Neurogenic Inflammation in Airways and Its Modulation by Peptidases“ JAY A. NADEL Cardiovascular Research Institute and Departments of Medicine and Physiology Box 0130 University of California San Francisco San Francisco, California 94143 INTRODUCTION
In addition to the parasympathetic and sympathetic nervous systems, a third neural pathway has been described. This pathway exists in the C fibers of the sensory nerves. Stimulation of these nerves electrically or by chemical means (using the drug capsaicin) results in a series of responses in the airways including plasma extravasation, vasodilation, neutrophil adhesion, gland secretion, smooth muscle contraction, cough and potentiation of cholinergic transmission. This constellation of events is known as “neurogenic inflammation.” Stimulation of the sensory nerves in the airways causes the release of neuropeptides, including substance P (SP) and calcitonin gene-related peptide (CGRP).* There is convincing evidence that these neuropeptides are responsible for the neurogenic inflammatory repsonses caused by sensory nerve stimulation. The evidence that substance P plays an important role in these responses is based on the following: First, substance P is released from the sensory nerves upon ~timulation.~ Second, substance P mimics most of the effects of neurogenic inflammation induced by nerve stimulation (reviewed in 1). Third, substance P antagonists substantially inhibit various neurogenic inflammatory responses. Substance P and other “tachykinins” released from sensory nerves are small peptides. Their action is limited by the presence of enzymes, called “peptidases” which cleave and thereby inactivate the peptides, thus modulating the effects of the released peptides. Present evidence suggests that two enzymes normally play major roles in this modulation: neutral endopeptidase (also called enkephalinase, EC 3.4.24.11) and kininase I1 (also called angiotensin converting enzyme; ACE). This conference examines neurohumoral and neuroepithelial interactions in the gastrointestinal system. The justification for a discusison of neurogenic inflammation in airways is twofold. First, the lungs and gut are closely related embryologically, the lungs originating as an outpouching of the foregut. Therefore, it is not surprising that the two organs share many common mechanisms. Second, many of the studies of neurogenic inflammation have been performed in airways, and the findings may find applications in the gut. I shall describe various aspects of neurogenic inflammation and their modulation by peptidases. I shall provide evidence that up- and downregulation of peptidases (especially neutral endopeptidase) have profound effects on neurogenic inflammatory responses and that the stimuli to up- and downregulation may be relevant to the pathogenesis and treatment of inflammatory diseases of the airways such as asthma and chronic bronchitis. a This work was supported in part by National Institutes of Health Program Project Grant HL 24136. 408
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Neutral Endopeptidase in Airways Neutral endopeptidase (NEP) is an enzyme which is normally bound to the surfaces of specific cells that respond to tachykinins4 and to other peptides such as kinins, endothelin and neurotensin. Although substance P can be degraded by various enzymes including serine proteases5*6and mast cell chymase,' most neurogenic inflammatory responses are normally largely modulated by NEP. Vascular neurogenic inflammatory responses also appear to be modulated by ACE.* Biochemical studies have demonstrated NEP activity in various organs including lungs and airway^^*'^ where it resides on the surfaces of cells such as epithelium, submucosal glands, and smooth muscle.IO Localization of NEP in these airway cells has been confirmed by immunocytochemical s t ~ d i e s . ~ * Enzyme "J~ immunoreactivity is found in every species studied, including man. If NEP in the airways normally cleaves and inhibits SP significantly as the peptide diffuses through the tissue from sites of release to the receptors on target cells, then we reasoned that inhibition of NEP by pharmacologic inhibitors should potentiate cellular responses to SP. We found that selective NEP inhibitors (e.g., phosphoramidon) increased the effects of SP on airway smooth muscle contraction, gland secretion, vascular extravasation, and neutrophil adhesion. Similarly, when the airway sensory nerves are stimulated, pretreatment with NEP inhibitors causes exaggerated responses. The airway epithelium plays a special role in peptidase modulation by NEP, because many of the sites for neuropeptide release are located in close proximity to the e p i t h e l i ~ m ,and ' ~ because the epithelium contains substantial NEP.'O Thus removal of the airway epithelium results in potentiated SP responses.'* However, after epithelial removal, SP responses of cells such as smooth muscle still show further increases in response after phosphoramidon. From these studies it is concluded that NEP modulates neurogenic inflammatory responses both at sites of neuropeptide release and at sites of neuropeptide action.'.l2
