Lectin Cytochemistry Reveals Differences between Hamster Trachea and Bronchus in the Composition of Epithelial Surface Glycoconjugates and in the Response of Secretory Cells to Neutrophil Elastase Thomas G. Christensen, Raphael Breuer, Edgar C. Lucey, Linda J. Hornstra, Phillip J. Stone, and Gordon L. Snider Departments of Pathology and Biochemistry, and Pulmonary Center, Boston University School of Medicine, Veterans Administration Medical Center, Boston, Massachusetts, and Hadassah University Hospital, Jerusalem, Israel
Hamsters exposed to an intratracheal instillation of human neutrophil elastase (HNE) accumulate an abnormally high number of secretory granules in bronchial but not tracheal epithelial cells. We employed lectin cytochemistry to investigate possible differences in the epithelial cell surface glycoconjugate layer in trachea compared to bronchus which might explain the regional dissimilarity in response to HNE. Portions of glutaraldehyde-fixed trachea and bronchi were incubated in one of several ferritin-labeled lectins prior to embedding for transmission electron microscopy. Lectins from Ricinus communis, Helix pomatia, and Triticum vulgaris bound to the surface of tracheal secretory cells in moderate to profuse amounts, while most bronchial secretory cells showed little or no label with these lectins. Gold-labeled Helix pomatia agglutinin (HPA), a lectin specific for secretory cells, showed a decrease in surface binding to all tracheal secretory cell types within 2 h of HNE instillation, compared to saline controls. In contrast, the majority of bronchial secretory cells showed an HNE-induced increase in surface label from extremely low levels in saline controls. The low levels of lectin binding to bronchial cells, in contrast to the trachea, may indicate the lack of a protective surface glycoconjugate coat, thus explaining the vulnerability of these cells to HNE. The rise in number of accessible HPA binding sites on the surface of bronchial secretory cells exposed to HNE may represent an important event in the pathologic accumulation of secretory granules by these cells.
The bronchial secretory cells of hamsters exposed to an intratracheal instillation of human neutrophil elastase (HNE) accumulate excessive numbers of secretory granules over a 3-wk period (1-4). This pathologic change is not seen in the trachea (5). The reason for this regional difference in response to HNE is unclear. Transmission electron microscopy (5, 6) has shown that similar types of secretory cells occur in trachea and bronchus but their relative proportions in these regions are different. We hypothesized that tracheal secretory cells might be invested with surface-associated glycoconjugates that differ Key ffiJrds: lectins, trachea, bronchus, neutrophil, elastase (Received in original form November 8, 1989 and in revised form February 13, 1990)
Addresscorrespondence to: T. G. Christensen, Mallory Institute of Pathology, 784 Massachusetts Avenue, Boston, MA 02118.
Abbreviations: Bandeiraeasimplicifolia agglutinin, BSA-II; Clara, C; gold particle, GP; human neutrophil elastase, HNE; Helix pomatia agglutinin, HPA; Lens culinaris agglutinin, LCA; mucous, M; nongranulated secretory cells, NGS cells; phosphate-buffered saline, PBS; Ricinus communis agglutinin, RCA-I; Triticum vulgaris agglutinin, WGA; Ulexeuropaeus agglutinin, UEA-I. Am. J. Respir. Cell Mol. BioI. Vol. 3. pp. 61-69, 1990
qualitatively or quantitatively from those in the bronchus. Such differences might yield insight into causes for the susceptibility or resistance of airway secretory cells to HNE. Accordingly, we screened a battery of ferritin-labeled lectins by electron microscopy to determine whether cell surface binding in the trachea differs from that in the bronchus. Using a secretory cell-specific lectin, we then probed for possible regional differences in surface binding caused by HNE instillation.
