs approach the distal lens of the bronchofiberscope, they appear larger. By standardizing the disc size, film projection factor, and filming speed, velocities can be computed from image size and frame number. This method is also applicable in unanesthetized animals (156) and human subjects (174) using topical anesthesia (lidocaine solution), which does not depress mucociliary transport rates (156). The accuracy of the original technique has since been improved by more careful data processing (175). Because bronchofiberscopy is invasive, the method was subsequently improved by impregnating the teflon discs with bismuth trioxide to render them radiopaque (108). The motion of the radio-

paque teflon discs can be observed by a fluoroscopic image intensifier, and tracheal mucous velocity can be calculated by recording both the fluoroscopic image and time on videotape. Because the teflon discs can be blown into the airway through the vocal cords without having to intubate the trachea with the bronchofiberscope, topical anesthesia in the trachea is not required. The various methods used to measure tracheobronchial mucociliary clearance in man are summarized in table 2. Mean tracheal mucous transport rates vary between 5 and 35 mm per min in anesthetized mammals (147) and between 7 and 16 mm per min in anesthetized healthy dogs (152, 173) (table 1). These values compare well with those obtained in conscious sheep and normal human subjects, in whom values ranging from 6 to 18 mm per min have been measured (156, 157, 175). Particle transport rates are slower in peripheral airways, but precise in vivo transport rates in humans have not yet been reported in these airways. Physico-chemical Factors

Humidity and Temperature The effects of relative humidity on mucociliary transport mechanisms have been extensively studied. During quiet breathing, the inspired air is warmed to body temperature and is saturated with water vapor by the time it reaches the carina. Therefore, mucous transport in the tracheobronchial tree should be relatively insensitive to changes in atmospheric temperature and humidity. This has been shown to be the

TABLE 2 METHODS USED TO MEASURE TRACHEOBRONCHIAL MUCOCILIARY FUNCTION IN MAN Method

Reference (137)

Advantage

Disadvantage

Clearance of inhaled radioactive particles from the lungs

Morrow eta/.

Airway clearance of insufflated tantalum powder

Wood eta/.

Tracheal transport of discrete markers

Freidman et a/. (108), Sackner et at. (173)

Short observation period; measurement in anatomically defined airway

Invasive; limited to large airways

Central airway clearance of inhaled boli of radioactive microspheres

Yeatesefa/. (145)

Noninvasive

Because of low rates, changes d i f f i c u l t to detect

(146)

Noninvasive

Measurement in anatomically defined airways (including smaller airways

Clearance depends on deposition pattern; long observation period (effects of coughing) Semiquantitative; invasive; long observation period

CLINICAL ASPECTS OF MUCOCILIARY TRANSPORT

case in the dog (153), but not in the chicken. In this animal species, low ambient temperature and vapor pressure both slow tracheal mucous flow (169), an effect that does not appear to be related to body temperature. The difference between the observations in the dog and chicken might be related to anatomic and physiologic differences in the upper airways of these animals. The sensitivity of the lower airway mucociliary transport to changes in ambient temperature and humidity has not been evaluated in man, but breathing of ambient air at 10 per cent relative humidity for 8 hours does not alter nasal mucous transport in normal subjects (176). Just as a decrease in ambient humidity probably does not impair lower airway mucous transport during normal breathing through the intact upper airways, enriching the inspired air with water vapor by the use of heated aerosol devices or ultrasonic nebulizers does not seem to stimulate mucociliary clearance as long as coughing is suppressed (177). Adding humidity to the inspired air in the form of aerosols, however, may increase the effectiveness of coughing, thereby contributing to the elimination of secretions (178). Although clianges in ambient temperature and humidity seem to have little or no effect on mucous transport in the lower airways, Bohning and associates (179) found that decreasing the temperature of inhaled aerosols resulted in more central particle deposition and slower over-all tracheobronchial clearance, presumably owing to bronchoconstriction. This phenomenon should be taken into consideration when using particle clearance techniques for the assessment of mucociliary transport. Water deprivation for 24 to 48 hours has been shown to decrease markedly nasal and tracheal mucous flow in the chicken (180, 181). Morphologically, this was associated with anchoring of the mucous sheet to the epithelial surface (181). Rehydration restored the impaired mucociliary function to normal. In patients requiring artificial airways (endotracheal tube or tracheostomy cannula) for respiratory management, or in patients with permanent tracheostomies, control of the temperature and humidity of the inspired air is important. Proetz (182) was probably the first to emphasize the detrimental effect of drying on ciliary function. Dalhamn (121) subsequently demonstrated that exposure to dry air slows mucociliary transport in the rat trachea. This im-

83

pairment of mucous transport in the isolated trachea by exposure to dry air may be restored by nebulized moisture within 15 min after cessation of motion (183). In an in vitro preparation in which an isolated rabbit trachea was exposed to to-and-fro ventilation using air of varying relative humidity, the slowing of ciliary beat frequency induced by dry air was restored by the use of an artificial heat and moisture exchanger (artificial nose) (184). In intact dogs, Hirsch and associates (185) found that breathing of dry air at room temperature through an uncuffed endotracheal tube to bypass the nasal passages produced almost complete cessation of tracheal mucous transport after 3 hours; but in contrast to the isolated trachea preparation, subsequent breathing of air at 38° C and 100 per cent relative humidity restored tracheal mucous velocity to baseline after 3 hours. Similarly, tracheal mucous transport decreases in most intubated dogs exposed to 50 per cent relative humidity and in all dogs breathing 25 per cent relative humidity (186). It has been calculated that to maintain a safe level of 75 per cent relative humidity at the distal end of the endotracheal tube, a nebulizer capable of delivering 100 per cent relative humidity must be warmed to at least 32° C. It has not been determined whether the observed detrimental effect of dry air on mucociliary transport results from a primary ciliary dysfunction, changes in the rheologic properties of mucus, or both. The direct influence of humidity and the level of tissue hydration on ciliary activity have not been evaluated. As far as changes Mi the physical properties of mucus are concerned, the beneficial effects of hydration and humidity on the viscoelastic properties (decrease of viscosity) have been controversial (187-191). Histologic changes of the respiratory mucosa produced by exposure to dry air have been reported by several investigators. These changes consist of submucosal inflammation (192), focal sloughing of the ciliated epithelium (185), and ultrastructural changes in the ciliated columnar cells. The observations of a fairly rapid restoration of mucous transport after its impairment by exposure to dry air, despite the associated structural changes of the mucosa, underscore the remarkable functional reserve of the ciliated epithelium. Although there is no scientific basis for the need of adequate hydration and humidification of inspired air in persons in whom the normal "air conditioning" function of the up-

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per airway is eliminated by artificial airways or tracheostomies, such therapeutic measures may be clinically beneficial in patients with lung disease who are able to breathe through their normal upper airways, possibly by stimulating cough (177). The use of an artificial heat and moisture exchanger in tracheostomized patients aids in the maintenance of humidity and temperature in the lower airways at a level that is sufficient to maintain adequate mucociliary transport rates (184, 185, 193). The optimal temperature range for maximal ciliary frequency as studied under both in vitro and in vivo conditions in various homeothermic animal species appears to lie between 20 and 40° C, with ciliary stasis occurring at less than approximately 5° C and more than 43 to 45° C (119, 194-198). Fever per se is therefore in most cases not expected to impair mucociliary transport. Mercke and associates (118, 194, 199) further showed that when humidity was maintained constant while temperature was changed, ciliary beat frequency decreased by 50 per cent as temperature decreased from 40 to 20° C. The optimal temperature for ciliary activity was at normal body temperature. Inorganic Salts and pH Most of the work relating to the effects of inorganic ions on ciliary activity has been performed in protozoa. Alterations of ciliary activity by inorganic salts appear to be related to the cations (200), with potassium and calcium being the most important. An effect on ciliary activity by potassium has been demonstrated in protozoa (201-204), and in single cilia preparations, potassium increases angular velocity and beating frequency (106, 107). Calcium and potassium have opposing actions, and ciliary stimulation relates best to the calcium and potassium ratio (168, 205). An increase in hydrogen ion concentration impairs and a decrease stimulates ciliary activity (1, 206). Hee and Guillerm (206) have reported an increase in ciliary activity between pH values of 7.4 and 10 units, but the potentially beneficial effect of neutral or alkaline salt solutions on impaired mucociliary transport in disease states has not been investigated. Ionized Air and Radiations Krueger and Smith (207, 208) reported that ciliary activity was decreased by negative air ions and was increased by positive ions as ob-

served in tracheal preparations in various animal species. They suggested as the underlying mechanism the interference of air ions with the metabolism of 5-hydroxytryptamine (208, 209), a known stimulator of ciliary motion (107). These findings were subsequently challenged by Hee and Guillerm (206), Guillerm and co-workers (210, 211), Badre and associates (212), and Kennsler and Battista (213), who by using similar and different techniques were unable to reproduce the observations of Krueger and Smith (207, 208). Exposure to X-rays (500 to 7,000 rads) has been reported both to stimulate (214) and to depress (215) mucociliary clearance. Possibly, a transient cilioacceleratory phase is followed by impairment of mucous transport mechanisms owing to inflammation of the mucosa. Impairment by Inhalants

Cigarette Smoke The irritant effects of cigarette smoke on mucociliary transport were recognized by Mendenhall and Shreeve 40 years ago (216). By observing the movement of carmine particles on the mucosa of excised cats' tracheas, they demonstrated a decrease in transport rate by bringing the mucosa in contact with cigarette smoke either directly or by dissolving it in a solution in which the trachea was immersed (216, 217). The findings of these early experiments were later confirmed by Hilding (218) and Dalhamn (219) approximately 2 decades ago. Since then, a number of investigators have assessed the effects of cigarette smoke on ciliary activity and mucociliary transport. Because a variety of experimental techniques have been used by different investigators with a wide range of exposure periods, the reported results and conclusions have been either contradictory or difficult to compare. Whole cigarette smoke is composed of volatile elements and particulate matter. It has become customary to distinguish between the gas phase and the aerosol phase of cigarette smoke; the gas phase, by definition, consists of the components that remain after cigarette smoke has been "effectively" filtered by passing it through a Cambridge filter or millipore and glasswool filters (220-222). The total particulate phase of an average cigarette amounts to 30 to 40 mg (223). Its major constituents are nicotine, phenols, hydrocarbons, aldehydes and ketones, or-

CLINICAL ASPECTS OF MUCOCILIARY TRANSPORT

ganic acids, and alcohols. Although 95 per cent of the gas phase (approximately 300 ml per cigarette) consists of combustion products (N 2 , 0 2 , Co 2 , and CO) in concentrations that do not affect mucociliary transport, some trace gases seem to be of considerable importance (224). These include nitrogen dioxide, ammonia, cyanides, aldehydes, ketones, acrolein, and acids. As will follow in the subsequent discussion, some controversies still remain as to whether the gas phase or the particulate phase of cigarette smoke is primarily responsible for its depressant effect on mucociliary activity. This problem is relevant when comparing the effects on mucociliary transport mechanisms of low-tar versus high-tar cigarettes and filtered cigarettes versus nonfiltered cigarettes. Before discussing the effects of cigarette smoke on mucociliary function, the associated morphologic lesions in the tracheobronchial mucosa should be considered. In dogs that inhale cigarette smoke through a tracheostomy, histologic changes have been observed in the bronchi after 229 to 421 days of exposure (225). These consist of epithelial hyperplasia, decreased numbers of ciliated cells, and areas of squamous metaplasia. One might argue that this is not a good model of cigarette smoking in humans because by bypassing the upper airway, which might absorb part of the smoke to decrease its toxic effects, the irritant effect on the tracheobronchial mucosa could be enhanced; however, chronic inflammatory changes in the airways have also been observed in mice and guinea pigs that inhaled cigarette smoke via their upper airway passages (226, 227). Bronchiolar epithelial hyperplasia has also been reported in dogs that inhaled cigarette smoke for prolonged periods (228). The histologic changes in the airways of cigarette smokers are similar to those produced in animal models, and consist of varying degrees of denudation of the ciliated epithelium, an increase in the number of goblet cells, and squamous metaplasia (229). Morphometric studies have revealed an increased quantity of mucus in the airway lumen without histologic evidence of coexistent emphysema or a history of obstructive lung disease, whereas this is not observed in the lungs of healthy nonsmokers (230, 231). In young cigarette smokers, inflammatory changes with denudation of the epithelium and the presence of mucous plugging has been demonstrated in the bronchioles (230). These few representative studies suggest that aft-

85

er long-term exposure to cigarette smoke, an abnormality in mucociliary transport may at least in part be related to destruction of the ciliated epithelium. It has not been determined whether the presence of excessive mucus in the airway lumen is the result of hypersecretion or a failure in its elimination. Similarly, the direct effect of cigarette smoke on the biochemical and rheologic properties of respiratory mucus is unknown, although histochemical and rheologic studies suggest specific changes of respiratory mucus in chronic bronchitis. Short-term exposure. The acute effects of cigarette smoke on ciliary activity and mucous transport have been extensively studied in vitro, in animal models and in human subjects. With a few exceptions (e.g., 232), most investigators have demonstrated an irritant effect of cigarette smoke on ciliated epithelium in vitro, usually characterized by ciliostasis or decrease in particle transport. Residue of cigarette smoke passed through an aqueous medium have been shown to produce ciliostasis in protozoa (233, 234) and fragments of human respiratory epithelium (235). In fragments of rat trachea, brief exposure to whole cigarette smoke appears to elicit a byphasic response with a short period of stimulation during 1 to 2 min followed by a marked decrease in ciliary beating frequency (223, 236). In an excised rabbit trachea model, 71 1-ml puffs or 35 10-ml puffs of whole cigarette smoke were necessary to produce ciliostasis; similar relationships were demonstrated in the tracheas of living cats (237). It has been suggested that the ciliotoxic effects of cigarette smoke is not a particularity of tobacco, because nontobacco cigarettes made of grass and lettuce have also been shown to produce stasis, a decrease in 0 2 consumption, and ultrastructural changes of the cilia in protozoans (238). The controversy about the separate effects of the gas phase and the aerosol phase on mucociliary function has been reviewed by Dalhamn (220). In protozoa, both phases of cigarette smoke have been shown to possess ciliotoxic properties (239). Falk and associates (222) reported that particle transport showed a byphasic response on exposure to irritant gases with a rapid initial stimulation followed by a phase of depression and a recovery phase. Exposure to whole cigarette smoke for 30 sec resulted in a marked decrease in transport rates during the second phase with a minimum after 15 min and a tendency toward recovery after 45 min. Short-

