Modulation of Neurogenic Inflammation by Neutral Endopeptidase 1 - 3 JAY A. NADEL and D. BENJAMIN BORSON4
Introduction Airway inflammation is a prominent feature of chronic airway diseases such as asthma, chronic bronchitis, and cystic fibrosis (for review, see 1).Earlier, investigators believedthat asthma was a disease triggered by allergens, and the primary focus of research on inflammatory mediators was the mast cell. However, multiple cells are likely to be involved in asthma, and these cells interact to produce the clinical responses (1). One of the systems that is implicated in airway inflammatory responses is the sensory nerves. Stimulation of these nerves causes a constellation of responses known as "neurogenic inflammation," and it is the modulation of this system that weare describing here. This nonadrenergic, noncholinergic sensory nervous system has only recently been characterized. Sensory nerves contain a novel class of molecules, the tachykinins, among which substance P (SP) is the best known (2, 3). The sensory nerves of the lower respiratory tract contain SP-immunoreactive fibers, including nerves in the airway epithelium, smooth muscle, and blood vessels (4). Substance P can also be detected in the airways by radioimmunoassay in several species (5), including humans (6). Release of SP from these nerves can be induced by antidromic electrical stimulation (7) or by chemical stimulation of the nerves by capsaicin (8). Exogenously delivered SP has potent inflammatory effects in airways, including cough (9), gland secretion (5, 10), muscle contraction (11, 12), increased vascular permeability (13) and neutrophil adhesion in vivo (14),and chemotaxis in vitro (15). These findings provide evidence that tachykinins contained in sensory nerves are powerful mediators of many cellular responses in airways. It was our idea that degradative mechanisms could exist in airway tissue and could playa potent role in modulating various effects of released tachykinins, just as acetylcholinesterase modulates the effects of acetylcholine that is released from nerves. Our major discovery is that an enzyme, neutral endopeptidase (NEP; also called enkephalinase; EC 3.4.24.11), bound to the surface of specific cells that are the sites of action of tachykinins (e.g., smooth muscle, epithelium, submucosal glands, blood vessels, neutrophils), by cleaving and thus inactivating tachykinins, modulates their effects. Our second hypothesis is that a decrease in tissue NEP increases neurogenic inflammatory responses and that an increase in tissue NEP diminishes or abolishes neurogenic inflammatory responses. These studies are the subject of this review.
SUMMARY The enzyme neutral endopeptidase (NEP)Is bound to the membranes of selected cells in the airways that have receptors for tachyklnlns. The location of the enzyme, along with its selectivity of substrates (tachykinins are a preferred substrate), allows the enzyme to cleave tachykinins that come close to the cell-surface receptors. By cleaving and thus inactivating tachykinins released during stimulation of the sensory nerves, NEP limits the degree of neurogenic inflammation. Neutral endopeptidase exists In the basal cells of the airway epithelium, nerves, smooth muscle, glands, blood vessels, and perhaps other cells. Thus, the enzyme modulates smooth muscle contraction, gland secretion, cough, vascular permeability, and neutrophil adhesion. Decreased NEP activity occurs with epithelial removal, during respiratory viral Infections, and during exposure to Irritants (e.g., cigarette smoke and toluene diisocyanate). Delivery of recombinant NEP (rNEP) by aerosol suppressed cough responses during neurogenic inflammation. We suggest that decreased NEP activity will result in exaggerated neurogenic Inflammation and may play an important role In Inflammatory diseases in airways. Furthermore, drugs that cause up-regulation of NEP may playa therapeutic role by suppressing neurogenic responses. Replacement therapy with rNEP may be useful in diseases where inflammatory peptides (e.g., tachykinins, bradykinin) playa role in pathogenesis. AM REV RESPIR DIS 1991; 143:S33-S36
Evidence that Neutral Endopeptidase Modulates Neurogenic Inflammation in Airways We have used several different kinds of evidence to support our hypothesis that membrane-bound NEP modulates neurogenic inflammation, including the demonstration (l) that NEP exists in specific airways cells, (2) that selective NEP inhibitors potentiate responses to exogenous tachykinins and to neurogenic inflammation, (3) that exogenous rNEP further inhibits these responses, and (4) that conditions that decrease NEP activity potentiate the neurogenic inflammatory responses.
Neutral Endopeptidase Is Present in Airways Neutral endopeptidase immunoreactivity has previously been reported to exist in peripheral tissues, including the airway epithelium (16, 17). Using polyclonal and monoclonal antibodies, we have investigated in detail the localization of NEP in airways. We have demonstrated immunoreactive NEP in the smooth muscle and epithelium (18), submucosal glands, sensory nerves, and blood vessels (1. Nadel, personal communication). In the airway epithelium, NEP is located conspicuously in the basal cells (1. Nadel, personal communication). Neutral endopeptidaselike biochemical activity has been known to exist in lungs, but its locations have not been described (17, 19). Recently, we have shown that this NEP-like activity is found specifically in epithelium, glands, nerves, and smooth muscle (12, 20, 21),locations identical to those where we have found NEP immunocytochemically.
Neutral Endopeptidase Inhibitors Potentiate Effects of Exogenous Tachykinins on Airway Responses It was reported previously that SP and other tachykinins are preferred substrates for NEP in vitro (22). We reasoned that if NEP is located on cells that are targets of tachykinin action, the enzyme, by cleaving the tachykinin close to the site of the tachykinin receptor (perhaps on the same cell), might inactivate a proportion of the tachykinin and thus decrease its action. Further, we reasoned that if NEP on cells normally limits effects of tachykinins, then selective inhibitors of NEP (e.g., phosphoramidon, thiorphan) should potentiate the effects of the tachykinins. For example, phosphoramidon has a nanomolar affinity constant for purified NEP (23)as well as for NEP-like activity in guinea pig airways (24). In fact, we first discovered the modulating role of NEP in neurogenic inflammation in an in vitro study of airway secretion: we found that inhibitors of NEP potentiated the effect of SP on mucus secretion in the ferret trachea, but inhibitors of other proteases had
1 From the Cardiovascular Research Institute and the Departments of Medicine and Physiology, University of California, San Francisco, San Francisco, California. 2 Supported in part by Program Project Grant HL-24136 from the National Institutes of Health. 3 Correspondence and requests for reprints should be addressed to Jay A. Nadel, M.D., Cardiovascular Research Institute, Box 0130, University of California, San Francisco, San Francisco, CA 94143-0130. 4 Recipient of First Independent Research Service and Transition Award HL-38947 from the National Institutes of Health.
