FUNCTIONAL MORPHOLOGY OF THE MUCOSA OF THE MIDDLE EAR AND EUSTACHIAN TUBE DAVID
J. LIM, M.D.
COLUMBUS, OHIO
SUMMARY - A review of available histological, histochemical and ultrastructural data on middle ear mucosa and the Eustachian tube was made to provide a broad cellular basis for understanding middle ear effusions. The presence of mucoeiliary defense system in a large part of the Eustachian tube and middle ear is seen as the first line of defense. Secretion by the mucosa has a profound biological significance. Immunoglobulins A, G, and even E and Mare produced locally by the mucosa and may contribute to the immunodefense of the middle ear. Secretory lysozyme is also produced by the mucosa and may contribute enzymatic defense of the ear. Mucosal immunoglobulins and lysozyme are significantly elevated in the effusions, which would imply that local defense systems are hyperactive in OME. It also appears thaI these increases are related to the increase of the secretory cell population. It is also suspected that auditory surface-active agent is produced locally and may facilitate normal function of the tube. The middle ear also can transport macromolecules very rapidly across intact mucosal epithelium. The large numbers of tissue and wandering macrophages found in the mucosa and effusions would also imply that the middle ear is capable of efficient phagocytosis, which may be involved in processing antigen.
The purpose of this presentation is to review available histological, histochemical and ultrastructural data on middle ear mucosa and the Eustachian tube and to provide a broad morphological and cellular basis for understanding middle ear effusions in perspective. Therefore, possible functional implications of cells present in these areas are included. This paper is condensed mainly from the author's previous review paper,' but with some modification and additions where new data have become available. This review is based primarily on a series of investigations conducted in this laboratory over the last ten years. Materials used include middle ear mucosa obtained from autopsy and mucosal biopsy of humans and from laboratory animals such as guinea pigs and squirrel monkeys. RESULTS
Morphology of the Eustachian Tube. It has often been suggested that the ana-
tomical structures of the tube, particularly in children, make them prone to frequent ear infections and also are the
underlying causes of otitis media with effusions (0ME). It is well established that the tube in laboratory animals and humans is formed by two anatomically distinct parts: bony and cartilaginous. These two parts are linked by a narrowing known as the tubal isthmus in the human and primate (Fig. 1). It is important to note that the tympanal orifice is located much higher than the hypotympanum. Therefore, when the head is kept upright, the tube cannot serve as a passively draining tube from the middle ear if it should develop effusions. The tympanal orifice of the tube is shorter in young individuals than in adults. In children, the tubal cartilage is found even in the bony portion, as in the rodent and feline. The epithelium of the bony portion of the Eustachian tube is formed by tall pseudostratified columnar cells. Approximately 80% of the epithelial cells are ciliated and the remaining 20% are secretory." The tubal isthmus occurs roughly at
From the Otological Research Laboratories, Department of Otolaryngology, The Ohio State University College of Medicine, Columbus, Ohio. This study is supported in part by a research grant from the NIH-NINCDS NS-08854·05. 36
FUNCTIONAL MORPHOLOGY OF MUCOSA
LeV&10r >leliplllalini
Fig. 1. An artist's view of the middle ear mastoid and the Eustachian tube demonstrating anatomical landmarks.
Fig. 2. Transmission electron micrograph of the guinea pig shows the demilune cells (D) and mucous cells (M) in the tubal gland. The insert shows a phase contrast micrograph of the demilune cells (D) and mucous cells (M).
37
38
DAVID L. LIM
Fig. 3. Scanning electron micrograph illustrates the ductal opening (DO) of the tubal gland from which the mucus strand (M) is expelled. Spherical mucus droplets ( MD) are seen on the surface of the epithelial cells. C - Cilia.
the junction between the bony portion and cartilaginous portion of the tube and represents the narrowest point in the whole tube. The diameter of this isthmus is smaller in children (about 2.4 mm x 0.8 mm) than in adults (about 4.3 mm x 1.7 mm ) and also varies from individual to individual. The cartilaginous portion of the tube, when cross-sectioned, shows a slit-like lumen which is reinforced partly by a cartilage resembling a shepherd's crook. The epithelial cells of this portion of the tube are formed mainly by pseudostratified tall columnar ciliated epithelium with secretory cells and nonsecretory cells. Occasionally, simple squamous mucosal epithelium covers the lumen, particularly near the pharyngeal orifice. The cartilaginous part of the tube is richly endowed with seromucous glands, which are composed of mucous parts and serous parts (demilune). The latter contain dark secretory granules (Fig. 2). Several glands open into a secretory duct, which in tum opens directly into the tub-
al lumen and the secreta pours out to bathe the epithelial lining (Fig. 3).
