Role of neutrophil elastase in allergen-induced lysozyme secretion in the dog trachea ELVAN

TABACHNIK,

ANTJE

SCHUSTER,

WARREN

M. GOLD,

AND

JAY A. NADEL

Cardiovascular Research Institute and Departments of Medicine and Physiology, University of California, San Francisco, California 94143-0130 TABACHNIK,ELVAN, ANTJE SCHUSTER,~ARRENM. GOLD, AND JAY A. NADEL. Role of neutrophil elastase in allergen-induced lysozyme secretion in the dog trachea. J. Appl. Physiol. 73(Z): 695-700, 1992.-To test our hypothesis that neutrophil elastaseplays a role in airway hypersecretion associatedwith the allergic late-phaseresponse,using an isolated tracheal segment system in vivo and measuring lysozyme activity in the perfusate of the lumen as a marker of submucosalgland secretion over 8 h, we studied the responseof five allergic dogs to ragweed.The dogswere exposedon separate occasionsto specific allergen, to allergen vehicle, and to allergen in the presence of a selective neutrophil elastaseinhibitor, ICI 200,355. Allergen exposure causeda marked increasein lysozyme secretion that was significantly increasedat 4,6, and 8 h compared with controls and ICI 200,355-treateddogs.Neutrophil elastase appearedin the perfusate after allergen exposure and waspositively correlated with lysozyme secretion at 8 h. These findings suggestthat neutrophil elastaseplays an important role as a secretagoguein the allergic late-phase response.

allergen in the presence of a selective neutrophil elastase inhibitor, ICI 200,355 (26). Our results indicate that allergen causes marked stimulation of lysozyme secretion into the airway segment, an effect that is associated with the appearance of neutrophil elastase activity in the luminal fluid. Lysozyme secretion after allergen is markedly inhibited when the airway is pretreated with the selective inhibitor of neutrophil elastase, ICI 200,355. METHODS

Ethical guidelines. The experimental protocol followed the published “Guiding Principles in the Care and Use of Animals” of the Council of the American Physiological Society and the NIH “Guide for the Care and Use of Laboratory Animals” and was approved by the Committee on Animal Research of the University of California, San Francisco. Preparation of animals. Five dogs (12-24 kg) were sesubmucosal gland; airway hypersecretion; ragweed; allergic lected from a colony of inbred ragweed-sensitized dogs late-phase response (6). These animals have previously been shown to possess some of the characteristics of human allergic asthma, including an early bronchoconstrictor response AIRWAY HYPERSECRETION is a Serious Symptom in allerafter the inhalation of ragweed aerosol and an increased gic airway disease. Thus, at least 80% of asthmatic pa- airway responsiveness to acetylcholine 6 h after allergen tients complain of increased secretions (32), and 10% challenge associated with an influx of inflammatory asthmatic patients undergoing an acute attack have cells, mainly neutrophils (3). bronchorrhea (> 100 ml sputum/day) (24). The dogs were anesthetized with pentobarbital sodium In bronchial asthma, allergen challenge induces an in(30 mg/kg iv), supplemented with doses of 65 mg/ml iv as flammatory response of the airways (5). The late-phase needed. The dogs were placed on a warming pad, and asthmatic response that usually develops 3-6 h after the their body temperature was maintained at 37OC during challenge is characterized by an infiltration of the airthe entire experiment. Heart rates of the dogs were monways with inflammatory cells including neutrophils (3, itored continuously during experiments and found to re4). Neutrophils are the source of the lysosomal enzyme main within the normal range after addition of allergen neutrophil elastase, which is one of the most potent se- to the perfusate. Intravenous infusions of 0.9% NaCl cretagogues ever described for airway submucosal gland (-75 ml/h) were given to prevent dehydration. Immediserous cells in permanent culture (27) as well as for sub- ately after induction of anesthesia, a- modified doublemucosal glands in tracheal tissue explants from various balloon endotracheal tube (see below) was inserted per OS species including humans (22). Therefore we hypothesize into the dog trachea and connected to a constant-volume that neutrophil infiltration of the airway wall and the ventilator (model 607A, Harvard Apparatus, Dover, MA) subsequent release of free elastase in the proximity of the set to deliver a tidal volume of 10 ml/kg at a frequency of submucosal glands may be a significant cause of airway 20 breathsimin. secretion after allergic stimulation of the airways. Isolated tracheal segment. A segment of the trachea was In the present study, using an isolated tracheal seg- isolated and perfused as described previously (10). ment system in vivo and measuring lysozyme activity as Briefly, the system allows for the continuous perfusion of an indicator of submucosal gland serous cell secretion the tracheal lumen in situ with physiological solutions (31), we have tested this hypothesis in dogs allergic to that contain test substances and allows for periodic samragweed. On separate occasions, the dogs were exposed. pling of the perfusate for analysis of tracheal secretions. to specific allergen alone, to allergen vehicle alone, or to A modified endotracheal tube (9.0 mm ID; Hi-Lo Jet 0161-7567/92 $2.00 Copyright 0 1992 the American Physiological Society

