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Transmission electron microscopy of the epithelium of distal airways and pulmonary parenchyma of the goat lung
Researchin VeterinaryScience1997,63, 49-56
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Transmission electron microscopy of the epithelium of distal airways and pulmonary parenchyma of the goat lung C. K. B. K A H W A , Department of Veterinary Anatomy, Sokoine University of Agriculture, PO Box 3016, Morogoro, Tanzania, O. S. ATWAL, Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Ontario, Canada, M. PURTON, Department of Veterinary Anatomy, Glasgow University Veterinary School, Bearsden,
Glasgow
SUMMARY Lungs from eight goats of mixed sexes and breeds (Cashmere, Nubian and Toggenburg) aged between 10 and 48 months were used in this study. Tissues from lung parenchyma were minced and routinely prepared for transmission electron microscopy (TEM)after using different methods of fixation. Thick sections were examined with a light microscope and samples, to include terminal bronchioles, respiratory bronchioles, alveolar ducts and alveolar membrane, were selected for ultrathin sectioning. Six cell types, ciliated, non-ciliated bronchiolar epithelial, mucus-producing, alveolar Type I, alveolar Type II and capillary endothelial cell were identified and characterised cytologically. It was established that the cell population in the distal airways is similar to that observed in other domestic mammals. The mucus-producing cell, which appears to be a common cell type in the distal airways of man and Rhesus monkey, was encountered particularly in adult goats in the present study. This study has also established that the Clara cell of the goat shows some cytological differences from those of some other mammalian species by having a large amount of SER,particularly in the apical region. Lipid vacuoles were seen to be a feature of the alveolar Type II cells; these do not appear to have been reported in other mammalian species. The study has provided a basic understanding of the morphological features of the cell population of the epithelium lining the distal airways in the goat's respiratory tract. The difference in junctional complexes between the various alveolar epithelial cells perhaps signify a different pattern of intercellular transport, thus influencing the pathogenesis and resolution of alveolar pulmonary edema.
D I S T A L airways, which are hereby defined as the regions of the respiratory system including the terminal bronchioles up to the alveoli (Davis et al 1984), are of physiological and clinical importance in the gaseous exchange mechanism, frequently showing the primary effects of pulmonary pathology caused by such factors as infectious agents, genetic disorders and inhaled toxic agents (Castleman et al 1980, Boucher et al 1983, Pirie 1990). Their morphological features may therefore be considered of prime importance. However, although ultrastructural studies of the distal airways in farm animals have been carried out in the pig (Epling 1964a, b, Baskerville 1970), ox (Mariassy et al 1975, Iovannitti et al 1985) and the horse (Pirie et al 1991), in the goat, such reports appear to be limited to the ultrastructure of the alveolar epithelial cells and to the descriptions of the transmission electron microscopic (TEM)anatomy of the pulmonary blood-air barrier (Atwal 1988, Atwal and Sweeny 1971, Atwal et al 1979, Atwal and Saldanha 1985). The purpose of this study therefore, was to use the transmission electron microscope to further characterise and identify, cytologically, those cell types previously observed by light and scanning electron microscopy to populate the distal airways (Kahwa i992), and to define the ultrastrucrural characteristics of the alveolar-capillary membrane.
MATERIALS AND METHODS Eight adult goats of mixed breeds (Cashmere, Nubian and Toggenburg) aged between 10 and 48 months (two being old and lactating) were used in this study. Animals were killed by an overdose of pentobarbital sodium. 0034-5288/97/040049 + 08 $18.00/0
Different methods of fixation were used in this study. The lungs of four goats were removed intact and each right lung perfused via the principal bronchus (airway instillation) with Karnovsky's fixative and tied off. Small portions of the lung parenchyma were then minced into 1 m m pieces and placed in chilled Karnovsky's fixative for 24 hours, before being immersed in 0-2 M cacodylate buffer for one hour and then post fixed with 1 per cent osmium tetroxide for one hour. After dehydration in a graded series of acetones, the specimens were put through two changes of propylene oxide, and embedded in Emix resin and left to polymerase. Ultrathin sections, cut on an LKB Mk III ultramicrotome, were stained with uranyl acetate and lead citrate and examined with a Hitachi HS8 transmission electron microscope. The lungs of two goats were fixed by vascular perfusion as soon as possible after death, the pulmonary vasculature being flushed via a cannula in the pulmonary artery with saline, followed by 2 per cent glutaraldehyde solution in phosphate buffer 0.1 M, pH 7-4. Samples were then taken from the dorsal and ventral regions of cranial, middle, and caudal lobes and minced into 1 m m 3 pieces for immersion fixation in the same fluid for two to four hours. Tissues were then washed in three changes of cold phosphate buffer, pH 7.4 and postfixed in 1 per cent OsO 4 solution in distilled water for one hour. The lungs of the other two animals were fixed, by airway instillation, by introducing 500 ml of fixative (2 per cent glutaraldehyde and 2.5 per cent paraformaldehyde in 0. I M HC1-Na-cacodylate buffer, pH 7.4) through a tracheal cannula; fixation in situ was carried out for 30 minutes to one hour. After fixation in situ specimens collected from caudal and middle lobes of the right lung were diced into small © 1997 W. B. Saunders CompanyLtd
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C. K. B. Kahwa, O. S. Atwal, M. Purton
pieces of about 1 mm 3, and fixation was continued by immersion in the fixative for 90 minutes. Tissue taken from the middle and caudal lobes was postfixed in 1 per cent OsO 4, and stained en bloc with 0.5 per cent tannic acid (also acting as an additional fixative) in 0.1 M HC1-Nacacodylate buffer. Tissues from the two latter groups of animals were processed through the two different methods of fixation, all were then dehydrated in ethanol and propylene oxide and finally embedded in Epon 812. Ultrathin sections were stained only with lead citrate in the case of tissue fixed by way of airway instillation and tannic acid, whereas the tissue fixed by vascular perfusion was stained with both uranyl acetate and lead citrate. The stained sections were examined with a JEOL-200 microscope at 80 kV.
RESULTS Six cell types were identified in the distal airways and pulmonary parenchyma (gas exchange area). Two cell types, the ciliated and non-ciliated bronchiolar epithelial cells, formed the major component of the cell population in the terminal bronchioles, with the mucus-producing cell being observed only occasionally. Non-ciliated bronchiolar epithelial cells (Clara cells) and a few ciliated cells bearing small numbers of cilia, together with the alveolar Type I, Type II cells and capillary endothelium, formed the cell population of the respiratory bronchiolar epithelium and pulmonary parenchyma respectively. The alveolar-capillary membrane of the goat was comprised of a continuous simple squamous epithelial lining and the endothelial lining of the associated capillary. The capillary was surrounded by alveolar interstitium, the amount of which differed from region to region. Two cell types were observed to constitute the epithelial lining of the alveolar spaces namely alveolar Type I and Type II cells.