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ening the exposure time resulted in a lesser degree of initial stimulation but comparable degrees of subsequent depression during the second phase. Removal of the particulate matter from cigarette smoke by passing it through filters decreased its depressant effect on particle transport, thereby indicating that the major effect on mucociliary transport is related to the particulate matter. In contrast, Kensler and Battista (221) incriminated the gas phase of cigarette smoke. They exposed strips of rabbit trachea to smoke from different cigarettes for 12 sec and identified hydrogen cyanide, ammonia, formaldehyde, acrolein, and nitrogen dioxide as gas phase constituents with a depressant effect on particle transport. These findings have been confirmed in intact animals (121, 240, 241). With regard to nicotine, the effects on mucociliary transport are less clear cut; however, more investigators have demonstrated a lack of effect (222, 236, 242) than a depression of mucociliary transport (241). Also, it has been suggested that there is a dose dependence, with stimulation at lower concentrations and depression at higher concentrations (243). Additives of cigarette tobacco such as menthol do not interfere with mucociliary transport (242). Phenols have been known to impair ciliary activity and mucous transport both in vitro (244-246) and in vivo (246) and Dalhamn and Legerstedt (244) and Dalhman (246) have even attempted to relate the toxicity of various phenols to their boiling points. The mechanism by which the various constituents of cigarette smoke interfere with mucociliary transport is unknown. Based on experiments in the fresh water mussel, it has been suggested that the ciliostatic effects of these components depend on their pH in solution, and that aliphatic acids are more ciliotoxic than phenols (247). It should be pointed out, however, that such in vitro experiments requiring an aqueous medium do not necessarily reflect the type of exposure that occurs in smokers in whom contact between cigarette smoke and the ciliated epithelium is made by impingement or bypass. In animal experiments, ciliary dysfunction and impairment of mucous transport by shortterm exposure to cigarette smoke have been demonstrated in rats (248, 249), rabbits (249, 250), cats (241, 249, 251, 252), dogs (236), and donkeys (240, 253) using a variety of different techniques. Only a few reports have appeared demonstrating a failure of short-term exposure

to cigarette smoke to depress mucociliary activity in animal models (254, 255). The reasons for the discrepancy between these and the previously listed studies are not quite clear but may well be related to methodology. The cigarette smoker inhales through the mouth, whereas many animals are nose breathers. The deposition pattern of cigarette smoke components in the upper airways is different for these 2 types of breathing. The pattern of cigarette smoke inhalation appears to be an important factor in the retention of cigarette smoke in the respiratory tract. Using a smoke-dosage apparatus that delivered standard puffs, Dalhamn and associates (256) reported that in human subjects there was 86 to 99 per cent retention of most components of the gas phase and particulate phase of cigarette smoke with the exception of CO, of which only 54 per cent was retained. They also examined the mouth absorption of various components of cigarette smoke in human subjects by having them hold cigarette smoke in the mouth for 2 sec (257) and found that 60 per cent of the water-soluble components and 20 per cent of the water-insoluble components of the gas phase, along with 16 per cent of particulate matter was absorbed and retained in the upper airways. This indicates a marked modification of cigarette smoke during its passage through the upper airways, which might decrease its ciliotoxic effect in the lower airways. This is supported by experiments using exposed cat tracheas showing that passing unfiltered cigarette smoke through a chamber with wet surfaces before bringing it in contact with the ciliated epithelium decreased the cilioinhibitory capacity of cigarette smoke (252). Similar observations have been made in miniature donkeys by passing cigarette smoke through a water trap (253). In the donkey, it has been also demonstrated that exposure to whole smoke of as many as 30 cigarettes does not impair nasal mucociliary transport (257), whereas the same number of cigarettes clearly altered the tracheobronchial deposition and clearance of radioactive aerosols (253), suggesting that with respect to cigarette smoke, nasal mucociliary transport might not be a good marker of tracheobronchial mucociliary transport. By using a nasal catheter for the delivery of cigarette smoke as a simulation of human smoking, 2 cigarettes produced acceleration of the clearance of radioactive aerosols from the lung periphery, whereas the inhalation of 10 to 20 cigarettes resulted in a transient generalized impairment of

CLINICAL ASPECTS OF MUCOCILIARY TRANSPORT

lung clearance (240). Exposure of guinea pigs to cigarette smoke of 1 to 4 cigarettes per day for 1 to 4 weeks has been shown to cause a decrease in particle transport and clearance of bacteria, with the impairment of mucous transport preceding the impairment of bacterial clearance (258). In these and other similar experiments (251, 259), the addition of an anti-inflammatory and antitussive agent, phenyl-methyl-oxadiazole (and chemically related substances) partially counteracted the toxic effects of cigarette smoke by allowing less decrease in mucous transport for a given period of exposure, or by increasing the number of puffs required for ciliostasis to occur. Measurements of mucociliary clearance in man immediately after the smoking of 1 or more cigarettes have shown conflicting results, i.e., increased (140, 260, 261) or unchanged (145) mucociliary clearance rates have been reported after inhaling aerosols of particles tagged with radionuclides. Transient stimulation of bronchial clearance has been reported both in smokers and nonsmokers (218). Comparison among the aerosol studies is difficult because the results depend on the deposition of the various aerosols in the lungs, which in turn is a function of the diameter and density of the particles and airflow and breadiing pattern during inhalation. Yergin and co-workers (262) showed that the transport of teflon discs in the trachea was increased, decreased, or unchanged in smokers within 10 min after smoking a cigarette. The type of response did not seem to be dependent on baseline tracheal mucous transport velocity, which was decreased in all smokers, nor did it make any difference whether or not there was physiologic evidence of minimal airway obstruction. One might conclude from the studies reported to date that, whereas short-term exposure to cigarette smoke depresses mucous transport in vitro and in animal models, the smoking of one or more cigarettes by smokers and nonsmokers has variable effects on mucous transport. Long-term exposure. The long-term effects of cigarette smoking have been studied in animals and human subjects. Wanner and associates (155) measured tracheal mucous velocity in purebred beagle dogs exposed to cigarette smoke (100 cigarettes per week) for 13.5 months by means of a mask through which cigarette smoke was administered through both the mouth and the nose for 1.5 hours twice daily (155).

87

Tracheal mucous velocity in dogs exposed to cigarette smoke was decreased to approximately 30 per cent of the velocity observed in control animals. Lung compliance and resistance, single-breath diffusing capacity, lung volumes, and arterial blood gases did not differ significantly between the 2 groups, suggesting that suppression of mucociliary activity associated with longterm cigarette smoking may precede abnormalities of pulmonary function as determined by conventional methods. Although no histologic studies were performed in these animals, secretions, increased vascular markings, and edema of the tracheal mucosa were observed in all but one of the animals exposed to cigarette smoke, whereas none of diese changes was seen in control animals. Similarly, Iravani and Melville (263) demonstrated a decrease of mucous transport and ciliary frequency in the airways of hamsters exposed to cigarette smoke for 1 year; however, in rats that were also exposed for 1 year under almost identical conditions, both mucous transport and ciliary frequency appeared to be increased, but qualitative analysis of mucociliary function revealed zones of ciliary inactivity, disturbance of ciliary coordination, and impaired mucous production. With regard to the long-term effects of cigarette smoking in human subjects, investigations using the aerosol clearance techniques reveal conflicting results. Thus, some investigators (143, 145, 264, 265) have not been able to demonstrate an abnormality in the clearance of inhaled radioactive aerosols in habitual cigarette smokers, whereas others (266-268) reported decreased clearance rates. Among the studies in which the abnormal deposition pattern and the effects of coughing on mucociliary clearance were taken into consideration, Louren^o and associates (266) found that the deposition pattern of 2-/um iron particles was comparable in healthy smokers and nonsmokers, whereas in the smokers there was delayed over-all clearance with accumulation of particles in central airways. Results of pulmonary function tests were within the normal range in the smokers. Bohning and co-workers (269) studied the deposition and clearance of 7-fim diameter particles in the tracheobronchial tree of 6 pairs of monozygotic twins, 4 of whom were discordant for cigarette smoking. They found comparable over-all mucociliary clearance in the smoking and nonsmoking pairs, but more central deposition and slower central clearance in the smokers. In a similar

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study Camner and Philipson (268) showed in 10 pairs of twins discordant for cigarette smoking a significantly lower average clearance rate in the smokers compared to the nonsmokers; in 5 pairs the smoker had a slower clearance rate than the nonsmoker, whereas in the remaining 5 pairs there was no difference between the smoker and the nonsmoker. By radiographically determining the motion of radiopaque teflon particles on the tracheal mucosa, Goodman and associates (96) found that tracheal mucous velocity was decreased in young healthy smokers to approximately one fifth of the value obtained in healthy nonsmokers. In the same study, they demonstrated comparable degrees of mucous transport impairment in young healthy smokers with normal pulmonary function tests, young healthy smokers with evidence of minimal airway obstruction, patients with simple bronchitis, and patients with obstructive bronchitis. Only one of 6 healthy smokers had a tracheal mucous velocity value in the normal range. The bulk of the evidence indicates that long-term cigarette smoking alters mucociliary transport mechanisms, that these changes can occur as early as 1 year after the start of cigarette smoking, and that impairment of mucous transport may precede cigarette smoke-associated abnormalities in the respiratory function of the lung. Once a cigarette smoker develops chronic bronchitis, mucous transport appears to be irreversibly damaged, because impairment has been demonstrated even in patients who have abstained from cigarette smoking for many years (174). On the other hand, partial recovery of mucociliary transport has been observed in cigarette smokers after cessation of 3 months or more but not after 1 week of cessation (267). Most of these subjects were asymptomatic without clinical signs and symptoms of chronic bronchitis. These observations have also been supported by animal experiments (270). Effects of filters. Because the toxic effect of cigarette smoke on mucociliary transport mechanisms seems to reside both in the gas phase and in the particulate matter, the filtering of cigarette smoke before inhalation may be protective. The histologic changes in the airways of guinea pigs exposed to unfiltered cigarette smoke for 4 to 8 weeks were not seen when cigarette smoke was passed through a Cambridge filter (227). Dalhamn (249) showed in cats that a longer exposure time was needed for ciliostasis to occur

with smoke from filter-tip than unfiltered cigarettes. Falk and associates (222) failed to demonstrate a difference between low- and high-tar cigarette residue with regard to mucociliary transport in vitro. In intact animals, the impairment of mucociliary clearance by 10 to 20 cigarettes was twice as severe with unfiltered smoke as with filtered cigarette smoke (glass-fiber filter). Kaminski and co-workers (252) showed that charcoal and cellulose acetate filters provided protection to mucociliary activity in the cat trachea, but there was no added protection when the toxicity of cigarette smoke was decreased by passing it through a chamber containing wet surfaces, a simulation of inhalation of cigarette smoke in humans. The depressant activity of whole cigarette smoke on mucociliary transport has been found to be markedly decerased by the use of filters containing activated charcoal granules (221). In exposed cat tracheas, short-term exposure to a standardized dose of cigarette smoke decreased particle transport rates by 50 per cent when unfiltered smoke was used, by 40 per cent when the cigarette smoke was passed through a cellulose acetate filter, and by 20 per cent when a carbon-cellulose filter was used (241). It appears that appropriate filters offer partial protection against the depressant effect of inhaled cigarette smoke on mucociliary transport. Better protection might be expected from filters that remove components of both the particulate and the gaseous phases of cigarette smoke, such as a combination of cellulose and charcoal (271). Atmospheric Pollutants A variety of atmospheric pollutants have been shown to exert an irritating effect on the respiratory mucosa that, for most irritants, is characterized by a depression of mucociliary function. The vulnerability of the mucociliary apparatus on exposure to exogenous chemical agents and irritant gases was demonstrated 35 to 40 years ago (272, 273). Since then, a voluminous literature has been accumulated on the effects of atmospheric pollutants, in both in vivo and in vitro preparations. This discussion will be limited to 3 major gaseous air pollutants, sulfur dioxide (S0 2 ), nitrogen dioxide (N0 2 ), and ozone, and inorganic particles, a group of potentially hazardous sulfate and nitrate salts that have recently received renewed interest as byproducts of catalytic converters. Camner and

CLINICAL ASPECTS OF MUCOCILIARY TRANSPORT

Philipson (274) were unable to demonstrate a difference in mucociliary clearance in a group of twins of whom one of the pair lived in an urban area and the other in a rural area of Sweden. Nevertheless, atmospheric pollutants should not be disregarded as being potentially harmful to mucociliary transport in the airways. Sulfur dioxide. Short-term exposure or exposure for a few weeks to high concentrations of S 0 2 has been shown to alter the mechanical properties of the lung by causing parenchymal damage (275-277). In addition, the airways of various animal species show squamous metaplasia, destruction of cilia, and goblet cell metaplasia in intermediate-sized and small bronchi with accumulation of mucus (278-280). Because S 0 2 stimulates the mucus-secreting structures of the bronchi, it has been used to produce an experimental model of chronic bronchitis in the rat (281-282). In 1956, Dalhamn (121, 283) reported ciliostatic effects in the tracheas of rats exposed to S 0 2 at a concentration of 10 ppm, and these findings were later confirmed by other investigators demonstrating the detrimental effects of short-term exposure on mucociliary activity by in vitro (163) and in vivo (284) methods. In healthy nonsmokers, short-term exposure to S 0 2 at a concentration of 5 ppm appears to cause a transient impairment of nasal (285) and tracheobronchial (286) clearance. The mechanisms by which S 0 2 interferes with mucociliary transport are not well understood. Both ciliostasis (121, 283) and the elaboration of abnormal mucus (281) have been demonstrated under experimental conditions using high-level exposures, but the temporal relationship between these 2 pathophysiologic processes is not known. Furthermore, exposure to such high concentrations of S 0 2 seems to produce early pathologic changes of the epithelium such as edema and desquamation, thereby obscuring the possibility of a primary functional impairment. It has been suggested that the irritant effect of S 0 2 results from its conversion to sulfuric acid, because the combination of aqueous aerosol with S 0 2 seems to potentiate its pulmonary toxicity (287). There are indications that the functional and structural abnormalities resulting from short-term exposure to S 0 2 and sulfuric acid might impair pulmonary defense mechanisms against bacterial and viral infections by decreasing the clearance of streptococci from the lungs (289) and favoring the development

