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Secretory Glycoconjugates of the Trachea and Bronchi
Chapter 5
Secretory Glycoconjugates of the Trachea and Bronchi Judith A. St George
Chapter Outline 1. 2. 3. 4.
Introduction Gel-Forming Mucin Histochemistry and Cytochemistry Lectin Histochemistry and Cytochemistry
53 53 54 55
1. INTRODUCTION The secretory cells of the tracheobronchial epithelium and submucosal glands collectively produce the mucous blanket of the conducting airways. There is, however, wide variation from species to species in the type of secretory cells at a given airway level, the numbers of secretory cells present, as well as the presence and abundance of submucosal glands. Some species, including the rat, mouse, rabbit, hamster, and guinea pig, have few if any glands; other species, such as the human, monkey, dog, cat, sheep, pig, and cow, have well-developed glands (Jeffery, 1983; Choi et al., 2000). These differences may affect the amount and properties of mucus available on the epithelial surface for mucociliary clearance. Mucociliary clearance is important in the innate defense of the lungs, where contaminants of inhaled air are trapped or dissolved in the mucous layer, then removed by ciliary transport. Optimal clearance is dependent on a complex interaction between the mucous layer, the underlying periciliary fluid, and ciliary action (Button et al., 2012). Although ciliated cells are present in relatively constant levels in most airway levels of most species, the content and sources of the mucous blanket vary considerably. The sources include mucous and serous cells of both the epithelial surface and submucosal glands. In some species, the Clara cell or noncilated bronchiolar epithelial cell may also contribute to the mucous layer (Plopper et al., 1984). This variability in sources of mucus suggests variation in the composition and properties of
Comparative Biology of the Normal Lung. http://dx.doi.org/10.1016/B978-0-12-404577-4.00005-9 Copyright © 2015 Elsevier Inc. All rights reserved.
5. Immunohistochemistry 6. Quantitation 7. Conclusions References
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mucus. Several methods have been used to define mucous composition, including carbohydrate histochemistry and cytochemistry (Spicer et al., 1983), autoradiography (Lamb and Reid, 1969), immunohistochemistry (St George et al., 1985), and biochemistry (Rose et al., 1979; Gendler and Spicer, 1995; Thornton et al., 2008; Rose and Voynow, 2006). All but the last of these methods have the advantage of localizing the constituents of mucus in situ. The goals of this chapter are to introduce the reader to the composition and sources of the mucous blanket, as well as to highlight the differences between species that may influence how experimental results in animal models can be extrapolated to humans.
2. GEL-FORMING MUCIN Mucous clearance is central to the innate defense of the lungs (Knowles and Boucher, 2002). However, overproduction and/or compromised clearance are characteristic of all airway inflammatory respiratory diseases, including chronic obstructive pulmonary disease, cystic fibrosis (CF), and asthma. In addition, the secretions from patients with these conditions are different due to differences in proportion of MUC gene products, glycosylation (Kirkham et al., 2002), the ratio of solids to liquid (Martens et al., 2011), or combinations of these (for a review, see Knowles and Boucher, 2002; Thornton et al., 2008). The gel-forming mucins are the key to proper function. The apical secretions covering the airway surfaces are not homogenous but rather
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SECTION | I Structural and Cellular Diversity of the Mammalian Respiratory System
a complex combination of polymeric mucins, proteins, lipids, water, ions, and more. The complexity of this fluid and the nature of mucins have made characterization difficult, in large part due to the challenges of isolating and purifying mucins. Since publication of the first edition of Comparative Biology of the Normal Lung, significant advances have been made in the understanding of mucins as molecular tools have become available. The most important of these has been the identification of the mucin genes (MUC genes in humans). More than 20 human and murine mucin genes have been described, but not all MUC gene products have similar protein backbones (Dekker et al., 2002; Rose and Voynow, 2006). The mucin family is broadly defined as proteins containing O-linked oligosaccharides as greater than 50% of the mass. Although several MUC genes are expressed in the lung, the two major gel-forming mucins are MUC5AC and MUC5B. In general, MUC5AC has been localized to the goblet cell of the epithelial surface, while MUC5B has been identified in the submucosal glands (Hovenberg et al., 1996). The protein backbones from these two genes are characterized by variable numbers of tandem repeats (TRs) that contain proline and are rich in serine and theronine, the sites for O-glycosylation (for a review, see Rose and Voynow, 2006). There are cysteine-rich domains that alternate with the TR regions. In addition, the N and C terminal ends contain cysteine-rich domains similar to domains contained within von Willebrand factor. TR domains are typically not conserved between species, but non-TRs appear highly conserved (Rose and Voynow, 2006). An individual mucin gene cannot identify a specific cell type as several MUC genes are expressed within the same or different cell types of the epithelia or glands (Audie et al., 1993). In summary, from this very brief overview, it is clear that mucin is a complex mixture of mucin gene products and other constituents that influence mucous properties. Infection and inflammation affect expression levels, changes in glycosylation, and the ratio of gene products. Knowledge of the animal model with regard to mucin gene expression will be important to relate data back to humans, for example, MUC5AC is a major component of normal mucus in humans; however, in normal rat airways, MUC5AC expression is very low to nonexistent, but it can be induced significantly by exposure to irritants (Borchers et al., 1998)
3. HISTOCHEMISTRY AND CYTOCHEMISTRY Given the large contribution of carbohydrates to the composition and properties of mucus, it is important to characterize this portion of mucin molecule. Carbohydrate histochemistry and cytochemistry have been used to