FIG 2: A ciliated cell (CC) shows a modest Golgi apparatus (G), basal bodies (asterisks) and a cilium (open arrow), Zonula occludente type of junctional complex (dark arrow), a mitochondrion. Immersion fixation method. Uranyl acetate and lead citrate staining, x 16,000 -
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Ciliated cells
FIG 1: A survey TEM view of a non-ciliated bronchiolar epithelial cell (NCB) and a ciliated cell (CC). Note the cuboidal nature of the Clara cell, it exhibits a more electron-dense cytoplasm than the adjacent ciliated cell (co). Intracytoplasmic inclusion bodies are seen in the ciliated cell (closed arrow). L - Airway lumen; basal lamina (open arrow). Airway instillation fixation. Uranyl acetate and lead citrate staining, x 5600
The cells were large and extended from the basal lamina to the airway lumen and were usually columnar in type. The luminal surfaces were seen to bear cilia and numerous microvilli. The cilia were seen to arise from the apical cytoplasm and were anchored onto the basal bodies (Fig 1). Microvilli were seen to have no obvious internal structure. The cytoplasm showed a profuse distribution of ribosomes and mitochondria in the apical portion of the cells (particularly in close association with the ciliary rootlets). Isolated vesicles of smooth endoplasmic reticulum (SER) were seen around the nucleus (Figs 1 and 2). The cytoplasm, containing an oval, basally situated nucleus, was much more electron-lucent than in the neighbouring non-ciliated and microvillous ceils (Fig 1). A welldeveloped Golgi apparatus was observed in the supranuclear region, and a number of mitochondria with relatively few cristae were seen to be present below the apical surface. A few intracytoplasmic inclusion bodies were observed. Profiles of SER were also present. A narrow intercellular space was seen surrounding the lateral cell surface where tight junctions with adjacent ceils were present (Fig 2). Occasionally, cells characterised by numerous microvilli and a number of basal bodies were encountered and these were presumed to be regenerating ciliated cells.
Epithelium of caprine airways
51
FIG 4: Mucus-producing cell. Numerous heterogeneous secretory granules (G). A large nucleus (N) with a prominent nucleolus (open arrow). Microvilli are seen on the luminal surface, Profiles of rough endoplasmic reticulum are distributed throughout the cytoplasm (closed arrow), x 5500
particularly in the apical half of the cell. The apex of this cell was unlike the Clara cells which were often seen projecting into the lumen of the airway (Fig 3).
Alveolar Type I cells These were observed to contain oval nuclei surrounded FIG 3: Non-ciliated bronchiolar epithelial (Clara) cell (NCB) shows its usual apical cap (arrow) packed with smooth endoplasmic reticulum (SER). Two electron-dense secretory granules (arrowheads) near the basal border, delicate euchromatin and a prominent nucleolus are also depicted. Airway instillation fixation. Uranyl acetate-lead citrate staining, x 10,500
Non-ciliated bronchiolar epithelial (Clara) cells These were observed in the terminal bronchioles. The cells were columnar to cuboidal (Fig 1) and often presented characteristic apical protuberances (Fig 3). Numerous short, thick stumpy microvilli were observed on the apical surface. The cells were attached apically to each other by tight junctions; in the basal region, numerous interdigitations with neighbouring cells were always observed. Accumulation of SER was observed in the apical region as well as in the basolateral part of the cell (Fig 3). A few osmiophilic inclusion bodies were also encountered near the basal lamina. Discrete, low electron-dense granules within a clear limiting membrane, were observed, mainly in the apical region. Mitochondria with an electron dense matrix were observed.
Mucus-producing cells These were occasionally observed in the terminal bronchioles (Fig 4). The cells were cuboidal in shape and were attached to adjacent cells by tight junctional complexes. The luminal surface carried a few short subdividing microvilli. A large nucleus with a prominent nucleolus was basally situated. The cytoplasm was more electron-lucent compared with that of the Clara cell, although of a higher electron-density than that of ciliated cells. Several electronlucent mucous granules were found in the sub- as well as in the supra-nuclear regions of the cells. The mucous granules were present as single membrane-bound structures containing mostly electron-lucent flocculent material. At times the mucous cells also contained medium density granules. Abundant PER and small amounts of SER were encountered,
FIG 5: A alveolar Type II cell (Epll) located at the junctional corner of three alveolar septa. The cell shows two microvillous surfaces (arrowheads) facing two different alveolar spaces (AS). Lamellar bodies (asterisks) loaded with surfactant lipid are seen near the microvillous surfaces. Mitochondda, SER and multivesicular bodies are also seen. Note the irregular contour of the nucleus with deep invaginations (arrows), Alveolar Type I cell (Epl) shows various perinuclear cell organelles. Endothelial cells of all capillaries show tight junctions and plasmalemmal vesicles also on the thin side of the alveolar septum (double arrows), s - surfactant; le interstitial cell, Airway instillation method of fixation. Tannic acid, uranyl acid and lead citrate staining, x 6000
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C. K. B. Kahwa, O. S. Atwal, M. Purton
FIG 7: A junctional complex between alveolar Type I and alveolar Type II cells. Arrowhead points to fusion of outer leaflets of the cell membrane near the luminal surface and below four distinct membranes can be delineated suggestive of an open gap junction, LB lamellar body of alveolar Type II cell; CP - contractile proteins in a microvillus. Airway instillation method of fixation; tannic acid, uranyl acetate and lead citrate staining, x 60,000 -
FIG 6: Portion of an alveolar Type cell shows a large lamellar structure which has sequestered several glycogen (g) granules. Portion of the cell junction (arrowhead) seems to suggest fusion of outer lipid leaflets of the cell membranes. The rest is predominantly an open gap junction. FM fibromuscular cell; AS - alveolar space. Perfusion fixation; uranyl acetate and lead citrate staining,x 35,000 -
by a limited perikaryon, and their very long cytoplasmic extensions covered most of the alveolar surface. The cytoplasm appeared to be devoid of cellular organelles except for numerous pinocytotic vesicles. Occasionally, short microvillous-like protrusions were observed projecting from the luminal surface of the cell (Figs 5 and 6). Intercellular clefts between these cells were of complex interdigitating open gap-junction type. Tubular endoplasmic reticulum was commonly seen, especially in old lactating goats. Multilayered lamellar structures were seen. Glycogen particles were observed in the portion of the cytoplasm sequestered by the lamellar structures, especially in the old lactating goats (Fig 6). Alveolar Type II cells
These cells were seen to occupy a position in the corner of alveolar spaces. Numerous microvilli were observed on both flanks of the free surface, leaving the central part devoid of microvilli. They formed junctional complexes (described in the next section of the text) with the neighbouring alveolar Type I cells. The electron-dense cytoplasm contained characteristic lamellated bodies. The latter consisted of multiple phospholipid bilayers surrounded by a membrane and appeared to vary in size and number from cell to cell. Mitochondria, large and well formed with an electron-dense matrix and narrow cristae, were observed, along with the SER, numerous ribosomes and multivesicular bodies, and lysosomes (Figs 5 and 7). A Golgi apparatus was occasionally observed. Lipid vacuoles of variable sizes were seen to be present.