Morplwlogy of the Middle Ear Mucosa. The mucosa of human and animal alike is formed by epithelium and subepithelial connective tissue. Subepithelial connective tissue of the middle ear, which is mainly formed by collagen fibers, contains numerous blood capillaries, lymph capillaries, and nerve fibers. The cell components in the submucosa are mainly fibrocytes, but occasional mast cells, macrophages, lymphocytes and plasma cells are also found (Fig. 4). The mast cell granules are known to contain histamine, serotonin and heparin. The mast cell degranulation in the mucosa was implicated as a possible chemical mediator involved in increased vascular permeability in human OME and also in experimental OME.I Whether the mucosal epithelium of the middle ear is a true respiratory epithelium or not has been argued, but it is now generally agreed that it is.' Electron
FUNCTIONAL MORPHOLOGY OF MUCOSA
39
cell
Non- secretory cell
I ··0
Fig. 4. Representative cells in the middle ear mucosa as illustrated by an artist. BM - Basement membrane.
microscopic study shows that a larger portion of the middle ear cavity is covered by tall columnar cells near the tube and hypotympanum, and by cuboidal cells and simple squamous mucosal cells near the promontory, whereas the mastoid antrum is covered by simple squamous mucosal epithelium. The morphology of the ciliated cell is identical to the description of other ciliated cells. Whether or not the differentiated mucosal epithelium can become a ciliated cell or secretory cell is not yet settled. It was suggested that the middle ear mucosa under certain pathological conditions undergoes "metaplasia" to become a more active secreting membrane" or even a keratinizing epithelium.' Lim and Klamer" suggested that the basal cell differentiates to become either a ciliated or secretory cell as soon as the injured epithelial cells degenerate and are sloughed off. The basal cells are
generally considered to be undifferentiated cells which become one of the three aforementioned epithelial cell types by differentiation. The secretory cells are more diverse, not only in their size and shape, but also in their morphological characteristics of the secretory granules. The most common secretory cell in the mucosa is the mucus secreting cell characterized by its distinct light secretory granules. But the less commonly occurring secretory cells are dark granulated and mixed granulated cells.' "Dark" denotes the osmiophilic nature of these secretory granules, similar to those found in the demilune. Often the core of the mucus granule may be osmiophilic. While these dark granules can be considered to be an immature form of mucigen granules, the possibility that they may contain enzymes or unidentified bioactive substances is raised
40
DAVID L. LIM
for the following reasons: 1) The dark granulated cells resemble the demilune cells of the tubal glands. These dark secretory granules are morphologically similar to the secretory granules of enzymesecreting pancreatic acini cells and gastric Paneth cells. 2) Autoradiographic studies? using isotope labeled leucin and glucose demonstrated that the mucous cell incorporated a large amount of glucose, while the dark granulated cell took up more leucin than the mucous cells. This finding suggests biochemical diversity of secretory granules. 3) The presence of surface-active substances in the middle ear and tubal washings was demonstrated by Rapport et al,7 and their localization appears to be related to the dark cells by cytochemical study.' Therefore, it is possible that the middle ear mucosa secretes some bioactive substances.
Mucociliary Transportation System. The ciliated mucosa is known to have an ability to transport foreign particles when attached to the mucus blanket by metachronal motion of motile cilia. This transportation mechanism is considered a protective function in the respiratory mucosa. The system comprises three basic components: ciliated cells, secretory cells and mucus blanket. Sade" demonstrated the presence of this transportation system in the middle ear cavity in patients with a large dry perforation. Our study of cadaver materials provided direct evidence of the presence of an active mucociliary system in the normal middle ear mucosa. 1 Indirect evidence of such a system in the middle ear came from morphological study, and it is important to note that the distribution of the ciliated cells in the middle ear is unequivocally parallel to that of the secretory cells.' This finding strongly supports the concept that a large part of the middle ear mucosa and Eustachian tube is protected by the mucociliary transportation system. In the middle ear cavity, this system is most well developed in the following descending order: nearest the tympanic orifice of the tube, hypotympanum, epitympanum and promontory. Whether the mastoid air cells are protected by this system
is not yet determined. Metaplastic proliferation of ciliated cells and secretory cells in the middle ear mucosa of certain cases of OME can be interpreted as an expression of the mucosa to improve its protective ability. The secreted mucus is presumably made up of mucopolysaccharides and glycoprotein and is the basic substance in forming the mucus blanket. As mentioned earlier, effective transportation of foreign particles attached to the blanket is brought about by coordinated beating of the cilia (metachronal motion). How this coordination is accomplished by the ciliated cells remains an enigma. A recent study reported by Sade and his coworkers" sheds some interesting light on this problem. When mucus of the frog palate is replaced by synthetic polymer with similar rheological consistency, the active cilia fail to transport the blanket. However, when the synthetic mucus is replaced by the cow's cervical mucus, the same ineffective but active cilia become effective. This phenomenon is interpreted as the coupling of biological mucus with cilia which is essential for the effective metachronal motion of cilia. This coupling is made possible, perhaps, by certain chemical substances present in biological mucus. Therefore, changes of biochemical contents of the secretion may impair the effectiveness of the mucociliary system even if the cilia may be actively beating. Whether or not this situation exists in a certain type of OME is a matter of conjecture.