89

of pneumonia after exposure to virus (290); however, it is unclear to what extent mucociliary dysfunction contributes to the abnormal bacterial clearance. Although the impairment of mucociliary transport by short-term high-level exposure to S 0 2 has been clearly established, the significance of long-term low-level exposure in man in its relation to morbidity is uncertain. In beagles exposed intermittently to 1 ppm of S 0 2 for 12 months, we found a slight but significant impairment of mucous transport, but no demonstrable changes in mechanics of breathing or gas exchange (92). Long-term exposure to low concentrations of S 0 2 might produce an impairment of mucociliary transport as one of the first signs of pulmonary dysfunction. These observations may be of more relevance to the potentially harmful effects of S 0 2 on mucociliary transport in man than short-term high-level experiments, because the concentration of S 0 2 in the canine experiment approximates the concentrations recorded during peak yearly measurements in major metropolitan areas with industrial as well as automobile pollution (0.35 to 1.69 ppm in Chicago, Cincinnati, Philadelphia, and Washington, D.C.), and the concentrations of S 0 2 measured 0.5 to 15 miles from point sources of S 0 2 (0.47 to 5 ppm during 1 hour), such as smelters and coal-burning power plants (291). It is likely that the actual concentration of S 0 2 to which the lower airway mucosa was directly exposed in these dogs was less than 1 ppm because of absorption of S 0 2 in the upper airways (292). Although these findings cannot necessarily be extrapolated to humans, they indicate that low-level exposure to a single air pollutant at a concentration commonly encountered in urban areas irritates the respiratory mucosa of the lower airways as evidenced by slight depression of mucociliary transport. Possibly more significant abnormalities could result from a combination of different air pollutants in low concentrations, the usual atmospheric condition of urban areas. Nitrogen dioxide. Nitrogen dioxide results from burning fossil fuels and tobacco (293). Inhalation of this gas for short periods produces bronchoconstriction in dogs (20 ppm) (294) and man (5 ppm) (295). The pulmonary lesions associated with both short-term and long-term exposure to N 0 2 in a variety of animal species have been well described, and al-

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though a multitude of morphologic changes have been described, including parenchymal lesions, bronchiolitis appears to be common to all studies (147, 296-301). The typical histologic features of bronchiolitis are loss of cilia in terminal bronchioles, epithelial hypertrophy, narrowing of the airway lumen, and excessive bronchial secretions. The long-term threshold concentration for the development of pulmonary lesions is approximately 5 ppm (302). Subtle histologic changes, however, have been produced by long-term exposure to concentrations as low as 2 ppm (301). Long-term low-level exposure to N 0 2 also appears to increase the susceptibility to bacterial infections in mice (303). No controversies have arisen regarding the effects of N 0 2 on mucociliary transport mechanisms. Short-term high-level exposure has been clearly shown to produce ciliostasis within 5 min (121, 273) and a decrease in mucociliary transport rates (163, 164). In vitro, concentrations higher than 30 ppm seem to produce irreversible ciliostasis (273). Exposure of rats to 6 ppm of N 0 2 during a 6-week period produced a decrease in mucociliary transport rates associated with mucosal edema (147); the recovery of mucociliary dysfunction occurred within a week after cessation of exposure. Although pathologic abnormalities have been observed with long-term exposure to concentrations as low as 2 ppm, the effects on the mucociliary function under conditions that may approximate real-life atmospheric concentrations have not, to our knowledge, been investigated. The concentrations in urban atmospheres are less than 1 ppm, even during heavy smog (304), with the air quality standard being 0.05 ppm (305). Ozone. Ozone is the major component of photochemical smog. In metropolitan Los Angeles, the median annual ozone concentration is approximately 0.07 ppm, with extreme values as high as 0.58 ppm. Although short-term exposure to near-ambient concentrations of ozone under experimental conditions produces mild abnormalities in pulmonary function (306), it has not been determined whether ozone at ambient concentrations encountered in high-risk areas should be considered as an etiologic factor in the development of chronic lung disease (307). Ozone is actively removed from the inhaled air in the upper and central airways, so that for a given ambient concentration, the concentration that reaches the smaller airways may be quite low (308). In contrast to normal sub-

jects, however, ozone may well be a significant risk to patients with hyperreactive airways. There have been few studies dealing with the effects of ozone on mucociliary transport mechanisms. Short-term exposure of the frog palate in a chamber by either impingement or bypass did not produce any changes in particle transport rates up to a total contact "dosage" of 20 /tg, whereas higher "dosages" resulted in transient stimulation of mucous transport for as long as 16 min (309). Similar observations have been made with other preparations (213, 310), but studies in intact animals or humans have not been reported. The pulmonary lesions associated with exposure to ozone have been more extensively studied (311). Both short-term and long-term exposures to concentrations of 1 ppm or more have been shown to produce mucosal lesions similar to those observed after exposure to N 0 2 but located more peripherally (312, 313). They are characterized by thickening of the terminal and respiratory bronchiolar epithelium, squamous metaplasia, epithelial hyperplasia, and ultrastructural changes in the cytoplasm. The latter has been taken as an indication of the activation of oxidative metabolic processes by ozone (314). Similarly, the development of squamous metaplasia has been related to a deficiency of vitamin A (313), because vitamin A is readily oxidizable (315). Although the pathologic lesions are most severe at the bronchiolar level, interference with ciliary activity and mucous production may also be expected in the central airways, considering the high metabolic activity of ozone. Studies of mucous transport in intact animals and in man will be needed to evaluate the effects of inhaled ozone at ambient concentrations. Inorganic sulfates and nitrates. Inorganic nitrate and sulfate particulate matter is released into the atmosphere by automobile engine combustion and catalytic and photochemical oxidation of the combustion products. Most physiologic investigations have been directed toward examining the pulmonary effect of their gaseous precursors, N 0 9 and S0 2 . Epidemiologic studies indicate that long-term exposure (5 to 10 years) to an atmospheric environment high in S0 2 and suspended particles of nitrate and sulfate salts may depress the pulmonary function of school children (316). These and other data (317) suggest that the inorganic particulate matter might have adverse pulmonary effects. In cities of the United States, concentra-

CLINICAL ASPECTS OF MUCOCILIARY TRANSPORT

tions of nitrate salts range as high as 4.3 fig per m 3 ; of sulfate salts, as high as 21 fig per m 3 . No long-term low-level exposure experiments with inorganic sulfates and nitrates have been conducted to date. In conscious sheep who breathed aerosols of sulfate and nitrate salts, tracheal mucous transport rates remained unchanged for a 90-min observation period (316). The concentrations used in these short-term experiments ranged from 340 to 950 fig per m 3 , concentrations that are approximately 100 times higher than those found under ambient conditions. Sulfuric acid mist, another byproduct of catalytic converters, also failed to impair tracheal mucous transport in conscious sheep who breathed 1,000 fig per m 3 for 20 min. Short-term exposure of normal subjects to sulfuric acid mist did not alter conventional tests of pulmonary function or cause subjective discomfort (318), but effects on mucous transport have not been investigated. These preliminary experiments will have to be extended to longer exposure periods to ascertain the lack of a deleterious effect on mucous transport when breathing these agents. Oxygen The influence on mucociliary transport mechanisms of clianges in 0 2 concentration have been extensively studied in vitro, in animals and in man. Hyperoxia. Although the vulnerability of the alveolar membrane to high 0 2 concentration is well known (319), the effects of 0 2 on the respiratory mucosa and mucociliary transport mechanisms have only recently been recognized. In vitro, high concentrations of 0 2 have been reported to decrease (320) or not affect (321) mucociliary function. In the former experiments, ciliary stasis and loss of particle transport were observed in explants of tracheal epithelium of neonates after 2- to 4-day exposure to 80 per cent 0 2 (320). During the first 2 to 3 days of exposure, there was an increase in mucin and lysozyme secretion followed by a decrease in secretory function. These functional abnormalities correlated with histologic evidence of squamous metaplasia, degeneration and sloughing of ciliated cells, and loss of goblet cells. Boat and associates (320) suggested that the secretory malfunction might have been related both to the loss of goblet cells and to dysfunction of submucosal glands. In contrast to this study, Guillerm and associates (321) did not

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find an impairment of ciliary activity in fragments of sheep tracheas exposed to 100 per cent 0 2 for "prolonged" periods even under hyperbaric conditions, but the exposure time was not specified. In vivo data have been less controversial. With the exception of one group's observation (154), studies in intact animals indicate that high concentrations of inspired 0 2 deleteriously affect the morphologic features and function of the respiratory mucosa (165, 322-328). Laurenzi and associates (165) demonstrated an inverse relationship between tracheal mucous flow and 0 2 concentration within an exposure time of 20 min in anesthetized cats. Exposure to 25, 40, and 100 per cent inspired 0 2 concentrations resulted in decreases of tracheal mucous flow by 16, 18, and 37 per cent, respectively. In anesthetized dogs, the depression of mucous transport caused by breathing high concentrations of 0 2 takes several hours. We found that tracheal mucous velocity decreased significantly, to 50 per cent of the baseline value, after 3 to 4 hours of breathing pure 0 2 (322). In a 30-hour exposure study using the same animal model, Sackner and associates (326) also showed that the severity of mucociliary dysfunction is related to both inspired 0 2 concentration and exposure time. Breathing an 0 2 concentration of 75 per cent for 9 hours or 50 per cent for 30 hours decreased mucous transport by 40 and 50 per cent, respectively. By taking a 50 per cent decrease from baseline tracheal mucous velocity as an indicator of 0 2 effect, they were able to demonstrate an inverse logarithmic relationship between inspired 0 2 concentration and exposure time required for this effect to occur. Although similar histologic lesions have been described by various investigators, the duration of exposure until such changes can be observed varies from investigator to investigator, and is possibly related to the choice of laboratory animal. The earliest morphologic abnormalities in mice breathing pure 0 2 have been reported to occur after 3 days and to consist of changes in the mitochondria and the cell membrane of ciliated cells, with an acute necrotizing tracheobronchitis occurring after several days (323, 327). In dogs, histologic evidence of acute tracheobronchitis has been observed as early as after 6 hours of breathing pure 0 2 or 12 hours of breathing 75 per cent 0 2 (326). Because the impairment of mucociliary function and the histologic changes appear to occur concomitantly, it

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is difficult to determine whether hyperoxia leads to mucociliary dysfunction directly and/or indirectly by producing structural changes in the ciliated epithelium. Whatever the mechanism or mechanisms might be, it appears that both morphologically and functionally, the damage to the ciliated airway epithelium precedes the parenchymal lesions (324). Retrosternal tickling and pain suggesting acute tracheobronchitis are the earliest clinical symptoms in man during pure 0 2 breathing (329, 330). These symptoms occur from 4 to 24 hours after the beginning of breathing pure 0 2 at 1 atmosphere (atm), and from 3 to 5 hours after start of breathing pure 0 2 at 2 atm. No associated changes in airway resistance have been observed with 0 2 breathing at 1 atm (331), and only borderline increases in airway resistance (+30 per cent) have been observed after 5 hours of breathing 0 2 at 2 atm (332). On the other hand, in normal subjects who breathed 90 to 95 per cent 0 2 for 6 hours, tracheal mucous velocity was found to decrease significantly to 50 per cent of baseline with endoscopic signs of tracheitis but no physiologic signs of bronchoconstriction (93). These observations confirm the conclusions drawn from animal experiments that mucociliary dysfunction might be one of the earliest signs of 0 2 toxicity. "Safe" limits of 0 2 therapy with regard to mucociliary function, for both duration and concentration of 0 2 supplementation, have not been established in patients with pulmonary disease who might be more resistant to high concentrations of 0 2 because of prior injury to the airways. Some of the pathophysiologic consequences of the 0 2 -related impairment in mucociliary transport mechanisms, such as possible bacterial colonization of the lower airways, is of clinical relevance but remains to be demonstrated. A shift of the upper airway flora from oropharyngeal commensals to potential pathogens such as Staphylococcus aureus and coliform bacilli has been reported after long-term inhalation of pure 0 2 (333), but to our knowledge, no such studies have been conducted in the lower airways. Hypoxia. Anoxia produces reversible ciliary stasis in vitro (321, 334-336); in lower animals as well as in excised mamalian tracheas, an 0 2 concentration between 1 and 3 per cent is sufficient to maintain normal ciliary activity (321, 334, 336). Dalhamn and Rosengren (335) found in an excised rabbit trachea preparation

that in the absence of 0 2 , ciliary stasis occurs after 60 min in most specimens, with a mean time interval for ciliary stasis of 20 sec. The effects of hypoxia on mucociliary transport in intact animals are less clear. Whereas 13 per cent 0 2 breathing for 10 min in dogs did not affect tracheal mucous transport rates (154), 10 per cent 0 2 breathing for 20 min decreased tracheal mucous flow in cats by 30 per cent (165). The mechanism by which anoxia or severe hypoxia might affect mucociliary activity has been studied in protozoa (337) and in the oyster gill (338-340). Inhibition of oxidative phosphorylation resulted in a decrease of ciliary activity by 50 per cent (340), and addition to the preparation of cyanide, which blocks uptake of 0 2 by the respiratory enzymes, decreased ciliary activity by 85 per cent (339). This suggests that although the energy requirements for ciliary movement are predominantly met by aerobic metabolism, a residual ciliary function can be maintained by anaerobic processes. This is further supported by the effects of inhibitors of anaerobic glycolysis such as monoiodoacetic acid and sodium fluoride, both of which slightly depress ciliary activity (338). Thus, extreme hypoxia impairs mucociliary activity, but an effect of moderate to severe degrees of hypoxia consistent with life has not been unequivocally demonstrated. Hypercapnia. Exposure of Anodonta gill to C 0 2 by bypass results in ciliary stasis (335). A negative correlation exists between Pco 2 and ciliary activity in fragments of sheep trachea (321), and breathing of a normoxic gas mixture containing 7.5 per cent C 0 2 decreases tracheal mucous transport rates in anesthetized dogs (154). It is unclear whether C 0 2 has a direct ciliotoxic effect or impairs mucociliary transport mechanisms by decreasing tissue pH. Inhalation Anesthetics General anesthetics depress mucociliary clearance in animals (156, 341); in human subjects, the combination of interventions associated with general anesthesia results in decreased tracheal mucous transport rates, as suggested by Lichtiger and associates (342). Although Lichtiger and associates demonstrated a progressive decrease in tracheal mucous velocity as measured with the cinebronchofiberscopic technique during a 90-min period in nonsmokers undergoing gynecologic surgery, they realized that the mechanical effect of the endotracheal tube, the pre-