The large centrally-placed nucleus was often observed to contain an occasional nucleolus. The vesicular nature of the nucleus was evident by the delicately dispersed euchromatin (Fig 5). In corners formed at the union of two or three alveolar septa, alveolar Type II cells contained microvilli on their two free surfaces (two apices), each facing a different alveolar space, thus defying the conventional configuration of an epithelial cell in conformity with the concept of dynamic polarity of differentiated apical and basal surfaces (Fig 5). Epithelial junction
Junctional complexes between alveolar Type I and Type II cells identified two components: (i) a proximal, continuous zonula occludens near the apical surfaces. The outer leaflets of the contacting cells sometimes fused to obliterate the intercellular space. (ii) a distal, button-like macula adherens or desmosome (Fig 8), forming a prominent electron-dense plaque on the cytoplasmic side of each contacting cell. The presence of transmembrane glycoproteins contributed to the electron-density of the intercellular space (Fig 7). Alveolar septa
Capillary endothelial cells in the alveolar septa, were attenuated except for the nuclear region which bulged into the lumen. The cytoplasmic extensions presented numerous plasmalemmal vesicles which could be classified into luminal, cytoplasmic (free), and abluminal types. Most of the cell organelles, such as plasmalemmal vesicles, endoplasmic reticulum, Golgi complex, transport vesicles (coated) and multivesicular bodies, were restricted to the more
Epithelium of caprine airways
FIG 8: A high magnification view of junctional complexes between alveolar Type I (Epl) and Type II (Epll) cells depicts a proximal continuous zonula occludens (arrowhead) and a desmosome (DES). AS - alveolar space. Airway instillation method of fixation; tannic acid, uranyl acetate and lead citrate staining, x 52,500
expansive paranuclear region of the endothelial cell Plasmalemmal vesicles distributed throughout the endothelial cells were present in overwhelming numbers compared with the other organelles, particularly in the alveolar capillaries. Endothelial cells were joined to one another by fight junctions (Figs 5 and 9). The connective tissue within the alveolar septa varied in amount from one region to another, and was found to lie between the basal lamina of the epithelial cell and that of the endothelium. Aggregates of collagen fibres were observed, and fibroblasts and occasional Mast cells were also encountered. In some areas, where the septal connective tissue was absent, epithelial and endothelial basal laminae were fused to form a composite basal lamina.
DISCUSSION The lining epithelium of the distal airways and alveolar sacs, as observed in the goat, was seen to be populated by six different cell types, identified and characterised by the use of the TEM. These were ciliate& non-ciliated bronchiolar epithelial (Clara), mucus~producing, alveolar Type I and Type II and endothelial cell types. The general structure and composition of terminal bronchioles and alveoli are similar to those described for other domestic species of mammals. Ciliated cells were observed at the level of the terminal and respiratory bronchioles. The cells were easily identified by the characteristic presence of cilia and also by the less opaque nature of the cytoplasm, as compared with that of
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FIG 9: A cross-sectional view of an alveolar capillary. Inter-endothelial clefts are represented by tight junctions (arrowheads). Endothelial cell (E) shows plasmalemmal vesicles in both limbs of the endothelium, extending into thick and thin side (AS) of the alveolar septum. Note the presence of plasmalemmal vesicles (arrows) in the so-called avesicular zone in other mammalian species. Vascular perfusion method; tannic acid, uranyl acetate and lead cit* rate staining, x 12,000
the adjacent cells (Okano and Sugawa 1965). Cilia presented a typical cytoskeletal arrangement of nine peripheral tubules and a central pair, usually described as 9 + 2 axonemic microtubular pattern, similar to those described by Rhodin and Dalhamn (1956) and as reported in other mammalian species (Breeze and Wheeldon 1977, Gall and Lenfant 1983). Glycogen rosettes, observed in the ciliated tracheobronchial epithelium of the dog (Frasca et al 1968), were not observed in the present study, neither were they reported in the horse (Pirie 1990) or in earlier studies in the dog (Majid 1986). Also intracytoplasmic tonofilaments reported in the ciliated cells of man (Rhodin 1966) were not observed in the goat. The location of the nucleus at the base, the Golgi apparatus in the central region and abundant mitochondria in the apical region of ciliated cells, conformed to the general pattern of differentiation described in other mammalian lungs (Hansell and Morreti 1969). The membrane-bound inclusion bodies observed in the ciliated ceils in the present study have also been observed in other mammalian species (Tyler and Plopper 1985); they are clear on photomicrographs provided for the horse (Pirie 1990). However, their exact nature and function remains unclear; whether these inclusions are related to the secretions of macromolecular glycoconjugates by ciliated cells, which has been established in vitro (Varsano et al 1987), can only be speculative. The function of microvilli seen on ciliated cells in the present study and previous observations remains elusive. Since there are reports that tracheal epithelial cells synthesise and transport sulfated macromolecular glycoconjugates (Varsano et al 1987), it would appear that the mierovilli might play a role in the eventual release of glycoconjugates by increasing the surface area.
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C. K. B. Kahwa, O. S. Atwal, M. Purton
The TEM appearance of Clara cells in mammalian species shows a considerable variation in their observed ultrastructure (Plopper et al 1991). Clara cells observed in the present study contained numerous profiles of SER. In older goats there was an abundance of SER, particularly in the apical region. This supports similar observations made in the Clara cells of horse and sheep, where SEN is a major cell constituent, but differs from observations made in the ox, dog, cat and man (Plopper et al 1989a, b) and non-human primates (Castleman et al 1975), where SEN is not a prominent intracytoplasmic feature. Secretory granules observed in the goat have also been reported in a number of other mammalian species (Plopper et al 1980). Glycogen granules were not observed in the Clara cell in the present study in the goat. Although this is in agreement with observations made in the guinea pig, rat, hamster and mouse (Plopper et al 1980b) and in the horse (Plopper et al 1987b, Pirie 1990), it contrasts with reports in the ox, cat and ferret (Plopper et al 1980b) and in the dog (Plopper et al 1980b, Majid 1986) where glycogen granules were abundant. Although the function of the Clara cell is uncertain and controversial, it is generally accepted that the cell is secretory in nature (Smith et al 1979, Plopper et al 1980a, b, Tyler and Plopper 1985, Majid 1986). There is now strong evidence that they contain cytochrome P-450-dependent mono-oxygenases (Boyd 1977, Serabjit-Singh et al 1980, Plopper et al 1991) which provide a major pathway for the oxidative metabolism of xenobiotics. These cells have also been associated with the metabolism of pulmonary toxins. The absence of basal cells in the distal airways in the present study further supports the view that Clara cells may also be responsible for cell renewal at this level, as suggested by Evans et al (1973, 1978). Studies in man have suggested that Clara cells, rather than being distinct cells, are varieties of mucus-producing cells or alveolar Type lI cells. These suggestions were based on histochemical observations of the respiratory bronchioles in primates (Tyler and Plopper 1985, Plopper et al 1989), and supported by immunocytochemical studies