Experimental Lung Research
ISSN: 0190-2148 (Print) 1521-0499 (Online) Journal homepage: http://www.tandfonline.com/loi/ielu20
Changes in the Carbohydrate Content of Airway Epithelium Induced by Human Neutrophil Elastase in the Hamster V. K. Dimitriadis, T. G. Christensen, E. C. Lucey, G. L. Snider & C. G. Plopper To cite this article: V. K. Dimitriadis, T. G. Christensen, E. C. Lucey, G. L. Snider & C. G. Plopper (1992) Changes in the Carbohydrate Content of Airway Epithelium Induced by Human Neutrophil Elastase in the Hamster, Experimental Lung Research, 18:5, 731-742, DOI: 10.3109/01902149209031704 To link to this article: http://dx.doi.org/10.3109/01902149209031704
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Changes in the Carbohydrate Content of Airway Epithelium Induced by Human Neutrophil Elastase in the Hamster
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V. K. Dimitriadis, T. G. Christensen, E. C. Lucey, G. L. Snider, and C. G. Plopper
ABSTRACT: Hamster airway epithelial secretory cells were investigated by light and electron microscopic cytochemistry to study possible changes in their carbohydrate content induced by human neutrophil elastase H ( ",) an agent known to cause replacement of Clara cells by mucous cells in hamster bronchi. Characterization of secretory cell carbohydrates by the AB/PAS, PA-TCH-SeHID-TCH-SI: and LID-TCH-SPsequences indicated the existence of periodate-reactive acidic glycoconjugates, but the absence of sulfated or carboxylated glycoconjugates in both treated and control animals. Dtffoences were seen in the quality and quantity of historeactive carbohydrates throughout various regions in the lower respiratory tract. This was especially evident in the HNE-treated animals. It is conclued that the HNE-induced expression of the mucoNs cell phenotype is associated with an increase in the amount of neutral and acidic nonsulfated and noncarboxylated polysaccharides stored in the secretory granules of these cells.
INTRODUCTION Secretory cell metaplasia, a condition observed in human chronic bronchitis and other pulmonary diseases, is induced by exposure to tobacco [l, 21, drugs [3], nitrogen dioxide [4],nitric, hydrochloric, or sulfuric acid [ 5 ] , and other agents. Light [6, 71 and electron microscopic observations of the airway epithelium [l, 81 often demonstrate a shift in the chemical composition of the secretory product from neutral to acidic. In the hamster, a single intratracheal instillation of human neutrophil elastase (HNE) induces the development of secretory cell metaplasia in the
From the Department of Anatomy, School of I.'eterinary Medicine, University of Cal$omia, Davis, und the Mallory Institute of pa tho log^ the Boston University School of Medicine, the Pulmonary Center, and the Boston Eterans Administration Medical Center, Boston, Massachusetts. Address correspondence to Dr. K K. Dimitriadis, DtQurtment of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Tbessaloniki, 54006, Greece.
Experimental Lung Research 18:731-742 (1992) Copyright 0 1992 by Hemisphere Publishing Corporation
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intrapulmonary bronchi [9]. After early discharge of secretory granules [lo], abundant, often abnormal mucous-type granules are produced in preexistent Clara cells within 3 weeks of enzyme treatment [11, 121. This conversion of Clara cells to a mucous cell phenotype does not occur in the trachea [13] or bronchioles [14], regions that appear to be resistant to the metaplastic effects of HNE. In the present study, we employed cytochemical methods to characterize the carbohydrate content in the airway epithelium of both untreated or HNE-treated hamsters. Specifically, the following question was addressed: Does the glycoconjugate content altered in the bronchi reflect the HNEinduced phenotypic change that occurs?