localize carbohydrates containing vicinal hydroxyl groups, carboxyl groups, and sulfate esters. Tables 1 and 2 summarize the histochemically defined content of the secretory cells of the epithelial surface (Table 1) and submucosal glands (Table 2). Human respiratory mucous cells of both the epithelial surface and glands were shown to contain acidic glycoconjugates due to the presence of sulfate and/or sialic acid, whereas neutral glycoconjugates were localized in serous cells of submucosal glands (Lamb and Reid, 1969). With the examination of additional species, it was found that although mucous cells generally contain acidic mucins, the predominance of either sulfo- or sialomucin varies with the species. For example, sulfomucins predominate in the mucous cells of surface epithelium in dogs (Spicer et al., 1971), cats (Jeffery, 1977), rabbits (Plopper et al., 1984), and macaque monkeys (St George et al., 1984a). In the epithelial surface of sheep (Mariassy et al., 1988a) and humans (Lamb and Reid, 1969 and Spicer et al., 1971), either sulfo- or sialomucin predominates, but this varies according to airway level. In sheep, mucous cells of airway generations greater than 14 in the left cranial lobe or 22 in the left caudal lobe contain sulfomucin (Mariassy et al., 1988a). More proximal generations are lined by mucous cells with either sialo- or sulfomucins. The nasal cavity and distal bronchioles of humans (Thaete et al., 1981) and the nasal cavity of macaque monkeys (Harkema et al., 1987) contain predominantly sulfomucins. By contrast, in both rats and mice (McCarthy and Reid, 1964), sialomucins predominate in epithelial mucous cells of airway surfaces when mucous cells are found. The predominant secretory cell of rat tracheobronchial epithelium, however, is a serous cell containing a neutral glycoconjugate (Spicer et al., 1980). Acidic mucins are found in the mucous cells of submucosal glands as well. Sulfomucins are contained in glandular mucous cells of most species, including the rat, mouse (McCarthy and Reid, 1964), dog (Spicer et al., 1971), sheep (Mariassy et al., 1988a), pig (Jones et al., 1975), and rhesus monkey (St George et al., 1986a). In rats, the distribution of acidic mucins varies with the location within the gland, with mucous tubules containing sulfomucins and mucous ducts containing sialomucins (Mochizuki et al., 1982). In submucosal glands of human respiratory airways, sialo- and sulfomucins are present in roughly equal proportions (Lamb and Reid, 1969). Ultrastructural studies using carbohydrate cytochemical methods have been used to examine the intracellular distribution of respiratory glycoconjugates. On the whole, these have confirmed and extended the observations made at the light microscopic level. Differences in carbohydrate content have been demonstrated within individual granules of serous and mucous cells. Both cell types may contain monophasic, biphasic, or triphasic granules. Mucous granules of rabbit trachea are biphasic, with an outer cortex
Secretory Glycoconjugates of the Trachea and Bronchi Chapter | 5
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TABLE 1 Carbohydrate Content of Tracheal Epithelium Carbohydrate Content Species a
Hamster
b
Rat
Cell Type
Abundance
PAS
AB
HID
Clara
þþþ
þ
Mucous
þ
þ
þ
Serous
þþþ
þ
Mucous
þ
þ
þ
Mouseb
Mucous
þ
þ
þ
c
Mucous
þ
þ
þ
þ
Clara
þþþ
þ/
Canine
Mucous
þþ
þ
þ
þ
e
Mucous
þþ
þ
þ
Serous
þ
ND
ND
ND
Mucous
þ
þ
þ and
þ and
þ
þ
þ and
Rabbit
d
Cat
f
Pig
Mucous
þþ
h
Rhesus
Mucous
þþ
þ
þ
þ
Humani
Mucous
þþþ
þ
þ
þ
g
Sheep
Note: PAS ¼ periodic acid Schiff, reacts with vicinal hydroxyl groups; AB ¼ alcian blue, at pH 2.6 reacts with acidic glycoconjugates; HID ¼ high-iron diamine, reacts with acidic glycoconjugates containing sulfate esters; ND ¼ not determined. a Emura and Mohr (1975). b McCarthy and Reid (1964). c Plopper et al. (1984). d Spicer et al. (1971). e Jeffery (1977). f Jones et al. (1975). g Mariassy et al. (1988a). h St George et al. (1984a). i Lamb and Reid (1969).
where the sulfated glycoconjugate is concentrated (Plopper et al., 1984). By contrast, in rhesus tracheal mucous cells, the sulfated material is concentrated within the inner one or two core regions (St George et al., 1984a). In human epithelial mucous cells, the cores have been described as invariably negative for carbohydrate (Thaete et al., 1981). The content of the nonciliated bronchiolar-cell or Claracell secretory granules has also been characterized using histochemical methods for glycoproteins and lipids (for a review, see Plopper, 1983). These studies have shown that Clara-cell granules do not contain acidic glycoconjugates in any species, including the human (Cutz and Conen, 1971), mouse (Luke and Spicer, 1966 and Pack et al., 1981), rat (Spicer et al., 1980), hamster and guinea pig (Luke and Spicer, 1966), rabbit (Plopper et al., 1984), or cat (Klika and Petrik, 1969). Carbohydrates containing vicinal hydroxyl groups have been demonstrated consistently in some species and variably in the mouse and rat (Plopper, 1983). There is considerable histochemical evidence suggesting that Clara cells synthesize lipids or phospholipids (Cutz and
Conen, 1971; Azzopardi and Thurlbeck, 1969), but whether these components are incorporated into secretory granules has yet to be convincingly documented. Chapter 7 in this volume discusses the immunochemical characterization of the secretory product of nonciliated bronchiolar epithelial cells.
4. LECTIN HISTOCHEMISTRY AND CYTOCHEMISTRY More specific information on the composition of the glycoconjugates contained within the mucous and serous cells of the respiratory system was provided with the use of lectins. Lectins, with specificity for sugars primarily in the terminal position, were used to localize these sugars in complex carbohydrates. A summary of lectin reactivity is presented in Table 3. The majority of the work in this area was done by Spicer and colleagues, and was reviewed for the human, rat, and mouse in 1983 (Spicer et al., 1983). The results from the lectin studies provided additional
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SECTION | I Structural and Cellular Diversity of the Mammalian Respiratory System
TABLE 2 Carbohydrate Content of Tracheal Submucosal Glands Carbohydrate Content Species
Abundance
Secretory Cell
PAS
AB
HID
þ/
Mucous
þ
þ
Rat
þ
Serous
þ
Mucous
þ
þ
Mouseb
þ/
Serous
þ
Mucous
þ
þ
þ and
þ/
Mucous
þ
þ
þ
þþ
Serous
þ
Mucous
þ
þ
þ and
a
Hamster b
c
Rabbit
d
Canine
þþþþ
e
Cat
þþ
Pigf
þþ
g
Sheep
Rhesus
þþ
Humani
þþþ
h
þ and
Serous
þ
þ
þ and
Mucous
þ
þ
þ
Serous
þ
Mucous
þ
þ
þ and
Serous
þ
Mucous
þ
þ
þ
Serous
þ
Mucous
þ
þ
þ
Serous
þ
Mucous
þ
þ
þ and
Note: PAS ¼ periodic acid Schiff, reacts with vicinal hydroxyl groups; AB ¼ alcian blue, at pH 2.6 reacts with acidic glycoconjugates; HID ¼ high-iron diamine, reacts with acidic glycoconjugates containing sulfate esters; ND ¼ not determined. a Emura and Mohr (1975). b McCarthy and Reid (1964). c Plopper et al. (1984). d Spicer et al. (1971). e Jeffery (1977). f Jones et al. (1975). g Mariassy et al. (1988a). h St George et al. (1984a). i Lamb and Reid (1969).