Materials and Methods Male Syrian hamsters weighing 110 to 130 g were used in these studies. In the first experiment, involving a battery of six lectins and their blocking.sugars (Table I), three hamsters per lectin were deeply anesthetized with pentobarbital and were perfused via the vasculature with 4% formalin-l % glutaraldehyde (4CF-IG) (7) in 0.1 M sodium cacodylate buffer. Portions of trachea and left lung segments containing first- and second-order bronchi exceeding 0.5 mm in diameter were further fixed overnight and washed thoroughly in buffer. To block free aldehyde groups, the samples were immersed for 30 min in 0.1 M ammonium chloride in 0.05 M cacodylate buffer. They were then rinsed in Dulbecco's
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990
phosphate-buffered saline (PBS) and incubated with a ferritin-conjugated lectin with or without its blocking sugar. Lectin-ferritin conjugates (Polysciences, Warrington, PA) were prepared by diluting stock solutions with PBS to yield a final concentration of 100 t-tg/rnl. The blocking sugars, dissolved in PBS, were added to duplicate incubation vials 5 min before the tissue samples to give a final concentration of 0.2 M. After a 60-min incubation with gentle agitation at room temperature, the samples were rinsed 5 times with PBS and postfixed for 1 h in 1% osmium tetroxide in 0.1 M cacodylate buffer. They were dehydrated through graded acetone solutions and embedded in Epon-Araldite. Thin sections of airway epithelium were examined unstained in a Philips 300 electron microscope operated at 80 kV. The degree of binding to individual ciliated and secretory cells was assessed by a subjective score as follows: none (0), trace (0.1), slight (1), moderate (2), and profuse (3). Figure 1 illustrates an example of profuse binding on the surface of a tracheal secretory cell. For each lectin, a median score was determined from the grouped values of three hamsters for secretory and for ciliated cells in trachea and bronchus. This score was based on a minimum of 15 ciliated and 15 secretory cells per block from one or more blocks per region per animal. In a second experiment, two groups of six animals each were anesthetized by CO 2 inhalation. Each animal in one group received an intratracheal instillation of 300 t-tg of HNE dissolved in 0.5 ml physiologic saline. The other group received saline alone as a control. The HNE was a highly purified preparation isolated from sputum, as previously described (1). Three animals from each group were killed 20 min and 2 h after instillation by pentobarbital overdose and exsanguination. The trachea and bronchi were fixed and prepared for incubation as described for the first experiment. The tissues were incubated with Helix pomatia agglutinin (HPA) conjugated to lO-nm colloidal gold particles (100 t-tg/ml; Polysciences) with or without its blocking sugar, N-acetylgalactosamine, at a concentration of 0.2 M. After incubation, the tissues were processed as before and thin sections, stained with uranyl acetate and lead citrate, were examined in the electron microscope. Areas of epithelium with overlying mucus were excluded. Secretory cells in trachea and bronchus with or without surface gold particles were chosen at random for analysis of HPA-gold binding. Secretory cells were subclassifiedinto Clara (C) cells (three types) , mucous (M) cells, and nongranulated secretory
Figure 1. Electron micrograph of tracheal secretory cell showing profuse binding with ferritin-labeled HPA (magnification: x36,300).
(NGS) cells based on previously definedcriteria (5, 6). Briefly, C cells, but not M cells, have obvious apical smooth endoplasmic reticulum. Rough endoplasmic reticulum is sparse or absent in Cl cells but is abundant in C2 cells. Cl and C2 cells have homogeneous, electron-dense granules, whereas C3 and M cells contain at least three mucous-like granules (electron-lucent or with a heterogeneous content) in the plane of section. NGS cells contain cytoplasmic features characteristic of granulated secretory cells such as abundant apical smooth endoplasmic reticulum or rough endoplasmic reticulum or both, but they lack secretory granules in the plane of section. To analyze surface HPA-gold label, micrographs were prepared at a final magnification of approximately x16,000. For each treatment group, at least 50 classifiable secretory cells were selected at random from the trachea and from the bronchus. For the treatment groups in this experiment, the number of secretory cells per region per animal ranged from 11 to 36. Under xlO magnification of the micrographs, and without knowledge of the treatment, gold particles on or above the cell surface (within 100 nm of the plasma membrane) were counted along three random portions of the cell TABLE 2
Relative binding of ferritin-conjugated lectins to the luminal surface of tracheobronchial epithelial cells* Tracheal Cells
TABLE 1
Lectins employed for ultrastructural analysis of surface binding to hamster tracheobronchial epithelium Abbreviation
Sugar Specificity
Bandeiraea simplicifolia* Helix pomatia Lens culinaris Ricinus communis Triticum vulgaris
BSA (II)t HPA LCA RCA (I)t WGA
Ulex europaeus
UEA (I)t
N-acetyl glucosamine N-acetyl galactosamine o-o-mannose, a-D-glucose n-galactose N-acetyl glucosamine, N-acetyl neuraminic acid L-fucose
Source of Lectin
* Also known as Griffonia simplicifolia. t Form of the lectin purchased from Polysciences.