CLINICAL ASPECTS OF MUCOCILIARY TRANSPORT

medication with atropine and barbiturates, the use of muscle relaxants, and changes in the temperature and relative humidity of the anesthetic gases, all may have contributed to the observed depression in mucous transport rates. Therefore, they were unable to separate out the role of halothane and nitrous oxide, which were used in these patients. The direct effects of inhalation anesthetics on ciliary activity have been examined by Nunn and associates (343) in protozoans. They observed a depression of ciliary activity by a variety of anesthetic gases. In dogs anesthetized with 25 mg of thiopental per kg of body weight, halothane has been shown to impair the transport of radioactive droplets in the trachea (344). In this study, a reversible depressant effect of halothane on mucous transport was suggested by changing the minimal alveolar concentration from 0.6 to 2.4 per cent and comparing the values for mucous transport rates obtained at these levels to the baseline values after thiopental anesthesia. The maximal depression of tracheal mucous transport rate to 27 per cent of baseline was observed at a minimal alveolar concentration of 2.4 per cent, with return toward baseline values as the minimal alveolar concentration was decreased. Although the results represent the combined effects of thiopental and halothane, the reversible changes in mucous transport rates induced by manipulating the minimal alveolar concentration for halothane strongly suggest an independent depressant effect of halothane on mucociliary activity. This has recently been confirmed in rats (345). Miscellaneous In addition to atmospheric pollutants, cigarette smoke, and 0 2 , all 3 of which have been extensively studied with regard to their effects on structure and function of the respiratory mucosa, a number of other gases and aerosols have also been demonstrated to produce mucociliary dysfunction. Thus, in vitro experiments using short-term exposure demonstrated ciliotoxic effects for gases of ammonia, chlorine, hydrochloric acid, sulfuric acid, formaldehyde, hydrogen sulfide, cyanide, and chloroform (121, 213, 345). Some of these irritant gases might exert their effect by changing the pH of the medium (213, 346). Carbon monoxide, even in high concentrations, does not alter mucociliary transport mechanisms (213, 334). Long-term exposure of workers to chromates

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in the chrome plating and chromate-producing industry has been implicated as an etiologic factor in chronic respiratory disease characterized by tracheobronchitis and respiratory tract infections (347, 348). Mass and Lane (349) examined the effects of chromates on the morphologic features and ciliary activity of the rat tracheal epithelium by adding calcium chromate and sodium chromate salts to the culture medium. They showed a differential effect on structure and function of the epithelial cells, in that ultrastructural evidence of cytotoxicity was already seen after a few hours at concentrations of 10 to 100 /jig per ml, whereas a concentration of 10 mg per ml was required to produce ciliostasis within 20 min. At this level of exposure, cell death occurred within 24 hours. The changes seemed to be related to surface damage of the epithelial cells. These observations are of particular interest, because they suggest that at least in this model, ultrastructural changes of the cell membrane can be present without a detectable compromise of ciliary function. Fluorochlorohydrocarbons (Freon®) are in widespread use as propellants in personal cosmetic sprays, household sprays, or proprietary bronchodilator sprays; the tracheobronchial mucosa is therefore exposed to Freon either by direct inhalation or as an indoor air pollutant. In contrast to the potential cardiovascular effects of Freon, which have been a matter of dispute (350, 351), inhalation of Freon does not impair mucociliary transport in animals (352, 353) or normal subjects (157). Of the various commercially available aerosol sprays, hair sprays have probably generated the most interest with respect to their relationship to lung disease. Infiltrative lung lesions have been repeatedly associated with hair sprays (353, 354), although several surveys of hairdressers have in general failed to uncover cases of hair spray-related respiratory problems (355). Similarly, experimental toxicologic studies in animals have not supported the hypothesis of hair spray-related lung disease (356, 357). The possible transient changes in the maximal expiratory flow-volume curve after exposure to various hair spray preparations are also controversial (358, 359). In contrast, in normal subjects exposed to a commercial hair spray by directing the aerosol to the hair for 20 sec, tracheal mucous velocity decreased by 60 per cent 1 hour after exposure (157). This effect was transient, as evidenced by restoration to baseline values

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after 3 hours. No changes were observed in pulmonary function even with tests capable of detecting minimal airway dysfunction. Because no changes in tracheal mucous velocity were observed after exposure to the Freon® propellant alone, the transien£ decrease in mucociliary transport was caused by solvants or active ingredients of the hair spray. The measurement of mucociliary transport appears to be a sensitive indicator of this type of airway irritation. It should be noted that the observations made in this investigation pertain only to otherwise normal subjects who had a single exposure to aerosol hair spray directed at the hair by a technique customarily used by personal hair spray users. Repeated long-term hair spray use might have prolonged deleterious effects on pulmonary defenses or give rise to adaptation. Impairment in Disease States

Chronic Bronchitis Chronic productive cough is the most important clinical manifestation of chronic bronchitis and forms the basis for its definition (360). Abnormal production and/or elimination of mucus might therefore be expected in this condition. Most patients with chronic bronchitis are cigarette smokers or have been cigarette smokers in the past, and an attempt should therefore be made to separate the direct effects of cigarette smoke on mucociliary transport from those related to the structural and pathophysiologic changes of chronic bronchitis. In this section, the discussion will be limited to the latter. The irritant effects of cigarette smoke on the mucosa have been considered separately. The histologic changes of chronic bronchitis consist of hyperemia and edema of the mucosa, infiltration with round cells, variable degrees of basement membrane thickening, and hypertrophy and hyperplasia of the submucosal glands. An increase in the number and a change in the distribution pattern of goblet cells with goblet cell metaplasia in smaller airways has also been observed (361). In addition, atrophy of the columnar epithelium (362) and spotty squamous metaplasia (363) have been reported. Although these pathologic abnormalities are common, they do not, with the exception of bronchial gland hypertrophy and hyperplasia, correlate well with the clinical symptomatology of chronic bronchitis (364). The presence of visible respiratory secretions

is a frequent endoscopic finding in patients with chronic bronchitis, and increased amounts of bronchial secretions can be seen on pathologic sections of the lung (363). Mucous plugging and organization of mucus is considered to be a typical feature of the peripheral airway disease associated with chronic bronchitis (365). Along with the accumulation of tracheobronchial secretions, morphologic changes of the cilia representing the other component of the mucociliary apparatus have also been observed. Auerbach and associates (366), in a large postmortem study of cigarette smokers, reported epithelial lesions with loss of cilia in up to 30 per cent of random sections, compared to approximately 15 per cent of sections from nonsmokers. Serafini and Michaelson (20) found that a patient who died with chronic obstructive lung disease had a decrease in both the number of ciliated cells (10 per cent versus 60 per cent in normal subjects) and the mean ciliary length (3 versus 6 ^m) in the larger airways. Electron microscopic examinations of the airway epithelium show subtle abnormalities in bronchial biopsy material from areas that appeared abnormal endoscopically in contrast to specimens obtained from microscopically normal mucosa (367). These ultrastructural changes consist of swelling and serration of the epithelium with transformation of the goblet cell granules. The capsule surrounding the cilia is irregular, with areas of breakage and outward projections; some cilia show fibrillar degeneration. Colonization of the lower airways with pathogens (368, 369) and recurrent bronchopulmonary infection (365, 370) are well-recognized features of chronic bronchitis and suggest an impairment of pulmonary defense mechanisms. Considering the pathologic changes (epithelial damage, mucous gland hypertrophy, and mucous hypersecretion), the bacterial colonization of the lower airways, and the notion that cigarette smoke impairs mucociliary transport, Hilding (371) theorized that the earliest abnormality in chronic bronchitis is mucociliary dysfunction. His hypothesis ties in with the theory that the bronchiolitis (characterized by mucous plugging, organization of mucous plugs, and obliteration of the airway lumen) observed in smokers represents an early stage of chronic bronchitis (372). In patients with symptomatic chronic bronchitis, mucous transport has been studied eith-

CLINICAL ASPECTS OF MUCOCILIARY TRANSPORT

er by direct or indirect observation of the motion of 0.6- to 1-mm diameter teflon particles placed on the tracheal mucosa (174, 373) or by the deposition pattern and clearance rates of inhaled radioactive aerosols (374-379). Santa Cruz and associates (174) found a marked slowing of tracheal mucous velocity in 15 patients with chronic obstructive lung disease who were 57 to 71 years of age. By clinical examination and pulmonary function tests, these patients had both chronic bronchitis and emphysema. The mean mucous velocity, measured with the patient supine, was 1.7 mm per min, compared to a mean velocity of 21.5 mm per min in normal control subjects 20 to 44 years of age. More recently, Goodman and co-workers (373), also using a surface marker (1-mm teflon discs impregnated with bismuth trioxide) but observing their motion radiographically with the patient seated, found that more than 50 per cent of the discs in the trachea lacked any motion or showed transverse to-and-fro oscillations without axial motion. It is of interest that in this study some young asymptomatic smokers with normal pulmonary function, including tests of minimal airway obstruction, showed degrees of mucous transport impairment similar to those of older patients (smokers or nonsmokers) with chronic obstructive lung disease. Mucociliary clearance of inhaled aerosols is altered in patients with chronic bronchitis. Because the clearance of inhaled particles from the lungs is influenced by the deposition pattern, which in turn depends on particle size and flow regime in the airways, clearance rates can only be interpreted if particle deposition is carefully monitored (380, 381). Deposition of inhaled particles is more central in patients with airway obstruction. In addition, coughing, which may be difficult to control in patients, may contribute to clearance of particles (382). For these reasons, it is not surprising that mucociliary clearance has been reported to be increased (142, 376) (particle size, 3 to 15 /urn), normal (379) (particle size, 5 Mm), or decreased (374, 375, 378, 382) (particle size, 1 to 70 /mi) in patients with chronic bronchitis compared to normal subjects. One of the best controlled studies is that of Lourenco (374), who compared tracheobronchial clearance of inhaled iron particles tagged with gold-198 (mass median diameter, 1 /im) in normal nonsmokers, smokers with mild bronchitis, patients with chronic bronchitis without bronchiectasis, and patients with

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bronchiectasis. Tidal volume and respiratory rate were controlled for comparable particle deposition, and only test subjects with similar deposition patterns were used for comparison. The fastest clearance rates were observed in the normal nonsmokers, followed by smokers with mild bronchitis, with the slowest rates in patients with chronic bronchitis and bronchiectasis. In another study, Camner and associates (375) found that the clearance of 6-fim. 99mTc_ tagged teflon particles was significantly slower in 10 of 15 patients with chronic obstructive lung disease who did not cough during the procedure, but similar to that of normal control subjects in the remaining 5 patients who could not suppress coughing. There was no difference in clearance rates between smokers and former smokers among these patients. Two patients, a 54-year-old female ex-smoker and a 55-year-old male lifetime nonsmoker, with homozygous a r antitrypsin deficiency had normal tracheobronchial clearance. The same group of investigators (378) also reported that in 3 smokers with symptoms of chronic bronchitis, the initially markedly decreased clearance rates improved but were still below the normal range 3 months after cessation of smoking. These in vivo observations strongly indicate that mucous transport is usually impaired in chronic bronchitis, but the clearance of inhaled aerosols may be normal or even increased for methodologic reasons. It is difficult to determine from the available information whether ciliary dysfunction, abnormal mucous secretions, or a combination thereof is responsible for the impairment of the mucociliary apparatus in chronic bronchitis. Lower airway ciliary function has not been evaluated in chronic bronchitis, nor has the distribution, amount, and rheologic properties of mucus within the airways been studied; however, there is an extensive literature on the biochemistry (383) and rheology of expectorated sputum from patients with chronic bronchitis. These results are difficult to interpret, partly due to sample contamination with saliva and the rapid physical alteration of expectorated sputum, and partly because normal respiratory secretions for comparison are virtually impossible to obtain. Thus, most investigators have correlated the biochemical and rheologic properties of sputum with its macroscopic appearance, clinical symptoms, or pulmonary function tests. Even this approach may be hampered by the large variations in the visco-