information and further complicated the interpretation of secretory cell content. For example, histochemical analysis had not indicated the heterogeneity of content of both serous and mucous cells that became apparent with lectin application. An important result not reflected in Table 3 is that although some cells of a specific type contain a sugar, not all will, nor will they contain it in the same concentration. Additionally, in human airways, the sugar content of mucous, but not serous, cells varies with blood group antigen and presumably with the secretory status of a given individual (Spicer et al., 1983). Reactivity with lectins was used to define a specific cell type or was used as a marker for a secretory product (Wasano et al., 1988a). With the characterization of lectin reactivity established in the
airway cells of healthy animals, investigators employed lectins to detect shifts in secretory products with injury or disease (Inai et al., 1987; Mariassy et al., 1989). Lectins applied at the level of the electron microscope further elucidated the nature of the airway secretory product(s). Wasano et al. (1988b) examined mucous cells in the hamster trachea and demonstrated that glycoconjugates with discrete terminal sugars are localized within distinct regions of the Golgi apparatus. Sugars appeared in a sequential fashion from cis-to trans-cistemae. N-acetyl galactosamine was the only sugar detected in the cis-cistemae; then N-acetyl glucosamine and galactose, and lastly fucose and sialic acid, were detected in the transcistemae. This sequence of sugars coincides with the order
TABLE 3 Lectin Reactivity in Airway Surface Epithelium Lectin Species/Cell Type
Sugar Specificity
LCA Man Glc Glc NAc
WGA NANA Glc NAc
BSAI Gal
DBA Gal NAc
SBA Gal-Gal Nac Gal Gal Nac
PNA Gal Gal NAc
RCA Gal Gal NAc
UEA Fuc
Human bronchia
Mucous
þþþþ
þ
þ
þþþ
þþþþ
þþþþ
þþþþ
Sheep tracheabronchioleb
Mucous M1
þþþ
þþ
þþþ
þþþþ
Mucous M2
þþþ
þþ
þþþ
þþþþ
þþþ
þ
þ
þ
(þþþ)
þþþþ
þþþþ
(þþþ)
Mucous M3 Rat tracheac Rhesus trachea
Mucous
Mucous
þþþþ
c
þ
(þþþ)
þþþ þ
Lectin Reactivity of Airway Submucosal Glands
Sheep
a
Mouse trachea
LCA
WGA
BSAI
DBA
SBA
PNA
RCA
UEA
Mucous
þ
þ
þ
þþþ
þþþþ
þþþ
þþþþ
Serous
þþþ
þþþ
þþ
þ
þþ
Mucous 4
þþþþ
þþ
þþ
Serous
þþþ
þþ
þ
(þþþ)
þþþ
þþþþ
þþþþ
þþþþ
þþþþ
-
þþ
þþ
(þþþ)
þþþ
Mucous
Serous þþ
þþ
Rat trachea
Mucous
(þþþþ)
Rhesus trachea
Mucous
þþþþ
þþþþ
þþþ
(þþþþ)
(þþþþ)
þþþþ
Serous
þþ
þþ
þ
(þþ)
(þþ)
þ
Serous
Note: LCA ¼ Lotus tetragonolobus; WGA ¼ wheat germ agglutinin; BSA I ¼ Bandeirea simplicifolia I; DBA ¼ Dolichos biflorus; SBA ¼ Glycine max; PNA ¼ Arachis hypogea; RCA ¼ Ricinus communis; UEA ¼ Ulex europeus; Man ¼ mannose; Glc ¼ glucose; GlcNAc ¼ N-acetyl glucosamine; NANA ¼ N-acetyl neuraminic acid (sialic acid); Gal ¼ galactose; Ga1NAc ¼ N-acetylgalactosamine; Fuc ¼ fucose. a Spicer et al. (1983). b Mariassy et al. (1988b). c Reaction in parentheses is after neuraminidase treatment.
Secretory Glycoconjugates of the Trachea and Bronchi Chapter | 5
Human bronchi
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SECTION | I Structural and Cellular Diversity of the Mammalian Respiratory System
of glycosylation of O-linked respiratory mucins demonstrated using biochemical methods (Rana et al., 1986).
5. IMMUNOHISTOCHEMISTRY Immunohistochemical methods can be used to define more precisely the variations in content within cells or granules. For example, the enzymes lysozyme (Bowes et al., 1981) and lactoferrin (Bowes and Corrin, 1977) have been localized within secretory cells of the respiratory tract. Lysozyme, which does not contain carbohydrates, has been localized within serous cells of submucosal glands, leading Spicer and colleagues to suggest that the carbohydratenegative portion of serous granules is lysozyme (Spicer et al., 1980). Immunohistochemical studies using monoclonal antibodies against respiratory secretory products have expanded the previously discussed heterogeneity (St George et al., 1984b, 1985; Basbaum et al., 1984). We have used monoclonal antibodies against rhesus or rabbit tracheal secretions to show several antigenically distinct subpopulations of mucous cells (St George et al., 1985). When serial sections of histochemically and immunohistochemically stained samples are compared, secretory cells are found that do not vary histochemically but vary immunohistochemically (St George et al., 1984b). Application of the monoclonal antibodies to four airway levels distal to the trachea in rhesus monkey indicate that the more distal the airway, the less the secretory content resembles that in trachea (St George et al., 1986b). This variation in content suggests variations in the biophysical properties of secretion at different airway levels. We have also used the panel of monoclonal antibodies to examine the appearance of secretory products in the airways of developing fetal monkey lungs. From that study, we have concluded that the antigens of adult trachea are present in neonatal trachea at birth but that the antigens appear sequentially rather than simultaneously. The results of these studies using monoclonal antibodies are probably detecting differences in the glycosylation of mucous glycoconjugates, because characterization of the epitopes of our antibodies (Lin et al., 1989) and those of others (Basbaum et al., 1986) have indicated a carbohydrate specificity. Antibodies to the protein portion of mucus have been used to localize and quantify mucins within normal human airways and compare the results to those from a patient with CF (Burgel et al., 2007). Although the secretory cells are thought to be the primary source of the mucous blanket, other cell types within the epithelial surface and glands may modify or supplement the fluid lining. Modification to the fluid lining includes the secretion of water and ions. These changes influence the properties of the mucous blanket and are therefore important to the function of mucociliary clearance. One other cell
type that can participate in this process is the ciliated cell. Several studies have demonstrated the presence of glycoprotein on the surface of ciliated cells (Spicer et al., 1971; Jeffery, 1977). We have observed vesicles or granules containing mucous antigens in ciliated cells (St George et al., 1984b), suggesting either endocytosis or exocytosis of components of the mucous blanket.