Lectin"
Ciliated
Secretory
BSA-II HPA LCA RCA-I WGA UEA-I
0(45) 0(45) 0(49) 2§ (59) I (50) 1.5* (56)
0(93) 3§ (50) 0.1 (79) 2§ (55) 3§ (63) 1.5 (71)
Bronchial Cells Ciliated
o (45) o (45) 0(45) 0.5 (60) 1 (54) 0.5 (47)
Secretory
0(93) 0(94) o (51) 0.1 (60) I (52) I (68)
* Median valu~s of scores for surface binding based on a scale of 0 (none), 0.1 (trace), I (shght), 2 (moderate), or 3 (profuse); the number of cells analyzed is indicated in parentheses. t Abbreviations of lectins listed in Table I. § Significantly different (P < 0.(01) from its bronchial counterpart by MannWhitney rank sum test. Significantly different (P < 0.01) from bronchial ciliated cells by MannWhitney rank sum test.
*
Christensen, Breuer, Lucey et al.: Airway Epithelial Cell Surface Lectin Binding
63
w u
L2 n::
300
.....J .....J
250
:::J VJ
D _
20 MIN SAUNE 20 MIN HNE
W
Figure 2. Comparison of HPA-gold particle label on the luminal cell surface of trachea and bronchial secretory cells 20 min after intratracheal instillation of saline or HNE in saline. Abbreviations: CI, C2, and C3 = Clara I, 2, and 3 cell types, respectively; M = mucous cell; NGS = nongranulated secretory cell; * = significant difference (P < O.oS) compared to the matched saline control by Student's t test.
U ::::;;: :::J
*
200
Hamsters exposed to an intratracheal instillation of human neutrophil elastase (HNE) accumulate an abnormally high number of secretory granules in bronchial but not tracheal epithelial cells. We employed lectin cytochemistry to investigate possible differences in the epithelial cell surface glycoconjugate layer in trachea compared to bronchus which might explain the regional dissimilarity in response to HNE. Portions of glutaraldehyde-fixed trachea and bronchi were incubated in one of several ferritin-labeled lectins prior to embedding for transmission electron microscopy. Lectins from Ricinus communis, Helix pomatia, and Triticum vulgaris bound to the surface of tracheal secretory cells in moderate to profuse amounts, while most bronchial secretory cells showed little or no label with these lectins. Gold-labeled Helix pomatia agglutinin (HPA), a lectin specific for secretory cells, showed a decrease in surface binding to all tracheal secretory cell types within 2 h of HNE instillation, compared to saline controls. In contrast, the majority of bronchial secretory cells showed an HNE-induced increase in surface label from extremely low levels in saline controls. The low levels of lectin binding to bronchial cells, in contrast to the trachea, may indicate the lack of a protective surface glycoconjugate coat, thus explaining the vulnerability of these cells to HNE. The rise in number of accessible HPA binding sites on the surface of bronchial secretory cells exposed to HNE may represent an important event in the pathologic accumulation of secretory granules by these cells.