Local Immunoglobulin Production. Most of the mucous membranes of the body are now known to produce secretory immunoglobulins. McMaster and Hudaek" suggested that the middle ear is endowed with a local immunodefense system. Recent immunochemical and immunofluorescent studies l o - 1 2 in OME clearly demonstrated the presence not only of secretory IgA but also of IgG, IgM and IgE. This finding indicated that secretion may play an immunological role in OME. It was suggested that normal middle ear mucosa and the Eustachian tube also produce such immunoglobulins. A separate study from our laboratory'? strongly supports this notion,
FUNCTIONAL MORPHOLOGY OF MUCOSA
because the normal middle ear mucosa and Eustachian tube of squirrel monkeys are capable of producing IgA, IgG, IgM and IgE by plasma cells located in the submucosa. When the tube was experimentally obstructed, the number of immunoglobulin-producing plasma cells increased considerably. Furthermore, the epithelial cells of the mucosa and Eustachian tube are distinctly stained with IgA antiserum. Other investigators'v!' confirm that this immunoglobulin is produced by the middle ear mucosa. Along with secretory IgA, IgE is also considered a mucosal immunoglobulin. IgE is capable of releasing histamine from leukocytes." IgE producing plasma cells are known to occur in the nasal mucosal tissue and are implicated in nasal allergic reaction. The presence of IgE producing plasma cells in the middle ear mucosa of the primate and human'" may support the notion that the middle ear mucosa is immunologically similar to the respiratory mucosa elsewhere. The occurrence of macrophages in certain types of middle ear effusions is well established." The tissue macrophages are also seen in normal middle ear mucosa of both humans and animals. It is of further interest that the presence of immunoglobulins enhances phagocytosis (opsonization) of leukocytes and macrophages. The macrophages are known to process antigens, which in tum would sensitize the lymphocytes. Miglets" demonstrated that the middle ear of the squirrel monkey can be sensitized by anti-ragweed serum. When this sensitized ear is challenged by the ragweed pollens, the ear reacts as a shock organ. The immunologic mechanisms involved in this reaction are yet to be investigated; however, it is suspected that the mechanisms involved in the above experiment may be similar ( if not identical) to those of respiratory allergy. The presence of numerous macrophages and immunoglobulin-producing cells in the middle ear mucosa of patients with OME may suggest involvement of an immunologic response in producing effusions. The presence of mast cells in the vicinity of the capillaries in
41
the submucosa further supports the notion that the vasoactive amines released by immunologic reaction may have caused increased capillary permeability.' Whether the presence of these immunocompetent cells in the middle ear mucosa of patients with OME and of experimental monkeys is a result of specific immunologic response or just a nonspecific response in inflammation requires further study.
Secretory Enzymes. It is well established that nasal secretion contains a large quantity of (secretory) lysozyme. It is also found in tears, in saliva, in urine, and in many other body fluids. This enzyme is known to have certain bactericidal properties, particularly in the presence of immunoglobulins and the complements. Veltri and Sprinkle" reported that lysozyme is found in large quantities in certain middle ear effusions. Liu et al1 2 further showed that mucoidtype effusions had significantly higher lysozyme concentration than serous- or leukocytic-type effusions, suggesting the secretory nature of the enzyme since the mucoid type also contained high IgA. The source of this enzyme has been controversial, but now it is known to originate from mucosal secretory granules, confirming the de novo synthesis hypothesis." Acid phosphatase is often used in cytochemistry as a marker for lysosomes, which contain numerous hydrolytic enzymes. It is well established that the lysosomal enzymes have a variety of biological properties that are essential for morphogenesis and homeostasis of an organ. The lysosomal enzyme is present in dark granules of leukocytes and also macrophages. The epithelial cells of the mucosa also contain numerous lysosomes following ear infection. Lim! demonstrated the presence of acid phosphatase in the secretory granules and suggested the possibility of secretion of lysosomal enzymes by the epithelial cells. Although a strong acid phosphatase reaction was observed in lysosomes, the most interesting finding was the consistent localization of this enzyme in the cytoplasm of secretory cells. It is possible that the mucosal secretory cells may process or in-
42
DAVID L. LIM
corporate part of the lysosomal enzymes into the secretion which partly contrihutes to the enzymatic defense of the middle ear. Auditory Surface-active Agents. The normal Eustachian tube is thought to be closed most of the time, but is opened ?ccasionally by tubal muscles to equalize the pressure. Therefore, it is logical that the tube and the middle ear mucosa are covered by (secreted) surface-active agent ( s) to facilitate efficient tubal function. Birken and Brookler'" initially observed that washings of the Eustachian tube of the dog contain surface tension lowering substances. Rapport et al' demonstrated physiological evidence of a surface-active agent in the guinea pig Eustachian tube. This agent(s) may have a similar function to the pulmonary surfactant. The pulmonary surfactant from mammals is composed of saturated phospholipids (lecithin), protein and mucopolysaccharides. Recent improvement of the cytochemical technique using the tricomplex flocculation procedure successfully preserved surfactant on the surface lining of the alveolus. It is generally believed that surfactant is produced by the alveolar secretory cell (Type II) which contains lamellar granules. We have demonstrated that the secreta of the tube and the middle ear mucosa in the guinea pig contain substances (phospholipids?) which reacted with tricomplex flocculation.' Numerous small granular substances, often covering dark cores of the secretory granules that are expelled into the glandular lumen, showed lamellar formation resembling pulmonary surfactant granules. Whether these lamellar granular substances found in the tube and middle ear are chemically identical to the pulmonary surfactant granules is not confirmed. However, it is tempting to speculate that the function of this material is to lower the surface tension which is necessary for the normal physiological function of the tube. The disturbance of the secretion of such substances may cause the malfunctioning of the Eustachian tube. . Macromolecular Transport by the Mucosal Epithelium. It is known that the
middle ear mucosa can rapidly resorb radioisotopes, suggesting that the middle ear mucosa has an effective resorptive function. The method of macromolecular transport through intact mucosal epithelium was also studied using tracer particles.' The majority of these particles are cleared through the tube, but when horseradish peroxidase (HRP) is used, large numbers of these macromolecules are taken up immediately by the mucosal epithelium through pinocytosis. The particles then pass through the basement membrane and are found in the submucosal connective tissue, where some of them are ingested by macrophages, however, the majority of them enter into the lymphatic capillaries. These particles are then eventually transported into the draining lymph nodes and can be found within five minutes in preauricular nodes and within 15 minutes in deep cervical nodes following the initial injection.' The initial ingestion of these particles is accomplished by all types of epithelial cells, which includes simple squamous cells, ciliated cells and secretory cells. Whether the failure to resorb fluid by the epithelial cells can be a contributing factor in OME is not yet established. However, obstruction of the lymphatic drainage from the middle ear and the tube was suggested to be the underlying cause of some cases of OME. A reverse transport of macromolecules by the mucosal epithelial cells was reported by several authors in experimental animals. Paparella and his associates" demonstrated increased capillary permeability of the middle ear mucosal vessels when the tube was obstructed. The carbon particles that were injected into the vessels readily passed through disrupted capillaries and eventually reached the middle ear effusions by going through separated tight junctions. These authors suggested that the middle ear effusion in their experimental model came from serum. In summary, it can be suggested that biological function of the mucosal membrane of the middle ear and Eustachian tube has the following defense systems: 1) mucociliary; 2) secretory (immunoglobulins, enzymes, and surface-active
FUNCTIONAL MORPHOLOGY OF MUCOSA
agents); and 3) phagocytic (macrophages). It is further suggested that
43
these defense systems are activated in OME.