6. QUANTITATION With certain toxicological insults or respiratory diseases, profound changes occur in the volume of secretion produced and stored in the airways. It has therefore been important to develop accurate methods to quantify the volume of stored secretory products under normal as well as abnormal conditions. Previous methods have included determining the mean gland-to-wall ratio (Reid Index, Reid, 1960) or point counting to quantify submucosal glands (Bedrossian et al., 1971). We have used a computerized morphometric method to assess the volume of the secretory product per unit surface area and have demonstrated that there is considerable variation at different airway levels in the amount of glycoconjugate stored in airway epithelium of the rhesus monkey (Heidsiek et al., 1987; Plopper et al., 1988). The product of the tracheal surface was predominantly acidic, while that in submucosal glands was neutral. The trachea stored at least twice as much per unit of surface area, as compared to distal airways (generation 11). Additionally, a change in secretory product from sulfomucin to sialomucin was detected distally. One surprising feature of these studies was the contribution to the total secretory product by submucosal glands versus surface epithelium, as suggested by the amount of stored product in either surface epithelium or submucosal glands. Several investigators have indicated that the overwhelming majority of secretion is from submucosal glands (Reid, 1960). However, morphometric analysis of stored product in rhesus trachea demonstrated that the contribution by the submucosal glands was less than 50% of the total product (Heidsiek et al., 1987). These same quantitative methods were used to immunochemically compare the secretory cells from normal airways with those from an asthma patient (Ordonez et al., 2001).
7. CONCLUSIONS Several methods, including biochemical, histochemical, and immunohistochemical techniques, have been used to define the content of secretory cells of airway epithelium. Application of these methods has demonstrated the extensive heterogeneity in content when comparing secretory cells from the epithelial surface to those in submucosal glands, in the same cell type at the same airway level, and in secretory cells from different airway levels, as well as
Secretory Glycoconjugates of the Trachea and Bronchi Chapter | 5
from one species to another. Many tools are now in place to more fully understand the secretory response and the airway secretory products; however, the investigator should consider the variations that occur at different airway levels in the same species, as well as differences between species. The influence of secretion by different sites (surface vs glands and proximal vs distal) on the properties of the mucous blanket and the importance in differences in control in these regions remains to be elucidated. Importantly, extrapolation of results from one species to another should be done, bearing in mind that the aforementioned differences may dictate a very different secretory response to a given toxicological insult.
REFERENCES Audie, J.P., et al., 1993. Expression of human mucin genes in respiratory, digestive, and reproductive tracts ascertained by in situ hybridization. J. Histochem. Cytochem. 41, 1479e1485. Azzopardi, A., Thurlbeck, W.M., 1969. The histochemistry of the nonciliated bronchiolar epithelial cells. Am. Rev. Respir. Dis. 99, 516. Basbaum, C.B., et al., 1984. Monoclonal antibodies as probes for unique antigens in secretory cells of mixed exocrine organs. Proc. Natl. Acad. Sci. USA 81, 4419. Basbaum, C.B., et al., 1986. Tracheal carbohydrate antigens identified by monoclonal antibodies. Arch. Biochem. Biophys. 249, 363. Bedrossian, C.W.M., Anderson, A.E., Foraker, A.G., 1971. Comparison of methods for quantitating bronchial morphology. Thorax 26, 406. Borchers, M.T., Wert, S.E., Leikauf, G.D., 1998. Acrolein-induced MUC5ac expression in rat airways. Am. J. Physiol. 274, L573eL581. Bowes, D., Corrin, B., 1977. Ultrastructural immunocytochemical localization of lysozyme in human bronchial glands. Thorax 32, 163. Burgel, P.R, Montani, D., Danel, C., Dusser, D.J., Nadel, J.A., 2007. A morphometric study of mucins and small airway plugging in cystic fibrosis. Thorax 62, 153e161. Bowes, D., Clark, A.E., Corrin, B., 1981. Ultrastructural localization of lactoferrin and glycoprotein in human bronchial glands. Thorax 36, 108. Button, B., et al., 2012. A periciliary brush promotes the lung health by separating the mucus layer from airway epithelia. Science 337, 937e994. Choi, H.K., Finkbeiner, W.E., Widdicombe, J.H., 2000. A comparative study of mammalian tracheal mucous glands. J. Anat. 197 (Pt 3), 361e372. Cutz, E., Conen, P.E., 1971. Ultrastructure and cytochemistry of Clara cells. Am. J. Pathol. 62 (1), 127. Dekker, J., et al., 2002. The MUC family: an obituary. Trends Biochem. Sci. 27, 126e131. Emura, M., Mohr, U., 1975. Morphological studies on the development of tracheal epithelium in the Syrian golden hamster. Z. Versuchstierk. 17 (1), 14e26. Gendler, S.J., Spicer, A.P., 1995. Epithelial mucin genes. Annu. Rev. Physiol. 57, 607e634. Harkema, J.R., et al., 1987. Regional differences in quantities of histochemically detectable mucosubstances in nasal, paranasal, and nasopharyngeal epithelium of the bonnet monkey. J. Histochem. Cytochem. 35, 279.
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Heidsiek, J.G., et al., 1987. Quantitative histochemistry of mucosubstance in tracheal epithelium of the macaque monkey. J. Histochem. Cytochem. 35, 435. Hovenberg, H.W., Davies, J.R., Carlstedt, I., 1996. Different mucins are produced by the surface epithelium and the submucosa in human trachea: identification of MUC5AC as a major mucin from the goblet cells. Biochem. J. 318 (Pt 1), 319e324. Inai, K., et al., 1987. An altered lectin binding to mucus glycoprotein in goblet cells of human tracheobronchial epithelium among former mustard-gas workers. Acta Pathol. Jpn. 37, 537. Jeffery, P.K., 1977. Structure and function of mucus-secreting cells of cat and goose airway epithelium. In: Respiratory Tract Mucus. ElsevierNorth Holland, New York, pp. 5e19. Jeffery, P.K., 1983. Morphologic features of airway surface epithelial cells and glands. Am. Rev. Resp. Dis. 128, S14. Jones, R.A., Baskerville, A., Reid, L.M., 1975. Histochemical identification of glycoproteins in pig bronchial epithelium: (a) normal and (b) hypertrophied from enzootic pneumonia. J. Pathol. 116, 1. Kirkham, S., et al., 2002. Heterogeneity of airways mucus: variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B. Biochem. J. 361, 537e546. Klika, E., Petrik, P., 1969. A study of the structure of the lung alveolus and bronchiolar epithelium. Acta Histochem. (Jena) 20, 331. Knowles, M.R., Boucher, R.C., 2002. Mucus clearance as a primary innate defense mechanism for mammalian airways. J. Clin. Invest. 109, 571e577. Lamb, D., Reid, L.M., 1969. Histochemical types of acidic glycoprotein produced by mucous cells of the tracheobronchial glands in man. J. Pathol. 98, 213. Lin, H., et al., 1989. An ELISA method for the quantitation of tracheal mucins from human and nonhuman primates. Am. J. Respir. Cell. Mol. Biol. 1, 41. Luke, J.L., Spicer, S.S., 1966. Histochemistry of surface epithelium and plural mucins in mammalian lung. Lab. Invest. 14, 2101. Mariassy, A.T., et al., 1988a. Tracheobronchial epithelium of the sheep. III. Carbohydrate histochemical and cytochemical characterization of secretory epithelial cells. Anat. Rec. 221, 540. Mariassy, A.T., et al., 1988b. Tracheobronchial epithelium of the sheep. IV. Lectin histochemical characterization of secretory epithelial cells. Anat. Rec. 222, 49. Mariassy, A.T., et al., 1989. Lectin-detectable effects of localized pneumonia on airway mucous cell populations: role of cyclooxygenase metabolites. Exp. Lung Res. 15, 113. Martens, C.J., et al., 2011. Mucous solids and liquid secretion by airways: studies with normal pig, cystic fibrosis human, and non-cystic fibrosis human bronchi. Am. J. Physiol. Lung Cell. Mol. Physiol. 301, L236eL246. McCarthy, C., Reid, L.M., 1964. Acid mucopolysaccharides in the bronchial tree in the mouse and rat (sialomucins and sulphate). Q. J. Exper. Physiol. 49, 81. Mochizuki, I., et al., 1982. Carbohydrate histochemistry of rat respiratory glands. Anat. Rec. 202, 45. Ordonez, C.L., et al., 2001. Mild and moderate asthma is associated with airway goblet cell hyperplasia and abnormalities in mucin gene expression. Am. J. Respir. Crit. Care Med. 163, 517e523. Pack, R.J., Al-Ugaily, L.H., Morris, G., 1981. The cells of the tracheobronchial epithelium of the mouse: a quantitative light and electron microscopic study. J. Anat. 132, 71.