The bronchial secretory cells of hamsters exposed to an intratracheal instillation of human neutrophil elastase (HNE) accumulate excessive numbers of secretory granules over a 3-wk period (1-4). This pathologic change is not seen in the trachea (5). The reason for this regional difference in response to HNE is unclear. Transmission electron microscopy (5, 6) has shown that similar types of secretory cells occur in trachea and bronchus but their relative proportions in these regions are different. We hypothesized that tracheal secretory cells might be invested with surface-associated glycoconjugates that differ Key ffiJrds: lectins, trachea, bronchus, neutrophil, elastase (Received in original form November 8, 1989 and in revised form February 13, 1990)
Addresscorrespondence to: T. G. Christensen, Mallory Institute of Pathology, 784 Massachusetts Avenue, Boston, MA 02118.
Abbreviations: Bandeiraeasimplicifolia agglutinin, BSA-II; Clara, C; gold particle, GP; human neutrophil elastase, HNE; Helix pomatia agglutinin, HPA; Lens culinaris agglutinin, LCA; mucous, M; nongranulated secretory cells, NGS cells; phosphate-buffered saline, PBS; Ricinus communis agglutinin, RCA-I; Triticum vulgaris agglutinin, WGA; Ulexeuropaeus agglutinin, UEA-I. Am. J. Respir. Cell Mol. BioI. Vol. 3. pp. 61-69, 1990
qualitatively or quantitatively from those in the bronchus. Such differences might yield insight into causes for the susceptibility or resistance of airway secretory cells to HNE. Accordingly, we screened a battery of ferritin-labeled lectins by electron microscopy to determine whether cell surface binding in the trachea differs from that in the bronchus. Using a secretory cell-specific lectin, we then probed for possible regional differences in surface binding caused by HNE instillation.
Materials and Methods Male Syrian hamsters weighing 110 to 130 g were used in these studies. In the first experiment, involving a battery of six lectins and their blocking.sugars (Table I), three hamsters per lectin were deeply anesthetized with pentobarbital and were perfused via the vasculature with 4% formalin-l % glutaraldehyde (4CF-IG) (7) in 0.1 M sodium cacodylate buffer. Portions of trachea and left lung segments containing first- and second-order bronchi exceeding 0.5 mm in diameter were further fixed overnight and washed thoroughly in buffer. To block free aldehyde groups, the samples were immersed for 30 min in 0.1 M ammonium chloride in 0.05 M cacodylate buffer. They were then rinsed in Dulbecco's
62
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990
phosphate-buffered saline (PBS) and incubated with a ferritin-conjugated lectin with or without its blocking sugar. Lectin-ferritin conjugates (Polysciences, Warrington, PA) were prepared by diluting stock solutions with PBS to yield a final concentration of 100 t-tg/rnl. The blocking sugars, dissolved in PBS, were added to duplicate incubation vials 5 min before the tissue samples to give a final concentration of 0.2 M. After a 60-min incubation with gentle agitation at room temperature, the samples were rinsed 5 times with PBS and postfixed for 1 h in 1% osmium tetroxide in 0.1 M cacodylate buffer. They were dehydrated through graded acetone solutions and embedded in Epon-Araldite. Thin sections of airway epithelium were examined unstained in a Philips 300 electron microscope operated at 80 kV. The degree of binding to individual ciliated and secretory cells was assessed by a subjective score as follows: none (0), trace (0.1), slight (1), moderate (2), and profuse (3). Figure 1 illustrates an example of profuse binding on the surface of a tracheal secretory cell. For each lectin, a median score was determined from the grouped values of three hamsters for secretory and for ciliated cells in trachea and bronchus. This score was based on a minimum of 15 ciliated and 15 secretory cells per block from one or more blocks per region per animal. In a second experiment, two groups of six animals each were anesthetized by CO 2 inhalation. Each animal in one group received an intratracheal instillation of 300 t-tg of HNE dissolved in 0.5 ml physiologic saline. The other group received saline alone as a control. The HNE was a highly purified preparation isolated from sputum, as previously described (1). Three animals from each group were killed 20 min and 2 h after instillation by pentobarbital overdose and exsanguination. The trachea and bronchi were fixed and prepared for incubation as described for the first experiment. The tissues were incubated with Helix pomatia agglutinin (HPA) conjugated to lO-nm colloidal gold particles (100 t-tg/ml; Polysciences) with or without its blocking sugar, N-acetylgalactosamine, at a concentration of 0.2 M. After incubation, the tissues were processed as before and thin sections, stained with uranyl acetate and lead citrate, were examined in the electron microscope. Areas of epithelium with overlying mucus were excluded. Secretory cells in trachea and bronchus with or without surface gold particles were chosen at random for analysis of HPA-gold binding. Secretory cells were subclassifiedinto Clara (C) cells (three types) , mucous (M) cells, and nongranulated secretory
Figure 1. Electron micrograph of tracheal secretory cell showing profuse binding with ferritin-labeled HPA (magnification: x36,300).