MATERIALS A N D METHODS Eighteen 12-week-old male Syrian hamsters (124-150 g) were used in this study. The animals were anesthetized with CO, and one group of six animals was injected intratracheally with 0.5 mL of physiological saline containing 300 pg human neutrophil elastase. Another group of six animals was injected with saline alone, and another group of six animals remained untreated. The enzyme was prepared as described previously [ 151. Thirty-two days after the enzyme instillation, the animals were killed by pentobarbital anesthesia followed by exsanguination. Following thoracotomy, the trachea and lungs were fixed by airway infusion via tracheal cannula at 30 cm of pressure with glutaraldehyde/paraformaldehyde fixative in 0.1 M cacodylate buffer (adjusted to 400 m O sm and pH 7.4) [16]. Cross sections from tracheal areas, usually taken over the cartilage (generation O), from the left primary bronchus (generation l), from the proximal three generations of bronchi in the axial pathway of the left lobe (generations 2-4), and from the distal bronchioles (generation 10 or greater) from six different hamsters were embedded in methacrylate [ 171. Additional samples were embedded for electron microscopy as described below, Serial methacrylate sections 1.7 pm thick were stained by the alcian blue/ periodic acid Schiff (AB/PAS) sequence [ 181. For electron microscopic cytochemistry, finely minced pieces of airways were incubated overnight in low iron diamine (LID) [19]or in high iron diamine (HID) [2O, 211, osmicated, and embedded in Araldite 502. Thin sections of these specimens were stained with the thiocarbohydrazide-silver proteinate (TCH-SP) sequence. Control tissue was exposed to 1 M MgC1, in place of LID or HID. Unosmicated specimens were used for the postembedding periodatethiocarbohydrazide-silver proteinate (PA-TCH-SP) method [22], Control sections were stained without the periodate treatment. Thin sections of buffer-treated osmicated tissues, post-stained with uranyl acetate and lead citrate, were used to characterize normal airway ultrastructure. Sections were cut with a diamond knife using a Sorvall MT 5000 ultramicrotome and
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were examined with a Zeiss EM 10 operating at 80 kV. Positive controls for the HID-TCH-SP and LID-TCH-SP sequences were positive reactions in granules of submucosal mast cells in hamster airways, as well as in mucous cells of rhesus monkey airway tissues processed at the same time. The proportion of different cell types within the airway epithelial population was determined by light microscopy using AB/PAS stained sections examined at a final magnification of 400 X . A length of 1 mm of basement membrane of each airway was demarked using an eyepiece reticle and then the nuclei of each of the cell type was counted. From the values obtained, the percentages of the various cell types were estimated. Mean values and standard deviations were calculated from six different hamsters and statistically compared using Student's t test (p < .05).
RESULTS Control Animals Light microscopic examination of intrapulmonary bronchial epithelium showed the existence of secretory cells with varying amounts of polysaccharides. The granules of some of the bronchial epithelial cells were stained weakly or were undetectable by AB/PAS. Some cells exhibited granules stained magenta or purple by AB/I'AS (Fig. l), indicating the presence of periodate-reactive neutral or acidic polysaccharides, respectively. Ultrastructural characterization of carbohydrate content of secretory granules by the PA-TCH-SP sequence amplified the light microscopic observations. There was a large variation in the PA-TCH-SP-reactive material of secretory cell granules (Fig. 2). Some of the granules of Clara cells lacked PA-TCH-SP reaction product (Fig. 2A), while others showed a fine layer of PA-TCH-SP positive material inside the limiting membrane (Fig. 2 4 . Occasionally, Clara cells appeared with granules reacted moderately or strongly with PA-TCH-SP (Fig. 2C). Mucous cells, on the other hand, reacted strongly for periodate-reactive polysaccharides (Fig. 20). The HID-TCH-SP technique failed to demonstrate positive reactivity either on the secretory granules or on the glycocalyx of the bronchial cells (not shown), despite a positive reaction in granules of mast cells in the same sections (Fig. 2 6 . The LID-TCH-SP sequence also yielded no reaction in secretory granules of both Clara and mucous cells (Fig. 2€). In contrast, the glycocalyx of all the secretory cells was stained with the LID-TCH-SP sequence (Figs. 2E,G). The glycocalyx of all the bronchial secretory cells also showed a positive PA-TCH-SP reaction. Control sections for all cytochemical stain techniques constantly lacked reaction product. In the trachea and primary extrapulmonary bronchus, light microscopic AB/PAB staining was similar to that observed in more distal bronchi. However, the number of cells that reacted positively to the AB/PAS sequence in
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Figure 1 Percentages of secrecory cells in airways of hamster: (A)cells with weakly stained o r unstained granules by the PAS/AB sequence; (B and C ) cells with granules stained magenta or purple by the PAS/AB sequence. Mean values and standard deviations were obtained from measurements on crachea (generation O), primary (main) bronchus (generation I), lobar bronchus of left lobe (generation 4, inrrapulmonary bronchi of left lobe (generations 3 and 4, and terminal bronchioles (generation 10 or greater). Asterisk indicates statistically significanc differences ('j < .05) between treated and control animals.
