Neutrophil elastase stimulates tracheal submucosal secretion that is inhibited by ICI 200,355
gland
ANTJE SCHUSTER, IRIS UEKI, AND JAY A. NADEL Cardiovascular Research Institute, and Departments of Medicine and Physiology, University of California, San Francisco, California 94143 Antje Schuster, Iris Ueki, and Jay A. Nadel. Neutrophil elastase stimulates tracheal submucosal gland secretion that is inhibited by ICI 200,355. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L86-L91, 1992.- To investigate whether human neutrophil elastase (HNE) stimulates airway submucosal gland secretion, we studied the effect of purified HNE on secretion of 35S-labeled macromolecules from isolated tracheal tissues from ferrets, dogs, and humans. HNE stimulated secretion in a concentration-dependent fashion, the secretory response being most pronounced in ferret tissues with a maximal response of 1,498 t 384% above baseline at 10V5 M. In dog tissues, maximal secretory responses (509 t 169%) to HNE were much greater than to bethanechol (80 t 26%). Human tissues obtained several hours postmortem still responded to HNE (179 t 48 % at 10s5 M) significantly more than to the combination of isoproterenol, phenylephrine, and bethanechol (23 t 10%). Morphometric analysis of canine tracheal tissues showed degranulation of submucosal gland cells after HNE. A specific inhibitor of HNE (ICI 200,355) potently inhibited secretory responses in a concentration-dependent fashion. We suggest that HNE in airways of patients may cause hypersecretion and that treatment with ICI 200,355 may provide a strategy for therapeutic intervention.
cell lines have shown that human neutrophil elastase is a very potent secretagogue; in fact, it is the most potent stimulus yet described (27). These cultured serous cells are useful in studying the secretory process (25). However, this permanent bovine cell line might not reflect adequately responses of intact tissues or of other species including humans. We designed the present study to determine whether human neutrophil elastase stimulates secretion of macromolecules in intact tissue from different species, including humans. In addition, we performed a morphometric analysis of submucosal gland cells to determine whether human neutrophil elastase causes degranulation. Our results indicate that human neutrophil elastase causes stimulation of macromolecule secretion in tracheal tissues from ferrets, dogs, and humans, that the effect is much greater than responses to other secretagogues, that this secretagogue effect is associated with degranulation of the airway submucosal glands, and that the responses are inhibited by a selective human neutrophi1 elastase inhibitor.
mucus; serine protease; gland cell degranulation
MATERIALS
elastase inhibitor;
human;
ferret; dog;
are found, often in large numbers, in the airways of patients with hypersecretory states, but the role of neutrophils in the pathogenesis of these conditions is unknown. Thus the number of neutrophils is greatly increased in the purulent respiratory tract secretions in chronic bronchitis, cystic fibrosis, and after exposure to environmental irritants (10, 11, 20-23, 32). Neutrophils are a source of the lysosomal enzyme elastase, which is released during phagocytosis and on cell death (16, 35). In healthy subjects, levels of catalytically active elastase in airway secretions are negligible, but in patients with cystic fibrosis, elastase levels in sputum may exceed 100 pg/ml (4.0 x lOa M) (8, 29). Increased neutrophil elastase activity has also been demonstrated in the airways of cigarette smokers and patients with bronchitis (12, 17, 28, 31, 36). In humans, airway secretions are believed to originate predominantly from airway submucosal gland secretory cells whose volume is 40 times that of goblet cells in normal airways (19); in cystic fibrosis and chronic bronchitis with gland hypertrophy and hyperplasia (19, 24), submucosal gland secretory cells are even more conspicuous. Thus secretagogues of gland cells are likely to play important roles in the hypersecretion present in these disease states. Known secretagogues (e.g., acetylcholine, arachidonate metabolites, etc.) are not very potent in the various airway test systems studied. However, recent studies in permanent bovine airway submucosal gland NEUTROPHILS
L86
1040-0605/92 $1.50 Copyright
AND
METHODS