in man (Ten Have-Opbroek et al 1991). The latter demonstrated the presence of glycoproteins and surfactant proteinA, suggesting that the two different population of Clara cells represent mucus-producing and alveolar Type II precursor cells respectively. The current active interest of researchers has focused on the Clara cell's role as a secretory cell for the centroacinar region. Secretory granules of Clara cells contain glycoproteins and lipids. Regardless of the species, Clara cells do not contain acidic or sulfated glycoprotein. However, they have been identified as a source of secretory proteins ranging in size from 200 kDa to less than 10 kDa. Furthermore, they are also a source of pulmonary surfactant apoproteins (Phelps and Floros, 1988, Plopper et al 1991). Mucus-producing cells were encountered in only one young animal although all mature female goats showed these cells consistently. This contrasts with observations in other mammalian species, including the dog (Majid 1986), ox (Mariassy et al 1975, Iovannitti et al 1985) and horse (Pirie 1990). It is worthwhile to point out that mucus-producing cells constitute the major secretory cell type in the distal airways of humans (Ten Have-Opbroek et al 1991) and Rhesus monkey (Tyler and Plopper 1985). The present study is the first TEM evidence on the occurrence of electron-lucent mucous granules as single-membrane bound structures containing reticulated flocculent material in the
goat lung. According to Gbadially (1989) mucous granules, being hydrophilic, tend to swell and coalesce, and thus rupture the ensheathing membranes during routine specimen preparation. Alveolar Type I cells in the present study showed similar ultrastructural characteristics to those already described for the goat (Atwal and Sweeny 1971), except for the frequently observed mitochondria and endoplasmic reticulum in the perinuclear region. Atwal (1988) reported the presence of tubular endoplasmic reticulum (TEN), which is a modified SER, and pinocytotic vesicles in the goat. Whereas the latter were consistently observed, the presence of TER could be seen only when tissues were fixed via the vascular perfusion method. Different methods of fixation could account for the differences noted in the morphology of alveolar Type I cells. According to Weibel (1984) to achieve 'ideal' results, different methods of fixation are required to meet the precise requirements during electron microscopy of the lung structure. The result of an earlier study by Atwal (1988) demonstrated the presence of TER in alveolar Type I cells by using fixation by vascular perfusion. This method allows the proper fixation of lining epithelium as well as the surfactant layer lining the alveoli. Large membrane-bound inclusions containing electronlucent material, and presumed to be lipid vacuoles, were observed in alveolar Type II cells in the present study, and are in agreement with a previous report (Atwal and Sweeny 1971). Such vacuoles do not appear to have been reported in the normal lungs of other mammalian species except in guinea pigs exposed to hypoxia (Valdivia et al 1966). It has been suggested that the presence of lipid vacuoles may be a result of the high respiratory quotient of the goat (Homer 1977), which favours the formation of fat. The mammalian lung is also known to participate in de novo synthesis of fatty acids (Mason 1976). The osmiophilic inclusion bodies (lamellar bodies) observed in the alveolar Type I cell of the goat lung are associated with the production and storage of surface-active phospholipids. The lamellar bodies were well preserved especially when tannic acid was employed as an additional fixative. Tannic acid reacts primarily with the choline base of phosphatidylcholine which is the predominant component of the pulmonary surfactant (Kalina and Pease 1977). The same findings have been reported by several workers (Kuhn 1976, King 1979, Morrison et al 1983, Kikkawa and Smith 1983, Breeze and Turk 1984). The alveolar Type II cell is also known to be a stem cell of the alveolar epithelium, involved in regenerating the epithelium following injury (Kanffman 1980). The use of tannic acid as an additional fixative also facilitated in clarifying membrane structures by rendering surface complexes highly electron-dense. Consequently, alveolar epithelial junctions showed two components, a proximal continuous zonula occludens and a distal button-like desmosome. This is at variance with the three components usually shown in laboratory rodents (Scheeberger 1991), the additional third one being an intermediate continuous zonula adherense which was absent in the goat lung. One could speculate that such arrangement in the goat lung might permit increased transepithelial permeability. Plasmalemmal vesicles extended equally into both limbs of the endothelial cells. Therefore, a distinction between an avesicular zone and a vesicular zone in the goat lung perhaps does not exist. In general, in the mammalian lung such a differentiation exists. The avesicular zone of the endothelium lies on the thin side of the septum where the alveolar
Epithelium of caprine airways
Type I cell is separated from the endothelial cell by an unexpanded interstitium, which is represented by their composite basal laminae (Simionescu and Simionescu 1991). The plasmalemmal vesicles of the endothelial cells are considered to be dynamic structures which actively respond to various experimental insults especially during pulmonary oedema (Simionescu and Simionescu 1991). It is conceivable that extensive fluid-shifts across the alveolar-capillary membrane in the goat lung takes place more than is generally assumed to exist in other mammalian lungs (Simionescu and Simionescu 1991, Atwal and Brown 1980, Atwal et al 1990). The present study, in characterising the cytology of the cell populations that line the distal airways and alveolar parenchyma in the goat lung, has provided additional information which, when combined with previous LM and SEM studies, provides a basic understanding of the morphological features and differentiation of the cell population of the epithelium lining the distal airways of the goat's respiratory tract. This information is essential before pathological lesions due to disease processes can be interpreted.
ACKNOWLEDGEMENTS This work was supported by a grant from the Danish International Development Agency (DANIDA)and Natural Sciences and Engineering Council of Canada. The authors wish to thank Mrs Jacqueline McPherson, Mrs Mary Reilly, Mrs Kanwal Minhas and Mrs Dorothy McKeown for the preparation of the photomicrographs and the manuscript. Some of the work presented here has also been reported earlier (Kahwa 1992), in particular, results obtained from specimens fixed by airway instillation and Figs 1 and 2.
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(1989) Ultrastructural Biology of Cell and Matrix. Toronto, Butterworth. pp 428-429 HANSELL, M. M. & MORETTI, R. L. (1969) Ultrastructure of the mouse tracheal epithelium. Journal of Morphology 128, 159-170 HOMER, E. D. (1977) Energy metabolism in domestic animals. In Dukes Physiology of Domestic Animals. Ed M. J. Swenson. Ithaca and London, Comstock Publishing Associates, Comell University Press. pp 368-377 IOVANNITTI, B., PIRIE, H. M. & WRIGHT, N. G. (1985) A scanning electron microscopic study of the lower respiratory tract in calves and adult cattle. Research in Veterinary Science 38, 80-87 KAHWA, C. K. B. (1992) An ultrastmctural and histological study of the epithelium of the respiratory tract in the neonatal and adult goat. PhD thesis, University of Glasgow, Glasgow KALINA, M. & PEASE, D. C. (1977) The probable role of phosphatidylcholine in the tannic acid enhancement of cytomembrane electron contrast. Journal of Cell Biology 74, 742-746 KAUFFMAN, S. L. (1980) Cell proliferation in the mammalian lung. International Review of Experimental Pathology 22, 131-191 KIKKAWA, Y. & SMITH, F. (1983). Cellular and biochemical aspects of pnlmonm-y snffactant in health and disease. Laboratory