REFERENCES 1. Lim OJ: Functional morphology of 12. Liu YS, Lim OJ, Lang RW, et al: the lining membrane of the middle ear and Chronic middle ear effusions: ImmunochemiEustachian tube: An overview. Ann Otol cal and bacteriological investigations. Arch Rhinol Laryngol 88:5-26, 1974 Otolaryngol 101 :278-286, 1975 2. Shimada T, Lim OJ: Distribution of 13. Hussl B, Lim OJ: Experimental midciliated cells in the human middle ear dle ear effusions - an immunofluorescent SEM, TEM, and light microscopic observastudy. Ann Otol Rhinol Laryngol 83:332-342, tion. Ann Otol Rhinol Laryngol 81 :203-211, 1974 1972 14. Ishizaka K, Ishizaka T, Tada T, et al: 3. Sade J, Eliezer N, Silverberg A, et al: Site of synthesis and function of gamma-E. The role of mucus in transport by cilia. Am in Dayton 0, et al (eds ): Secretory ImmunoRev Respir Dis 102:48-52, 1970 logic System. Bethesda, Maryland, US HEW PHS NIH National Institutes of Child Health 4. Greenberg SO, Dickey JR, Neely JG: and Human Development, 1969, pp 71-80 Effects of tobacco on the ear. Ann Otol Rhinol Laryngol 82:311-317, 1973 15. Phillips MJ, Knight NJ, Manning H, 5. Lim OJ, Klainer A: Cellular reactions et al: IgE and secretory otitis media. Lancet in acute otitis media - scanning and trans- 2:1176-1179, 1974 mission electron microscopy. Laryngoscope 16. Lim OJ, Birck H: Ultrastructural pa81:1772-1786, 1971 thology of the middle ear mucosa in serous 6. Lim OJ: Protein secreting cells in the otitis media. Ann Otol Rhinol Laryngol 80: normal middle ear mucosa of the guinea pig. 838-853, 1971 Ann Otol Rhinol Laryngol 79:82-94, 1970 17. Miglets A: The experimental produc7. Rapport PN, Lim OJ, Weiss HS: Surtion of allergic middle ear effusions. Larynface-active agent in eustachian tube function. goscope 88:1355-1384, 1973 Arch Otolaryngol 101 :305-311, 1975 18. Veltri RW, Sprinkle PM: Serous otitis 8. Sade J: Ciliary activity and middle ear media - immunoglobulin and lysozyme levels clearance. Arch Otolaryngol 86: 128-135, 1967 in middle ear fluids and serum. Ann Otol Rhinol Laryngol 82:297-301, 1973 9. McMaster P, Hudack S, cited by Humphrey JH, White RG: Immunology for 19. Lim OJ, Liu YS, Birck H: Secretory Students of Medicine, ed 3. Philadelphia, FA lysozyme of the human middle ear mucosa Davis Company, 1970, p 263 immunocytochemical localization. Ann Otol 10. Bernstein JM, Hayes ER, Ishikawa T, Rhinol Laryngol 85:50-60, 1976 et al: Secretory otitis media - a histopatho20. Birken EA, Brookler KH: Surface tenlogic and immunochemical report. Trans Am sion lowering substance of the canine EustaAcad Ophthalmol Otolaryngol 76: 1305-1318, chian tube. Ann Otol Rhinol Laryngol 81: 1972 268-271, 1972 11. Mogi G, Honjo S, Maeda S, et al: Se21. Paparella M, Hiraide F, Juhn S, et al: cretory immunoglobulin A (SIgA) in middle Cellular events involved in middle ear fluid ear effusions. A further report. Ann Otol production. Ann Otol Rhinol Laryngol 79: Rhinol Laryngol 83:92-101, 1974. 766-780, 1970 REPRINTS - David J. Lim, M.D., Otological Research Laboratories, Department of Otolaryngology, The Ohio State University College of Medicine, 456 Clinic Dr., Columbus, OH 43210. ACKNOWLEDGMENT - Ilija Karanfilov, Joan Osborne, Jonathan Darby, and Katherine Adamson provided invaluable assistance. Illustration was made by Nancy Sally.