MateriaZs. Ham’s F-12 medium was obtained from the Cell Culture Facility of the University of California, San Francisco (UCSF). Carrier-free Na235S04 (sp act 43 Ci/mg) was purchased from ICN Radiochemicals (Irvine, CA). Human neutrophil elastase isolated from human purulent sputum was purchased from Elastin Products (Pacific, MO); the lyophilized powder was diluted in a buffer containing 0.05 M sodium acetate plus 0.1 M NaCl plus 0.02% NaN3, pH 5.0. Bethanechol (carbamyl,&methyl-choline chloride), isoproterenol, and phenylephrine were purchased from Sigma (St. Louis, MO). Platelet-activating factor (PAF), isolated from egg yolk, was purchased from Calbiochem (La Jolla, CA). The selective inhibitor of human neutrophil elastase 4-(4bromophenylsulfonylcarbamoyl) benzoyl-L-valyl-L-proline 1 (RS)-(1-trifluroacetyl-2-methylpropyl)amide (ICI 200,355) was generously provided by the Department of Medical Chemistry, ICI Americas, Wilmington, DE. Preparation of tissue. The experimental animal protocol was approved by the Committee on Animal Research of UCSF. Seven adult ferrets were killed by intraperitoneal injection of pentobarbital sodium (150 mg/kg body wt) and bilateral thoracotomy. The trachea was removed and opened longitudinally. To study secretion that is derived exclusively from submucosal glands, the epithelium was removed mechanically. Square tissue pieces consisting of four tracheal rings were obtained and transferred into a beaker of Ham’s F-12 medium, placed in a Dubnoff shaker at 37OC, and provided with 95% 02-5% CO,. Thirteen mongrel dog tracheas were obtained from the Tissue Sharing Program of UCSF. The trachea was incised longitudinally along the posterior membrane, and the epithelium was removed mechanically. The intercartilaginous areas containing the submucosal glands were dissected out and trans-
0 1992 th:
American
Physiological
Society
Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 15, 2019.
INHIBITION
OF
TRACHEAL
ferred into a beaker of Ham’s F-12 medium, placed in a Dubnoff shaker at 37°C and provided with 95% 02-5% CO,. Human tracheal tissue from informed donors was obtained 5-36 h postmortem from the Department of Pathology at UCSF. The tracheas were treated identically to the dog tracheas. Human tissues were only included in the study if they showed a secretory response to a combination of known secretagogues (isoproterenol, phenylephrine, and bethanechol, 10s5 M each). Release of 35S-labeled macromolecules. Tissues were incubated for 2 h in Ham’s F-12 medium containing 20 ,&i/ml Naz3’S04. The medium containing the radiolabel was removed, and the tissues were washed 10 times with phosphate-buffered saline. The protocol was as follows: the tissue pieces were incubated in vials containing 2 ml of fresh drug-free medium. After 120 min of rest, the medium was collected and replaced every 30 min. Baseline samples were collected at 150, 180, and 210 min. Between 210 and 240 min, the tissue pieces were incubated either in fresh medium alone (control), in medium containing the vehicle of elastase (sham experiment), or in medium containing the secretagogues at different concentrations. In the experiments with the human neutrophil elastase inhibitor ICI 200,355, elastase-containing medium was preincubated with the inhibitor for 10 min before addition to the tissues. At 240 min, the medium was collected again. The harvested samples were dialyzed exhaustively (SpectraPor tubing, molecular mass cutoff 12,000-14,000 Da) against distilled water containing 10 mg/l sodium azide to remove unincorporated radiolabeled sulfate. After addition of scintillation fluid (ReadySafe, Beckman Instruments, Fullerton, CA), nondialyzable 35S-labeled macromolecules were counted by scintillation spectroscopy (accuracy, 2%; beta counter model LS7500, Beckman Instruments). Secretion is expressed as percent increase of release of 35Slabeled macromolecules during incubation with the agonists over the release during the immediately preceding time period and is corrected for the declining baseline. Morphometric studies. In parallel to the radiolabeled tissues, unlabeled tracheal tissues from five dogs were incubated either in fresh medium alone or in the presence of human neutrophil elastase (lo-” M) for 30 min. Then, the tissues were fixed in 5% glutaraldehyde-3% paraformaldehyde, embedded in glycomethacrylate, cut into sections of 2.5~pm thickness, placed on glass slides, and stained with p-aminosalicylic acid (PAS)alcian blue. The volume density of secretory granules in submucosal gland cells (V,), defined by standard stereologic principles (34) as the ratio of the total area of all the granule profiles in the cells to the area of the cell profiles, was assessed by a point-counting procedure with an IBM computer and graphics tablet interfaced with a Sony TV camera to a Zeiss microscope (magnification, x 3,725; 2,000 points per slide with a grid of 51 points per screen to achieve a standard error of mean V, of