Investigation 49, 122-139 KING, R. J. (1979) Utilization of alveolar Type II cells for the study of surfactant. Federation Proceedings 38, 2637-2643 KUHN, C. (1976) The cells of the lung and their organelles. In The Biochemical Basis of Pulmonary Function. Ed R. G. Crystal. New York and Basel. Marcel Decker Inc. pp 3-48 MAJID, A. (1986) A combined histological, histochemical and scanning electron microscopical study of the canine respiratory tract. PhD thesis, University of Glasgow, Glasgow MARIASSY, A. T., PLOPPER, C. G. & DUNGWORTH, D. L. (1975) Characteristics of bovine lung as observed by scanning electron microscope. Anatomical Record 183, 13-26 MASON, R. J. (1976) Lipid metabolism. In The Biochemical Basis of Pulmonary Function. Ed R. G. Crystal. New York and Basel, Marcel Dekker Inc. pp 127169 MORRISON, E. E., LEIPOLD, H. W. & KRUCKENBERG, S. M. (1983) The micro-anatomy of the coyote's (Canis latrans) respiratory system. Anatomia, Histologia, Embryologia 12, 325-340 OKANO, M. & SUGAWA, Y. (1965) Ultrastmcture of the respiratory mucous epithelium of the canine nasal cavity. Archives of Histology (Japan) 26, 1-21 PHELPS, D. S & FLOROS, J. (1988) Localization of pulmonary suffactant proteins using immunocytochemistry and tissue in sire hybridization. Experimental Lung Research 17, 985-995 PIRIE, M. E. S. (1990) An ultrastructural and histological study of the equine respiratory tract in health and disease. PhD thesis, University of Glasgow, Glasgow PIRIE, M., PIRIE, H. M., CRANSTOUN, S. & WRIGHT, N. G. (1991) A scanning electron microscopic study of the equine lower respiratory tract. Equine Veterinary Journal 22, 333-337 PLOPPER, C. G., HAIDSIEK, J. G , WEIR, A. J., ST. GEORGE, J. A. & HYDE, D. M. (1989) Tracheobronchial epithelium in the adult Rhesus Monkey: A quantitative histochemieal and ultrastmcmral study. American Journal of Anatomy 184, 31-40 PLOPPER, C. G., HILL, L. H. & MARIASSY, A. T. (1980a) Ultrastructure of the non-ciliated bronchiolar epithelial (Clara) cell of mammalian lung. III. A study of man with comparison of 15 mammalian species. Experimental Lung Research 1, 171-180 PLOPPER, C. G., MARIASSY, A. T. & HILL, L. H. (1980b) Ultrastructure of the non-ciliated bronchiolar epithelial (Clara) cell of mammalian lung. II. A comparison of horse, steer, sheep, dog and cat. Experimental Lung Research 1, 155-169 PLOPPER, C. G., HYDE, D. M. & BUCKPITT, A. R. (1991) Clara cells. In The Lung. Eds R. G. Crystal, J. B. West. Vol 1. New York, Raven Press. pp 215-227 RHODIN, J. (1966) The ultrastmcture and function of the human tracheal mucosa. American Review of Respiratory Disease 93, 1-15 RHODIN, J. & DALHAMN, T. (1956) Electron microscopy of the tracheal ciliated mucosa in the rat. Zeitschriftfiir Zellfurschung 40, (Supplement) 345-412 SCHEEBERGER, E. E. (1991) Airway and alveolar epithelial junctions. In The Lung, Vol 1. Eds R. G. Crystal, J. B. West. New York, Raven Press. pp 205-214 SERABJIT-SINGH, C. J., WOLF, C. R. & PHILPOT, R. M. (1980) Cytochrome P450: Localization in rabbit lung. Science 207, 1469-1470 SIMIONESCU, M. & S1MIONESCU, N. (1991) Endothelial transport of macromolecules: transcytosis and endocytosis. A look from cell biology. Cell Biology Reviews 25, 1-78 SMITH, M. N., GREENBERG, S. D. & SPJUT, J. H. (1979) The Clara cell: A comparative ultrastructural study in mammals. American Journal of Anatomy 155, 15-29 TEN HAVE-OPBROEK, A. A. W., oTrO-VERBERNE, C. J. M., DUBBELDAM,
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J. A. & DYKMAN, J. H. (1991) Proximal border of the human respiratory unit, as shown by scanning and transmission electron microscopy and light microscopical cytochemistry. Anatomical Record 229, 339-354 TYLER, N. K. & PLOPPER, C. G. (1985) Morphology of the distal conducting airways in Rhesus monkey lungs. Anatomical Record 211, 295-303 VALDIVIA, E., SONNAD, J. & AMATO, J. (1966) Fatty changes of pneumocytes. Science 151, 213-214 VARSANO, S., BASBAUM, C. B., FORSBERG, L. S., BORSON, D. G., CAUGHEY, G. & NADEL, J. A. (1987) Dog tracheal epithelial cells in culture synthe-
sise sulfated macromolecular glycoconjugates and release them from the cell surface upon exposure to extraceIlular proteinases. Experimental Lung Research 13, 157-184 WEIBEL, E. R. (1984) Morphometric and stereological methods in respiratory physiology including fixation tectmiques. In Techniques in Life Sciences, Respiratory Physiology Ireland, Elsevier Scientific Publishers. pp 1-35 Received September 6, 1995 Accepted January 14, 1997
G ' ~
Transmission electron microscopy of the epithelium of distal airways and pulmonary parenchyma of the goat lung C. K. B. K A H W A , Department of Veterinary Anatomy, Sokoine University of Agriculture, PO Box 3016, Morogoro, Tanzania, O. S. ATWAL, Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Ontario, Canada, M. PURTON, Department of Veterinary Anatomy, Glasgow University Veterinary School, Bearsden,
Glasgow
SUMMARY Lungs from eight goats of mixed sexes and breeds (Cashmere, Nubian and Toggenburg) aged between 10 and 48 months were used in this study. Tissues from lung parenchyma were minced and routinely prepared for transmission electron microscopy (TEM)after using different methods of fixation. Thick sections were examined with a light microscope and samples, to include terminal bronchioles, respiratory bronchioles, alveolar ducts and alveolar membrane, were selected for ultrathin sectioning. Six cell types, ciliated, non-ciliated bronchiolar epithelial, mucus-producing, alveolar Type I, alveolar Type II and capillary endothelial cell were identified and characterised cytologically. It was established that the cell population in the distal airways is similar to that observed in other domestic mammals. The mucus-producing cell, which appears to be a common cell type in the distal airways of man and Rhesus monkey, was encountered particularly in adult goats in the present study. This study has also established that the Clara cell of the goat shows some cytological differences from those of some other mammalian species by having a large amount of SER,particularly in the apical region. Lipid vacuoles were seen to be a feature of the alveolar Type II cells; these do not appear to have been reported in other mammalian species. The study has provided a basic understanding of the morphological features of the cell population of the epithelium lining the distal airways in the goat's respiratory tract. The difference in junctional complexes between the various alveolar epithelial cells perhaps signify a different pattern of intercellular transport, thus influencing the pathogenesis and resolution of alveolar pulmonary edema.
D I S T A L airways, which are hereby defined as the regions of the respiratory system including the terminal bronchioles up to the alveoli (Davis et al 1984), are of physiological and clinical importance in the gaseous exchange mechanism, frequently showing the primary effects of pulmonary pathology caused by such factors as infectious agents, genetic disorders and inhaled toxic agents (Castleman et al 1980, Boucher et al 1983, Pirie 1990). Their morphological features may therefore be considered of prime importance. However, although ultrastructural studies of the distal airways in farm animals have been carried out in the pig (Epling 1964a, b, Baskerville 1970), ox (Mariassy et al 1975, Iovannitti et al 1985) and the horse (Pirie et al 1991), in the goat, such reports appear to be limited to the ultrastructure of the alveolar epithelial cells and to the descriptions of the transmission electron microscopic (TEM)anatomy of the pulmonary blood-air barrier (Atwal 1988, Atwal and Sweeny 1971, Atwal et al 1979, Atwal and Saldanha 1985). The purpose of this study therefore, was to use the transmission electron microscope to further characterise and identify, cytologically, those cell types previously observed by light and scanning electron microscopy to populate the distal airways (Kahwa i992), and to define the ultrastrucrural characteristics of the alveolar-capillary membrane.