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elastic characteristics of expectorated sputum in the same patient during short periods of time (384). Mucoid sputum in patients with chronic bronchitis is biochemically similar to respiratory secretions in normal subjects induced by hypertonic saline aerosol, with the exception of slightly higher fucose and neuraminic acid contents in the former (385). In purulent sputum from these patients, biochemical changes typical of inflammatory conditions such as increases in dry weight and deoxyribonucleic acid content, and increased crosslinking by hydrogen bonding in the sputum gel were observed. The increased amounts of bronchial secretions in chronic bronchitis may result from hypersecretion of mucus by the submucosal glands and goblet cells, increased serum transudation (386), or prolonged bronchial residence time due to impaired removal by the mucociliary apparatus. Possibly some patients have a productive cough that is not related to bronchial hypersecretion, but is an expression of a defective mucociliary transport system that under normal circumstances would be capable of removing respiratory secretions of comparable quantities unnoticed by the subject. Using neuraminic acid as a marker of mucopolysaccharides, Reid (387) showed that the neuraminic acid content per ml of sputum is increased in chronic bronchitis, suggesting augmented secretion by the mucus-producing structures. Hypersecretion of mucus is further suggested by mucous gland hypertrophy and hyperplasia and an increase in the number of goblet cells (388). Histochemical studies indicate distended acini of the submucosal glands in patients with chronic bronchitis compared to normal subjects, with an increase in the volume of both the acid mucopolysaccharide- and neutral mucopolysaccharide-producing acini (387). Rheologic abnormalities of respiratory secretions may contribute to the impaired mucous transport mechanisms in chronic bronchitis. Because low viscosity and high elasticity appear to be the ideal combination for an optimal interaction between cilia and mucus (99, 101), deviation from this should result in decreased mucous transport rates. Dulfano and associates (389) used the capillary tube method to measure sputum viscosity and elasticity at low shear rates in patients with chronic bronchitis. They observed during periods of clinical stability lower viscosity and higher elastic recoil values than during acute exacerbations. Expectoration was more

difficult, with sputum exhibiting high viscosity and low elastic recoil. Mucoid sputum obtained from patients with chronic bronchitis and bronchiectasis appears to be more viscous than that from patients with cystic fibrosis, but less viscous than sputum from asthmatic patients (390, 391). Purulent sputum has a higher viscosity than mucoid sputum regardless of clinical diagnosis. In a longitudinal study involving patients with chronic bronchitis, the dry weight, neuraminic acid content, and viscosity of sputum were found to be inversely related to the 1-sec forced expiratory volume (381). The interrelationships between ciliary function and mucous secretions, and consequently the mechanisms underlying the observed mucociliary dysfunction have not been elucidated in human chronic bronchitis. Mucous flow and ciliary activity, however, have been studied in detail in rats after exposure to irritant gases (121) and in experimental bronchitis of rats (42). Iravani and van As (105) and Iravani (149) examined mucociliary interactions in rats with spontaneous chronic bronchitis or bronchitis induced by exposure to cold, moist air. By using a whole lung preparation and separating the surrounding lung tissue from the trachea and bronchial tree, they observed microscopically the transport of mucous droplets, flakes, and cellular debris, and ciliary motion (via incident light technique) through the membranous wall of the airways. In rats with chronic bronchitis, the ciliary system showed discoordination, zonal akinesia, and a decrease in over-all cephalad transport rate. In addition to these changes, reversal of transport direction, whirlpool formations, and inactive zones without ciliary motion as large as 2 mm by several hundred fim were seen. The amount of mucus was increased, but paradoxically, over-all mucous transport rates were increased in the rats with chronic bronchitis unless extensive epithelial damage was present. These experiments support the circumstantial evidence derived from pathologic, biochemical, and rheologic studies in man that both ciliary dysfunction and "abnormal" respiratory secretions are responsible for the failing mucous transport system in patients with chronic bronchitis. Many questions regarding the precise mechanisms underlying the mucociliary dysfunction in chronic bronchitis remain open. In particular, the production and transport of mucus in peripheral airways and their relationship to early

CLINICAL ASPECTS OF MUCOCILIARY TRANSPORT

obstructive lung disease deserve thorough investigation. Although mucous transport is impaired in chronic bronchitis, the elimination of respiratory secretions and inhaled particles from the tracheobronchial tree is aided by coughing and more central particle deposition. Cystic Fibrosis Although airway disease is probably not present in neonates and the onset of respiratory symptoms may appear as late as months or years after birth, chronic lung disease eventually develops in all patients with cystic fibrosis. Early pulmonary lesions are characterized by hypertrophy of bronchial glands, goblet cell metaplasia, and mucous plugging of peripheral airways (392). In addition, chronic infection leads to bronchiolitis, bronchitis, bronchiectasis, and eventually to chronic airway obstruction. The chronic inflammatory process results in destruction of the ciliated epithelium and the development of squamous metaplasia (393). At this stage of the disease, the role of ciliary dysfunction and altered mucus in the pathogenesis of the chronic airway disease is difficult to establish. The malfunction of the mucociliary apparatus might be a consequence of the chronic inflammatory airway disease, or the abnormalities in ciliary function and mucous secretion might precede the obstructive airway disease. Because cystic fibrosis is characterized by a generalized abnormality in exocrine function, it is not surprising that many investigators have directed their studies at airway mucus and ciliary function in an attempt to understand the pathogenesis of the pulmonary disease. A number of papers have dealt with the biochemical composition and the structure of expectorated sputum (394397). These studies have shown that, although airway secretions from both patients with chronic bronchitis and patients with cystic fibrosis are biochemically different from normal airway secretions, nasal and tracheobronchial secretions of patients with cystic fibrosis also differ from those in patients with chronic bronchitis in that the former contain larger amounts of acid mucoglycoproteins that can be extracted from the gel phase. Despite the distinguishing biochemical characteristics of airway secretions in cystic fibrosis, Charman and Reid (390), Sturgess and Reid (398), and Feather and Russell (399) have not been able to detect any significant differences in rheologic properties among sputa obtained from patients with cystic fibrosis, chronic bron-

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chitis, or bronchiectasis. It should be noted that in addition to the viscoelastic properties, die amount and distribution of respiratory secretions, and the thickness of die mucous layer have a bearing on mucociliary transport. No studies, to date, have dealt with this aspect of mucous transport in different types of chronic airway diseases. Although an abnormality of mucous production has been well established by these pathologic, biochemical, and rheologic investigations, the contribution of abnormal mucus to the pathogenesis of cystic fibrosis has not yet been determined. Another mechanism for retention of respiratory secretions in cystic fibrosis could be primary impairment of cilia. In 1967, Spock and co-workers (400) observed that serum from patients with cystic fibrosis disorganized the ciliary rhythm in explants of rabbit respiratory epithelium. After incubating the tracheal mucosa using standard tissue culture techniques for 4 to 6 days, small specimens containing beating cilia were observed at room temperature under a phase contrast microscope after exposure to a drop of test serum, and dyskinesia was qualitatively assessed. Dyskinesia was produced by the sera of all of the patients with cystic fibrosis, but in heterozygotes, native serum caused discoordination only in two thirds of the cases. Subsequently, it was suggested that the ciliotoxic effect was a property of the euglobulin fraction of the serum; if the euglobulin fraction of heterozygote serum was concentrated approximately 3fold, dyskinesia could be detected in all of them. On the other hand, the addition to the preparation of a 10-fold concentration of euglobulin from normal control patients failed to disorganize the ciliary beat. Spock and co-workers (400) postulated the presence of a cilio-inhibitory serum factor in patients with cystic fibrosis and their parents, with the activity contained in the euglobulin fraction. Subsequently, numerous investigations dealt with the cilioinhibitory factor, and new bioassay systems were developed using the cilia of oyster gills (401). Although Bowman and associates (401) and a number of other investigators using the rabbit tracheal explant or oyster gill preparation confirmed the original observations of Spock and co-workers (400), others were unable to reproduce the results (402, 403). In addition, Conover and associates (404) reported inhibition of ciliary activity with sera from patients with allergic lung disorders in addition to patients with cystic fibro-

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sis. It has been suggested that the cilioinhibitory factor might be released in cell cultures of fibroblasts, lymphocytes, and amniotic cells derived from patients with cystic fibrosis and in heterozygotes for cystic fibrosis; that only metachromatic cells release this factor; that the factor is bound to IgG; and that it might be related to serum complement components. The variable results of these studies and the resulting controversies have been reviewed by Lederberg (405). Recently, Wood and diSant-Agnese (406) tested the reliability of the oyster gill and rabbit trachea assays and concluded that they could not differentiate sera of patients with cystic fibrosis from sera of normal subjects. Therefore, the presence of a cilioinhibitory serum factor in patients with cystic fibrosis still awaits final proof, because in vitro techniques currently used may not be sufficiently discriminatory. A single in vivo study has been reported that attempted to evaluate the effects of serum from patients with cystic fibrosis on nasal mucociliary clearance; because an inhibitory effect of cystic fibrosis serum when placed locally on the nasal mucosa was only detectable in the presence of mucosal inflammation, this observation is difficult to interpret (407). Unlike the cilioinhibitory factor whose existence and role in the padiogenesis of cystic fibrosis is still in dispute, and unlike the uncertainties regarding the mechanisms by which the abnormal mucus in cystic fibrosis interferes with mucociliary transport, it has now been established that mucous transport is usually slowed. Wood and associates (408) found that 13 of 14 adult patients with cystic fibrosis had a markedly slowed tracheal mucous velocity, with a mean of approximately 15 per cent of that in normal control subjects. No correlation could be established between tracheal mucous transport rates and the prognostic score in these patients. Indeed, the highest transport rate, which was within 1 SD of the mean value of normal control subjects, was observed in a patient with one of the lowest prognostic scores. Although considerable amounts of thick, tenacious secretions were macroscopically observed in die central airways of most patients, the amount of secretions did not correlate with the velocity of tracheal mucous flow. Subsequently, Yeates and co-workers (409) examined mucociliary transport rates in die trachea of adolescent patients with cystic fibrosis by having them inhale an aqueous aerosol containing albumin microspheres labeled

with 9 9 m Tc; by a special breathing maneuver, the bolus was deposited preferentially in the large airways. With this technique, they found a large variability of tracheal transport rates, ranging from 0 to 12.8 mm per min. In a number of them, the transport rates were comparable to those in normal persons, whereas in others there was partial or total impairment of mucociliary transport. Patients widi a low Schwachman score tended to have lower transport rates. Paradoxically, serum from patients with the higher transport rates produced more ciliary dyskinesia in the rabbit trachea assay. In contrast to these 2 studies investigating mucous transport rates in the trachea, lung clearance of inhaled radioactive aerosols containing 9»mTclabeled albumin was found to be normal or even increased in children and adult patients with cystic fibrosis (410, 411). These observations might be explained by a more central deposition of the inhaled aerosol particles (mass median diameter, 3 to 5 fim) in these patients with obstructive airway disease resulting in a more rapid clearance than that in normal control subjects, because clearance rates are faster in central than peripheral airways. In addition, coughing could not be controlled in these studies in which the clearance curves were constructed from the radioactivity remaining in the lung over several hours. The information available at present strongly suggests that mucociliary transport, at least in central airways, is impaired in most patients with cystic fibrosis; however, it is still unclear whether this disorder is a nonspecific feature of the chronic inflammatory airway disease or represents an essential factor in the pathogenesis of the pulmonary abnormalities. Bronchial Asthma Inflammatory changes of the bronchial mucosa accompanied by increased amounts of tracheobronchial secretions are characteristic pathologic features of bronchial asthma (412). One might therefore expect an impairment of mucociliary function in addition to the well-known abnormalities in mechanics of breathing. Formation of mucous plugs leading to atelectasis is not uncommonly observed in asthmatics, and several postmortem studies indicate that widespread mucous plugging of the airways is a major cause of death in status aschmaticus (413, 414). This is circumstantial evidence that tracheobronchial mucous transport is decreased in

CLINICAL ASPECTS OF MUCOCILIARY TRANSPORT

bronchial asthma. Similarly, Hilding's (415) postulate of a deficient ciliary mechanism in fatal cases of asthma was based on morphologic observations (decreased number of ciliated epithelial cells, goblet cell metaplasia, and excessive viscid mucous). In stable asthmatic patients, a marked slowing of tracheal mucous velocity has been reported (observation of 3 patients) (416), whereas in another study normal tracheobronchial clearance of 6-^m teflon particles tagged with 99mT;c w a s founc| j n 12 patients with stable bronchial asthma (417). As pointed out by Mossberg and associates (417), normal clearance does not necessarily mean that the mucociliary apparatus is intact in bronchial asthma, because the presence of bronchial obstruction will result in the deposition of the inhaled aerosol in more central airways where clearance rates are naturally faster, thereby compensating for a decrease in over-all clearance rates. Mucociliary activity has not yet been studied during acute asthmatic attacks in man. Theoretically, mucociliary transport may be impaired in bronchial asthma by the following mechanisms. (1) Structural and/or functional changes of the cilia might lead to the accumulation of bronchial secretions in the airways. (2) The normal interaction between cilia and mucus might be impaired by abnormalities in the amount, distribution, and rheologic properties of mucus. (3) The decrease in airway cross-sectional area resulting from bronchoconstriction might lead to inspissation of mucus already present in the bronchi. Denudation of ciliated epithelium is part of the airway pathologic findings seen in patients dying from bronchial asthma (415). Using explants of nasal and tracheal mucosa from rabbits sensitized with horse serum, Ballenger (418) was unable to detect qualitative changes in ciliary function when the specimen was brought in contact with the antigen. He further observed that histamine given either as an intravenous dose before the animals were killed or by local application to the preparation also failed to change ciliary function. It should be noted that these studies with specific antigen and a major chemical mediator of the asthmatic response were not done in an intact animal; furthermore, they relied on qualitative assessment of ciliary function. A variety of physico-chemical abnormalities characterize the respiratory secretions in bronchial asthma. Although it has been stated that the average asthmatic does not expectorate more