the trachea and main bronchus was less than that seen in the intrapulmonary bronchi (Fig. 1). With the PA-TCH-SP sequence, the electron microscopic appearance of secretory cells in these regions showed the same variability in the type and amount of carbohydrates in their secretory granules as was found in the intrapulmonary bronchi. In addition, the secretory
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granules did not react with HID-TCH-SP or LID-TCH-SP, whereas the giycocalyx was positive with the PA-TCH-SP and LID-TCH-SP staining. Bronchioles were not stained with the AB/PAS sequence (Fig. 1). By electron microscopy, none of the bronchiolar secretory granules was positive with PA-TCH-SP, LID-TCH-SP, or HID-TCH-SP. The glycocalyx reacted positively with PA-TCH-SP and LID-TCH-SP, but lacked reaction product with HID-TCH-SP
Figure 2 Bronchial cells. (A-c) Secretory granules of Clara cells indicate lack of reaction product (A. arrows), a weak peripheral positive reaction (E, arrows), or a peripheral or central positive reaction (C, arrows) with the PA-TCH-SP sequence. (A) x 12,500, (B) x 12,500, x 10,000; (0)Some of the
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secretory granules of a Clara cell (asterisks) show a positive PA-TCH-SP reaction very similar t o that observed on the secretory granules of a mucous cell (MC), x 16,000. (E) A Clara (CIC) and a mucous cell (MC) of a bronchus with lacking LID-TCH-SP reaction product. A positive reaction is noticed on the glycocalyx of the Clara cell (arrow), x 10,000. (9Mast cells show a positive HID-TCH-SP reaction in their granules. These positive controls were treated under the same conditions as the secretory epithelial cells, x 12,500. (G) Enlarged micrograph of LID-TCH-SP positive reaction on the glycocalyx of secretory and ciliated bronchial cells, x 20,000.
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Human Neutrophil Elastase Treated Animals As expected, human neutrophil elastase treatment induced intense alterations in the intrapulmonary bronchi, but failed to cause significant change in the trachea or terminal bronchioles. In the intrapulmonary bronchi, the number of nonciliated secretory cells with granules that reacted strongly with AB/PAS was greater in treated than in control animals (Fig. 1). This was especially true with respect to the cells with purple reaction product indicating the presence of acidic glycoconjugates. In addition, the percentage of secretory as well as of basal or ciliated cells was not altered in the HNEtreated hamsters compared to untreated or saline-treated hamsters (Fig. 3). The ultrastructural examination of the HNE-treated animals showed a different type of granules in the secretory cells (Fig. 4), which were originally described in HNE-treated bronchi [ 121. These granules were elongated and displayed a filamentous matrix (Fig. 4A) surrounded by a single membrane. In certain cells the filamentous material formed very large structures which were also covered by a single membrane. This occupied most of the cytoplasm in a small number of cells (Fig. 4B). The material inside the granule and the filamentous structures was in crystalline arrays with a periodicity of approximately 9 nm (Figs. 4C,D). This material lacked reaction product with PA-TCH-SP (Figs. 5A,B) or HID-TCH-SP and LID-TCH-SP sequences. In the HNE-treated hamsters, the PA-TCH-SP staining indicated a strong periodate reactivity in granules of mucous cells on the bronchi of treated animals (Fig. 5A). There was less reactivity in granules of Clara cells. As in controls, the secretory granules of treated hamsters lacked reaction product with HID-TCH-SP and LID-TCH-SP (Figs. SC,D).
DISCUSSION The carbohydrate cytochemistry of the normal hamster tracheobronchial surface epithelium shows some similarities to that of the rat [23]. The rat tracheobronchial airways contain a variable number of cells with periodatepositive secretory granules. This was believed to result from the existence of four distinct types of serous-Clara cells and three types of mucous cells [23]. In the present study, the hamster airways present periodate-unreactive or weakly reactive Clara and periodate-reactive mucous cells. Like the rat airways, many intermediate classes were present among the epithelial cells with weakly reactive or unreactive granules and among those secretory cells with strongly periodate-reative granules. In the hamster, the secretory granules of both the mucous and Clara cells were negative with both HID-TCHSP and LID-TCH-SP reactions, suggesting absence of sulfated and carboxylated glycoconjugates [19, 231. This was unexpected since the light microscopic AB/PAS staining clearly indicated the presence of acidic glyco-
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conjugates in many of the secretory cells as evidenced by the purple color. This discrepancy may be due to differences in the sensitivity of the methods or to procedural difficulties inherent in a pre-embedding cytochemical reaction that attempts to localize intracellular constituents. The positive LID-TCH-SP reaction in the glycocalyx of Clara cells of hamsters indicates the presence of cell surface-associated acidic glycoconjugates. This material probably arises from the synthesis and transportation of coat material to the apical plasma membrane by a route separate from that
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Figure 4 Elastase-treated animals. A secretory Clara cell (A) shows elongated granules (arrow) in its basal cytoplasm. Ciliated cell (CC), x 8000. (E) A bronchial Clara cell shows formations of filamentous material, which occupy large areas of its cytoplasm (asterisk). Secretory granule (SG), x 12,500. (C, 0) High magnifications of the filamentous material observed inside the elongated granules (c) o r inside filamentous structures (0).Both show a crystalline composition (arrows), (C) X50,000, (0)x 50,000.