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Plopper, C.G., 1983. Comparative morphologic features of bronchiolar epithelial cells: the Clara cell. Am. Rev. Respir. Dis. 128, S37. Plopper, C.G., et al., 1984. Carbohydrate cytochemistry of tracheobronchial airway epithelium of the rabbit. J. Histochem. Cytochem. 32, 209. Plopper, C.G., et al., 1988. Tracheobronchial epithelium in the adult rhesus monkey: a quantitative histochemical and ultrastructural study. Am. J. Anat. 184, 31. Rana, S.S., Chandrasekaran, E.V., Mendicino, J., 1986. Structures of the sialylated oligosaccharide chains in swine trachea mucin glycoproteins. J. Biol. Chem. 262, 3654. Reid, L.M., 1960. Measurement of the bronchial mucous gland layer: a diagnostic yardstick in chronic bronchitis. Thorax 15, 132. Rose, M.C., Lynn, W.S., Kaufman, B., 1979. Resolution of the major components of human lung mucosal gel and their capabilities for reaggregation and gel formation. Biochemistry 18, 4030. Rose, M.C., Voynow, J.A., 2006. Respiratory tract mucin genes and mucin glycoproteins in health and disease. Physiol. Rev. 86, 245e278. Spicer, S.S., et al., 1971. Histochemistry of mucosubstances in the canine and human respiratory tract. Lab. Invest. 25, 483. Spicer, S.S., et al., 1980. Complex carbohydrates of rat tracheobronchial surface epithelium visualized ultrastructurally. Am. J. Anat. 158, 93. Spicer, S.S., Schulte, B.A., Thomopoulos, G.N., 1983. Histochemical properties of the respiratory tract in different species. Am. Rev. Respir. Dis. 128, S20.
St George, J.A., Nishio, S.J., Plopper, C.G., 1984a. Carbohydrate cytochemistry of the rhesus monkey tracheal epithelium. Anat. Rec. 210, 293. St George, J.A., et al., 1984b. An immunocytochemical/histochemical approach to tracheobronchial mucus characterization in the rabbit. Am. Rev. Respir. Dis. 130, 124. St George, J.A., et al., 1985. An immunohistochemical characterization of rhesus monkey respiratory secretions using monoclonal antibodies. Am. Rev. Respir. Dis. 132, 556. St George, J.A., et al., 1986a. Carbohydrate cytochemistry of rhesus monkey tracheal submucosal glands. Anat. Rec. 216, 60. St George, J.A., et al., 1986b. Variation of respiratory mucins with airway level in rhesus monkey. Am. Rev. Respir. Dis. 133, A294. Thaete, L.C., Spicer, S.S., Spock, A., 1981. Histology, ultrastructure, and carbohydrate cytochemistry of surface and glandular epithelium of human nasal mucosa. Am. J. Anat. 162, 243. Thornton, D.J., Rousseau, K., McGuckin, M.A., 2008. Structure and function of the polymeric mucins in airways mucus. Annu. Rev. Physiol. 70, 459e486. Wasano, K., et al., 1988a. Membrane differentiation markers of airway epithelial secretory cells. J. Histochem. Cytochem 36 (2), 167. Wasano, K., Nakamura, K., Yamamoto, T., 1988b. Lectin-gold cytochemistry of mucin oligosaccharide biosynthesis in golgi apparatus of airway secretory cells of the hamster. Anat. Rec. 221, 635.
Secretory Glycoconjugates of the Trachea and Bronchi Judith A. St George
Chapter Outline 1. 2. 3. 4.
Introduction Gel-Forming Mucin Histochemistry and Cytochemistry Lectin Histochemistry and Cytochemistry
53 53 54 55
1. INTRODUCTION The secretory cells of the tracheobronchial epithelium and submucosal glands collectively produce the mucous blanket of the conducting airways. There is, however, wide variation from species to species in the type of secretory cells at a given airway level, the numbers of secretory cells present, as well as the presence and abundance of submucosal glands. Some species, including the rat, mouse, rabbit, hamster, and guinea pig, have few if any glands; other species, such as the human, monkey, dog, cat, sheep, pig, and cow, have well-developed glands (Jeffery, 1983; Choi et al., 2000). These differences may affect the amount and properties of mucus available on the epithelial surface for mucociliary clearance. Mucociliary clearance is important in the innate defense of the lungs, where contaminants of inhaled air are trapped or dissolved in the mucous layer, then removed by ciliary transport. Optimal clearance is dependent on a complex interaction between the mucous layer, the underlying periciliary fluid, and ciliary action (Button et al., 2012). Although ciliated cells are present in relatively constant levels in most airway levels of most species, the content and sources of the mucous blanket vary considerably. The sources include mucous and serous cells of both the epithelial surface and submucosal glands. In some species, the Clara cell or noncilated bronchiolar epithelial cell may also contribute to the mucous layer (Plopper et al., 1984). This variability in sources of mucus suggests variation in the composition and properties of
Comparative Biology of the Normal Lung. http://dx.doi.org/10.1016/B978-0-12-404577-4.00005-9 Copyright © 2015 Elsevier Inc. All rights reserved.