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no effect (5, 20). In addition, we showed that the secretagogue effects of all tachykinins tested were also potentiated by NEP inhibitors (25). Inhibitors of NEP potentiated the effects of different tachykinins to variable degrees. These findings indicate that differential effects of tachykinins on a specific tissue may depend not only on the tachykinin receptor subtype present on the specific cell but also on the presence of NEP and its rate of cleavage of the particular tachykinin in situ. A major site of cleavage of SP by NEP is between the 9 and 10 positions, generating the N-terminal fragment SP l - 9 (22). The fact that this fragment does not produce gland secretion provides further evidence that NEP inactivates SP in this system (5). Neutral endopeptidase inhibitors also potentiate other effects of SP in vitro in airways, including smooth muscle contraction (12) and potentiation of cholinergic transmission (12). Like gland secretion, relative effects of various tachykinins on airway smooth muscle are affected differently. Thus, before the NEP inhibitor leucine-thiorphan, the rank order of tachykinin potency on airway smooth muscle was neurokinin A (NKA) > SP >neurokinin B (NKB). After leucine-thiorphan, the rank order of potency changed to NKA = NKB > SP (18). Thus, the rank order of potency may depend not only on receptor subtype but also on the presence of NEP on the cells and its efficiency in cleaving the tachykinino In fact, Iwamoto and coworkers (26) showed that the measured binding of SP to receptors is influenced by NEP-degrading effects. Effects of tachykinins are also potentiated in vivo. Thus, submucosal gland secretion (27) and smooth muscle contraction (24, 28, 29) induced by SP are potentiated selectively by NEP inhibitors. Most interestingly, NEP inhibitors potentiate SP-induced cough, and do so at remarkedly low concentrations of SP (9).
Neutral Endopeptidase Inhibitors Potentiate Neurogenic Inflammation We define "neurogenic inflammation" as the cascade of effects subsequent to the release of tachykinins from the sensory nerves by electrical stimulation of the nerves or by chemical stimulation by capsaicin (8). Thus, electrical field stimulation of ferret tracheal smooth muscle caused contraction that was potentiated by an NEP inhibitor, an effect that was prevented by an SP antagonist (12). Similarly, in vivo bronchoconstrictor responses to vagus nerve stimulation were potentiated by an NEP inhibitor, and these effects were abolished by a tachykinin antagonist (24). These studies implicate endogenous NEP in modulation of airway smooth muscle responses to endogenously released tachykinins. Neutral endopeptidase inhibitors also potentiate cough responses to capsaicin aerosols (9). They also potentiate the increase in vascular permeability and in neutrophil adhe-
sion to postcapillary venular endothelium induced by vagus nerve stimulation and by capsaicin in rats (30). Thus, all of the described responses caused by neurogenic inflammation are potentiated selectively by NEP inhibitors.
Recombinant Neutral Endopeptidase (rNEP) Inhibits Cough Induced by Neurogenic Inflammation Prolonged, uncontrolled, or nonproductive cough is an important symptom of respiratory disease, but the exact mechanisms of cough stimulation are unknown. As mentioned above, cough can be provoked by very low concentrations of SP and of capsaicin, and these effects are potentiated by aerosols of the NEP inhibitor phosphoramidon. The recent cloning of the enzyme (31) now allows us to study the therapeutic uses of rNEP. In awake guinea pigs, rNEP (120 ug by aerosol) inhibited cough caused by SP and to capsaicin. Inactivation of rNEP by boiling and acidification prevented the suppressive effect of the inhaled enzyme on cough. Prior administration of an NEP inhibitor also prevented the suppressive effect of rNEP on cough (32). These results suggest that when aerosolized rNEP reaches the sites of release or actions of endogenously released tachykinins, it degrades them and thus prevents effects (e.g., cough) induced by their release. In addition, our studies suggest that rNEP might be useful in the treatment of diseases involving peptides cleaved by this enzyme.
Effects of Conditions that Decrease Neutral Endopeptidase If NEP strategically located on airway cells, by cleaving and thus inactivating tachykinins, reduces the severity of neurogenic inflammation, we speculated that a reduction in tissue NEP should increase neurogenic inflammatory responses. The following studies suggest that a link exists between the amount ofNEP activity of tissues and the degree of neurogenic inflammation: (l) removal of airway epithelium; (2) respiratory viral infections; (3) exposure to inhaled irritants.