J. LIM, M.D.
COLUMBUS, OHIO
SUMMARY - A review of available histological, histochemical and ultrastructural data on middle ear mucosa and the Eustachian tube was made to provide a broad cellular basis for understanding middle ear effusions. The presence of mucoeiliary defense system in a large part of the Eustachian tube and middle ear is seen as the first line of defense. Secretion by the mucosa has a profound biological significance. Immunoglobulins A, G, and even E and Mare produced locally by the mucosa and may contribute to the immunodefense of the middle ear. Secretory lysozyme is also produced by the mucosa and may contribute enzymatic defense of the ear. Mucosal immunoglobulins and lysozyme are significantly elevated in the effusions, which would imply that local defense systems are hyperactive in OME. It also appears thaI these increases are related to the increase of the secretory cell population. It is also suspected that auditory surface-active agent is produced locally and may facilitate normal function of the tube. The middle ear also can transport macromolecules very rapidly across intact mucosal epithelium. The large numbers of tissue and wandering macrophages found in the mucosa and effusions would also imply that the middle ear is capable of efficient phagocytosis, which may be involved in processing antigen.
The purpose of this presentation is to review available histological, histochemical and ultrastructural data on middle ear mucosa and the Eustachian tube and to provide a broad morphological and cellular basis for understanding middle ear effusions in perspective. Therefore, possible functional implications of cells present in these areas are included. This paper is condensed mainly from the author's previous review paper,' but with some modification and additions where new data have become available. This review is based primarily on a series of investigations conducted in this laboratory over the last ten years. Materials used include middle ear mucosa obtained from autopsy and mucosal biopsy of humans and from laboratory animals such as guinea pigs and squirrel monkeys. RESULTS
Morphology of the Eustachian Tube. It has often been suggested that the ana-
tomical structures of the tube, particularly in children, make them prone to frequent ear infections and also are the
underlying causes of otitis media with effusions (0ME). It is well established that the tube in laboratory animals and humans is formed by two anatomically distinct parts: bony and cartilaginous. These two parts are linked by a narrowing known as the tubal isthmus in the human and primate (Fig. 1). It is important to note that the tympanal orifice is located much higher than the hypotympanum. Therefore, when the head is kept upright, the tube cannot serve as a passively draining tube from the middle ear if it should develop effusions. The tympanal orifice of the tube is shorter in young individuals than in adults. In children, the tubal cartilage is found even in the bony portion, as in the rodent and feline. The epithelium of the bony portion of the Eustachian tube is formed by tall pseudostratified columnar cells. Approximately 80% of the epithelial cells are ciliated and the remaining 20% are secretory." The tubal isthmus occurs roughly at
From the Otological Research Laboratories, Department of Otolaryngology, The Ohio State University College of Medicine, Columbus, Ohio. This study is supported in part by a research grant from the NIH-NINCDS NS-08854·05. 36
FUNCTIONAL MORPHOLOGY OF MUCOSA
LeV&10r >leliplllalini
Fig. 1. An artist's view of the middle ear mastoid and the Eustachian tube demonstrating anatomical landmarks.
Fig. 2. Transmission electron micrograph of the guinea pig shows the demilune cells (D) and mucous cells (M) in the tubal gland. The insert shows a phase contrast micrograph of the demilune cells (D) and mucous cells (M).
37
38
DAVID L. LIM
Fig. 3. Scanning electron micrograph illustrates the ductal opening (DO) of the tubal gland from which the mucus strand (M) is expelled. Spherical mucus droplets ( MD) are seen on the surface of the epithelial cells. C - Cilia.
the junction between the bony portion and cartilaginous portion of the tube and represents the narrowest point in the whole tube. The diameter of this isthmus is smaller in children (about 2.4 mm x 0.8 mm) than in adults (about 4.3 mm x 1.7 mm ) and also varies from individual to individual. The cartilaginous portion of the tube, when cross-sectioned, shows a slit-like lumen which is reinforced partly by a cartilage resembling a shepherd's crook. The epithelial cells of this portion of the tube are formed mainly by pseudostratified tall columnar ciliated epithelium with secretory cells and nonsecretory cells. Occasionally, simple squamous mucosal epithelium covers the lumen, particularly near the pharyngeal orifice. The cartilaginous part of the tube is richly endowed with seromucous glands, which are composed of mucous parts and serous parts (demilune). The latter contain dark secretory granules (Fig. 2). Several glands open into a secretory duct, which in tum opens directly into the tub-
al lumen and the secreta pours out to bathe the epithelial lining (Fig. 3).