of the secretory granule production as proposed by Kim et al. [24] from in vitro studies on hamster tracheal cells, A noteworthy effect of H N E treatment on the hamster airways was the appearance of the aberrant form of secretory granules. Some of these granules contained filamentous structures with a crystalline organization. The presence of crystals inside secretory granules has been also reported in secretory granules of other species [25-281. In contrast to the form of granules described in the literature, the elongated form observed in the present study is highly unusual, Abnormal secretory granules with long thin profiles were also observed in hamster bronchial epithelium, 16 days after H N E treatment [12]. However, in the present study many more of these granules appeared 32 days after H N E instillation. In some cases, they occupied most of the cytoplasmic area of certain secretory cells. The lack of staining of these granules by PA-TCH-SP, HID-TCH-SP, and LID-TCH-SP sequences suggests that they do not contain significant amounts of carbohydrates. Whether these granules are totally proteinaceous or contain masked carbohydrates is uncertain. However, our study has clearly demonstrated that their chemical composition significantly differs from that found in the secretory granules of hamster airway epithelium under steady-state conditions.
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The major effect of HNE treatment on the hamster airways was an increase in the number of mucous cells, which is consistent with earlier reports [9, 12, 131. AB/PAS staining indicated a significant increase in the number of bronchial epithelial cells stained purple, which we interpret to suggest a rise in the acidic character of the intracellular secretory product after HNE treatment. Secretory cell metaplasia is induced in mammalian airways by tobacco smoke [8, 291, chemicals such as sulfur, nitrogen dioxide, and chlorine [4, 30, 311, drugs [3], and other agents [32-341. In hamsters, secretory cell metaplasia has been produced by inhalation of sulfur dioxide [35], by intratracheal instillation of nitric, hydrochloric, or sulfuric acid [5], or pancreatic elastase [36]. A single intratracheal instillation of human neutrophil elastase caused secretory cell metaplasia in the large bronchi of the hamster 21 days after the enzyme treatment [9]. In addition, the changes attributed to elastase persisted throughout an 18-month study [37]. Furthermore, many Clara cells developed ultrastructural characteristics of mucous cells. They contained large numbers of normal and abnormal mucous granules 16 days after human neutrophil elastase instillation on hamster airways [ll]. The mechanism by which these factors modulate the proportion of the
Figure 5 Elastase-treated animals. (A) The granules of a secretory cell exhibit a strong PA-TCH-SP positive reaction, while the elongated granules (arrow) were not stained, x 8000. (B) Lack of PA-TCH-SP reaction product indicates the filamentous structures of a secretory cell (asterisk), x 6300. (C, D)Mucous cells lack HID-TCH-SP (C') o r LID-TCH-SP (0)reaction product. Ciliated cell (CC), nucleus (N), and secretory granules (SG), (q X 8000, (0)X 10,000.
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different cell types within the airway epithelial population is obscure. Some reports suggest an increase in mitotic activity 16, 29, 38, 391 and others suggest the conversion of Clara and/or serous cells to mucous cells [2, 8, 12, 40,411. In the present study, the percentage of airway secretory cells did not alter in the HNE-treated hamsters compared to controls (Fig. 3B). Therefore, the induced changes were probably related to rnetaplastic rather than hyperplastic phenomena of secretory cells. We conclude that the HNEinduced changes represent a replacement of Clara by mucous cells with a more acid carbohydrate content stored in their secretory granules than normally present. Why they failed to react with LID-TCH-SP sequence is obscure. The epithelial changes in animal models of secretory cell metaplasia often involve chemical changes in the secretory products of these cells. A change in the cellular secretion from neutral to acidic carbohydrates has been reported by light [6, 71 and electron microscopy [l, 81. In rats, the shift from neutral to acidic mucus occurs within 24 h of exposure to smoke [2]. The present study suggests that the lesion in hamster bronchi induced by HNE involves a change in the amount rather than the chemical type of polysaccharides. However, further work will be necessary to rule out HNE-induced change in chain length or branching structure of airway carbohydrates, which may be undetectable by the procedures employed in this study.