5. Immunohistochemistry 6. Quantitation 7. Conclusions References
58 58 58 59
mucus. Several methods have been used to define mucous composition, including carbohydrate histochemistry and cytochemistry (Spicer et al., 1983), autoradiography (Lamb and Reid, 1969), immunohistochemistry (St George et al., 1985), and biochemistry (Rose et al., 1979; Gendler and Spicer, 1995; Thornton et al., 2008; Rose and Voynow, 2006). All but the last of these methods have the advantage of localizing the constituents of mucus in situ. The goals of this chapter are to introduce the reader to the composition and sources of the mucous blanket, as well as to highlight the differences between species that may influence how experimental results in animal models can be extrapolated to humans.
2. GEL-FORMING MUCIN Mucous clearance is central to the innate defense of the lungs (Knowles and Boucher, 2002). However, overproduction and/or compromised clearance are characteristic of all airway inflammatory respiratory diseases, including chronic obstructive pulmonary disease, cystic fibrosis (CF), and asthma. In addition, the secretions from patients with these conditions are different due to differences in proportion of MUC gene products, glycosylation (Kirkham et al., 2002), the ratio of solids to liquid (Martens et al., 2011), or combinations of these (for a review, see Knowles and Boucher, 2002; Thornton et al., 2008). The gel-forming mucins are the key to proper function. The apical secretions covering the airway surfaces are not homogenous but rather
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SECTION | I Structural and Cellular Diversity of the Mammalian Respiratory System
a complex combination of polymeric mucins, proteins, lipids, water, ions, and more. The complexity of this fluid and the nature of mucins have made characterization difficult, in large part due to the challenges of isolating and purifying mucins. Since publication of the first edition of Comparative Biology of the Normal Lung, significant advances have been made in the understanding of mucins as molecular tools have become available. The most important of these has been the identification of the mucin genes (MUC genes in humans). More than 20 human and murine mucin genes have been described, but not all MUC gene products have similar protein backbones (Dekker et al., 2002; Rose and Voynow, 2006). The mucin family is broadly defined as proteins containing O-linked oligosaccharides as greater than 50% of the mass. Although several MUC genes are expressed in the lung, the two major gel-forming mucins are MUC5AC and MUC5B. In general, MUC5AC has been localized to the goblet cell of the epithelial surface, while MUC5B has been identified in the submucosal glands (Hovenberg et al., 1996). The protein backbones from these two genes are characterized by variable numbers of tandem repeats (TRs) that contain proline and are rich in serine and theronine, the sites for O-glycosylation (for a review, see Rose and Voynow, 2006). There are cysteine-rich domains that alternate with the TR regions. In addition, the N and C terminal ends contain cysteine-rich domains similar to domains contained within von Willebrand factor. TR domains are typically not conserved between species, but non-TRs appear highly conserved (Rose and Voynow, 2006). An individual mucin gene cannot identify a specific cell type as several MUC genes are expressed within the same or different cell types of the epithelia or glands (Audie et al., 1993). In summary, from this very brief overview, it is clear that mucin is a complex mixture of mucin gene products and other constituents that influence mucous properties. Infection and inflammation affect expression levels, changes in glycosylation, and the ratio of gene products. Knowledge of the animal model with regard to mucin gene expression will be important to relate data back to humans, for example, MUC5AC is a major component of normal mucus in humans; however, in normal rat airways, MUC5AC expression is very low to nonexistent, but it can be induced significantly by exposure to irritants (Borchers et al., 1998)
3. HISTOCHEMISTRY AND CYTOCHEMISTRY Given the large contribution of carbohydrates to the composition and properties of mucus, it is important to characterize this portion of mucin molecule. Carbohydrate histochemistry and cytochemistry have been used to
localize carbohydrates containing vicinal hydroxyl groups, carboxyl groups, and sulfate esters. Tables 1 and 2 summarize the histochemically defined content of the secretory cells of the epithelial surface (Table 1) and submucosal glands (Table 2). Human respiratory mucous cells of both the epithelial surface and glands were shown to contain acidic glycoconjugates due to the presence of sulfate and/or sialic acid, whereas neutral glycoconjugates were localized in serous cells of submucosal glands (Lamb and Reid, 1969). With the examination of additional species, it was found that although mucous cells generally contain acidic mucins, the predominance of either sulfo- or sialomucin varies with the species. For example, sulfomucins predominate in the mucous cells of surface epithelium in dogs (Spicer et al., 1971), cats (Jeffery, 1977), rabbits (Plopper et al., 1984), and macaque monkeys (St George et al., 1984a). In the epithelial surface of sheep (Mariassy et al., 1988a) and humans (Lamb and Reid, 1969 and Spicer et al., 1971), either sulfo- or sialomucin predominates, but this varies according to airway level. In sheep, mucous cells of airway generations greater than 14 in the left cranial lobe or 22 in the left caudal lobe contain sulfomucin (Mariassy et al., 1988a). More proximal generations are lined by mucous cells with either sialo- or sulfomucins. The nasal cavity and distal bronchioles of humans (Thaete et al., 1981) and the nasal cavity of macaque monkeys (Harkema et al., 1987) contain predominantly sulfomucins. By contrast, in both rats and mice (McCarthy and Reid, 1964), sialomucins predominate in epithelial mucous cells of airway surfaces when mucous cells are found. The predominant secretory cell of rat tracheobronchial epithelium, however, is a serous cell containing a neutral glycoconjugate (Spicer et al., 1980). Acidic mucins are found in the mucous cells of submucosal glands as well. Sulfomucins are contained in glandular mucous cells of most species, including the rat, mouse (McCarthy and Reid, 1964), dog (Spicer et al., 1971), sheep (Mariassy et al., 1988a), pig (Jones et al., 1975), and rhesus monkey (St George et al., 1986a). In rats, the distribution of acidic mucins varies with the location within the gland, with mucous tubules containing sulfomucins and mucous ducts containing sialomucins (Mochizuki et al., 1982). In submucosal glands of human respiratory airways, sialo- and sulfomucins are present in roughly equal proportions (Lamb and Reid, 1969). Ultrastructural studies using carbohydrate cytochemical methods have been used to examine the intracellular distribution of respiratory glycoconjugates. On the whole, these have confirmed and extended the observations made at the light microscopic level. Differences in carbohydrate content have been demonstrated within individual granules of serous and mucous cells. Both cell types may contain monophasic, biphasic, or triphasic granules. Mucous granules of rabbit trachea are biphasic, with an outer cortex
Secretory Glycoconjugates of the Trachea and Bronchi Chapter | 5
55
TABLE 1 Carbohydrate Content of Tracheal Epithelium Carbohydrate Content Species a
Hamster
b
Rat
Cell Type
Abundance
PAS
AB
HID
Clara
þþþ
þ
Mucous
þ
þ
þ
Serous
þþþ
þ
Mucous
þ
þ
þ
Mouseb
Mucous
þ
þ
þ
c
Mucous
þ
þ
þ
þ
Clara
þþþ
þ/
Canine
Mucous
þþ
þ
þ
þ
e
Mucous
þþ
þ
þ
Serous
þ
ND
ND
ND
Mucous
þ
þ
þ and
þ and
þ
þ
þ and
Rabbit
d
Cat
f
Pig
Mucous
þþ
h
Rhesus
Mucous
þþ
þ
þ
þ
Humani
Mucous
þþþ
þ
þ
þ
g
Sheep
Note: PAS ¼ periodic acid Schiff, reacts with vicinal hydroxyl groups; AB ¼ alcian blue, at pH 2.6 reacts with acidic glycoconjugates; HID ¼ high-iron diamine, reacts with acidic glycoconjugates containing sulfate esters; ND ¼ not determined. a Emura and Mohr (1975). b McCarthy and Reid (1964). c Plopper et al. (1984). d Spicer et al. (1971). e Jeffery (1977). f Jones et al. (1975). g Mariassy et al. (1988a). h St George et al. (1984a). i Lamb and Reid (1969).