Morplwlogy of the Middle Ear Mucosa. The mucosa of human and animal alike is formed by epithelium and subepithelial connective tissue. Subepithelial connective tissue of the middle ear, which is mainly formed by collagen fibers, contains numerous blood capillaries, lymph capillaries, and nerve fibers. The cell components in the submucosa are mainly fibrocytes, but occasional mast cells, macrophages, lymphocytes and plasma cells are also found (Fig. 4). The mast cell granules are known to contain histamine, serotonin and heparin. The mast cell degranulation in the mucosa was implicated as a possible chemical mediator involved in increased vascular permeability in human OME and also in experimental OME.I Whether the mucosal epithelium of the middle ear is a true respiratory epithelium or not has been argued, but it is now generally agreed that it is.' Electron
FUNCTIONAL MORPHOLOGY OF MUCOSA
39
cell
Non- secretory cell
I ··0
Fig. 4. Representative cells in the middle ear mucosa as illustrated by an artist. BM - Basement membrane.
microscopic study shows that a larger portion of the middle ear cavity is covered by tall columnar cells near the tube and hypotympanum, and by cuboidal cells and simple squamous mucosal cells near the promontory, whereas the mastoid antrum is covered by simple squamous mucosal epithelium. The morphology of the ciliated cell is identical to the description of other ciliated cells. Whether or not the differentiated mucosal epithelium can become a ciliated cell or secretory cell is not yet settled. It was suggested that the middle ear mucosa under certain pathological conditions undergoes "metaplasia" to become a more active secreting membrane" or even a keratinizing epithelium.' Lim and Klamer" suggested that the basal cell differentiates to become either a ciliated or secretory cell as soon as the injured epithelial cells degenerate and are sloughed off. The basal cells are
generally considered to be undifferentiated cells which become one of the three aforementioned epithelial cell types by differentiation. The secretory cells are more diverse, not only in their size and shape, but also in their morphological characteristics of the secretory granules. The most common secretory cell in the mucosa is the mucus secreting cell characterized by its distinct light secretory granules. But the less commonly occurring secretory cells are dark granulated and mixed granulated cells.' "Dark" denotes the osmiophilic nature of these secretory granules, similar to those found in the demilune. Often the core of the mucus granule may be osmiophilic. While these dark granules can be considered to be an immature form of mucigen granules, the possibility that they may contain enzymes or unidentified bioactive substances is raised
40
DAVID L. LIM
for the following reasons: 1) The dark granulated cells resemble the demilune cells of the tubal glands. These dark secretory granules are morphologically similar to the secretory granules of enzymesecreting pancreatic acini cells and gastric Paneth cells. 2) Autoradiographic studies? using isotope labeled leucin and glucose demonstrated that the mucous cell incorporated a large amount of glucose, while the dark granulated cell took up more leucin than the mucous cells. This finding suggests biochemical diversity of secretory granules. 3) The presence of surface-active substances in the middle ear and tubal washings was demonstrated by Rapport et al,7 and their localization appears to be related to the dark cells by cytochemical study.' Therefore, it is possible that the middle ear mucosa secretes some bioactive substances.
Mucociliary Transportation System. The ciliated mucosa is known to have an ability to transport foreign particles when attached to the mucus blanket by metachronal motion of motile cilia. This transportation mechanism is considered a protective function in the respiratory mucosa. The system comprises three basic components: ciliated cells, secretory cells and mucus blanket. Sade" demonstrated the presence of this transportation system in the middle ear cavity in patients with a large dry perforation. Our study of cadaver materials provided direct evidence of the presence of an active mucociliary system in the normal middle ear mucosa. 1 Indirect evidence of such a system in the middle ear came from morphological study, and it is important to note that the distribution of the ciliated cells in the middle ear is unequivocally parallel to that of the secretory cells.' This finding strongly supports the concept that a large part of the middle ear mucosa and Eustachian tube is protected by the mucociliary transportation system. In the middle ear cavity, this system is most well developed in the following descending order: nearest the tympanic orifice of the tube, hypotympanum, epitympanum and promontory. Whether the mastoid air cells are protected by this system
is not yet determined. Metaplastic proliferation of ciliated cells and secretory cells in the middle ear mucosa of certain cases of OME can be interpreted as an expression of the mucosa to improve its protective ability. The secreted mucus is presumably made up of mucopolysaccharides and glycoprotein and is the basic substance in forming the mucus blanket. As mentioned earlier, effective transportation of foreign particles attached to the blanket is brought about by coordinated beating of the cilia (metachronal motion). How this coordination is accomplished by the ciliated cells remains an enigma. A recent study reported by Sade and his coworkers" sheds some interesting light on this problem. When mucus of the frog palate is replaced by synthetic polymer with similar rheological consistency, the active cilia fail to transport the blanket. However, when the synthetic mucus is replaced by the cow's cervical mucus, the same ineffective but active cilia become effective. This phenomenon is interpreted as the coupling of biological mucus with cilia which is essential for the effective metachronal motion of cilia. This coupling is made possible, perhaps, by certain chemical substances present in biological mucus. Therefore, changes of biochemical contents of the secretion may impair the effectiveness of the mucociliary system even if the cilia may be actively beating. Whether or not this situation exists in a certain type of OME is a matter of conjecture.