gland
ANTJE SCHUSTER, IRIS UEKI, AND JAY A. NADEL Cardiovascular Research Institute, and Departments of Medicine and Physiology, University of California, San Francisco, California 94143 Antje Schuster, Iris Ueki, and Jay A. Nadel. Neutrophil elastase stimulates tracheal submucosal gland secretion that is inhibited by ICI 200,355. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L86-L91, 1992.- To investigate whether human neutrophil elastase (HNE) stimulates airway submucosal gland secretion, we studied the effect of purified HNE on secretion of 35S-labeled macromolecules from isolated tracheal tissues from ferrets, dogs, and humans. HNE stimulated secretion in a concentration-dependent fashion, the secretory response being most pronounced in ferret tissues with a maximal response of 1,498 t 384% above baseline at 10V5 M. In dog tissues, maximal secretory responses (509 t 169%) to HNE were much greater than to bethanechol (80 t 26%). Human tissues obtained several hours postmortem still responded to HNE (179 t 48 % at 10s5 M) significantly more than to the combination of isoproterenol, phenylephrine, and bethanechol (23 t 10%). Morphometric analysis of canine tracheal tissues showed degranulation of submucosal gland cells after HNE. A specific inhibitor of HNE (ICI 200,355) potently inhibited secretory responses in a concentration-dependent fashion. We suggest that HNE in airways of patients may cause hypersecretion and that treatment with ICI 200,355 may provide a strategy for therapeutic intervention.
cell lines have shown that human neutrophil elastase is a very potent secretagogue; in fact, it is the most potent stimulus yet described (27). These cultured serous cells are useful in studying the secretory process (25). However, this permanent bovine cell line might not reflect adequately responses of intact tissues or of other species including humans. We designed the present study to determine whether human neutrophil elastase stimulates secretion of macromolecules in intact tissue from different species, including humans. In addition, we performed a morphometric analysis of submucosal gland cells to determine whether human neutrophil elastase causes degranulation. Our results indicate that human neutrophil elastase causes stimulation of macromolecule secretion in tracheal tissues from ferrets, dogs, and humans, that the effect is much greater than responses to other secretagogues, that this secretagogue effect is associated with degranulation of the airway submucosal glands, and that the responses are inhibited by a selective human neutrophi1 elastase inhibitor.
mucus; serine protease; gland cell degranulation
MATERIALS
elastase inhibitor;
human;
ferret; dog;
are found, often in large numbers, in the airways of patients with hypersecretory states, but the role of neutrophils in the pathogenesis of these conditions is unknown. Thus the number of neutrophils is greatly increased in the purulent respiratory tract secretions in chronic bronchitis, cystic fibrosis, and after exposure to environmental irritants (10, 11, 20-23, 32). Neutrophils are a source of the lysosomal enzyme elastase, which is released during phagocytosis and on cell death (16, 35). In healthy subjects, levels of catalytically active elastase in airway secretions are negligible, but in patients with cystic fibrosis, elastase levels in sputum may exceed 100 pg/ml (4.0 x lOa M) (8, 29). Increased neutrophil elastase activity has also been demonstrated in the airways of cigarette smokers and patients with bronchitis (12, 17, 28, 31, 36). In humans, airway secretions are believed to originate predominantly from airway submucosal gland secretory cells whose volume is 40 times that of goblet cells in normal airways (19); in cystic fibrosis and chronic bronchitis with gland hypertrophy and hyperplasia (19, 24), submucosal gland secretory cells are even more conspicuous. Thus secretagogues of gland cells are likely to play important roles in the hypersecretion present in these disease states. Known secretagogues (e.g., acetylcholine, arachidonate metabolites, etc.) are not very potent in the various airway test systems studied. However, recent studies in permanent bovine airway submucosal gland NEUTROPHILS
L86
1040-0605/92 $1.50 Copyright
AND
METHODS