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Oral, Mallinckrodt, Argyle, NY), containing two standard low-pressure inflatable balloons (6 cm apart), was inserted per OS into the dog trachea, and then the balloons were inflated. The wall of the modified endotracheal tube contained two small-bore Silastic tubes that open to the surface between the two balloons and provide conduits for flow of fluid to and from the interballoon tracheal segment. The volume in contact with the trachea was -10 ml. A polypropylene 20-ml syringe barrel served as a reservoir from which physiological solution was pumped to and from the interballoon tracheal segment via Silastic tubing. Initially, 30 ml of solution were placed in the closed system (isolated tracheal segment, reservoir, and Silastic tubing). A peristaltic roller pump (Buchler, Fort Lee, NJ) was used to achieve a constant flow of fluid (14 ml/min) to and from the reservoir. The perfusate was composed of sterile Hanks’ balanced salt solution (HBSS) containing penicillin (final concn, 100 U/ml) and streptomycin (final concn, 100 mg/ml) to prevent bacterial growth. The substances tested [short ragweed pollen extract, neutrophil elastase inhibitor ICI 200,355, and pollen extract vehicle (50% glycerin)] were diluted in HBSS and added to the perfusate at the start of the experiment. Short ragweed pollen extract stock solution was diluted in HBSS (l:lOO, wt/vol) and added to the perfusate in the reservoir to give a final concentration of 42 protein nitrogen units (PNU)/ml. This concentration of ragweed allergen was chosen because onetenth of this concentration (4.2 PNU/ml) elicited a wheal >15 mm diam after intradermal injection in each dog and caused lysozyme secretion into the tracheal segment in preliminary experiments. In control experiments, allergen vehicle (glycerin) was diluted in HBSS and added to the perfusate in the reservoir, giving a final concentration of 0.05% (wt/vol). In experiments designed to test the effect of a selective inhibitor of neutrophil elastase, ICI 200,355 (26) was added to the perfusate to produce a final concentration of 10B5 M. Experimental design. Fifteen experiments were conducted in five dogs, each being submitted to an experimental protocol on three occasions and each serving as its own control. In each dog, short ragweed pollen extract, allergen vehicle (control experiment) or short ragweed pollen plus the selective neutrophil elastase inhibitor ICI 200,355 was added to the perfusate in the reservoir after a 3-ml sample was withdrawn for baseline measurements (0 h) of lysozyme activity and neutrophil elastase activity. The perfusate was allowed to circulate for 10 min before baseline measurements were made. When ICI 200,355 was tested, it was instilled into the perfusate 5 min before allergen exposure. Perfusate (1.5 ml) was then removed from the reservoir 2,4,6, and 8 h after the start of the challenge for analysis of lysozyme activity and neutrophil elastase activity. The dogs were rested for 22 wk between exposures; the sequence of experiments was performed in random order. Lysozyme activity assay. A modification of the radialdiffusion plate method (16) was used to measure lysozyme concentration. Briefly, 150 mg of Micrococcus Zysodeicticus were suspended in 1% melted agarose in 0.07 M triphosphate buffer (pH 7.0). A fixed volume of medium