where the sulfated glycoconjugate is concentrated (Plopper et al., 1984). By contrast, in rhesus tracheal mucous cells, the sulfated material is concentrated within the inner one or two core regions (St George et al., 1984a). In human epithelial mucous cells, the cores have been described as invariably negative for carbohydrate (Thaete et al., 1981). The content of the nonciliated bronchiolar-cell or Claracell secretory granules has also been characterized using histochemical methods for glycoproteins and lipids (for a review, see Plopper, 1983). These studies have shown that Clara-cell granules do not contain acidic glycoconjugates in any species, including the human (Cutz and Conen, 1971), mouse (Luke and Spicer, 1966 and Pack et al., 1981), rat (Spicer et al., 1980), hamster and guinea pig (Luke and Spicer, 1966), rabbit (Plopper et al., 1984), or cat (Klika and Petrik, 1969). Carbohydrates containing vicinal hydroxyl groups have been demonstrated consistently in some species and variably in the mouse and rat (Plopper, 1983). There is considerable histochemical evidence suggesting that Clara cells synthesize lipids or phospholipids (Cutz and
Conen, 1971; Azzopardi and Thurlbeck, 1969), but whether these components are incorporated into secretory granules has yet to be convincingly documented. Chapter 7 in this volume discusses the immunochemical characterization of the secretory product of nonciliated bronchiolar epithelial cells.
4. LECTIN HISTOCHEMISTRY AND CYTOCHEMISTRY More specific information on the composition of the glycoconjugates contained within the mucous and serous cells of the respiratory system was provided with the use of lectins. Lectins, with specificity for sugars primarily in the terminal position, were used to localize these sugars in complex carbohydrates. A summary of lectin reactivity is presented in Table 3. The majority of the work in this area was done by Spicer and colleagues, and was reviewed for the human, rat, and mouse in 1983 (Spicer et al., 1983). The results from the lectin studies provided additional
56
SECTION | I Structural and Cellular Diversity of the Mammalian Respiratory System
TABLE 2 Carbohydrate Content of Tracheal Submucosal Glands Carbohydrate Content Species
Abundance
Secretory Cell
PAS
AB
HID
þ/
Mucous
þ
þ
Rat
þ
Serous
þ
Mucous
þ
þ
Mouseb
þ/
Serous
þ
Mucous
þ
þ
þ and
þ/
Mucous
þ
þ
þ
þþ
Serous
þ
Mucous
þ
þ
þ and
a
Hamster b
c
Rabbit
d
Canine
þþþþ
e
Cat
þþ
Pigf
þþ
g
Sheep
Rhesus
þþ
Humani
þþþ
h
þ and
Serous
þ
þ
þ and
Mucous
þ
þ
þ
Serous
þ
Mucous
þ
þ
þ and
Serous
þ
Mucous
þ
þ
þ
Serous
þ
Mucous
þ
þ
þ
Serous
þ
Mucous
þ
þ
þ and
Note: PAS ¼ periodic acid Schiff, reacts with vicinal hydroxyl groups; AB ¼ alcian blue, at pH 2.6 reacts with acidic glycoconjugates; HID ¼ high-iron diamine, reacts with acidic glycoconjugates containing sulfate esters; ND ¼ not determined. a Emura and Mohr (1975). b McCarthy and Reid (1964). c Plopper et al. (1984). d Spicer et al. (1971). e Jeffery (1977). f Jones et al. (1975). g Mariassy et al. (1988a). h St George et al. (1984a). i Lamb and Reid (1969).
information and further complicated the interpretation of secretory cell content. For example, histochemical analysis had not indicated the heterogeneity of content of both serous and mucous cells that became apparent with lectin application. An important result not reflected in Table 3 is that although some cells of a specific type contain a sugar, not all will, nor will they contain it in the same concentration. Additionally, in human airways, the sugar content of mucous, but not serous, cells varies with blood group antigen and presumably with the secretory status of a given individual (Spicer et al., 1983). Reactivity with lectins was used to define a specific cell type or was used as a marker for a secretory product (Wasano et al., 1988a). With the characterization of lectin reactivity established in the
airway cells of healthy animals, investigators employed lectins to detect shifts in secretory products with injury or disease (Inai et al., 1987; Mariassy et al., 1989). Lectins applied at the level of the electron microscope further elucidated the nature of the airway secretory product(s). Wasano et al. (1988b) examined mucous cells in the hamster trachea and demonstrated that glycoconjugates with discrete terminal sugars are localized within distinct regions of the Golgi apparatus. Sugars appeared in a sequential fashion from cis-to trans-cistemae. N-acetyl galactosamine was the only sugar detected in the cis-cistemae; then N-acetyl glucosamine and galactose, and lastly fucose and sialic acid, were detected in the transcistemae. This sequence of sugars coincides with the order
TABLE 3 Lectin Reactivity in Airway Surface Epithelium Lectin Species/Cell Type
Sugar Specificity
LCA Man Glc Glc NAc
WGA NANA Glc NAc
BSAI Gal
DBA Gal NAc
SBA Gal-Gal Nac Gal Gal Nac
PNA Gal Gal NAc
RCA Gal Gal NAc
UEA Fuc
Human bronchia
Mucous
þþþþ
þ
þ
þþþ
þþþþ
þþþþ
þþþþ
Sheep tracheabronchioleb
Mucous M1
þþþ
þþ
þþþ
þþþþ
Mucous M2
þþþ
þþ
þþþ
þþþþ
þþþ
þ
þ
þ
(þþþ)
þþþþ
þþþþ
(þþþ)
Mucous M3 Rat tracheac Rhesus trachea
Mucous
Mucous
þþþþ
c
þ
(þþþ)
þþþ þ
Lectin Reactivity of Airway Submucosal Glands
Sheep
a
Mouse trachea
LCA
WGA
BSAI
DBA
SBA
PNA
RCA
UEA
Mucous
þ
þ
þ
þþþ
þþþþ
þþþ
þþþþ
Serous
þþþ
þþþ
þþ
þ
þþ
Mucous 4
þþþþ
þþ
þþ
Serous
þþþ
þþ
þ
(þþþ)
þþþ
þþþþ
þþþþ
þþþþ
þþþþ
-
þþ
þþ
(þþþ)
þþþ
Mucous
Serous þþ
þþ
Rat trachea
Mucous
(þþþþ)
Rhesus trachea
Mucous
þþþþ
þþþþ
þþþ
(þþþþ)
(þþþþ)
þþþþ
Serous
þþ
þþ
þ
(þþ)
(þþ)
þ
Serous
Note: LCA ¼ Lotus tetragonolobus; WGA ¼ wheat germ agglutinin; BSA I ¼ Bandeirea simplicifolia I; DBA ¼ Dolichos biflorus; SBA ¼ Glycine max; PNA ¼ Arachis hypogea; RCA ¼ Ricinus communis; UEA ¼ Ulex europeus; Man ¼ mannose; Glc ¼ glucose; GlcNAc ¼ N-acetyl glucosamine; NANA ¼ N-acetyl neuraminic acid (sialic acid); Gal ¼ galactose; Ga1NAc ¼ N-acetylgalactosamine; Fuc ¼ fucose. a Spicer et al. (1983). b Mariassy et al. (1988b). c Reaction in parentheses is after neuraminidase treatment.