Local Immunoglobulin Production. Most of the mucous membranes of the body are now known to produce secretory immunoglobulins. McMaster and Hudaek" suggested that the middle ear is endowed with a local immunodefense system. Recent immunochemical and immunofluorescent studies l o - 1 2 in OME clearly demonstrated the presence not only of secretory IgA but also of IgG, IgM and IgE. This finding indicated that secretion may play an immunological role in OME. It was suggested that normal middle ear mucosa and the Eustachian tube also produce such immunoglobulins. A separate study from our laboratory'? strongly supports this notion,
FUNCTIONAL MORPHOLOGY OF MUCOSA
because the normal middle ear mucosa and Eustachian tube of squirrel monkeys are capable of producing IgA, IgG, IgM and IgE by plasma cells located in the submucosa. When the tube was experimentally obstructed, the number of immunoglobulin-producing plasma cells increased considerably. Furthermore, the epithelial cells of the mucosa and Eustachian tube are distinctly stained with IgA antiserum. Other investigators'v!' confirm that this immunoglobulin is produced by the middle ear mucosa. Along with secretory IgA, IgE is also considered a mucosal immunoglobulin. IgE is capable of releasing histamine from leukocytes." IgE producing plasma cells are known to occur in the nasal mucosal tissue and are implicated in nasal allergic reaction. The presence of IgE producing plasma cells in the middle ear mucosa of the primate and human'" may support the notion that the middle ear mucosa is immunologically similar to the respiratory mucosa elsewhere. The occurrence of macrophages in certain types of middle ear effusions is well established." The tissue macrophages are also seen in normal middle ear mucosa of both humans and animals. It is of further interest that the presence of immunoglobulins enhances phagocytosis (opsonization) of leukocytes and macrophages. The macrophages are known to process antigens, which in tum would sensitize the lymphocytes. Miglets" demonstrated that the middle ear of the squirrel monkey can be sensitized by anti-ragweed serum. When this sensitized ear is challenged by the ragweed pollens, the ear reacts as a shock organ. The immunologic mechanisms involved in this reaction are yet to be investigated; however, it is suspected that the mechanisms involved in the above experiment may be similar ( if not identical) to those of respiratory allergy. The presence of numerous macrophages and immunoglobulin-producing cells in the middle ear mucosa of patients with OME may suggest involvement of an immunologic response in producing effusions. The presence of mast cells in the vicinity of the capillaries in
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the submucosa further supports the notion that the vasoactive amines released by immunologic reaction may have caused increased capillary permeability.' Whether the presence of these immunocompetent cells in the middle ear mucosa of patients with OME and of experimental monkeys is a result of specific immunologic response or just a nonspecific response in inflammation requires further study.
Secretory Enzymes. It is well established that nasal secretion contains a large quantity of (secretory) lysozyme. It is also found in tears, in saliva, in urine, and in many other body fluids. This enzyme is known to have certain bactericidal properties, particularly in the presence of immunoglobulins and the complements. Veltri and Sprinkle" reported that lysozyme is found in large quantities in certain middle ear effusions. Liu et al1 2 further showed that mucoidtype effusions had significantly higher lysozyme concentration than serous- or leukocytic-type effusions, suggesting the secretory nature of the enzyme since the mucoid type also contained high IgA. The source of this enzyme has been controversial, but now it is known to originate from mucosal secretory granules, confirming the de novo synthesis hypothesis." Acid phosphatase is often used in cytochemistry as a marker for lysosomes, which contain numerous hydrolytic enzymes. It is well established that the lysosomal enzymes have a variety of biological properties that are essential for morphogenesis and homeostasis of an organ. The lysosomal enzyme is present in dark granules of leukocytes and also macrophages. The epithelial cells of the mucosa also contain numerous lysosomes following ear infection. Lim! demonstrated the presence of acid phosphatase in the secretory granules and suggested the possibility of secretion of lysosomal enzymes by the epithelial cells. Although a strong acid phosphatase reaction was observed in lysosomes, the most interesting finding was the consistent localization of this enzyme in the cytoplasm of secretory cells. It is possible that the mucosal secretory cells may process or in-
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DAVID L. LIM
corporate part of the lysosomal enzymes into the secretion which partly contrihutes to the enzymatic defense of the middle ear. Auditory Surface-active Agents. The normal Eustachian tube is thought to be closed most of the time, but is opened ?ccasionally by tubal muscles to equalize the pressure. Therefore, it is logical that the tube and the middle ear mucosa are covered by (secreted) surface-active agent ( s) to facilitate efficient tubal function. Birken and Brookler'" initially observed that washings of the Eustachian tube of the dog contain surface tension lowering substances. Rapport et al' demonstrated physiological evidence of a surface-active agent in the guinea pig Eustachian tube. This agent(s) may have a similar function to the pulmonary surfactant. The pulmonary surfactant from mammals is composed of saturated phospholipids (lecithin), protein and mucopolysaccharides. Recent improvement of the cytochemical technique using the tricomplex flocculation procedure successfully preserved surfactant on the surface lining of the alveolus. It is generally believed that surfactant is produced by the alveolar secretory cell (Type II) which contains lamellar granules. We have demonstrated that the secreta of the tube and the middle ear mucosa in the guinea pig contain substances (phospholipids?) which reacted with tricomplex flocculation.' Numerous small granular substances, often covering dark cores of the secretory granules that are expelled into the glandular lumen, showed lamellar formation resembling pulmonary surfactant granules. Whether these lamellar granular substances found in the tube and middle ear are chemically identical to the pulmonary surfactant granules is not confirmed. However, it is tempting to speculate that the function of this material is to lower the surface tension which is necessary for the normal physiological function of the tube. The disturbance of the secretion of such substances may cause the malfunctioning of the Eustachian tube. . Macromolecular Transport by the Mucosal Epithelium. It is known that the
middle ear mucosa can rapidly resorb radioisotopes, suggesting that the middle ear mucosa has an effective resorptive function. The method of macromolecular transport through intact mucosal epithelium was also studied using tracer particles.' The majority of these particles are cleared through the tube, but when horseradish peroxidase (HRP) is used, large numbers of these macromolecules are taken up immediately by the mucosal epithelium through pinocytosis. The particles then pass through the basement membrane and are found in the submucosal connective tissue, where some of them are ingested by macrophages, however, the majority of them enter into the lymphatic capillaries. These particles are then eventually transported into the draining lymph nodes and can be found within five minutes in preauricular nodes and within 15 minutes in deep cervical nodes following the initial injection.' The initial ingestion of these particles is accomplished by all types of epithelial cells, which includes simple squamous cells, ciliated cells and secretory cells. Whether the failure to resorb fluid by the epithelial cells can be a contributing factor in OME is not yet established. However, obstruction of the lymphatic drainage from the middle ear and the tube was suggested to be the underlying cause of some cases of OME. A reverse transport of macromolecules by the mucosal epithelial cells was reported by several authors in experimental animals. Paparella and his associates" demonstrated increased capillary permeability of the middle ear mucosal vessels when the tube was obstructed. The carbon particles that were injected into the vessels readily passed through disrupted capillaries and eventually reached the middle ear effusions by going through separated tight junctions. These authors suggested that the middle ear effusion in their experimental model came from serum. In summary, it can be suggested that biological function of the mucosal membrane of the middle ear and Eustachian tube has the following defense systems: 1) mucociliary; 2) secretory (immunoglobulins, enzymes, and surface-active
FUNCTIONAL MORPHOLOGY OF MUCOSA
agents); and 3) phagocytic (macrophages). It is further suggested that
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these defense systems are activated in OME.