(NGS) cells based on previously definedcriteria (5, 6). Briefly, C cells, but not M cells, have obvious apical smooth endoplasmic reticulum. Rough endoplasmic reticulum is sparse or absent in Cl cells but is abundant in C2 cells. Cl and C2 cells have homogeneous, electron-dense granules, whereas C3 and M cells contain at least three mucous-like granules (electron-lucent or with a heterogeneous content) in the plane of section. NGS cells contain cytoplasmic features characteristic of granulated secretory cells such as abundant apical smooth endoplasmic reticulum or rough endoplasmic reticulum or both, but they lack secretory granules in the plane of section. To analyze surface HPA-gold label, micrographs were prepared at a final magnification of approximately x16,000. For each treatment group, at least 50 classifiable secretory cells were selected at random from the trachea and from the bronchus. For the treatment groups in this experiment, the number of secretory cells per region per animal ranged from 11 to 36. Under xlO magnification of the micrographs, and without knowledge of the treatment, gold particles on or above the cell surface (within 100 nm of the plasma membrane) were counted along three random portions of the cell TABLE 2
Relative binding of ferritin-conjugated lectins to the luminal surface of tracheobronchial epithelial cells* Tracheal Cells
TABLE 1
Lectins employed for ultrastructural analysis of surface binding to hamster tracheobronchial epithelium Abbreviation
Sugar Specificity
Bandeiraea simplicifolia* Helix pomatia Lens culinaris Ricinus communis Triticum vulgaris
BSA (II)t HPA LCA RCA (I)t WGA
Ulex europaeus
UEA (I)t
N-acetyl glucosamine N-acetyl galactosamine o-o-mannose, a-D-glucose n-galactose N-acetyl glucosamine, N-acetyl neuraminic acid L-fucose
Source of Lectin
* Also known as Griffonia simplicifolia. t Form of the lectin purchased from Polysciences.
Lectin"
Ciliated
Secretory
BSA-II HPA LCA RCA-I WGA UEA-I
0(45) 0(45) 0(49) 2§ (59) I (50) 1.5* (56)
0(93) 3§ (50) 0.1 (79) 2§ (55) 3§ (63) 1.5 (71)
Bronchial Cells Ciliated
o (45) o (45) 0(45) 0.5 (60) 1 (54) 0.5 (47)
Secretory
0(93) 0(94) o (51) 0.1 (60) I (52) I (68)
* Median valu~s of scores for surface binding based on a scale of 0 (none), 0.1 (trace), I (shght), 2 (moderate), or 3 (profuse); the number of cells analyzed is indicated in parentheses. t Abbreviations of lectins listed in Table I. § Significantly different (P < 0.(01) from its bronchial counterpart by MannWhitney rank sum test. Significantly different (P < 0.01) from bronchial ciliated cells by MannWhitney rank sum test.
*
Christensen, Breuer, Lucey et al.: Airway Epithelial Cell Surface Lectin Binding
63
w u
L2 n::
300
.....J .....J
250
:::J VJ
D _
20 MIN SAUNE 20 MIN HNE
W
Figure 2. Comparison of HPA-gold particle label on the luminal cell surface of trachea and bronchial secretory cells 20 min after intratracheal instillation of saline or HNE in saline. Abbreviations: CI, C2, and C3 = Clara I, 2, and 3 cell types, respectively; M = mucous cell; NGS = nongranulated secretory cell; * = significant difference (P < O.oS) compared to the matched saline control by Student's t test.
U ::::;;: :::J
*
200