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than 25 ml of sputum per day (419), it is generally accepted that the amount of mucus residing in the airways is increased in this condition. The total amount of water in mucus from asthmatics does not appear to be markedly decreased when compared to "normal" tracheobronchial secretions obtained from tracheostomized patients, but the water binding in asthmatic mucus is probably different from that in normal secretions (190). This may result in an abnormal water-glycoprotein structure of mucus, thereby possibly increasing its viscosity and decreasing its permeability to water (420). Glycoproteins seem to be responsible for the rheologic properties of mucus, especially by the crosslinking of glycoproteins (421). Increased amounts of unusual polysaccharides (422) and crosslinking between transudated serum proteins and secretory IgA, both of which appear to be increased in bronchial asthma (423-425), have been found in asthmatic secretions. In addition, changes in electrolyte concentrations including increases in calcium have been reported (426), and serum proteins appear to accumulate in the sol phase of asthmatic sputum (427). Despite these distinguishing biochemical characteristics and their possible effect on the viscoelastic properties of mucus, only a few studies examining the viscoelastic properties of respiratory secretions of patients with bronchial asthma have been reported. In addition, the most relevant observations have been made on expectorated sputum, not on lower airway secretions. Charman and Reid (390) using a Ferranti-Shirley viscometer measured viscosity over a wide range of shear rates of mucoid specimens from patients with cystic fibrosis, chronic bronchitis, bronchiectasis, and asthma. In general, sputum from asthmatic patients tended to be more viscous than that obtained from patients with other types of obstructive airway disease. Keal and Reid (428) demonstrated that a marked increase in viscosity at low shear rates was characteristic of the sputum obtained from asthmatic patients. Dulfano and Adler (99, 101), who placed sputum on an isolated frog palate, found that the combination of high elasticity with low viscosity represented the best condition for a fast transport rate, although the change in elasticity was a more important determinant. Therefore, high viscosity of mucus in bronchial asthma would tend to decrease mucous transport rates. Stasis of mucus within the airway has been shown to lead to changes in the composition and

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structure of mucus, which may further compromise its removal (429). As stagnant mucus gets older, its fibrin content increases and appears as lamina within the mucus. The mechanisms responsible for this organization of stagnant mucus are not known, but an enzyme resembling blood clotting factor 13 (7-glutamine transpeptidase) found in sputum facilitates the conversion of fibrinogen to fibrin (79). Thus, clinical experience and pathologic, biochemical, and rheologic investigations strongly suggest that mucous transport is impaired in bronchial asthma, and direct measurement of tracheal mucous transport revealed a slower rate in patients with stable asthma (416). Wanner and associates (430) conducted experiments in an experimental canine asthma model to examine mucous transport in relation to changes in pulmonary function during induced asthma, and to evaluate the possible effects of known chemical mediators of the asthmatic response on mucous transport. Mongrel dogs with natural sensitivity to Ascaris suum were challenged with aerosols of A. suum extract. Only 50 per cent of the animals responded with transient bronchospasm, which was no longer detectable after 2 hours; tracheal mucous velocity measured with the cinebronchofiberscopic technique decreased in all dogs to a mean of 30 per cent of the baseline value within 30 to 45 min of antigen challenge, regardless of whether or not bronchospasm occurred. Furthermore, tracheal mucous velocity was still significantly decreased after the end of the 2-hour observation period. Aerosols of both histamine and acetylcholine in concentrations that produced a degree of bronchospasm comparable to that observed in the antigen-challenged animals increased tracheal mucous velocity, thereby excluding them as mediators of the impairment of mucous transport in experimental asthma. Pretreatment with an antagonist of slow-reacting substance of anaphylaxis not only prevented the expected decrease in tracheal mucous velocity but produced a transient increase to 250 to 300 per cent of baseline after antigen challenge without preventing the development of bronchospasm. Because no changes occurred in tracheal mucous velocity after inhalation of the antagonist alone, this "overshoot" phenomenon might have been related to a modified mediator response. Possibly, slowreacting substance of anaphylaxis, which is elaborated in the reagin-mediated airway response, depressed mucociliary transport, and its inhibi-

tion unmasked the stimulating effects of other mediators such as histamine, or of a vagally mediated reflex. Obviously, these experiments were not capable of determining whether ciliary dysfunction or an alteration in the physical characteristics of mucus was responsible for the observed decrease in tracheal mucous transport. Furthermore, the findings cannot necessarily be extrapolated to human asthma, particularly the nonallergic type, and must await confirmation in man. It is also not known whether the impairment of mucous transport in the larger airways also signifies mucociliary dysfunction in peripheral airways. This seems of importance not only with regard to generalized mucous plugging in status asthmaticus, but also to asthma in remission, in which excessive mucus may contribute to the residual peripheral airway obstruction. Respiratory Infections The normal lower respiratory tract distal to the carina is considered to be free of bacteria (431, 432), and along with other host defense mechanisms, mucociliary transport may also play a role in keeping the lower respiratory tract sterile. An impairment of mucociliary transport mechanisms in the respiratory tract could therefore allow the retention of inhaled particles containing microorganisms, thereby leading to bacterial colonization in the airways. Indeed, Laurenzi and Guarneri (433) suggested on the basis of experiments in which they determined the lung clearance of bacterial aerosols in mice and mucous transport rates in the transluminated exposed cat trachea, that the increased retention of bacteria after administration of ethyl alcohol was related to both impaired macrophage function and a decrease in mucous transport rate. To our knowledge, this is the only study that demonstrated that after damage to the functional integrity of the ciliated respiratory epithelium, the pulmonary host defense mechanisms against bacterial invasion were impaired. It does not, however, answer the question of whether an a priori normal mucociliary apparatus is impaired in its function during an acute respiratory infection. The bronchial mucosa shows marked changes in acute bacterial bronchitis and pneumonia (434, 435), and it has been clearly shown that the ciliated epithelium is destroyed in influenza-A infections (435-439) and in Mycoplasma pneumoniae respiratory infections (440, 441). In addition, distinct bio-

CLINICAL ASPECTS OF MUCOCILIARY TRANSPORT

chemical abnormalities of sputum that seem to be related to the destruction of leukocytes and increased turnover rate of epithelial cells and increased ratio of albumin to secretory IgA (442), have been correlated with acute infectious exacerbations of chronic airway disease (443-445), but their effect on the rheologic properties and mucociliary clearance has not been established. Mucociliary transport has not been studied in acute bacterial respiratory tract infections. In viral infections, however, impairment of mucous transport has been clearly established. Bang and associates (446) evaluated both structure and function of the nasal mucosa of chickens infected with a virulent strain, of Newcastle disease virus. They found extensive sloughing of the nasal mucosa by the virus with an associated decrease of mucous transport. They also showed that if the mucous layer is intact, a relatively small numbers of viruses could penetrate the mucus and attach themselves to the epithelial cells. Pretreatment of the animals with pilocarpine, which disorganizes the mucous layer, or cocaine, which paralyzes ciliary beat, increased the number of cells infected by a given virus load (446). Louren^o and associates (447) found abnormal deposition in the large airways (not specified) and over-all slow disappearance and regional retention of inhaled 2-fim iron oxide particles labeled with 198 Au in patients with acute respiratory tract infections caused by respiratory viruses. This abnormality in mucociliary transport persisted as long as 6 weeks after the disappearance of symptoms. Similarly, Camner and associates (448), using somewhat larger particles (6 /xm) of inhaled radiolabeled aerosol showed a marked impairment of lung clearance in influenza-A infection 1 week after the onset of symptoms. Two and 3 months later, the clearance rates had returned to values obtained in normal control subjects. Although Noscapine, a mucociliary depressant, had to be administered to some of the patients to suppress coughing, careful examination of the individual data suggests a true impairment of the particle clearance in the acute phase of influenza-A infection. The same group of investigators (449) also found an impaired lung clearance of inhaled 6-/mi particles in 9 patients 10 to 15 days after the onset of M. pneumoniae pneumonia. Three weeks, 3 months, and 1 year later, the clearance was markedly improved but was still

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somewhat slower than that in age-matched normal control subjects. In summary, the pathologic lesions in the tracheobronchial tree of patients with bacterial, viral, and mycoplasmal respiratory tract infections suggest an impairment of mucociliary transport, which thus far has only been demonstrated in nonbacterial infections. It seems likely that the impairment of mucociliary transport mechanisms produced by viral respiratory tract infections will compromise pulmonary defenses and facilitate the retention of bacteria in the lungs. Miscellaneous Endotracheal intubation and bronchoscopy. Although prolonged endotracheal intubation has been used since the early 1940s, its traumatizing effect on the tracheal mucosa and the development of tracheal stenosis and tracheal malacia resulting from endotracheal tubes and tracheostomy cannulas have only relatively recently been recognized. This subject has been carefully reviewed by Lindholm (450). It was subsequently shown that tracheal and bronchial lesions can also result from suction catheters, which are frequently used in intubated patients (451). These lesions, consisting of mucosal denudation, edema, and inflammatory exudates, can in most cases be prevented by an improved design of the suction catheter, which has a terminal lead to minimize contact of the catheter with the airway mucosa (452). The extent of epithelial desquamation and ulceration in the larynx and trachea produced by intubation has been demonstrated by Hilding (453) using methylene blue to stain the lesions. It is likely that endotracheal tubes provided with lowpressure, high-compliance cuffs not only decrease the incidence of tracheal malacia and tracheal stenosis, but are also associated with a lower degree of epithelial damage as judged by a lesser degree of squamous metaplasia than the conventional high-pressure, low-compliance cuffs (454). Therefore, persistent localized impairment of mucous transport after extubation would be decreased by using high-compliance cuffs. This seems to be supported by a study of Klainer and associates (455) who examined the surface of the trachea by scanning electron microscopy in laboratory animals and in humans after endotracheal intubation. Linear areas of ciliary denudation were observed in dogs after 2 hours of endotracheal intubation with the cuff inflated

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in the regular fashion, whereas by intubation with uncuffed tubes or using tubes with cuffs inflated with a minimal occlusion volume, the mucosal damage was much less extensive. Regeneration of cilia was seen by 2 days, and after 7 days recovery of the mucosa was anatomically nearly complete. Postmortem studies in patients with prolonged intubation with the same 2 cuff systems showed good correlation with the animal experiments. A potentiation by a cuffed endotracheal tube of the impairment in tracheal mucous velocity induced by breathing pure 0 2 has been shown in anesthetized dogs (456). Inflation of the endotracheal tube cuff in the subglottic area produced a significant decrease in tracheal mucous velocity in the distal trachea as early as 1 hour after cuff inflation, regardless of whether a high-compliance or a low-compliance cuff system was used (457). This suggests depression of mucociliary function or pile up of mucus during intubation. How long this impairment persists after extubation has not been assessed by functional tests; however, based on anatomic studies, the mucociliary apparatus may not regain its integrity for quite some time, especially in areas of granulation tissue formation (152). The vulnerability of the tracheobronchial mucosa was first emphasized by Hilding (458, 459), who showed extensive desquamation of the mucosa produced by a routine endoscopic examination with a rigid bronchoscope and by rubbing the airway mucosa with such innocuous objects as gauze and cotton swabs (460). Although this type of airway manipulation may produce localized mucosal damage, routine rigid and fiberoptic bronchoscopic examinations have not been shown to impair over-all tracheal mucous velocity for as long as 3 hours after termination of the procedure in dogs (Landa, J.: Personal communication). Contamination of the mucosa with blood, however, appears to impair mucous transport (89). Tracheal resection and lung transplantation. Simple transection of a major airway with endto-end anastomosis, and allotransplantation or autotransplantation of the lung have been shown to decrease temporarily mucociliary clearance in the lung, with return to baseline values within 5 days to 1 month (461-463). Likewise, tracheal resection (7 rings) with subsequent end-to-end anastomosis in beagle dogs resulted in a marked decrease of tracheal mucociliary transport as measured with 99m Tc-labeled sodi-

um pertechnetate 3 days after the procedure (152). After 31 days, the clearance rates had returned to preoperative values, with restoration of normal ciliated epithelium within 6 months. Thus, tracheal resection is associated with a transient impairment of mucous transport; however, if tracheal stenosis develops, the decrease in tracheal mucociliary clearance may persist. Postoperative retention of secretions. The development of postoperative atelectasis has been attributed to airway closure in the dependent zones of the lung, retention of airway secretions resulting from inefficient cough due to pain, and the presence of obstructive airway disease (464-466). Mucociliary dysfunction due to general anesthesia, endotracheal intubation, long-term cigarette smoking, and administration of 0 2 , may also contribute to the postoperative retention of secretions and subsequent development of atelectasis. Gamsu and co-workers (467) studied mucociliary transport in large and small airways by observing the clearance of insufflated tantalum powder with a semiquantitative radiographic technique in the postoperative period. In patients who underwent orthopedic surgery there was complete clearance of tantalum within 48 hours, and no radiographically detectable atelectasis occurred. On the other hand, atelectasis was seen in most patients after abdominal surgery. Regional pooling of tantalum seemed to precede the development of atelectasis. Relationship of impaired mucociliary transport to carcinogenesis. By placing droplets of India ink on the tracheobronchial mucosa of cows soon after death, Hilding (89) observed that ciliary streaming was unstable around the bronchial opening with slowing, whirlpool formation, and areas of stasis. The distribution of these areas was reminiscent of the distribution pattern of squamous cell carcinoma in man (468). Because the relationship between squamous metaplasia and bronchogenic carcinoma has been known for some time (469, 470), and because the ciliary streaming would be expected to be interrupted in diese areas, Hilding theorized that prolonged residence time of inhaled carcinogens in cigarette smoke at these locations might favor the development of bronchogenic carcinoma. A somewhat different but equally provocative possibility was suggested by Macklin (464). He reasoned that although inhaled carcinogenic material might be deposited throughout the tracheobronchial tree, concen-