Removal of Airway Epithelium Studies performed by removing the vascular endothelium were used to provide evidence for an endothelium-derived relaxing factor (33). Similarly, investigators have removed airway epithelium, and their results have also suggested that the epithelium contains a relaxing factor (34, 35). We have shown that the epithelium contains NEP-like activity both by immunocytochemical (20) and by biochemical (18) criteria. Therefore, we were not surprised to find that stripping of the epithelium shifted the concentration-response curves of ferret airway smooth muscle to NKA and to SP by 0.5 to 1.0 log units (18). These effects could have been due to the removal of a broad spectrum "epithelial relaxing factor," but the following evidence suggests that the effect was due specifically to the removal of the NEP
activity that resided in the epithelium. First, removal of the epithelium did not affect acetylcholine-induced contraction in the ferret tissue. Second, we repeated the study in the presence of an NEP inhibitor. Wereasoned that in the presence of this inhibitor, the concentration of NEP in the tissue should not affect bronchomotor responses to tachykinins. Thus, the fact that tachykinin-induced contractions in the presence of an NEP inhibitor were not different whether the epithelium was intact or removed is strong evidence that the "relaxing factor" in this case was NEP. Similarly, in guinea-pig bronchi we showed that removal of the epithelium approximately doubled the smooth muscle response to capsaicin (36). That the epithelium is not the sole source of NEP is evidenced by the fact that an NEP inhibitor still potentiated capsaicininduced contractions. However, the fact that in the presence of the NEP inhibitor, capsaicin-induced contractions were not different whether or not the epithelium was present is evidence that the removal of epithelial NEP was the cause of the exaggerated response to SP and to capsaicin that occurred when the epithelium was removed. These studies provide a simple demonstration that NEP in multiple locations can affect neurogenic inflammation and that the strategic 10cation(s) of the enzyme playa major role in modulating these responses. We question whether a single epithelial relaxing factor explains the various findings related to epithelial removal and airway smooth muscle (and other airway functions). Rather, we suggest that multiple factors may playa role, one of which is NEP. Another potent relaxing factor is prostaglandin E 2 , which is released from the airway epithelium and which inhibits neural transmission to airway smooth muscle as well as relaxing the muscle directly (37). Furthermore, we suggest that "removal" of the epithelium may do more than eliminate epithelial cells. For example, damage (even subtle damage) may cause the
release oj mediators rather than just the removalojmediators. The epithelium, located at the interface of the internal milieu and the external environment must playa key role in the defense of the body (38). Thus, damage to the airway epithelium leads to multiple effects. Alteration of the NEP content is one of these consequences.
Respiratory Viral Injections These infections frequently cause exacerbations of asthma (39). Some of the viral effects could be due to neurogenic inflammation, so we studied the effect of experimental respiratory viral infections on several tissue responses to exogenous and endogenous tachykinins. First, we studied ferret tracheal segments infected in vitro with influenza viruses (21). Viral infection caused a tripling of the response to SP, but the response to acetylcholine was unaffected. Pretreatment with an NEP inhibitor (but not with other protease inhibitors) increased the response to SP
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to the same final level in both infected and control tissues. These findings suggest that influenza virus infection in vitro potentiates tachykinin responses by decreasing the degradation of the peptide by tissue NEP. Measurement of NEP activity confirmed a decrease in infected tissues. Respiratory infection with parainfluenza virus in guinea pigs in vivo led to a similar increase in bronchomotor responsiveness to SP and to capsaicin, and this infection was also associated with a decrease in NEP activity (40). Because viral infections potentiate neurogenic inflammatory responses in smooth muscle and decrease NEP activity, we investigated whether other elements of neurogenic inflammation (e.g., vascular permeability and neutrophil adhesion) are similarly affected. First, we compared pathogen-free rats with rats of the same age and strain that had acquired respiratory infections (41). Previously infected rats had a greater susceptibility to increased vascular permeability in the airway upon stimulation with SP and, as in previous studies in vitro, the infected animals had a decrease in NEP activity in the airways. Similarly, experimental infection with Sendai virus potentiated the neurogenic inflammatory responses (vascular permeability and neutrophil adhesion to the postcapillary venular endothelium) to capsaicin compared with control, pathogen-free rats (42). All of these studies indicate that respiratory viral infections potentiate many neurogenic inflammatory responses, and they suggest that the associated decrease in tissue NEP is the cause of the increased responses.
Exposure to Inhaled Irritants Toluene diisocyanate (TDI) is a widely used industrial chemical that can cause asthma in exposed workers. In guinea pigs exposed acutely to TDI, bronchomotor responses to inhaled SP increased, and this was associated with a decrease in airway tissue NEP activity (29). Similarly, inhalation of cigarette smoke in anesthetized guinea pigs increased bronchomotor responses to SP (43). The results suggested that free radicals from cigarette smoke caused the enhanced responses by inactivating airway NEP. Summary
Neutral endopeptidase, located on the surfaces of epithelial, smooth muscle, gland, vascular, and neural cells, cleavesand inactivates tachykinins and thereby limits cough, bronchospasm, increased vascular permeability, and neutrophil accumulation during neurogenic inflammation. In addition to tachykinins, NEP modulates airway responses to bradykinin (44), neurotensin (45), and other peptides. The enzyme modulates responses in other tissues such as gut (46) and skin (47). Decreased NEP is associated with increased tissue responses during neurogenic inflammation. Replacement therapy with recombinant NEP may provide useful therapy in diseases of airways and in other tissues where peptides