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ISSN: 0190-2148 (Print) 1521-0499 (Online) Journal homepage: http://www.tandfonline.com/loi/ielu20
Changes in the Carbohydrate Content of Airway Epithelium Induced by Human Neutrophil Elastase in the Hamster V. K. Dimitriadis, T. G. Christensen, E. C. Lucey, G. L. Snider & C. G. Plopper To cite this article: V. K. Dimitriadis, T. G. Christensen, E. C. Lucey, G. L. Snider & C. G. Plopper (1992) Changes in the Carbohydrate Content of Airway Epithelium Induced by Human Neutrophil Elastase in the Hamster, Experimental Lung Research, 18:5, 731-742, DOI: 10.3109/01902149209031704 To link to this article: http://dx.doi.org/10.3109/01902149209031704
Published online: 02 Jul 2009.
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Changes in the Carbohydrate Content of Airway Epithelium Induced by Human Neutrophil Elastase in the Hamster
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V. K. Dimitriadis, T. G. Christensen, E. C. Lucey, G. L. Snider, and C. G. Plopper
ABSTRACT: Hamster airway epithelial secretory cells were investigated by light and electron microscopic cytochemistry to study possible changes in their carbohydrate content induced by human neutrophil elastase H ( ",) an agent known to cause replacement of Clara cells by mucous cells in hamster bronchi. Characterization of secretory cell carbohydrates by the AB/PAS, PA-TCH-SeHID-TCH-SI: and LID-TCH-SPsequences indicated the existence of periodate-reactive acidic glycoconjugates, but the absence of sulfated or carboxylated glycoconjugates in both treated and control animals. Dtffoences were seen in the quality and quantity of historeactive carbohydrates throughout various regions in the lower respiratory tract. This was especially evident in the HNE-treated animals. It is conclued that the HNE-induced expression of the mucoNs cell phenotype is associated with an increase in the amount of neutral and acidic nonsulfated and noncarboxylated polysaccharides stored in the secretory granules of these cells.
INTRODUCTION Secretory cell metaplasia, a condition observed in human chronic bronchitis and other pulmonary diseases, is induced by exposure to tobacco [l, 21, drugs [3], nitrogen dioxide [4],nitric, hydrochloric, or sulfuric acid [ 5 ] , and other agents. Light [6, 71 and electron microscopic observations of the airway epithelium [l, 81 often demonstrate a shift in the chemical composition of the secretory product from neutral to acidic. In the hamster, a single intratracheal instillation of human neutrophil elastase (HNE) induces the development of secretory cell metaplasia in the
From the Department of Anatomy, School of I.'eterinary Medicine, University of Cal$omia, Davis, und the Mallory Institute of pa tho log^ the Boston University School of Medicine, the Pulmonary Center, and the Boston Eterans Administration Medical Center, Boston, Massachusetts. Address correspondence to Dr. K K. Dimitriadis, DtQurtment of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Tbessaloniki, 54006, Greece.
Experimental Lung Research 18:731-742 (1992) Copyright 0 1992 by Hemisphere Publishing Corporation
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intrapulmonary bronchi [9]. After early discharge of secretory granules [lo], abundant, often abnormal mucous-type granules are produced in preexistent Clara cells within 3 weeks of enzyme treatment [11, 121. This conversion of Clara cells to a mucous cell phenotype does not occur in the trachea [13] or bronchioles [14], regions that appear to be resistant to the metaplastic effects of HNE. In the present study, we employed cytochemical methods to characterize the carbohydrate content in the airway epithelium of both untreated or HNE-treated hamsters. Specifically, the following question was addressed: Does the glycoconjugate content altered in the bronchi reflect the HNEinduced phenotypic change that occurs?