MateriaZs. Ham’s F-12 medium was obtained from the Cell Culture Facility of the University of California, San Francisco (UCSF). Carrier-free Na235S04 (sp act 43 Ci/mg) was purchased from ICN Radiochemicals (Irvine, CA). Human neutrophil elastase isolated from human purulent sputum was purchased from Elastin Products (Pacific, MO); the lyophilized powder was diluted in a buffer containing 0.05 M sodium acetate plus 0.1 M NaCl plus 0.02% NaN3, pH 5.0. Bethanechol (carbamyl,&methyl-choline chloride), isoproterenol, and phenylephrine were purchased from Sigma (St. Louis, MO). Platelet-activating factor (PAF), isolated from egg yolk, was purchased from Calbiochem (La Jolla, CA). The selective inhibitor of human neutrophil elastase 4-(4bromophenylsulfonylcarbamoyl) benzoyl-L-valyl-L-proline 1 (RS)-(1-trifluroacetyl-2-methylpropyl)amide (ICI 200,355) was generously provided by the Department of Medical Chemistry, ICI Americas, Wilmington, DE. Preparation of tissue. The experimental animal protocol was approved by the Committee on Animal Research of UCSF. Seven adult ferrets were killed by intraperitoneal injection of pentobarbital sodium (150 mg/kg body wt) and bilateral thoracotomy. The trachea was removed and opened longitudinally. To study secretion that is derived exclusively from submucosal glands, the epithelium was removed mechanically. Square tissue pieces consisting of four tracheal rings were obtained and transferred into a beaker of Ham’s F-12 medium, placed in a Dubnoff shaker at 37OC, and provided with 95% 02-5% CO,. Thirteen mongrel dog tracheas were obtained from the Tissue Sharing Program of UCSF. The trachea was incised longitudinally along the posterior membrane, and the epithelium was removed mechanically. The intercartilaginous areas containing the submucosal glands were dissected out and trans-
0 1992 th:
American
Physiological
Society
Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 15, 2019.
INHIBITION
OF
TRACHEAL
ferred into a beaker of Ham’s F-12 medium, placed in a Dubnoff shaker at 37°C and provided with 95% 02-5% CO,. Human tracheal tissue from informed donors was obtained 5-36 h postmortem from the Department of Pathology at UCSF. The tracheas were treated identically to the dog tracheas. Human tissues were only included in the study if they showed a secretory response to a combination of known secretagogues (isoproterenol, phenylephrine, and bethanechol, 10s5 M each). Release of 35S-labeled macromolecules. Tissues were incubated for 2 h in Ham’s F-12 medium containing 20 ,&i/ml Naz3’S04. The medium containing the radiolabel was removed, and the tissues were washed 10 times with phosphate-buffered saline. The protocol was as follows: the tissue pieces were incubated in vials containing 2 ml of fresh drug-free medium. After 120 min of rest, the medium was collected and replaced every 30 min. Baseline samples were collected at 150, 180, and 210 min. Between 210 and 240 min, the tissue pieces were incubated either in fresh medium alone (control), in medium containing the vehicle of elastase (sham experiment), or in medium containing the secretagogues at different concentrations. In the experiments with the human neutrophil elastase inhibitor ICI 200,355, elastase-containing medium was preincubated with the inhibitor for 10 min before addition to the tissues. At 240 min, the medium was collected again. The harvested samples were dialyzed exhaustively (SpectraPor tubing, molecular mass cutoff 12,000-14,000 Da) against distilled water containing 10 mg/l sodium azide to remove unincorporated radiolabeled sulfate. After addition of scintillation fluid (ReadySafe, Beckman Instruments, Fullerton, CA), nondialyzable 35S-labeled macromolecules were counted by scintillation spectroscopy (accuracy, 2%; beta counter model LS7500, Beckman Instruments). Secretion is expressed as percent increase of release of 35Slabeled macromolecules during incubation with the agonists over the release during the immediately preceding time period and is corrected for the declining baseline. Morphometric studies. In parallel to the radiolabeled tissues, unlabeled tracheal tissues from five dogs were incubated either in fresh medium alone or in the presence of human neutrophil elastase (lo-” M) for 30 min. Then, the tissues were fixed in 5% glutaraldehyde-3% paraformaldehyde, embedded in glycomethacrylate, cut into sections of 2.5~pm thickness, placed on glass slides, and stained with p-aminosalicylic acid (PAS)alcian blue. The volume density of secretory granules in submucosal gland cells (V,), defined by standard stereologic principles (34) as the ratio of the total area of all the granule profiles in the cells to the area of the cell profiles, was assessed by a point-counting procedure with an IBM computer and graphics tablet interfaced with a Sony TV camera to a Zeiss microscope (magnification, x 3,725; 2,000 points per slide with a grid of 51 points per screen to achieve a standard error of mean V, of