hydrolyzed by NEP playa pathogenetic role. Although our evidence suggests that NEP plays a dominant role in modulating neurogenic inflammation in normal airways, other enzymes also cleave tachykinins. When they are available in close proximity to released tachykinins, they may also play modulatory roles. References 1. Bigby TD, Nadel JA. Asthma. In: Gallin 11, Goldstein 1M, Snyderman R, eds. Inflammation: basic principles and clinical correlates. New York: Raven Press, 1988; 679-94. 2. Hokfelt T, Kellerth JO, Nilsson G, Pernow B. Substance P: localization in the central nervous system and in some primary sensory neurons. Science 1975; 190:889-90. 3. Ogawa T, Kanazawa I, Kimura S. Regional distribution of substance P, neurokinin a and neurokinin ~ in rat spinal cord, nerve roots and dorsal root ganglia, and the effects of dorsal root section or spinal transection. Brain Res 1985; 359:152. 4. Lundberg JM, Hokfelt T, Martling C-R, Saria A, Cuello S. Substance P-immunoreactive sensory nervesin the lower respiratory tract of various mammals including man. Cell Tissue Res 1984; 235: 251-61. 5. Borson DB, Corrales R, Varsano S, et af. Enkephalinase inhibitors potentiate substance P-induced secretion of 35S04-macromolecules from ferret trachea. Exp Lung Res 1987; 12:21-36. 6. Lundberg JM, Martling CR, Saria A. Substance P and capsaicin-induced contraction of human bronchi. Acta Physiol Scand 1983; 119:49-53. 7. Olgart L, Gazelius B, Brodin E, Nilsson G. Release of substance P-like immunoreactivity from the dental pulp. Acta Physiol Scand 1977; 101:510-2. 8. Saria A, Theodorsson-Norheim E, Gamse R, Lundberg JM. Release of substance P- and substance K-like immunoreactivities from the isolated perfused guinea-pig lung. Eur J Pharmacol1985; 106:207-8. 9. Kohrogi H, Graf PD, Sekizawa K, Borson DB, Nadel JA. Neutral endopeptidase inhibitors potentiate substance P- and capsaicin-induced cough in awake guinea pigs. J Clin Invest 1988; 82:2063-8. 10. Gashi AA, Borson DB,Finkbeiner WE, Nadel JA, Basbaum CB. Neuropeptides degranulate serous cells of ferret tracheal glands. Am J Physiol 251:C223-9. 11. Tanaka DT, Grunstein MM. Mechanisms of substance P-induced contraction of rabbit airway smooth muscle. J Appl Physiol 1984; 57:1551-7. 12. SekizawaK, Tamaoki J, Nadel JA, Borson DB. Enkephalinase inhibitor potentiates substance P-and electrically-induced contraction in ferret trachea. J Appl Physiol 1987; 63:1401-5. 13. Lundberg JM, Saria A, Brodin E, Rosell S, Folkers K. A substance P antagonist inhibits vagally induced increase in vascular permeability and bronchial smooth muscle contraction in the guinea pig. Proc Natl Acad Sci USA 1983; 80:1120-4. 14. McDonald DM. Respiratory tract infections increase susceptibility to neurogenic inflammation in the rat trachea. Am Rev Respir Dis 1988; 137: 1432-40. 15. Marasco WA, Showell HJ, Becker EL. Substance P binds to the formylpeptide chemotaxis receptor on the rabbit neutrophil. Biochem Biophys Res Commun 1981; 99:1065-72. 16. Johnson AR, Ashton J, Schulz WW, Erdos EG. Neutral metalloendopeptidase in human lung tissue and cultured cells. Am Rev Respir Dis 1985; 132:564-8.
835 17. Llorens C, Schwartz J-C. Enkephalinase activity in rat peripheral organs. Eur J Pharmacol 1981; 69:113-6. 18. Sekizawa K, Tamaoki J, Graf PD, Basbaum CB, Borson DB, Nadel JA. Enkephalinase inhibitor potentiates mammalian tachykinin-induced contraction in ferret trachea. J Pharmacol Exp Ther 1987; 243:1211-7. 19. Kenny AJ, Bowes MA, Gee NS, Matsas R. Endopeptidase-24.l1: a cell-surface enzyme for metabolizing regulatory peptides. Biochem Soc Trans 1985; 13:293-5. 20. Borson DB, Malfroy B, Gold M, Ramachandran J, Nadel JA. Tachykininsinhibit enkephalinase activity from tracheas and lungs of ferrets. Physiologist 1986; 29:174. 21. Jacoby DB, Tamaoki J, Borson DB, Nadel JA. Influenza infection increases airway smooth muscle responsiveness to substance P in ferrets by decreasing enkephalinase. J Appl Physiol 1988; 64:2653-8. 22. Skidgel RA, Engelbrecht A, Johnson AR, Erdos EG. Hydrolysis of substance P and neurotensin by converting enzyme and neutral endoproteinase. Peptides 1984; 5:769-76. 23. Hudgin RL, Charleson SE, Zimmerman M, Mumford R, Wood PL. Enkephalinase: selective peptide inhibitors. Life Sci 1981; 29:2593-2601. 24. Dusser DJ, Umeno E, Graf PD, Djokic T, Borson DB, Nadel JA. Airway neutral endopeptidaselike enzyme modulates tachykinin-induced bronchoconstriction in vivo. J Appl Physiol 1988; 65:2585-91. 25. Borson DB, Gold M, Varsano S, Caughey G, Ramachandran J, Nadel JA. Enkephalinase inhibitors potentiate tachykinin-induced releaseof 35S04labeled macromolecules from ferret trachea. Fed Proc 1986; 45:626. 26. Iwamoto I, Ueki I, Nadel JA. Effect of neutral endopeptidase inhibitors on 3H-substance P binding in rat ileum. Neuropeptides 1988; 11:185-93. 27. Haxhiu MA, Tseng HC, Davis B. Thiorphan enhances baseline and substance P induced tracheal gland secretion in dogs. Clin Res 1988; 36:506A. 28. Stimler-Gerard NP. Neutral endopeptidase-like enzymecontrols the contractile activity of substance P in guinea pig lung. J Clin Invest 1987;79:1819-25. 29. Sheppard D, Thompson JE, Scypinski L, Dusser D, Nadel JA, Borson DB. Toluene diisocyanate increases airway responsiveness to substance P and decreases airway enkephalinase. J Clin Invest 1988; 81:1111-5. 30. Umeno E, Nadel JA, Huang H-T,McDonald DM. Inhibition of neutral endopeptidase potentiates neurogenic inflammation in the rat trachea. J Appl Physiol 1989; 2647-52. 31. Malfroy B, Schofield PR, Kuang W-J,Seeburg PH, Mason AJ, Henzel WJ. Molecular cloning and amino acid sequence of rat enkephalinase. Biochem Biophys Res Commun 1987; 144:59-66. 32. Kohrogi H, Nadel JA, Malfroy B, et af. Recombinant human enkephalinase (neutral endopeptidase) prevents cough induced by tachykinins in awake guinea pigs. J Clin Invest 1989; 84:781-6. 33. Furchgott RF. The role of endothelium in the responsesof vascular smooth muscle to drugs. Annu Rev Pharmacol Toxicol 1984; 24:175-97. 34. Flavahan NA, Aarhus LL, Rimele TJ, Vanhoutte PM. Respiratory epithelium inhibits bronchial smooth muscle tone. J Appl Physiol 1985; 58:834-8. 35. Barnes PJ, Cuss FM, Palmer JB. The effect of airway epithelium on smooth muscle contractility in bovine trachea. Br J Pharmacol 1985; 86:685-91. 36. Djokic TD, Nadel JA, Dusser DJ, Sekizawa K, Graf PD, Borson DB. Inhibitors of neutral en-
536 dopeptidase potentiate electrically and capsaicininduced noncholinergic contraction in guinea pig bronchi. 1 Pharmacol Exp Ther 1989; 248:7-11. 37. Barnett K, lacoby DB, Lazarus SC, Nadel lA. The effects of epithelial cell supernatant on contractions of isolated canine tracheal smooth muscle. Am Rev Respir Dis 1988; 138:780-3. 38. lacoby DB, Nadel lA. Airway epithelial metabolism and airway smooth muscle hyperresponsiveness. In: Coburn RF, ed. Airway smooth muscle in health and disease. New York: Plenum Publishing Corp. 1989; 237-65. 39. Welliver RC. Upper respiratory infections in asthma. 1 Allergy Clin Immunol 1983; 72:341-6. 40. Dusser Dl, lacoby DB, Dickie TD, Borson DB, Nadel lA. Virus induces airway hyperresponsiveness to tachykinins: role of neutral endopeptidase. 1 Appl Physiol 1989; 67:1504-11.