MATERIALS A N D METHODS Eighteen 12-week-old male Syrian hamsters (124-150 g) were used in this study. The animals were anesthetized with CO, and one group of six animals was injected intratracheally with 0.5 mL of physiological saline containing 300 pg human neutrophil elastase. Another group of six animals was injected with saline alone, and another group of six animals remained untreated. The enzyme was prepared as described previously [ 151. Thirty-two days after the enzyme instillation, the animals were killed by pentobarbital anesthesia followed by exsanguination. Following thoracotomy, the trachea and lungs were fixed by airway infusion via tracheal cannula at 30 cm of pressure with glutaraldehyde/paraformaldehyde fixative in 0.1 M cacodylate buffer (adjusted to 400 m O sm and pH 7.4) [16]. Cross sections from tracheal areas, usually taken over the cartilage (generation O), from the left primary bronchus (generation l), from the proximal three generations of bronchi in the axial pathway of the left lobe (generations 2-4), and from the distal bronchioles (generation 10 or greater) from six different hamsters were embedded in methacrylate [ 171. Additional samples were embedded for electron microscopy as described below, Serial methacrylate sections 1.7 pm thick were stained by the alcian blue/ periodic acid Schiff (AB/PAS) sequence [ 181. For electron microscopic cytochemistry, finely minced pieces of airways were incubated overnight in low iron diamine (LID) [19]or in high iron diamine (HID) [2O, 211, osmicated, and embedded in Araldite 502. Thin sections of these specimens were stained with the thiocarbohydrazide-silver proteinate (TCH-SP) sequence. Control tissue was exposed to 1 M MgC1, in place of LID or HID. Unosmicated specimens were used for the postembedding periodatethiocarbohydrazide-silver proteinate (PA-TCH-SP) method [22], Control sections were stained without the periodate treatment. Thin sections of buffer-treated osmicated tissues, post-stained with uranyl acetate and lead citrate, were used to characterize normal airway ultrastructure. Sections were cut with a diamond knife using a Sorvall MT 5000 ultramicrotome and
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were examined with a Zeiss EM 10 operating at 80 kV. Positive controls for the HID-TCH-SP and LID-TCH-SP sequences were positive reactions in granules of submucosal mast cells in hamster airways, as well as in mucous cells of rhesus monkey airway tissues processed at the same time. The proportion of different cell types within the airway epithelial population was determined by light microscopy using AB/PAS stained sections examined at a final magnification of 400 X . A length of 1 mm of basement membrane of each airway was demarked using an eyepiece reticle and then the nuclei of each of the cell type was counted. From the values obtained, the percentages of the various cell types were estimated. Mean values and standard deviations were calculated from six different hamsters and statistically compared using Student's t test (p < .05).
RESULTS Control Animals Light microscopic examination of intrapulmonary bronchial epithelium showed the existence of secretory cells with varying amounts of polysaccharides. The granules of some of the bronchial epithelial cells were stained weakly or were undetectable by AB/PAS. Some cells exhibited granules stained magenta or purple by AB/I'AS (Fig. l), indicating the presence of periodate-reactive neutral or acidic polysaccharides, respectively. Ultrastructural characterization of carbohydrate content of secretory granules by the PA-TCH-SP sequence amplified the light microscopic observations. There was a large variation in the PA-TCH-SP-reactive material of secretory cell granules (Fig. 2). Some of the granules of Clara cells lacked PA-TCH-SP reaction product (Fig. 2A), while others showed a fine layer of PA-TCH-SP positive material inside the limiting membrane (Fig. 2 4 . Occasionally, Clara cells appeared with granules reacted moderately or strongly with PA-TCH-SP (Fig. 2C). Mucous cells, on the other hand, reacted strongly for periodate-reactive polysaccharides (Fig. 20). The HID-TCH-SP technique failed to demonstrate positive reactivity either on the secretory granules or on the glycocalyx of the bronchial cells (not shown), despite a positive reaction in granules of mast cells in the same sections (Fig. 2 6 . The LID-TCH-SP sequence also yielded no reaction in secretory granules of both Clara and mucous cells (Fig. 2€). In contrast, the glycocalyx of all the secretory cells was stained with the LID-TCH-SP sequence (Figs. 2E,G). The glycocalyx of all the bronchial secretory cells also showed a positive PA-TCH-SP reaction. Control sections for all cytochemical stain techniques constantly lacked reaction product. In the trachea and primary extrapulmonary bronchus, light microscopic AB/PAB staining was similar to that observed in more distal bronchi. However, the number of cells that reacted positively to the AB/PAS sequence in
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Figure 1 Percentages of secrecory cells in airways of hamster: (A)cells with weakly stained o r unstained granules by the PAS/AB sequence; (B and C ) cells with granules stained magenta or purple by the PAS/AB sequence. Mean values and standard deviations were obtained from measurements on crachea (generation O), primary (main) bronchus (generation I), lobar bronchus of left lobe (generation 4, inrrapulmonary bronchi of left lobe (generations 3 and 4, and terminal bronchioles (generation 10 or greater). Asterisk indicates statistically significanc differences ('j < .05) between treated and control animals.