CLINICAL ASPECTS OF MUCOCILIARY TRANSPORT

tration of this material would occur in the central airways due to convergence of mucus originating from the large surface area of the peripheral airways to the central airways, thereby precipitating the development of a bronchogenic carcinoma in the major bronchi. The considerable acceleration of the transport of mucus toward the trachea might explain why the trachea is less involved in lung cancer. Depression by Pharmacologic Agents

A depressant effect on mucociliary transport has been claimed for a variety of pharmacologic agents that are commonly administered in clinical practice. The results of many such investigations have been conflicting, probably because of methodologic differences. The following review will limit itself to 4 groups of pharmacologic agents for which different experimental techniques have shown rather consistent results: general anesthetics, local anesthetics, anticholinergics, and narcotics. General Anesthetics Marin and Morrow (154) failed to demonstrate a dose-dependent effect of intravenously administered barbiturates on tracheal mucociliary transport in dogs; increments in the dosage of barbiturates were not associated with changes in tracheal mucous transport. Asmundsson and Kilburn (39) found comparable mucous transport rates in the exposed canine trachea regardless of whether the animals were killed with a blow to the head or with pentobarbital. More recent studies strongly suggest, however, a depressant effect of general anesthetics on mucociliary transport. Thus, mucociliary transport in the trachea as determined by the motion of a radioactive tracer was found to cease during deep pentobarbital or thioamylal anesthesia, whereas some motion was observed during light anesthesia with the same barbiturates (341). In isolated preparations of the bronchial tree of rats, addition of pentobarbital to the perfusate produced a dose-dependent depression of ciliary beat frequency (471). Finally, in intact sheep in which tracheal mucous velocity can be determined without sedation or topical anesthesia of the airways, intravenous administration of 30 mg of pentobarbital or thioamylal per kg of body weight decreased tracheal mucous velocity by 35 per cent from the preanesthesia control value after 30 min (156). Similar observations have been made in rats (345). It appears,

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therefore, that both systemic administration and topical application of barbiturates impair mucociliary transport, and that this impairment can be observed in commonly used animal models. This should be taken into consideration in the design of mucociliary transport studies using animal preparations that require general anesthesia. Local Anesthetics In vitro experiments and in vivo studies suggest that the impairment of mucociliary transport by some local anesthetics results, at least in part, from their ciliotoxic effect. Corssen and Allen (472) and Corssen (473) studied the effects of various local anesthetics by placing explants from punch biopsies of human tracheal and bronchial epithelium in a tissue culture medium. With special manipulations, some of the explants could be made to curl up so as to form an epithelial globe with the cilia located on the outside; these tissue globes rotated in the perfusion chamber because of ciliary activity. Reversible stoppage of rotation was found for solutions of different local anesthetics greater than the following concentrations: lidocaine, 5 per cent; procaine, 5 per cent; chloroprocaine, 0.5 per cent; cocaine, 10 per cent; tetracaine, 0.1 per cent; and dibucaine, 0.05 per cent. For most of these agents, mild stimulation of ciliary activity was observed at lower concentrations. Local instillation of 0.5 per cent tetracaine, 2 per cent carbocaine, and 4 per cent lidocaine had no effect on the transport rate of India ink in the chicken nose, whereas 10 per cent cocaine, 20 per cent cocaine, and 5 per cent cyclaine decreased transport rates 3-fold (474). Histologic studies indicated that this effect was probably not due to changes in secretions of the epithelium but to abnormalities in ciliary activity. Also, the local instillation of 5 per cent cyclaine produced sloughing of the mucosa. Depression of nasal mucociliary activity in intact animals by local application of 4 per cent cocaine (158), and in human subjects of 2 per cent tetracaine (475) has also been demonstrated. In the latter study, cessation of mucociliary function was observed immediately after the application of 2 per cent tetracaine, and this effect lasted for at least 45 min. On the other hand, 4 per cent lidocaine in most instances had no effect. The lack of mucociliary depression by lidocaine has been confirmed in normal subjects by another group of investiga-

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tors (476), although they demonstrated a mild depression in smokers. The significance of this latter observation is not quite clear. In unanesthetized or anesthetized sheep, 10 ml of 2 per cent lidocaine solution instilled into the trachea during a 1-hour observation period did not affect tracheal mucous velocity (156). Similar observations were made in human subjects (108). These few studies indicate a considerable variability among different local anesthetics with regard to their effect on mucociliary transport; some of them, such as lidocaine, seem to lack any effect, at least at conventionally used concentrations (1 to 4 per cent). Anticholinergic Agents Although early workers found that atropine did not affect mucociliary transport on the frog palate (topical application) (477) or in the turtle trachea (intravenous administration) (478), more recent investigations have demonstrated impairment of mucociliary transport with atropine administration. Burton (192) showed a decrease in tracheal mucous transport of anesthetized dogs by intravenous administration of atropine, and Annis and associates (479) found that during routine anesthesia in women undergoing gynecologic surgery atropine might have a depressant effect on mucous transport. Orally administered atropine (0.8 mg) and hyoscine (0.008 mg per kg) appear to impair mucociliary clearance in the lungs of normal subjects (480, 481). Blair and Woods (482) demonstrated a slowing of mucous transport in the trachea of anesthetized cats by topically applied atropine. Atropine inhibits the production of tracheobronchial secretions and the volume of sputum (in part by decreasing the secretion of saliva) (483, 484), and thus might impair mucociliary transport, but the possibility of a direct action of atropine on ciliary activity has not been excluded (485, 486). Examination of

such a mechanism might be of particular interest, because in vitro experiments suggest a direct action of acetylcholine on ciliary activity (487, 488). In contrast to atropine, ipratropium bromide (SCH-1000, Atrovent®), a new aerosol bronchodilator agent, does not slow mucous transport in animals (489, 490) or mucociliary clearance in human subjects when therapeutic doses are used (142). Narcotics With a few exceptions (e.g., 478), opiates appear to depress mucociliary transport. Various narcotics impair ciliary activity of the oyster gill (491) and there is a dose-dependent depression of ciliary beat frequency by direct exposure to solutions of codeine and normethadone in the rat trachea (471). Likewise, subcutaneous injection of 5 mg of codeine per kg of body weight and 0.5 mg of morphine per kg of body weight have been shown to impair the tracheobronchial clearance of insufflated barium sulfate powder in cats for several hours (492, 493). Ethyl alcohol inhibits tracheal mucous transport, and the degree of inhibition correlates with blood alcohol concentration (494). The depressant effects of the pharmacologic agents described are summarized in table 3. Stimulation by Pharmacologic Agents and Physical Therapy

From what is known about the physiologic and pathophysiologic characteristics of the mucociliary apparatus, pharmacologic agents theoretically might exert stimulating effects on mucociliary transport by improving ciliary activity, changing the volume and physical properties of periciliary fluid and mucus thereby improving mucociliary interaction, or a combination thereof. The mechanisms of drug action have been partially elucidated for some pharmacologic agents by using experimental designs that

TABLE 3 DEPRESSION OF MUCOCILIARY ACTIVITY BY PHARMOCOLOGIC AGENTS Agent(s) General anesthetics

Ciliary Activity

Mucous Production

Mucociliary Activity

?

?

Depression ( 3 4 1 , 4 7 1 , 156)

?

Depression (158, 474, 475)

Depression (483, 484)

Depression (192, 479-482)

?

Depression (471, 492, 493)

Certain local anesthetics

Depression (472, 473)

Anticholinergic agents

Probable depression (485, 386)

Narcotics

Depression (491)

CLINICAL ASPECTS OF MUCOCILIARY TRANSPORT

allow separate assessment of the individual functions in vitro and in vivo. Examination of ciliary preparations in the absence of mucus, administration of pharmacologic agents either topically or by intravenous injection, or histologic studies of mucous production have all contributed to a better understanding of underlying mechanisms. Drugs that are known to increase intracellular cyclic adenosine monophosphate (cyclic AMP) typically stimulate mucociliary transport, although it is not quite clear how cyclic AMP interrelates with ciliary energetics (488). Stimulation of mucociliary transport by adrenergic agents does not appear to be related to the bronchodilator effect (increase in airway circumference) or increased blood flow to the bronchial mucosa (93, 351, 480). Although a stimulating effect on mucociliary transport has been attributed to a great number of different pharmacologic agents, it would not be practical to list all of them. Four groups of drugs have been extensively studied: adrenergic agents, cholinergic agents, biologically active amines, and methyl-xandiines. Many of these drugs may be of therapeutic value in disease states associated with impairment of mucociliary transport. Adrenergic Agents A direct effect of epinephrine or isoproterenol on ciliary activity has not been established (482, 488, 495). Although an increase in ciliary beat frequency has been observed in intact bronchial preparations in response to direct exposure to /3-adrenergic agonists (496, 497), this finding does not necessarily imply direct ciliary excitation, because die observed increase in ciliary beat frequency might have been secondary to changes in respiratory secretions. Indeed, in the same experiments using bronchial preparations of rats, hamsters, and cats, incubation of the preparation for 30 min with a ^-adrenergic agonist seemed to stimulate mucous production, as evidenced by increased amounts of mucus and differentiation of goblet cells (496, 497). Similar observations have been made by others after intravenous administration in rats (498). A difference appears to exist between in vivo and in vitro preparations, because adrenergic agonists have not been found to stimulate the secretions from submucosal glands in organ cultures (499). The mechanism by which adrenergic agents stimulate mucociliary transport may be related to the rate of chloride and water flux across the

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tracheal epithelium (500). Systemic administration of terbutaline caused active transport of chloride. With this chloride pump, water moves from the tracheal wall into the lumen and may alter the rheologic properties of the periciliary fluid and mucus. This factor may be an important mechanism by whicli adrenergic agonists stimulate mucociliary function. In animal preparations, a stimulating effect of adrenergic agents on over-all mucociliary transport has been clearly demonstrated. Both in lower animals (477, 486) and in mammals (165, 351, 482, 489, 501, 502), adrenergic agonists administered topically or systemically increase mucociliary transport rates. This effect is observed with epinephrine, isoproterenol, or £adrenergic agonists with predominant /32 action such as fenoterol (489), salbutamol (501), carbuterol (351), and terbutaline (93). Norepinephrine has no effect on mucous transport (486). Sackner and co-workers (351) have shown a dose-dependent effect of inhaled isoproterenol and carbuterol delivered by metereddose aerosol in anesthetized dogs. Three hundred micrograms of isoproterenol and 400 fig of carbuterol produced increases in tracheal mucous velocity of 70 and 80 per cent, respectively, whereas 1,500 /ig of isoproterenol and 2,000 /mg of carbuterol both increased tracheal mucous velocity by 100 per cent. The peak effect occurred between 10 and 30 min after drug administration; however, with these doses, cardiac output increased, suggesting that the stimulation of mucous velocity might have been related in part to the drugs' acting by systemic distribution to the trachea. /3-adrenergic agents have been shown to stimulate mucociliary transport in normal subjects (145, 480, 503, 504) after impairment of mucociliary transport by pure 0 2 breathing in normal subjects (93) and in patients with bronchial asthma (505), chronic bronchitis (174, 504), or cystic fibrosis (408). In human subjects, stimulating effects have been observed with all forms of administration (oral, sublingual, parenteral, or inhalation). By the mucociliary clearance technique, 0.25 mg of terbutaline by subcutaneous injection has been shown to improve clearance by as much as 50 per cent in normal subjects (503), whereas both with the mucociliary clearance technique and the tracheal particle transport technique, the effects of comparable doses of terbutaline are much less dramatic in patients with asthma (505), chronic bronchitis

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(174, 504), and cystic fibrosis (408), at least as far as absolute values are concerned. A 50 per cent improvement of clearance in normal subjects represents a considerable stimulation of mucociliary transport, whereas in patients with obstructive lung disease, a 100 per cent increase in tracheal mucous velocity from 10 to 20 per cent of normal control values may not be of therapeutic significance. Prevention of mucociliary depression by prior administration of adrenergic agents has thus far only been demonstrated for pure 0 2 breathing (93, 165). From the available information, the following conclusions can be drawn with regard to the effect of adrenergic agents on mucociliary transport: (1) mucociliary transport is increased by ^-adrenergic agonists both under normal conditions and under conditions of impaired mucociliary activity; (2) this effect is also exerted by ^-adrenergic agonists widi /32 selectivity; (3) /3adrenergic agonists are effective in all forms of administration; and (4) the major effect of these agents may result from alterations in the physical characteristics of respiratory secretions. Although the clinical significance of mucociliary stimulation still remains to be demonstrated, it is likely that in obstructive lung disease ^-adrenergic agonists, in addition to their direct bronchodilator effect, may also benefit the patient by stimulating mucociliary transport. Cholinergic Agents In contrast to adrenergic agents, cholinergic agents have consistently been shown to stimulate ciliary activity directly (485, 487, 488, 506) in in vitro preparations. A 0.1 to 1 per cent solution of acetylcholine increases ciliary activity in explants of human respiratory epithelium, and pretreatment with eserine further enhances this effect (487). Cholinergic stimulation also stimulates mucous secretions. Vagal stimulation increases the secretions from mucous glands in die airways (507), and explants of submucosal tissue containing submucosal glands maintained in organ culture have been shown to respond to cholinergic agents with an increased production and secretion of mucus (499). Chronic cholinergic stimulation of the canine trachea in vitro stimulates the release of macromolecules and increases the concentration of enzymes involved in respiratory mucin synthesis (508, 509). Pilocarpine, a postganglionic cholinergic stimulant, increases the secretion of submucosal glands and

goblet cells (498, 510) and histochemical studies reveal an increase in the number of both acid- and neutral glycoprotein-producing goblet cells at moderate dosages, with emptying of die secretory cells (498) at higher dosages. Bang and Bang (474) observed a marked increase in mucous transport rates with pilocarpine, and related this effect to the marked increase in respiratory secretions. Similar observations have been made by other investigators using different experimental techniques (471, 477). Cholinergic agents have also been shown to stimulate mucociliary transport both in lower and higher animals (161, 430, 477, 486, 511, 512). Depending on the experimental design, the concentrations required for mucociliary stimulation to occur varied among the different investigations. In anesthetized dogs who inhaled 4 to 8 ml of nebulized acetylcholine (1 per cent solution), there was a transient increase in tracheal mucous velocity with a peak at approximately 150 per cent of baseline after 5 min and a return to baseline values after 30 min. This response in mucous transport correlated well with the concomitant decrease in respiratory system conductance (430). Subcutaneous administration of cholinergic agents also stimulates mucociliary clearance in normal human subjects (513). Cholinergic agents are potent stimulators of ciliary activity, mucous secretion, and mucociliary transport. Their bronchoconstrictor effect, however, precludes their therapeutic use in patients with impaired mucociliary transport mechanisms. Biologically Active Amines The effects of histamine and serotonin on mucociliary transport have been carefully studied in vitro and in vivo. Although these agents are not used therapeutically, they are of considerable importance as chemical mediators in certain pathologic processes in the lung, such as bronchial asthma and pulmonary embolism. Serotonin stimulates ciliary beating frequency in the lamellibrancli gill at low concentrations and might be the endogenous pacemaker substance in the cilia of this species (514-516). In contrast to this clear-cut effect, the effect of serotonin in intact animals is conflicting. In the transluminated cat trachea, the flow of mucus has been shown to increase after administration of serotonin or its precursors (5-hydroxytryptophan and 5-hydroxyindole-acetic acid) in aerosol form