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41. Borson DB, Brokaw 1, Sekizawa, McDonald D, Nadel lA. Viral infection increases tracheal permeability response to substance P (SP) in rats by decreasingneutral endopeptidase (NEP). 1 Cell Biochern Suppl 1988; 12B:295. 42. PiedimonteG, Nadell, UmenoE, McDonald DM, Nadel lA. Sendai virus infection potentiates neurogenic inflammation in the rat trachea. 1 Appl Physiol 1990; 68:754-60. 43. Dusser Dl, Djokic TD, Borson DB, Nadel lA. Cigarette smoke induces bronchoconstrictor hyperresponsiveness to substance P and inactivates airway neutral endopeptidase in the guinea pig. Possible role of free radicals. 1 Clin Invest 1989; 84:900-6. 44. Dusser Dl, Nadel lA, Sekizawa K, GrafPD, Borson DB. Neutral endopeptidase and angiotensin converting enzyme inhibitors protentiate kinin-
induced contraction of ferret trachea. 1 Pharmacol Exp Ther 1988; 244:531-6. 45. Djokic TD, Dusser Dl, Borson DB, Nadel lA. Neutral endopeptidase modulates neurotensininduced airway contraction in guinea pigs. 1 Appl Physiol 1989; 66:2338-43. 46. Djokic TD, Sekizawa K, Borson DB, Nadel lA. Neutral endopeptidase inhibitors potentiate substanceP-inducedcontraction in gut smooth muscle. Am 1 Physiol 1989; 256:G39-43. 47. Iwamoto I, Ueki IF, Borson DB, Nadel lA. Neutral endopeptidase modulates tachykinininduced increase in vascular permeability in guinea pig skin. Int Arch Allergy Appl Immunol 1989; 88:288-93.
Introduction Airway inflammation is a prominent feature of chronic airway diseases such as asthma, chronic bronchitis, and cystic fibrosis (for review, see 1).Earlier, investigators believedthat asthma was a disease triggered by allergens, and the primary focus of research on inflammatory mediators was the mast cell. However, multiple cells are likely to be involved in asthma, and these cells interact to produce the clinical responses (1). One of the systems that is implicated in airway inflammatory responses is the sensory nerves. Stimulation of these nerves causes a constellation of responses known as "neurogenic inflammation," and it is the modulation of this system that weare describing here. This nonadrenergic, noncholinergic sensory nervous system has only recently been characterized. Sensory nerves contain a novel class of molecules, the tachykinins, among which substance P (SP) is the best known (2, 3). The sensory nerves of the lower respiratory tract contain SP-immunoreactive fibers, including nerves in the airway epithelium, smooth muscle, and blood vessels (4). Substance P can also be detected in the airways by radioimmunoassay in several species (5), including humans (6). Release of SP from these nerves can be induced by antidromic electrical stimulation (7) or by chemical stimulation of the nerves by capsaicin (8). Exogenously delivered SP has potent inflammatory effects in airways, including cough (9), gland secretion (5, 10), muscle contraction (11, 12), increased vascular permeability (13) and neutrophil adhesion in vivo (14),and chemotaxis in vitro (15). These findings provide evidence that tachykinins contained in sensory nerves are powerful mediators of many cellular responses in airways. It was our idea that degradative mechanisms could exist in airway tissue and could playa potent role in modulating various effects of released tachykinins, just as acetylcholinesterase modulates the effects of acetylcholine that is released from nerves. Our major discovery is that an enzyme, neutral endopeptidase (NEP; also called enkephalinase; EC 3.4.24.11), bound to the surface of specific cells that are the sites of action of tachykinins (e.g., smooth muscle, epithelium, submucosal glands, blood vessels, neutrophils), by cleaving and thus inactivating tachykinins, modulates their effects. Our second hypothesis is that a decrease in tissue NEP increases neurogenic inflammatory responses and that an increase in tissue NEP diminishes or abolishes neurogenic inflammatory responses. These studies are the subject of this review.