the trachea and main bronchus was less than that seen in the intrapulmonary bronchi (Fig. 1). With the PA-TCH-SP sequence, the electron microscopic appearance of secretory cells in these regions showed the same variability in the type and amount of carbohydrates in their secretory granules as was found in the intrapulmonary bronchi. In addition, the secretory
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granules did not react with HID-TCH-SP or LID-TCH-SP, whereas the giycocalyx was positive with the PA-TCH-SP and LID-TCH-SP staining. Bronchioles were not stained with the AB/PAS sequence (Fig. 1). By electron microscopy, none of the bronchiolar secretory granules was positive with PA-TCH-SP, LID-TCH-SP, or HID-TCH-SP. The glycocalyx reacted positively with PA-TCH-SP and LID-TCH-SP, but lacked reaction product with HID-TCH-SP
Figure 2 Bronchial cells. (A-c) Secretory granules of Clara cells indicate lack of reaction product (A. arrows), a weak peripheral positive reaction (E, arrows), or a peripheral or central positive reaction (C, arrows) with the PA-TCH-SP sequence. (A) x 12,500, (B) x 12,500, x 10,000; (0)Some of the
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secretory granules of a Clara cell (asterisks) show a positive PA-TCH-SP reaction very similar t o that observed on the secretory granules of a mucous cell (MC), x 16,000. (E) A Clara (CIC) and a mucous cell (MC) of a bronchus with lacking LID-TCH-SP reaction product. A positive reaction is noticed on the glycocalyx of the Clara cell (arrow), x 10,000. (9Mast cells show a positive HID-TCH-SP reaction in their granules. These positive controls were treated under the same conditions as the secretory epithelial cells, x 12,500. (G) Enlarged micrograph of LID-TCH-SP positive reaction on the glycocalyx of secretory and ciliated bronchial cells, x 20,000.
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Human Neutrophil Elastase Treated Animals As expected, human neutrophil elastase treatment induced intense alterations in the intrapulmonary bronchi, but failed to cause significant change in the trachea or terminal bronchioles. In the intrapulmonary bronchi, the number of nonciliated secretory cells with granules that reacted strongly with AB/PAS was greater in treated than in control animals (Fig. 1). This was especially true with respect to the cells with purple reaction product indicating the presence of acidic glycoconjugates. In addition, the percentage of secretory as well as of basal or ciliated cells was not altered in the HNEtreated hamsters compared to untreated or saline-treated hamsters (Fig. 3). The ultrastructural examination of the HNE-treated animals showed a different type of granules in the secretory cells (Fig. 4), which were originally described in HNE-treated bronchi [ 121. These granules were elongated and displayed a filamentous matrix (Fig. 4A) surrounded by a single membrane. In certain cells the filamentous material formed very large structures which were also covered by a single membrane. This occupied most of the cytoplasm in a small number of cells (Fig. 4B). The material inside the granule and the filamentous structures was in crystalline arrays with a periodicity of approximately 9 nm (Figs. 4C,D). This material lacked reaction product with PA-TCH-SP (Figs. 5A,B) or HID-TCH-SP and LID-TCH-SP sequences. In the HNE-treated hamsters, the PA-TCH-SP staining indicated a strong periodate reactivity in granules of mucous cells on the bronchi of treated animals (Fig. 5A). There was less reactivity in granules of Clara cells. As in controls, the secretory granules of treated hamsters lacked reaction product with HID-TCH-SP and LID-TCH-SP (Figs. SC,D).
DISCUSSION The carbohydrate cytochemistry of the normal hamster tracheobronchial surface epithelium shows some similarities to that of the rat [23]. The rat tracheobronchial airways contain a variable number of cells with periodatepositive secretory granules. This was believed to result from the existence of four distinct types of serous-Clara cells and three types of mucous cells [23]. In the present study, the hamster airways present periodate-unreactive or weakly reactive Clara and periodate-reactive mucous cells. Like the rat airways, many intermediate classes were present among the epithelial cells with weakly reactive or unreactive granules and among those secretory cells with strongly periodate-reative granules. In the hamster, the secretory granules of both the mucous and Clara cells were negative with both HID-TCHSP and LID-TCH-SP reactions, suggesting absence of sulfated and carboxylated glycoconjugates [19, 231. This was unexpected since the light microscopic AB/PAS staining clearly indicated the presence of acidic glyco-
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conjugates in many of the secretory cells as evidenced by the purple color. This discrepancy may be due to differences in the sensitivity of the methods or to procedural difficulties inherent in a pre-embedding cytochemical reaction that attempts to localize intracellular constituents. The positive LID-TCH-SP reaction in the glycocalyx of Clara cells of hamsters indicates the presence of cell surface-associated acidic glycoconjugates. This material probably arises from the synthesis and transportation of coat material to the apical plasma membrane by a route separate from that
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Figure 4 Elastase-treated animals. A secretory Clara cell (A) shows elongated granules (arrow) in its basal cytoplasm. Ciliated cell (CC), x 8000. (E) A bronchial Clara cell shows formations of filamentous material, which occupy large areas of its cytoplasm (asterisk). Secretory granule (SG), x 12,500. (C, 0) High magnifications of the filamentous material observed inside the elongated granules (c) o r inside filamentous structures (0).Both show a crystalline composition (arrows), (C) X50,000, (0)x 50,000.