MATERIALS AND METHODS Eight adult goats of mixed breeds (Cashmere, Nubian and Toggenburg) aged between 10 and 48 months (two being old and lactating) were used in this study. Animals were killed by an overdose of pentobarbital sodium. 0034-5288/97/040049 + 08 $18.00/0
Different methods of fixation were used in this study. The lungs of four goats were removed intact and each right lung perfused via the principal bronchus (airway instillation) with Karnovsky's fixative and tied off. Small portions of the lung parenchyma were then minced into 1 m m pieces and placed in chilled Karnovsky's fixative for 24 hours, before being immersed in 0-2 M cacodylate buffer for one hour and then post fixed with 1 per cent osmium tetroxide for one hour. After dehydration in a graded series of acetones, the specimens were put through two changes of propylene oxide, and embedded in Emix resin and left to polymerase. Ultrathin sections, cut on an LKB Mk III ultramicrotome, were stained with uranyl acetate and lead citrate and examined with a Hitachi HS8 transmission electron microscope. The lungs of two goats were fixed by vascular perfusion as soon as possible after death, the pulmonary vasculature being flushed via a cannula in the pulmonary artery with saline, followed by 2 per cent glutaraldehyde solution in phosphate buffer 0.1 M, pH 7-4. Samples were then taken from the dorsal and ventral regions of cranial, middle, and caudal lobes and minced into 1 m m 3 pieces for immersion fixation in the same fluid for two to four hours. Tissues were then washed in three changes of cold phosphate buffer, pH 7.4 and postfixed in 1 per cent OsO 4 solution in distilled water for one hour. The lungs of the other two animals were fixed, by airway instillation, by introducing 500 ml of fixative (2 per cent glutaraldehyde and 2.5 per cent paraformaldehyde in 0. I M HC1-Na-cacodylate buffer, pH 7.4) through a tracheal cannula; fixation in situ was carried out for 30 minutes to one hour. After fixation in situ specimens collected from caudal and middle lobes of the right lung were diced into small © 1997 W. B. Saunders CompanyLtd
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C. K. B. Kahwa, O. S. Atwal, M. Purton
pieces of about 1 mm 3, and fixation was continued by immersion in the fixative for 90 minutes. Tissue taken from the middle and caudal lobes was postfixed in 1 per cent OsO 4, and stained en bloc with 0.5 per cent tannic acid (also acting as an additional fixative) in 0.1 M HC1-Nacacodylate buffer. Tissues from the two latter groups of animals were processed through the two different methods of fixation, all were then dehydrated in ethanol and propylene oxide and finally embedded in Epon 812. Ultrathin sections were stained only with lead citrate in the case of tissue fixed by way of airway instillation and tannic acid, whereas the tissue fixed by vascular perfusion was stained with both uranyl acetate and lead citrate. The stained sections were examined with a JEOL-200 microscope at 80 kV.
RESULTS Six cell types were identified in the distal airways and pulmonary parenchyma (gas exchange area). Two cell types, the ciliated and non-ciliated bronchiolar epithelial cells, formed the major component of the cell population in the terminal bronchioles, with the mucus-producing cell being observed only occasionally. Non-ciliated bronchiolar epithelial cells (Clara cells) and a few ciliated cells bearing small numbers of cilia, together with the alveolar Type I, Type II cells and capillary endothelium, formed the cell population of the respiratory bronchiolar epithelium and pulmonary parenchyma respectively. The alveolar-capillary membrane of the goat was comprised of a continuous simple squamous epithelial lining and the endothelial lining of the associated capillary. The capillary was surrounded by alveolar interstitium, the amount of which differed from region to region. Two cell types were observed to constitute the epithelial lining of the alveolar spaces namely alveolar Type I and Type II cells.
FIG 2: A ciliated cell (CC) shows a modest Golgi apparatus (G), basal bodies (asterisks) and a cilium (open arrow), Zonula occludente type of junctional complex (dark arrow), a mitochondrion. Immersion fixation method. Uranyl acetate and lead citrate staining, x 16,000 -
-
Ciliated cells
FIG 1: A survey TEM view of a non-ciliated bronchiolar epithelial cell (NCB) and a ciliated cell (CC). Note the cuboidal nature of the Clara cell, it exhibits a more electron-dense cytoplasm than the adjacent ciliated cell (co). Intracytoplasmic inclusion bodies are seen in the ciliated cell (closed arrow). L - Airway lumen; basal lamina (open arrow). Airway instillation fixation. Uranyl acetate and lead citrate staining, x 5600
The cells were large and extended from the basal lamina to the airway lumen and were usually columnar in type. The luminal surfaces were seen to bear cilia and numerous microvilli. The cilia were seen to arise from the apical cytoplasm and were anchored onto the basal bodies (Fig 1). Microvilli were seen to have no obvious internal structure. The cytoplasm showed a profuse distribution of ribosomes and mitochondria in the apical portion of the cells (particularly in close association with the ciliary rootlets). Isolated vesicles of smooth endoplasmic reticulum (SER) were seen around the nucleus (Figs 1 and 2). The cytoplasm, containing an oval, basally situated nucleus, was much more electron-lucent than in the neighbouring non-ciliated and microvillous ceils (Fig 1). A welldeveloped Golgi apparatus was observed in the supranuclear region, and a number of mitochondria with relatively few cristae were seen to be present below the apical surface. A few intracytoplasmic inclusion bodies were observed. Profiles of SER were also present. A narrow intercellular space was seen surrounding the lateral cell surface where tight junctions with adjacent ceils were present (Fig 2). Occasionally, cells characterised by numerous microvilli and a number of basal bodies were encountered and these were presumed to be regenerating ciliated cells.
Epithelium of caprine airways
51
FIG 4: Mucus-producing cell. Numerous heterogeneous secretory granules (G). A large nucleus (N) with a prominent nucleolus (open arrow). Microvilli are seen on the luminal surface, Profiles of rough endoplasmic reticulum are distributed throughout the cytoplasm (closed arrow), x 5500
particularly in the apical half of the cell. The apex of this cell was unlike the Clara cells which were often seen projecting into the lumen of the airway (Fig 3).
Alveolar Type I cells These were observed to contain oval nuclei surrounded FIG 3: Non-ciliated bronchiolar epithelial (Clara) cell (NCB) shows its usual apical cap (arrow) packed with smooth endoplasmic reticulum (SER). Two electron-dense secretory granules (arrowheads) near the basal border, delicate euchromatin and a prominent nucleolus are also depicted. Airway instillation fixation. Uranyl acetate-lead citrate staining, x 10,500
Non-ciliated bronchiolar epithelial (Clara) cells These were observed in the terminal bronchioles. The cells were columnar to cuboidal (Fig 1) and often presented characteristic apical protuberances (Fig 3). Numerous short, thick stumpy microvilli were observed on the apical surface. The cells were attached apically to each other by tight junctions; in the basal region, numerous interdigitations with neighbouring cells were always observed. Accumulation of SER was observed in the apical region as well as in the basolateral part of the cell (Fig 3). A few osmiophilic inclusion bodies were also encountered near the basal lamina. Discrete, low electron-dense granules within a clear limiting membrane, were observed, mainly in the apical region. Mitochondria with an electron dense matrix were observed.