SUMMARY The enzyme neutral endopeptidase (NEP)Is bound to the membranes of selected cells in the airways that have receptors for tachyklnlns. The location of the enzyme, along with its selectivity of substrates (tachykinins are a preferred substrate), allows the enzyme to cleave tachykinins that come close to the cell-surface receptors. By cleaving and thus inactivating tachykinins released during stimulation of the sensory nerves, NEP limits the degree of neurogenic inflammation. Neutral endopeptidase exists In the basal cells of the airway epithelium, nerves, smooth muscle, glands, blood vessels, and perhaps other cells. Thus, the enzyme modulates smooth muscle contraction, gland secretion, cough, vascular permeability, and neutrophil adhesion. Decreased NEP activity occurs with epithelial removal, during respiratory viral Infections, and during exposure to Irritants (e.g., cigarette smoke and toluene diisocyanate). Delivery of recombinant NEP (rNEP) by aerosol suppressed cough responses during neurogenic inflammation. We suggest that decreased NEP activity will result in exaggerated neurogenic Inflammation and may play an important role In Inflammatory diseases in airways. Furthermore, drugs that cause up-regulation of NEP may playa therapeutic role by suppressing neurogenic responses. Replacement therapy with rNEP may be useful in diseases where inflammatory peptides (e.g., tachykinins, bradykinin) playa role in pathogenesis. AM REV RESPIR DIS 1991; 143:S33-S36
Evidence that Neutral Endopeptidase Modulates Neurogenic Inflammation in Airways We have used several different kinds of evidence to support our hypothesis that membrane-bound NEP modulates neurogenic inflammation, including the demonstration (l) that NEP exists in specific airways cells, (2) that selective NEP inhibitors potentiate responses to exogenous tachykinins and to neurogenic inflammation, (3) that exogenous rNEP further inhibits these responses, and (4) that conditions that decrease NEP activity potentiate the neurogenic inflammatory responses.
Neutral Endopeptidase Is Present in Airways Neutral endopeptidase immunoreactivity has previously been reported to exist in peripheral tissues, including the airway epithelium (16, 17). Using polyclonal and monoclonal antibodies, we have investigated in detail the localization of NEP in airways. We have demonstrated immunoreactive NEP in the smooth muscle and epithelium (18), submucosal glands, sensory nerves, and blood vessels (1. Nadel, personal communication). In the airway epithelium, NEP is located conspicuously in the basal cells (1. Nadel, personal communication). Neutral endopeptidaselike biochemical activity has been known to exist in lungs, but its locations have not been described (17, 19). Recently, we have shown that this NEP-like activity is found specifically in epithelium, glands, nerves, and smooth muscle (12, 20, 21),locations identical to those where we have found NEP immunocytochemically.
Neutral Endopeptidase Inhibitors Potentiate Effects of Exogenous Tachykinins on Airway Responses It was reported previously that SP and other tachykinins are preferred substrates for NEP in vitro (22). We reasoned that if NEP is located on cells that are targets of tachykinin action, the enzyme, by cleaving the tachykinin close to the site of the tachykinin receptor (perhaps on the same cell), might inactivate a proportion of the tachykinin and thus decrease its action. Further, we reasoned that if NEP on cells normally limits effects of tachykinins, then selective inhibitors of NEP (e.g., phosphoramidon, thiorphan) should potentiate the effects of the tachykinins. For example, phosphoramidon has a nanomolar affinity constant for purified NEP (23)as well as for NEP-like activity in guinea pig airways (24). In fact, we first discovered the modulating role of NEP in neurogenic inflammation in an in vitro study of airway secretion: we found that inhibitors of NEP potentiated the effect of SP on mucus secretion in the ferret trachea, but inhibitors of other proteases had
1 From the Cardiovascular Research Institute and the Departments of Medicine and Physiology, University of California, San Francisco, San Francisco, California. 2 Supported in part by Program Project Grant HL-24136 from the National Institutes of Health. 3 Correspondence and requests for reprints should be addressed to Jay A. Nadel, M.D., Cardiovascular Research Institute, Box 0130, University of California, San Francisco, San Francisco, CA 94143-0130. 4 Recipient of First Independent Research Service and Transition Award HL-38947 from the National Institutes of Health.
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no effect (5, 20). In addition, we showed that the secretagogue effects of all tachykinins tested were also potentiated by NEP inhibitors (25). Inhibitors of NEP potentiated the effects of different tachykinins to variable degrees. These findings indicate that differential effects of tachykinins on a specific tissue may depend not only on the tachykinin receptor subtype present on the specific cell but also on the presence of NEP and its rate of cleavage of the particular tachykinin in situ. A major site of cleavage of SP by NEP is between the 9 and 10 positions, generating the N-terminal fragment SP l - 9 (22). The fact that this fragment does not produce gland secretion provides further evidence that NEP inactivates SP in this system (5). Neutral endopeptidase inhibitors also potentiate other effects of SP in vitro in airways, including smooth muscle contraction (12) and potentiation of cholinergic transmission (12). Like gland secretion, relative effects of various tachykinins on airway smooth muscle are affected differently. Thus, before the NEP inhibitor leucine-thiorphan, the rank order of tachykinin potency on airway smooth muscle was neurokinin A (NKA) > SP >neurokinin B (NKB). After leucine-thiorphan, the rank order of potency changed to NKA = NKB > SP (18). Thus, the rank order of potency may depend not only on receptor subtype but also on the presence of NEP on the cells and its efficiency in cleaving the tachykinino In fact, Iwamoto and coworkers (26) showed that the measured binding of SP to receptors is influenced by NEP-degrading effects. Effects of tachykinins are also potentiated in vivo. Thus, submucosal gland secretion (27) and smooth muscle contraction (24, 28, 29) induced by SP are potentiated selectively by NEP inhibitors. Most interestingly, NEP inhibitors potentiate SP-induced cough, and do so at remarkedly low concentrations of SP (9).