REFERENCES 1. Lim OJ: Functional morphology of 12. Liu YS, Lim OJ, Lang RW, et al: the lining membrane of the middle ear and Chronic middle ear effusions: ImmunochemiEustachian tube: An overview. Ann Otol cal and bacteriological investigations. Arch Rhinol Laryngol 88:5-26, 1974 Otolaryngol 101 :278-286, 1975 2. Shimada T, Lim OJ: Distribution of 13. Hussl B, Lim OJ: Experimental midciliated cells in the human middle ear dle ear effusions - an immunofluorescent SEM, TEM, and light microscopic observastudy. Ann Otol Rhinol Laryngol 83:332-342, tion. Ann Otol Rhinol Laryngol 81 :203-211, 1974 1972 14. Ishizaka K, Ishizaka T, Tada T, et al: 3. Sade J, Eliezer N, Silverberg A, et al: Site of synthesis and function of gamma-E. The role of mucus in transport by cilia. Am in Dayton 0, et al (eds ): Secretory ImmunoRev Respir Dis 102:48-52, 1970 logic System. Bethesda, Maryland, US HEW PHS NIH National Institutes of Child Health 4. Greenberg SO, Dickey JR, Neely JG: and Human Development, 1969, pp 71-80 Effects of tobacco on the ear. Ann Otol Rhinol Laryngol 82:311-317, 1973 15. Phillips MJ, Knight NJ, Manning H, 5. Lim OJ, Klainer A: Cellular reactions et al: IgE and secretory otitis media. Lancet in acute otitis media - scanning and trans- 2:1176-1179, 1974 mission electron microscopy. Laryngoscope 16. Lim OJ, Birck H: Ultrastructural pa81:1772-1786, 1971 thology of the middle ear mucosa in serous 6. Lim OJ: Protein secreting cells in the otitis media. Ann Otol Rhinol Laryngol 80: normal middle ear mucosa of the guinea pig. 838-853, 1971 Ann Otol Rhinol Laryngol 79:82-94, 1970 17. Miglets A: The experimental produc7. Rapport PN, Lim OJ, Weiss HS: Surtion of allergic middle ear effusions. Larynface-active agent in eustachian tube function. goscope 88:1355-1384, 1973 Arch Otolaryngol 101 :305-311, 1975 18. Veltri RW, Sprinkle PM: Serous otitis 8. Sade J: Ciliary activity and middle ear media - immunoglobulin and lysozyme levels clearance. Arch Otolaryngol 86: 128-135, 1967 in middle ear fluids and serum. Ann Otol Rhinol Laryngol 82:297-301, 1973 9. McMaster P, Hudack S, cited by Humphrey JH, White RG: Immunology for 19. Lim OJ, Liu YS, Birck H: Secretory Students of Medicine, ed 3. Philadelphia, FA lysozyme of the human middle ear mucosa Davis Company, 1970, p 263 immunocytochemical localization. Ann Otol 10. Bernstein JM, Hayes ER, Ishikawa T, Rhinol Laryngol 85:50-60, 1976 et al: Secretory otitis media - a histopatho20. Birken EA, Brookler KH: Surface tenlogic and immunochemical report. Trans Am sion lowering substance of the canine EustaAcad Ophthalmol Otolaryngol 76: 1305-1318, chian tube. Ann Otol Rhinol Laryngol 81: 1972 268-271, 1972 11. Mogi G, Honjo S, Maeda S, et al: Se21. Paparella M, Hiraide F, Juhn S, et al: cretory immunoglobulin A (SIgA) in middle Cellular events involved in middle ear fluid ear effusions. A further report. Ann Otol production. Ann Otol Rhinol Laryngol 79: Rhinol Laryngol 83:92-101, 1974. 766-780, 1970 REPRINTS - David J. Lim, M.D., Otological Research Laboratories, Department of Otolaryngology, The Ohio State University College of Medicine, 456 Clinic Dr., Columbus, OH 43210. ACKNOWLEDGMENT - Ilija Karanfilov, Joan Osborne, Jonathan Darby, and Katherine Adamson provided invaluable assistance. Illustration was made by Nancy Sally.