Mucus-producing cells These were occasionally observed in the terminal bronchioles (Fig 4). The cells were cuboidal in shape and were attached to adjacent cells by tight junctional complexes. The luminal surface carried a few short subdividing microvilli. A large nucleus with a prominent nucleolus was basally situated. The cytoplasm was more electron-lucent compared with that of the Clara cell, although of a higher electron-density than that of ciliated cells. Several electronlucent mucous granules were found in the sub- as well as in the supra-nuclear regions of the cells. The mucous granules were present as single membrane-bound structures containing mostly electron-lucent flocculent material. At times the mucous cells also contained medium density granules. Abundant PER and small amounts of SER were encountered,
FIG 5: A alveolar Type II cell (Epll) located at the junctional corner of three alveolar septa. The cell shows two microvillous surfaces (arrowheads) facing two different alveolar spaces (AS). Lamellar bodies (asterisks) loaded with surfactant lipid are seen near the microvillous surfaces. Mitochondda, SER and multivesicular bodies are also seen. Note the irregular contour of the nucleus with deep invaginations (arrows), Alveolar Type I cell (Epl) shows various perinuclear cell organelles. Endothelial cells of all capillaries show tight junctions and plasmalemmal vesicles also on the thin side of the alveolar septum (double arrows), s - surfactant; le interstitial cell, Airway instillation method of fixation. Tannic acid, uranyl acid and lead citrate staining, x 6000
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C. K. B. Kahwa, O. S. Atwal, M. Purton
FIG 7: A junctional complex between alveolar Type I and alveolar Type II cells. Arrowhead points to fusion of outer leaflets of the cell membrane near the luminal surface and below four distinct membranes can be delineated suggestive of an open gap junction, LB lamellar body of alveolar Type II cell; CP - contractile proteins in a microvillus. Airway instillation method of fixation; tannic acid, uranyl acetate and lead citrate staining, x 60,000 -
FIG 6: Portion of an alveolar Type cell shows a large lamellar structure which has sequestered several glycogen (g) granules. Portion of the cell junction (arrowhead) seems to suggest fusion of outer lipid leaflets of the cell membranes. The rest is predominantly an open gap junction. FM fibromuscular cell; AS - alveolar space. Perfusion fixation; uranyl acetate and lead citrate staining,x 35,000 -
by a limited perikaryon, and their very long cytoplasmic extensions covered most of the alveolar surface. The cytoplasm appeared to be devoid of cellular organelles except for numerous pinocytotic vesicles. Occasionally, short microvillous-like protrusions were observed projecting from the luminal surface of the cell (Figs 5 and 6). Intercellular clefts between these cells were of complex interdigitating open gap-junction type. Tubular endoplasmic reticulum was commonly seen, especially in old lactating goats. Multilayered lamellar structures were seen. Glycogen particles were observed in the portion of the cytoplasm sequestered by the lamellar structures, especially in the old lactating goats (Fig 6). Alveolar Type II cells
These cells were seen to occupy a position in the corner of alveolar spaces. Numerous microvilli were observed on both flanks of the free surface, leaving the central part devoid of microvilli. They formed junctional complexes (described in the next section of the text) with the neighbouring alveolar Type I cells. The electron-dense cytoplasm contained characteristic lamellated bodies. The latter consisted of multiple phospholipid bilayers surrounded by a membrane and appeared to vary in size and number from cell to cell. Mitochondria, large and well formed with an electron-dense matrix and narrow cristae, were observed, along with the SER, numerous ribosomes and multivesicular bodies, and lysosomes (Figs 5 and 7). A Golgi apparatus was occasionally observed. Lipid vacuoles of variable sizes were seen to be present.
The large centrally-placed nucleus was often observed to contain an occasional nucleolus. The vesicular nature of the nucleus was evident by the delicately dispersed euchromatin (Fig 5). In corners formed at the union of two or three alveolar septa, alveolar Type II cells contained microvilli on their two free surfaces (two apices), each facing a different alveolar space, thus defying the conventional configuration of an epithelial cell in conformity with the concept of dynamic polarity of differentiated apical and basal surfaces (Fig 5). Epithelial junction
Junctional complexes between alveolar Type I and Type II cells identified two components: (i) a proximal, continuous zonula occludens near the apical surfaces. The outer leaflets of the contacting cells sometimes fused to obliterate the intercellular space. (ii) a distal, button-like macula adherens or desmosome (Fig 8), forming a prominent electron-dense plaque on the cytoplasmic side of each contacting cell. The presence of transmembrane glycoproteins contributed to the electron-density of the intercellular space (Fig 7). Alveolar septa
Capillary endothelial cells in the alveolar septa, were attenuated except for the nuclear region which bulged into the lumen. The cytoplasmic extensions presented numerous plasmalemmal vesicles which could be classified into luminal, cytoplasmic (free), and abluminal types. Most of the cell organelles, such as plasmalemmal vesicles, endoplasmic reticulum, Golgi complex, transport vesicles (coated) and multivesicular bodies, were restricted to the more
Epithelium of caprine airways
FIG 8: A high magnification view of junctional complexes between alveolar Type I (Epl) and Type II (Epll) cells depicts a proximal continuous zonula occludens (arrowhead) and a desmosome (DES). AS - alveolar space. Airway instillation method of fixation; tannic acid, uranyl acetate and lead citrate staining, x 52,500
expansive paranuclear region of the endothelial cell Plasmalemmal vesicles distributed throughout the endothelial cells were present in overwhelming numbers compared with the other organelles, particularly in the alveolar capillaries. Endothelial cells were joined to one another by fight junctions (Figs 5 and 9). The connective tissue within the alveolar septa varied in amount from one region to another, and was found to lie between the basal lamina of the epithelial cell and that of the endothelium. Aggregates of collagen fibres were observed, and fibroblasts and occasional Mast cells were also encountered. In some areas, where the septal connective tissue was absent, epithelial and endothelial basal laminae were fused to form a composite basal lamina.
DISCUSSION The lining epithelium of the distal airways and alveolar sacs, as observed in the goat, was seen to be populated by six different cell types, identified and characterised by the use of the TEM. These were ciliate& non-ciliated bronchiolar epithelial (Clara), mucus~producing, alveolar Type I and Type II and endothelial cell types. The general structure and composition of terminal bronchioles and alveoli are similar to those described for other domestic species of mammals. Ciliated cells were observed at the level of the terminal and respiratory bronchioles. The cells were easily identified by the characteristic presence of cilia and also by the less opaque nature of the cytoplasm, as compared with that of
53
FIG 9: A cross-sectional view of an alveolar capillary. Inter-endothelial clefts are represented by tight junctions (arrowheads). Endothelial cell (E) shows plasmalemmal vesicles in both limbs of the endothelium, extending into thick and thin side (AS) of the alveolar septum. Note the presence of plasmalemmal vesicles (arrows) in the so-called avesicular zone in other mammalian species. Vascular perfusion method; tannic acid, uranyl acetate and lead cit* rate staining, x 12,000
the adjacent cells (Okano and Sugawa 1965). Cilia presented a typical cytoskeletal arrangement of nine peripheral tubules and a central pair, usually described as 9 + 2 axonemic microtubular pattern, similar to those described by Rhodin and Dalhamn (1956) and as reported in other mammalian species (Breeze and Wheeldon 1977, Gall and Lenfant 1983). Glycogen rosettes, observed in the ciliated tracheobronchial epithelium of the dog (Frasca et al 1968), were not observed in the present study, neither were they reported in the horse (Pirie 1990) or in earlier studies in the dog (Majid 1986). Also intracytoplasmic tonofilaments reported in the ciliated cells of man (Rhodin 1966) were not observed in the goat. The location of the nucleus at the base, the Golgi apparatus in the central region and abundant mitochondria in the apical region of ciliated cells, conformed to the general pattern of differentiation described in other mammalian lungs (Hansell and Morreti 1969). The membrane-bound inclusion bodies observed in the ciliated ceils in the present study have also been observed in other mammalian species (Tyler and Plopper 1985); they are clear on photomicrographs provided for the horse (Pirie 1990). However, their exact nature and function remains unclear; whether these inclusions are related to the secretions of macromolecular glycoconjugates by ciliated cells, which has been established in vitro (Varsano et al 1987), can only be speculative. The function of microvilli seen on ciliated cells in the present study and previous observations remains elusive. Since there are reports that tracheal epithelial cells synthesise and transport sulfated macromolecular glycoconjugates (Varsano et al 1987), it would appear that the mierovilli might play a role in the eventual release of glycoconjugates by increasing the surface area.