Neutral Endopeptidase Inhibitors Potentiate Neurogenic Inflammation We define "neurogenic inflammation" as the cascade of effects subsequent to the release of tachykinins from the sensory nerves by electrical stimulation of the nerves or by chemical stimulation by capsaicin (8). Thus, electrical field stimulation of ferret tracheal smooth muscle caused contraction that was potentiated by an NEP inhibitor, an effect that was prevented by an SP antagonist (12). Similarly, in vivo bronchoconstrictor responses to vagus nerve stimulation were potentiated by an NEP inhibitor, and these effects were abolished by a tachykinin antagonist (24). These studies implicate endogenous NEP in modulation of airway smooth muscle responses to endogenously released tachykinins. Neutral endopeptidase inhibitors also potentiate cough responses to capsaicin aerosols (9). They also potentiate the increase in vascular permeability and in neutrophil adhe-
sion to postcapillary venular endothelium induced by vagus nerve stimulation and by capsaicin in rats (30). Thus, all of the described responses caused by neurogenic inflammation are potentiated selectively by NEP inhibitors.
Recombinant Neutral Endopeptidase (rNEP) Inhibits Cough Induced by Neurogenic Inflammation Prolonged, uncontrolled, or nonproductive cough is an important symptom of respiratory disease, but the exact mechanisms of cough stimulation are unknown. As mentioned above, cough can be provoked by very low concentrations of SP and of capsaicin, and these effects are potentiated by aerosols of the NEP inhibitor phosphoramidon. The recent cloning of the enzyme (31) now allows us to study the therapeutic uses of rNEP. In awake guinea pigs, rNEP (120 ug by aerosol) inhibited cough caused by SP and to capsaicin. Inactivation of rNEP by boiling and acidification prevented the suppressive effect of the inhaled enzyme on cough. Prior administration of an NEP inhibitor also prevented the suppressive effect of rNEP on cough (32). These results suggest that when aerosolized rNEP reaches the sites of release or actions of endogenously released tachykinins, it degrades them and thus prevents effects (e.g., cough) induced by their release. In addition, our studies suggest that rNEP might be useful in the treatment of diseases involving peptides cleaved by this enzyme.
Effects of Conditions that Decrease Neutral Endopeptidase If NEP strategically located on airway cells, by cleaving and thus inactivating tachykinins, reduces the severity of neurogenic inflammation, we speculated that a reduction in tissue NEP should increase neurogenic inflammatory responses. The following studies suggest that a link exists between the amount ofNEP activity of tissues and the degree of neurogenic inflammation: (l) removal of airway epithelium; (2) respiratory viral infections; (3) exposure to inhaled irritants.
Removal of Airway Epithelium Studies performed by removing the vascular endothelium were used to provide evidence for an endothelium-derived relaxing factor (33). Similarly, investigators have removed airway epithelium, and their results have also suggested that the epithelium contains a relaxing factor (34, 35). We have shown that the epithelium contains NEP-like activity both by immunocytochemical (20) and by biochemical (18) criteria. Therefore, we were not surprised to find that stripping of the epithelium shifted the concentration-response curves of ferret airway smooth muscle to NKA and to SP by 0.5 to 1.0 log units (18). These effects could have been due to the removal of a broad spectrum "epithelial relaxing factor," but the following evidence suggests that the effect was due specifically to the removal of the NEP
activity that resided in the epithelium. First, removal of the epithelium did not affect acetylcholine-induced contraction in the ferret tissue. Second, we repeated the study in the presence of an NEP inhibitor. Wereasoned that in the presence of this inhibitor, the concentration of NEP in the tissue should not affect bronchomotor responses to tachykinins. Thus, the fact that tachykinin-induced contractions in the presence of an NEP inhibitor were not different whether the epithelium was intact or removed is strong evidence that the "relaxing factor" in this case was NEP. Similarly, in guinea-pig bronchi we showed that removal of the epithelium approximately doubled the smooth muscle response to capsaicin (36). That the epithelium is not the sole source of NEP is evidenced by the fact that an NEP inhibitor still potentiated capsaicininduced contractions. However, the fact that in the presence of the NEP inhibitor, capsaicin-induced contractions were not different whether or not the epithelium was present is evidence that the removal of epithelial NEP was the cause of the exaggerated response to SP and to capsaicin that occurred when the epithelium was removed. These studies provide a simple demonstration that NEP in multiple locations can affect neurogenic inflammation and that the strategic 10cation(s) of the enzyme playa major role in modulating these responses. We question whether a single epithelial relaxing factor explains the various findings related to epithelial removal and airway smooth muscle (and other airway functions). Rather, we suggest that multiple factors may playa role, one of which is NEP. Another potent relaxing factor is prostaglandin E 2 , which is released from the airway epithelium and which inhibits neural transmission to airway smooth muscle as well as relaxing the muscle directly (37). Furthermore, we suggest that "removal" of the epithelium may do more than eliminate epithelial cells. For example, damage (even subtle damage) may cause the
release oj mediators rather than just the removalojmediators. The epithelium, located at the interface of the internal milieu and the external environment must playa key role in the defense of the body (38). Thus, damage to the airway epithelium leads to multiple effects. Alteration of the NEP content is one of these consequences.