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was poured into Petri dishes (15 X 20 mm) and allowed to set. Using an LKB gel puncher, 3-mm wells were made in the plates. The wells were filled with 10 ~1 of perfusate samples plus various concentrations of egg white lysozyme (0.1-20 ,uglml). The plates were then incubated at 37OC for 14 h. During this time, zones of lysis appeared. A standard curve was obtained by measuring the diameters of these lytic zones and plotting the diameters against the semilogarithmic concentrations of reference standards. In 12 assays (n = 48 samples), the within-assay variability was 0.3% and the between-assay variability was 7%. Lysozyme concentrations of perfusate samples were calculated from the standard curve. The lower limit of detection of the assay was 0.25 pglml. Incubation of short ragweed pollen extract, HBSS, allergen vehicle, and ICI 200,355 elastase inhibitor showed no detectable lysozyme activity. Neutrophil elastaseactivity. The activity of free neutrophi1 elastase was determined with a chromogenic substrate specific for neutrophil elastase (methoxysuccinyl~-alanyl-~-alanyl-Z-prolyl-~-valyl-p-nitroanilide). Perfusate samples from 0,2,4,6, and 8 h reacted with 1 mM of the substrate dissolved in l-methyl-2-pyrrol idinone. The change in absorbance per minute was recorded at 410 nm in a spectrophotometer (Titertek Multiscan MC, Flow Laboratories, Helsinki). This assay is capable of measuring neutrophil elastase concentrations as low as lo-l1 M reliably; concentrations lO-fold higher than the response to histamine (27). In that study, the threshold concentration of neutrophil elastase required to induce a secretory response was 10sl' M, which is two orders of magnitude less than the levels we measured in the tracheal perfusates after allergen exposure in the present study. The secretagogue effect of neutrophil elastase for airway submucosal glands has also been demonstrated in tracheal tissue explants from different species including humans (22). In both the cultured gland serous cells (26) and the tracheal tissues (22), ICI 200,355 has been shown to potently inhibit neutrophil elastase-induced secretion. In the present studies, we found that a drug, ICI 200,355, markedly inhibited the late secretagogue responses that occurred after the introduction of allergen into the isolated tracheal segment. Because we use this as evidence suggesting that allergen-induced hypersecretion is due to human neutrophil elastase, it is necessary to review the previous findings. In the original studies demonstrating that pure neutrophil elastase causes potent secretion from airway submucosal gland cells in culture, we showed that phenylmethylsulfonyl fluoride, N-methyoxipuccinyl-Ala-Ala-Pro-Val-chloromethylketone, and a,-proteinase inhibitor blocked this response, indicating that the secretagogue effect of human neutro-

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phi1 elastase requires the active catalytic site of the enzyme (27). Further studies in whole tissue confirmed the marked secretagogue effect of neutrophil elastase (21). In the latter studies, we tested the effect of ICI 200,355, and we found that it is a selective inhibitor of neutrophil elastase (21). The drug was shown to be a potent (Ki = 0.6 nM) inhibitor of neutrophil elastase and a much weaker inhibitor of other hydrolases. It also inhibited the ongoing destruction of insoluble elastin by human neutrophil elastase and produced a concentration-dependent inhibition of the secretory response induced by human neutrophi1 elastase. It had no effect on baseline secretion or on the secretory response to chymase, cathepsin G, or Pseudomonas aeruginosa elastase. It was ineffective in inhibiting bovine pancreatic chymotrypsin, bovine pancreatic trypsin, human plasma thrombin, acetylcholinesterase, or papain. The drug also has the advantage of being a tight-binding reversible inhibitor. For these reasons, we chose to use this inhibitor in the present study. Lysozyme secretion induced by allergen was markedly but not completely attenuated by the neutrophil elastase inhibitor ICI 200,355. In addition to elastase, neutrophils also contain cathepsin G, which also stimulates gland secretion potently (27) and has recently been shown to contribute to a minor degree to the secretagogue activity of purulent sputum from patients with cystic fibrosis (21). Several other mediators and substances released in response to allergen, such as histamine, prostaglandins, leukotrienes, mast cell chymase, platelet-activating factor, eosinophil cationic protein, and even plasma, have secretagogue activity (13, 25, 27). Some of these agents may be responsible for the small residual secretagogue activity not blocked by ICI 200,355 in the present study. The most potent of these secretagogues are the two proteases, neutrophil cathepsin G and mast cell chymase. Alternatively, the remaining secretory response may be due to involvement of the autonomic nervous system or neural reflexes after allergen challenge. It is known that parasympathomimetic and also sympathetic stimulation as well as C-fiber reflexes can cause secretion from submucosal airway glands (13), and it is feasible that the presence of antigen in the trachea induces such systemic or neural responses either directly or indirectly. Possible interactions between neutrophil elastase and other potential secretagogues in the system have not been studied. Airway secretions are produced by airway epithelial cells, goblet cells, and the submucosal glands (which contain both mucous and serous cells). Because the submucosal glands occupy a substantial proportion of the volume of the large airways, it has been estimated that they are the major source of tracheal secretions (19). Lysozyme is an antimicrobial protein that has been demonstrated immunocytochemically within the secretory granules of the submucosal glands (11). Because the primary cellular source of lysozyme in the airways is the serous cells of the submucosal glands (11,31), it has been used as a marker of serous cell secretion in the trachea (31), in the lower respiratory tract (30), and in the nose (18). In our present study, because of the inflammatory response evoked by allergen with an influx of neutrophils