107

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(517). In rat bronchi, direct exposure to serotonin had no effect on ciliary activity (471). These contradictory results may be related to differences in exposure time and drug concentrations, because stimulation has been demonstrated at low and impairment, at high concentrations in the isolated rabbit trachea (243). Histamine (1 per cent solution) does not affect ciliary activity in tracheal explants (159), and both histamine and bradykinin appear to lack an effect on mucous gland secretions (485, 499). Intravenous injection of 0.1 per cent histamine or local application of histamine to the rabbit trachea does not affect mucociliary activity (159, 418). In the preceding experiments, however, a dose- and time-dependent suppression of mucociliary transport was observed after administration of antihistaminics, and this effect was related to changes in mucous secretions (159). In another preparation, the direct application of histamine to the ciliated bronchial epithelium had no effect in low concentrations but produced ciliary discoordination in high concentrations (471). In contrast to these observations, Wanner and associates (430) found that histamine stimulates mucociliary transport. Dogs who inhaled 4 to 8 ml of nebulized histamine (1 per cent solution) increased tracheal mucous velocity to 190 per cent after 30 min and this effect was sustained for at least 2 hours, at which time tracheal mucous velocity was still 160 per cent of baseline. This effect seemed to be independent of the bronchoconstriction produced by histamine, because specific respiratory system conductance had returned to baseline values at the time when tracheal mucous velocity was still significantly increased. The inconsistencies in the results obtained with histamine and serotonin may well be related to differences in experimental techniques,

animal species, drug concentration, and mode and duration of drug administration. The effects of these agents on mucociliary transport have not been evaluated in normal subjects or patients. Methyl-Xanthines Direct in vitro exposure to methyl-xanthines (theophylline and aminophylline) increases the frequency of ciliary beat and stimulates mucous production (488, 471). Serafini and co-workers (19) also observed an increase of mucous transport in intact dogs to 64 per cent of baseline immediately after intravenous infusion of 4 mg of aminophylline per kg of body weight during a 15- to 30-min period. To our knowledge, no studies have been done in human subjects on mucociliary transport as influenced by methyl-xanthines, nor has the potentially synergistic effect of adrenergic agents and methyl-xanthines been demonstrated. As is the case for /3-adrenergic agents, methyl-xanthines may be of therapeutic value as stimulators of mucociliary transport. A list of pharmacologic agents with established stimulatory effects on mucociliary activity is presented in table 4. Miscellaneous Pharmacologic Agents The susceptibility of the mucociliary apparatus to selected pharmacologic agents that either are respiratory drugs or are otherwise commonly used in daily clinical practice is considered to be of sufficient interest to be included in this review. Antimicrobial drugs. Topical application of penicillin or streptomycin to ciliated epithelium both in vitro and in vivo has either no effect or slightly stimulates mucociliary transport (518, 519). The effects of systemically administered antimicrobial drugs have not been examined.

TABLE 4 PHARMOCOLOGIC AGENTS WITH ESTABLISHED STIMULATORY EFFECT ON MUCOCILIARY ACTIVITY Agent(s)

Ciliary Activity

Mucous Production

j3-adrenergic agonists

No effect

Stimulation (496-498, 500)

Cholinergic agents

Stimulation (485, 487, 488, 506) Stimulation (488)

Stimulation ( 4 7 1 , 474, 477, 498, 499, 507-510) Stimulation (471)

Methyl-xanthines

Mucociliary Activity Stimulation (93, 145, 165, 174, 3 5 1 , 4 0 8 , 4 7 7 , 4 8 0 , 482, 486, 489, 5 0 1 - 5 0 5 , 507) Stimulation ( 1 6 1 , 430, 477, 486, 511-513) Stimulation (19)

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Immunopharmacologic agents. Prostaglandin E p when administered as an aerosol, is a potent bronchodilator in asthmatic patients (520). Iravani and associates (496) observed in tracheal preparations from rats and cats that were incubated for 2 min that prostaglandin E x increased mucous production and promoted differentiation of goblet cells, but did not alter ciliary beating frequency. The effect of prostaglandin E1 on mucociliary transport in intact animals and human subjects remains to be demonstrated. Topical application of cromolyn sodium to intact normal tracheal mucosa has no effect on mucociliary transport (482); however, this and other immunopharmacologic agents might protect mucociliary transport in patients with extrinsic asthma (430). Corticosteroids. Although systemic corticosteroids decrease bronchial obstruction and facilitate expectoration in patients with asthma and chronic bronchitis, they do not alter sputum viscosity (521). Direct exposure of bronchial mucosa to prednisolone results in mild cilioexcitation (471), whereas 4 puffs (300 fig) of the topical corticosteroid beclamethasone diproprionate had no effect on tracheal mucous velocity in conscious sheep (522). An effect of systemic corticosteroids on mucociliary transport has not been excluded. Mucolytic agents. The clinical value of expectorants and how they affect the output and composition of respiratory secretions has been extensively reviewed by Boyd (523) and Perry and Boyd (524). The effects of mucolytic agents such as iV-acetyl-cysteine and S-carboxymethylcysteine on the rheologic properties of respiratory secretions have been clearly demonstrated in vitro (525, 526), and proteolytic properties have also been attributed to iodides in vitro (527). In addition, clinical studies suggest that the administration of mucolytic aerosols and of oral bromhexine (Bisolvon®) decrease sputum viscosity in patients with chronic bronchitis (528, 529). Also, S-carboxymethylcysteine has been suggested to facilitate expectoration in patients with obstructive lung disease when administered for long periods of time, and this effect has been related to fluidification of sputum (530); however, specific studies on the effects of mucolytic agents and expectorants on mucociliary transport are more controversial. iV-acetylcysteine has no effect on the secretion of mucous glands in vitro (499); in the exposed cat trachea

it does not alter mucociliary transport at low concentrations and even causes depression of mucociliary function at higher concentrations (151, 502). In contrast, direct exposure of bronchial epithelium in other preparations to bromhexine metabolites has been shown to cause increases in the frequency of ciliary beating and in stimulation of mucous secretions (497, 531). In the same and similar preparations, expectorants such as ammonium chloride, potassium iodide, and a terpin hydrate-related compound (ozothin) also appear to stimulate ciliary activity and the secretion of respiratory mucus (471, 502, 532). The effects of mucolytic agents and expectorants on mucociliary transport in man have been investigated with aerosol clearance techniques. A lack of effect or slight stimulation of mucociliary clearance has been reported for bromhexine in patients with chronic bronchitis (533). Glyceryl guaiacolate has no effect on mucociliary clearance in normal subjects, but improves it in patients with chronic bronchitis without affecting the deposition pattern of the inhaled particles (534). On the other hand, oral administration of S-carboxymethylcysteine does not appear to stimulate mucociliary clearance in patients with chronic obstructive lung disease after a few days of therapy (535). A critical assessment of the action of mucolytic agents and expectorants is rather difficult. Although mucolytic agents undoubtedly change the rheologic properties of respiratory secretions in vitro, their effects on mucous secretion and mucociliary transport in vivo is not clear. The demonstration of a beneficial effect of expectorants in patients with obstructive lung disease has been based on subjective symptoms and rheologic studies of expectorated sputum, whereas the few studies which attempted actually to measure mucociliary transport in these patients have not convincingly demonstrated a beneficial effect. Further studies are obviously needed to understand better the action of mucolytic agents and expectorants in patients with pulmonary disease. Cardiac glycosides. Digitoxin, digitamine, and strophanthin all stimulate ciliary beating frequency in lower animals (536, 537). Laurenzi and Yin (538) also demonstrated a stimulating effect of acetylouabain by intravenous injection on tracheal mucous transport in cats. A dosedependent increase in particle transport rates occurred between 15 and 50 jug of acetylouabain per kg of body weight and its administration

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109

also reversed or prevented a decrease in mucous transport resulting from b r e a t h i n g 0 2 . Although this observation merely represents a physiologic curiosity, it suggests a functional similarity between the contractile elements of cilia a n d cardiac muscle.

mucociliary a p p a r a t u s may protect against the development of certain l u n g disorders.

Physical Therapy Besides pharmacologic interventions, physical therapy such as chest clapping and postural drainage may also have beneficial effects on mucociliary transport in patients with l u n g disease (539). T h e effectiveness of such therapeutic modalities in improving mucociliary clearance awaits further confirmation.

References 1. Sleigh, M. A.: T h e Biology of Cilia and Flagella, Pergamon Press, Oxford, England, 1962. 2. Gray, C : Ciliary Movement, Cambridge University Press, London, England, 1928. 3. Rivera, J. A.: Cilia, Ciliated Epithelium and Ciliary activity, Pergamon Press, Oxford, England, 1962. 4. Kinosita, H., and Murakami, A.: Control of ciliary motion, Physiol Rev, 1967, 47, 53. 5. Kilburn, K. H., and Salzano, J. V., ed.: Symposium on structure, function and measurement of respiratory cilia, Am Rev Respir Dis, 1966, 93 (Part 2), 184. 6. Hilding, A. C : The role of the respiratory mucosa, Minn Med, 1967,50, 915. 7. Kilburn, K. H.: Theory and models for cellular injury and clearance failure in the lung, Yale J Biol Med, 1968, 40, 339. 8. Kilburn, K. H.: A hypothesis for pulmonary clearance and its implications, Am Rev Respir Dis, 1968,98,449. 9. Okeson, G. C , and Divertie, M. B.: Cilia and bronchial clearance: The effects of pharmacologic agents and disease, Mayo Clin Proc, 1970,45,361. 10. Asmundsson, T., and Kilburn, K. H.: Mechanisms of respiratory tract clearance, in Sputum, M. J. Dulfano, ed., Charles C Thomas, Springfield, 111., 1973, p. 107. 11. Delahunty, J. E., and Cherry, J.: T h e laryngeal saccule, J Laryngol Otol, 1969, 83, 803. 12. Leeson, T. S., and Leeson, C. R.: A light and electron microscope study of developing respiratory tissue in the rat, J Anat, 1964,98,183. 13. Von Hayek, H.: The Human Lung, Hafner, New York, 1960. 14. Gillespie, J. R., and Tyler, W. S.: Quantitative electron microscopy of the interalveolar septa of the horse lung, Am Rev Respir Dis, 1967, 95, 477. 15. Rhodin, J.: Ultrastructure of the tracheal ciliated mucosa in rat and man, Ann Otol Rhinol Laryngol, 1959,^,964. 16. Alexander, I., Ritchie, B. C , Maloney, J. E., and Hunter, C. R.: Epithelial surfaces of the trachea and principal bronchi in the rat, Thorax, 1975,30,171. 17. Rhodin, J. A. G.: Ultrastructure and function of the human tracheal mucosa, Am Rev Respir

Summary Mucociliary transport in the l u n g is capable of removing inhaled particulate m a t t e r from the ciliated airways; however, the importance of mucous transport as a p u l m o n a r y defense mechanism has not been demonstrated experimentally. N o r m a l mucociliary function d e p e n d s on a morphologically a n d functionally intact ciliated epithelium as well as n o r m a l rheologic properties a n d q u a n t i t y of respiratory secretions for optimal mucociliary interaction. T h e pathophysiologic features of the mucociliary apparatus and the presence of impaired mucous transport mechanisms u n d e r various conditions both demonstrate the extreme vulnerability of this nonrespiratory function of the lung. T h u s , decreases in t e m p e r a t u r e a n d humidity, exposure to inhalants such as cigarette smoke, atmospheric pollutants, a n d s u p p l e m e n t a l 0 2 or certain pharmacologic agents all depress mucous transport. Likewise, mucociliary dysfunction appears to be associated with obstructive l u n g diseases a n d acute respiratory infections. Stimulation of mucociliary transport by drugs, notably ^-adrenergic agonists, cholinergic agents, and methyl-xanthines has been clearly observed and may be of clinical relevance in patients with retained tracheobronchial secretions. Because several studies have demonstrated that i m p a i r m e n t of mucous transport may represent the earliest detectable sign of l u n g injury, it is n o t surprising that mucociliary dysfunction has been implicated as a pathogenetic factor in a n u m b e r of pathologic conditions of the lung. Although this has n o t been d o c u m e n t e d experimentally, the possibility remains that an intact

Acknowledgment The author wishes to thank Ms. Isabel Rodriguez for her devotion in preparing this manuscript.

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Clinical aspects of mucociliary transport.

State of the Art Clinical Aspects of Mucociliary Transport1 ADAM WANNER Contents Introduction Normal Structure and Function Structure Function Cilia...
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