Respiratory Viral Injections These infections frequently cause exacerbations of asthma (39). Some of the viral effects could be due to neurogenic inflammation, so we studied the effect of experimental respiratory viral infections on several tissue responses to exogenous and endogenous tachykinins. First, we studied ferret tracheal segments infected in vitro with influenza viruses (21). Viral infection caused a tripling of the response to SP, but the response to acetylcholine was unaffected. Pretreatment with an NEP inhibitor (but not with other protease inhibitors) increased the response to SP
MODULATION OF NEUROGENIC INFLAMMATION BY NEUTRAL ENDOPEPTIDASE
to the same final level in both infected and control tissues. These findings suggest that influenza virus infection in vitro potentiates tachykinin responses by decreasing the degradation of the peptide by tissue NEP. Measurement of NEP activity confirmed a decrease in infected tissues. Respiratory infection with parainfluenza virus in guinea pigs in vivo led to a similar increase in bronchomotor responsiveness to SP and to capsaicin, and this infection was also associated with a decrease in NEP activity (40). Because viral infections potentiate neurogenic inflammatory responses in smooth muscle and decrease NEP activity, we investigated whether other elements of neurogenic inflammation (e.g., vascular permeability and neutrophil adhesion) are similarly affected. First, we compared pathogen-free rats with rats of the same age and strain that had acquired respiratory infections (41). Previously infected rats had a greater susceptibility to increased vascular permeability in the airway upon stimulation with SP and, as in previous studies in vitro, the infected animals had a decrease in NEP activity in the airways. Similarly, experimental infection with Sendai virus potentiated the neurogenic inflammatory responses (vascular permeability and neutrophil adhesion to the postcapillary venular endothelium) to capsaicin compared with control, pathogen-free rats (42). All of these studies indicate that respiratory viral infections potentiate many neurogenic inflammatory responses, and they suggest that the associated decrease in tissue NEP is the cause of the increased responses.
Exposure to Inhaled Irritants Toluene diisocyanate (TDI) is a widely used industrial chemical that can cause asthma in exposed workers. In guinea pigs exposed acutely to TDI, bronchomotor responses to inhaled SP increased, and this was associated with a decrease in airway tissue NEP activity (29). Similarly, inhalation of cigarette smoke in anesthetized guinea pigs increased bronchomotor responses to SP (43). The results suggested that free radicals from cigarette smoke caused the enhanced responses by inactivating airway NEP. Summary
Neutral endopeptidase, located on the surfaces of epithelial, smooth muscle, gland, vascular, and neural cells, cleavesand inactivates tachykinins and thereby limits cough, bronchospasm, increased vascular permeability, and neutrophil accumulation during neurogenic inflammation. In addition to tachykinins, NEP modulates airway responses to bradykinin (44), neurotensin (45), and other peptides. The enzyme modulates responses in other tissues such as gut (46) and skin (47). Decreased NEP is associated with increased tissue responses during neurogenic inflammation. Replacement therapy with recombinant NEP may provide useful therapy in diseases of airways and in other tissues where peptides
hydrolyzed by NEP playa pathogenetic role. Although our evidence suggests that NEP plays a dominant role in modulating neurogenic inflammation in normal airways, other enzymes also cleave tachykinins. When they are available in close proximity to released tachykinins, they may also play modulatory roles. References 1. Bigby TD, Nadel JA. Asthma. In: Gallin 11, Goldstein 1M, Snyderman R, eds. Inflammation: basic principles and clinical correlates. New York: Raven Press, 1988; 679-94. 2. Hokfelt T, Kellerth JO, Nilsson G, Pernow B. Substance P: localization in the central nervous system and in some primary sensory neurons. Science 1975; 190:889-90. 3. Ogawa T, Kanazawa I, Kimura S. Regional distribution of substance P, neurokinin a and neurokinin ~ in rat spinal cord, nerve roots and dorsal root ganglia, and the effects of dorsal root section or spinal transection. Brain Res 1985; 359:152. 4. Lundberg JM, Hokfelt T, Martling C-R, Saria A, Cuello S. Substance P-immunoreactive sensory nervesin the lower respiratory tract of various mammals including man. Cell Tissue Res 1984; 235: 251-61. 5. Borson DB, Corrales R, Varsano S, et af. Enkephalinase inhibitors potentiate substance P-induced secretion of 35S04-macromolecules from ferret trachea. Exp Lung Res 1987; 12:21-36. 6. Lundberg JM, Martling CR, Saria A. Substance P and capsaicin-induced contraction of human bronchi. Acta Physiol Scand 1983; 119:49-53. 7. Olgart L, Gazelius B, Brodin E, Nilsson G. Release of substance P-like immunoreactivity from the dental pulp. Acta Physiol Scand 1977; 101:510-2. 8. Saria A, Theodorsson-Norheim E, Gamse R, Lundberg JM. Release of substance P- and substance K-like immunoreactivities from the isolated perfused guinea-pig lung. Eur J Pharmacol1985; 106:207-8. 9. Kohrogi H, Graf PD, Sekizawa K, Borson DB, Nadel JA. Neutral endopeptidase inhibitors potentiate substance P- and capsaicin-induced cough in awake guinea pigs. J Clin Invest 1988; 82:2063-8. 10. Gashi AA, Borson DB,Finkbeiner WE, Nadel JA, Basbaum CB. Neuropeptides degranulate serous cells of ferret tracheal glands. Am J Physiol 251:C223-9. 11. Tanaka DT, Grunstein MM. Mechanisms of substance P-induced contraction of rabbit airway smooth muscle. J Appl Physiol 1984; 57:1551-7. 12. SekizawaK, Tamaoki J, Nadel JA, Borson DB. Enkephalinase inhibitor potentiates substance P-and electrically-induced contraction in ferret trachea. J Appl Physiol 1987; 63:1401-5. 13. Lundberg JM, Saria A, Brodin E, Rosell S, Folkers K. A substance P antagonist inhibits vagally induced increase in vascular permeability and bronchial smooth muscle contraction in the guinea pig. Proc Natl Acad Sci USA 1983; 80:1120-4. 14. McDonald DM. Respiratory tract infections increase susceptibility to neurogenic inflammation in the rat trachea. Am Rev Respir Dis 1988; 137: 1432-40. 15. Marasco WA, Showell HJ, Becker EL. Substance P binds to the formylpeptide chemotaxis receptor on the rabbit neutrophil. Biochem Biophys Res Commun 1981; 99:1065-72. 16. Johnson AR, Ashton J, Schulz WW, Erdos EG. Neutral metalloendopeptidase in human lung tissue and cultured cells. Am Rev Respir Dis 1985; 132:564-8.
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