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ALLERGEN-INDUCED

into the tracheal wall, it is possible that at least some of the lysozyme measured in the tracheal perfusate was released from neutrophils. Neutrophils contain -4 ,ug of lysozyme per lo6 neutrophils (7). However, the likelihood of significant contamination of the perfusate with neutrophi1 lysozyme is small, because in allergic dogs the magnitude of neutrophil and mononuclear cell airway influx was only in the order of lo4 cells/ml (3). Moreover the ability to block lysozyme secretion with a specific inhibitor of neutrophil elastase implicates neutrophil elastaseinduced secretion as the main source of lysozyme in the perfusate. In previous studies, allergen exposure led to increased glycoprotein secretion in sensitized human bronchus (23), in allergic sheep tracheal explants (17), and in the cat trachea in vivo (20). In the allergic sheep trachea, both high- and low-molecular-fraction glycoproteins were reported to be increased after allergen challenge, probably representing mucin and smaller proteins such as lysozyme; the majority of the radioactive labels (35S and 3H) was taken up by the submucosal glands, suggesting that these glands were in fact the major source of the radiolabeled glycoproteins (17). Our results confirm the observation that specific allergen markedly stimulates submucosal gland secretion. Our present study may explain, at least in part, a pathophysiological mechanism involved in hypersecretion associated with the allergic late-phase response. The mechanism by which neutrophi1 elastase induces a secretory response remains unknown, but, as a hypothesis, it is feasible that elastase cleaves a secretion-inhibiting mediator or activates a channel on the gland cell surface. In summary, we have shown that specific allergen exposure in allergic dogs results in lysozyme secretion, associated with the appearance of neutrophil elastase. The ability to attenuate secretion by a selective neutrophil elastase inhibitor implicates neutrophil elastase in the secretion of lysozyme into the airway lumen. We suggest that neutrophil elastase may play a significant role in the pathophysiology of hypersecretion that occurs in inflammatory diseases of the airways, including asthma, and that selective neutrophil elastase inhibitors might provide a therapeutic strategy for the treatment of airway hypersecretion associated with inflammatory states such as the late-phase allergic response. NOTE

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Since submissionof this manuscript, an enzyme with the samephysical and immunocytochemical properties as the major elastaseisozyme of human neutrophils has been described in eosinophils (Lungarella et al. Arch. Biochem. Biophys. 292: 12%135,1992). We suggestthat eosinophil elastasemay play a role in the responsedescribedhere. The authors are grateful to Paul Graf and Iris Ueki for assistance with the experiments and Terry Peura for preparation of the manuscript. This study was supported in part by a Cystic Fibrosis Foundation Research Development Program Grant. A. Schuster is the recipient of a fellowship from the Deutsche Forschungsgemeinschaft.

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Address for reprint requests: J. A. Nadel, Box 0130, Cardiovascular Research Institute, University of California, San Francisco, CA 941430130. Received 17 September 1991; accepted in final form 26 February 1992. REFERENCES

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Role of neutrophil elastase in allergen-induced lysozyme secretion in the dog trachea.

To test our hypothesis that neutrophil elastase plays a role in airway hypersecretion associated with the allergic late-phase response, using an isola...
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