Secretory Glycoconjugates of the Trachea and Bronchi Chapter | 5
Human bronchi
57
58
SECTION | I Structural and Cellular Diversity of the Mammalian Respiratory System
of glycosylation of O-linked respiratory mucins demonstrated using biochemical methods (Rana et al., 1986).
5. IMMUNOHISTOCHEMISTRY Immunohistochemical methods can be used to define more precisely the variations in content within cells or granules. For example, the enzymes lysozyme (Bowes et al., 1981) and lactoferrin (Bowes and Corrin, 1977) have been localized within secretory cells of the respiratory tract. Lysozyme, which does not contain carbohydrates, has been localized within serous cells of submucosal glands, leading Spicer and colleagues to suggest that the carbohydratenegative portion of serous granules is lysozyme (Spicer et al., 1980). Immunohistochemical studies using monoclonal antibodies against respiratory secretory products have expanded the previously discussed heterogeneity (St George et al., 1984b, 1985; Basbaum et al., 1984). We have used monoclonal antibodies against rhesus or rabbit tracheal secretions to show several antigenically distinct subpopulations of mucous cells (St George et al., 1985). When serial sections of histochemically and immunohistochemically stained samples are compared, secretory cells are found that do not vary histochemically but vary immunohistochemically (St George et al., 1984b). Application of the monoclonal antibodies to four airway levels distal to the trachea in rhesus monkey indicate that the more distal the airway, the less the secretory content resembles that in trachea (St George et al., 1986b). This variation in content suggests variations in the biophysical properties of secretion at different airway levels. We have also used the panel of monoclonal antibodies to examine the appearance of secretory products in the airways of developing fetal monkey lungs. From that study, we have concluded that the antigens of adult trachea are present in neonatal trachea at birth but that the antigens appear sequentially rather than simultaneously. The results of these studies using monoclonal antibodies are probably detecting differences in the glycosylation of mucous glycoconjugates, because characterization of the epitopes of our antibodies (Lin et al., 1989) and those of others (Basbaum et al., 1986) have indicated a carbohydrate specificity. Antibodies to the protein portion of mucus have been used to localize and quantify mucins within normal human airways and compare the results to those from a patient with CF (Burgel et al., 2007). Although the secretory cells are thought to be the primary source of the mucous blanket, other cell types within the epithelial surface and glands may modify or supplement the fluid lining. Modification to the fluid lining includes the secretion of water and ions. These changes influence the properties of the mucous blanket and are therefore important to the function of mucociliary clearance. One other cell
type that can participate in this process is the ciliated cell. Several studies have demonstrated the presence of glycoprotein on the surface of ciliated cells (Spicer et al., 1971; Jeffery, 1977). We have observed vesicles or granules containing mucous antigens in ciliated cells (St George et al., 1984b), suggesting either endocytosis or exocytosis of components of the mucous blanket.
6. QUANTITATION With certain toxicological insults or respiratory diseases, profound changes occur in the volume of secretion produced and stored in the airways. It has therefore been important to develop accurate methods to quantify the volume of stored secretory products under normal as well as abnormal conditions. Previous methods have included determining the mean gland-to-wall ratio (Reid Index, Reid, 1960) or point counting to quantify submucosal glands (Bedrossian et al., 1971). We have used a computerized morphometric method to assess the volume of the secretory product per unit surface area and have demonstrated that there is considerable variation at different airway levels in the amount of glycoconjugate stored in airway epithelium of the rhesus monkey (Heidsiek et al., 1987; Plopper et al., 1988). The product of the tracheal surface was predominantly acidic, while that in submucosal glands was neutral. The trachea stored at least twice as much per unit of surface area, as compared to distal airways (generation 11). Additionally, a change in secretory product from sulfomucin to sialomucin was detected distally. One surprising feature of these studies was the contribution to the total secretory product by submucosal glands versus surface epithelium, as suggested by the amount of stored product in either surface epithelium or submucosal glands. Several investigators have indicated that the overwhelming majority of secretion is from submucosal glands (Reid, 1960). However, morphometric analysis of stored product in rhesus trachea demonstrated that the contribution by the submucosal glands was less than 50% of the total product (Heidsiek et al., 1987). These same quantitative methods were used to immunochemically compare the secretory cells from normal airways with those from an asthma patient (Ordonez et al., 2001).
7. CONCLUSIONS Several methods, including biochemical, histochemical, and immunohistochemical techniques, have been used to define the content of secretory cells of airway epithelium. Application of these methods has demonstrated the extensive heterogeneity in content when comparing secretory cells from the epithelial surface to those in submucosal glands, in the same cell type at the same airway level, and in secretory cells from different airway levels, as well as
Secretory Glycoconjugates of the Trachea and Bronchi Chapter | 5
from one species to another. Many tools are now in place to more fully understand the secretory response and the airway secretory products; however, the investigator should consider the variations that occur at different airway levels in the same species, as well as differences between species. The influence of secretion by different sites (surface vs glands and proximal vs distal) on the properties of the mucous blanket and the importance in differences in control in these regions remains to be elucidated. Importantly, extrapolation of results from one species to another should be done, bearing in mind that the aforementioned differences may dictate a very different secretory response to a given toxicological insult.
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SECTION | I Structural and Cellular Diversity of the Mammalian Respiratory System
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