of the secretory granule production as proposed by Kim et al. [24] from in vitro studies on hamster tracheal cells, A noteworthy effect of H N E treatment on the hamster airways was the appearance of the aberrant form of secretory granules. Some of these granules contained filamentous structures with a crystalline organization. The presence of crystals inside secretory granules has been also reported in secretory granules of other species [25-281. In contrast to the form of granules described in the literature, the elongated form observed in the present study is highly unusual, Abnormal secretory granules with long thin profiles were also observed in hamster bronchial epithelium, 16 days after H N E treatment [12]. However, in the present study many more of these granules appeared 32 days after H N E instillation. In some cases, they occupied most of the cytoplasmic area of certain secretory cells. The lack of staining of these granules by PA-TCH-SP, HID-TCH-SP, and LID-TCH-SP sequences suggests that they do not contain significant amounts of carbohydrates. Whether these granules are totally proteinaceous or contain masked carbohydrates is uncertain. However, our study has clearly demonstrated that their chemical composition significantly differs from that found in the secretory granules of hamster airway epithelium under steady-state conditions.
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The major effect of HNE treatment on the hamster airways was an increase in the number of mucous cells, which is consistent with earlier reports [9, 12, 131. AB/PAS staining indicated a significant increase in the number of bronchial epithelial cells stained purple, which we interpret to suggest a rise in the acidic character of the intracellular secretory product after HNE treatment. Secretory cell metaplasia is induced in mammalian airways by tobacco smoke [8, 291, chemicals such as sulfur, nitrogen dioxide, and chlorine [4, 30, 311, drugs [3], and other agents [32-341. In hamsters, secretory cell metaplasia has been produced by inhalation of sulfur dioxide [35], by intratracheal instillation of nitric, hydrochloric, or sulfuric acid [5], or pancreatic elastase [36]. A single intratracheal instillation of human neutrophil elastase caused secretory cell metaplasia in the large bronchi of the hamster 21 days after the enzyme treatment [9]. In addition, the changes attributed to elastase persisted throughout an 18-month study [37]. Furthermore, many Clara cells developed ultrastructural characteristics of mucous cells. They contained large numbers of normal and abnormal mucous granules 16 days after human neutrophil elastase instillation on hamster airways [ll]. The mechanism by which these factors modulate the proportion of the
Figure 5 Elastase-treated animals. (A) The granules of a secretory cell exhibit a strong PA-TCH-SP positive reaction, while the elongated granules (arrow) were not stained, x 8000. (B) Lack of PA-TCH-SP reaction product indicates the filamentous structures of a secretory cell (asterisk), x 6300. (C, D)Mucous cells lack HID-TCH-SP (C') o r LID-TCH-SP (0)reaction product. Ciliated cell (CC), nucleus (N), and secretory granules (SG), (q X 8000, (0)X 10,000.
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different cell types within the airway epithelial population is obscure. Some reports suggest an increase in mitotic activity 16, 29, 38, 391 and others suggest the conversion of Clara and/or serous cells to mucous cells [2, 8, 12, 40,411. In the present study, the percentage of airway secretory cells did not alter in the HNE-treated hamsters compared to controls (Fig. 3B). Therefore, the induced changes were probably related to rnetaplastic rather than hyperplastic phenomena of secretory cells. We conclude that the HNEinduced changes represent a replacement of Clara by mucous cells with a more acid carbohydrate content stored in their secretory granules than normally present. Why they failed to react with LID-TCH-SP sequence is obscure. The epithelial changes in animal models of secretory cell metaplasia often involve chemical changes in the secretory products of these cells. A change in the cellular secretion from neutral to acidic carbohydrates has been reported by light [6, 71 and electron microscopy [l, 81. In rats, the shift from neutral to acidic mucus occurs within 24 h of exposure to smoke [2]. The present study suggests that the lesion in hamster bronchi induced by HNE involves a change in the amount rather than the chemical type of polysaccharides. However, further work will be necessary to rule out HNE-induced change in chain length or branching structure of airway carbohydrates, which may be undetectable by the procedures employed in this study.
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