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C. K. B. Kahwa, O. S. Atwal, M. Purton
The TEM appearance of Clara cells in mammalian species shows a considerable variation in their observed ultrastructure (Plopper et al 1991). Clara cells observed in the present study contained numerous profiles of SER. In older goats there was an abundance of SER, particularly in the apical region. This supports similar observations made in the Clara cells of horse and sheep, where SEN is a major cell constituent, but differs from observations made in the ox, dog, cat and man (Plopper et al 1989a, b) and non-human primates (Castleman et al 1975), where SEN is not a prominent intracytoplasmic feature. Secretory granules observed in the goat have also been reported in a number of other mammalian species (Plopper et al 1980). Glycogen granules were not observed in the Clara cell in the present study in the goat. Although this is in agreement with observations made in the guinea pig, rat, hamster and mouse (Plopper et al 1980b) and in the horse (Plopper et al 1987b, Pirie 1990), it contrasts with reports in the ox, cat and ferret (Plopper et al 1980b) and in the dog (Plopper et al 1980b, Majid 1986) where glycogen granules were abundant. Although the function of the Clara cell is uncertain and controversial, it is generally accepted that the cell is secretory in nature (Smith et al 1979, Plopper et al 1980a, b, Tyler and Plopper 1985, Majid 1986). There is now strong evidence that they contain cytochrome P-450-dependent mono-oxygenases (Boyd 1977, Serabjit-Singh et al 1980, Plopper et al 1991) which provide a major pathway for the oxidative metabolism of xenobiotics. These cells have also been associated with the metabolism of pulmonary toxins. The absence of basal cells in the distal airways in the present study further supports the view that Clara cells may also be responsible for cell renewal at this level, as suggested by Evans et al (1973, 1978). Studies in man have suggested that Clara cells, rather than being distinct cells, are varieties of mucus-producing cells or alveolar Type lI cells. These suggestions were based on histochemical observations of the respiratory bronchioles in primates (Tyler and Plopper 1985, Plopper et al 1989), and supported by immunocytochemical studies in man (Ten Have-Opbroek et al 1991). The latter demonstrated the presence of glycoproteins and surfactant proteinA, suggesting that the two different population of Clara cells represent mucus-producing and alveolar Type II precursor cells respectively. The current active interest of researchers has focused on the Clara cell's role as a secretory cell for the centroacinar region. Secretory granules of Clara cells contain glycoproteins and lipids. Regardless of the species, Clara cells do not contain acidic or sulfated glycoprotein. However, they have been identified as a source of secretory proteins ranging in size from 200 kDa to less than 10 kDa. Furthermore, they are also a source of pulmonary surfactant apoproteins (Phelps and Floros, 1988, Plopper et al 1991). Mucus-producing cells were encountered in only one young animal although all mature female goats showed these cells consistently. This contrasts with observations in other mammalian species, including the dog (Majid 1986), ox (Mariassy et al 1975, Iovannitti et al 1985) and horse (Pirie 1990). It is worthwhile to point out that mucus-producing cells constitute the major secretory cell type in the distal airways of humans (Ten Have-Opbroek et al 1991) and Rhesus monkey (Tyler and Plopper 1985). The present study is the first TEM evidence on the occurrence of electron-lucent mucous granules as single-membrane bound structures containing reticulated flocculent material in the
goat lung. According to Gbadially (1989) mucous granules, being hydrophilic, tend to swell and coalesce, and thus rupture the ensheathing membranes during routine specimen preparation. Alveolar Type I cells in the present study showed similar ultrastructural characteristics to those already described for the goat (Atwal and Sweeny 1971), except for the frequently observed mitochondria and endoplasmic reticulum in the perinuclear region. Atwal (1988) reported the presence of tubular endoplasmic reticulum (TEN), which is a modified SER, and pinocytotic vesicles in the goat. Whereas the latter were consistently observed, the presence of TER could be seen only when tissues were fixed via the vascular perfusion method. Different methods of fixation could account for the differences noted in the morphology of alveolar Type I cells. According to Weibel (1984) to achieve 'ideal' results, different methods of fixation are required to meet the precise requirements during electron microscopy of the lung structure. The result of an earlier study by Atwal (1988) demonstrated the presence of TER in alveolar Type I cells by using fixation by vascular perfusion. This method allows the proper fixation of lining epithelium as well as the surfactant layer lining the alveoli. Large membrane-bound inclusions containing electronlucent material, and presumed to be lipid vacuoles, were observed in alveolar Type II cells in the present study, and are in agreement with a previous report (Atwal and Sweeny 1971). Such vacuoles do not appear to have been reported in the normal lungs of other mammalian species except in guinea pigs exposed to hypoxia (Valdivia et al 1966). It has been suggested that the presence of lipid vacuoles may be a result of the high respiratory quotient of the goat (Homer 1977), which favours the formation of fat. The mammalian lung is also known to participate in de novo synthesis of fatty acids (Mason 1976). The osmiophilic inclusion bodies (lamellar bodies) observed in the alveolar Type I cell of the goat lung are associated with the production and storage of surface-active phospholipids. The lamellar bodies were well preserved especially when tannic acid was employed as an additional fixative. Tannic acid reacts primarily with the choline base of phosphatidylcholine which is the predominant component of the pulmonary surfactant (Kalina and Pease 1977). The same findings have been reported by several workers (Kuhn 1976, King 1979, Morrison et al 1983, Kikkawa and Smith 1983, Breeze and Turk 1984). The alveolar Type II cell is also known to be a stem cell of the alveolar epithelium, involved in regenerating the epithelium following injury (Kanffman 1980). The use of tannic acid as an additional fixative also facilitated in clarifying membrane structures by rendering surface complexes highly electron-dense. Consequently, alveolar epithelial junctions showed two components, a proximal continuous zonula occludens and a distal button-like desmosome. This is at variance with the three components usually shown in laboratory rodents (Scheeberger 1991), the additional third one being an intermediate continuous zonula adherense which was absent in the goat lung. One could speculate that such arrangement in the goat lung might permit increased transepithelial permeability. Plasmalemmal vesicles extended equally into both limbs of the endothelial cells. Therefore, a distinction between an avesicular zone and a vesicular zone in the goat lung perhaps does not exist. In general, in the mammalian lung such a differentiation exists. The avesicular zone of the endothelium lies on the thin side of the septum where the alveolar
Epithelium of caprine airways
Type I cell is separated from the endothelial cell by an unexpanded interstitium, which is represented by their composite basal laminae (Simionescu and Simionescu 1991). The plasmalemmal vesicles of the endothelial cells are considered to be dynamic structures which actively respond to various experimental insults especially during pulmonary oedema (Simionescu and Simionescu 1991). It is conceivable that extensive fluid-shifts across the alveolar-capillary membrane in the goat lung takes place more than is generally assumed to exist in other mammalian lungs (Simionescu and Simionescu 1991, Atwal and Brown 1980, Atwal et al 1990). The present study, in characterising the cytology of the cell populations that line the distal airways and alveolar parenchyma in the goat lung, has provided additional information which, when combined with previous LM and SEM studies, provides a basic understanding of the morphological features and differentiation of the cell population of the epithelium lining the distal airways of the goat's respiratory tract. This information is essential before pathological lesions due to disease processes can be interpreted.
ACKNOWLEDGEMENTS This work was supported by a grant from the Danish International Development Agency (DANIDA)and Natural Sciences and Engineering Council of Canada. The authors wish to thank Mrs Jacqueline McPherson, Mrs Mary Reilly, Mrs Kanwal Minhas and Mrs Dorothy McKeown for the preparation of the photomicrographs and the manuscript. Some of the work presented here has also been reported earlier (Kahwa 1992), in particular, results obtained from specimens fixed by airway instillation and Figs 1 and 2.
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