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Neuropeptides Causing Vascular Neurogenic Responses Inflammation is characterized by vasodilation, edema and movement of inflammatory cells (e.g., neutrophils) into the involved tissue. Stimulation of the unmyelinated sensory nerves in the respiratory mucosa causes vasodilation, increased vascular permeability, leading to plasma extravasation and inflammatory edema, and neutrophil adhesion (reviewed in 1). Stimulation of the sensory nerves leads to the co-release of several peptide neurotransmitters including the tachykinins, substance P and neurokinin A, and calcitonin gene-related peptide (CGRP).2 The fact that phosphoramidon potentiates neurogenic vascular extravasation and neutrophil adhesion in rats suggests that tachykinins mediate these response^.'^,'^ Recent application of a microsphere technique to the study of microvascular blood flow in small circulations enabled the study of the regulation of blood flow in the tracheobronchial circulation.I6 Using this technique, Piedimonte er al. have shown that stimulation of the sensory nerves of the airways with capsaicin increases microvascular blood flow in rats, effects that are mimicked by administration of SP but not of CGRP.I6 This suggested that SP released from the nerves was the cause of the vasodilation. Biological effects of tachykinins are mediated by three different receptor subtypes, denoted as NK-1, NK-2, and NK-3." Recently, selective antagonists of NK-1 receptors have been developed. The vasodilator effect
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of capsaicin was completely abolished by pretreatment with an NK-1 receptor antagonist, confirming that neurogenic extravasation is due to tachykinin release from the nerves and indicating that NK-1 receptors mediate the vasodilator responses.'* Similarly, substance P- and capsaicin-induced plasma extravasation in rat airways is abolished by pretreatment with an NK-1 receptor antag~nist.'~ The increased vascular extravasation due to hypertonic salineZoand to cigarette smoke19 was also prevented by pretreatment with a selective NK-1 receptor antagonist. Selective agonists of the three tachykinin receptor subtypes also exist.2',2z Administration of the NK-1 selective agonist increased airway blood while NK-2 and NK-3 selective agonists were without effect. From these studies we conclude that neurogenic vascular responses are due to NK-1 receptor activation. It is interesting that SP had potent vasodilator effects on airway blood vessels in rats but CGRP was without effect.I6This contrasts with findings of blood flow in the stomach, where CGRP appears to be the predominant vasodilator in neurogenic responses. Thus, the roles of different neuropeptides may vary in different organs and in different species.
Neurogenic InJlammatory Responses to Inhaled Irritants and Effects of NEP Inactivation The airway surface is exposed to the external environment, so inhalation of foreign materials (e.g., irritants) could lead to inflammatory responses. For example, chronic inhalation of cigarette smoke is known to produce inflammatory changes in the airway tissue and is the most important cause of chronic obstructive pulmonary disease.24Recently, it has been shown that cigarette smoke stimulates airway sensory nerves,25with the subsequent release of tachykinins causing plasma extravasation via an effect on NK-1 receptors.19 Increased osmolarity of airway lining fluid due to evaporative water loss is thought to be the principal stimulus for exercise-induced asthma,26and aerosolized hypertonic saline triggers bronchospasm in asthmatic^^^.^^ and cough.Z8Inhalation of aerosols of hypertonic saline results in plasma extravasation in rats.z9 This response is abolished by capsaicin pretreatment which selectively prevents neuropeptide release from the sensory nerves and is potentiated by NEP inhibitor^,^^ suggesting that a peptide (such as SP) normally cleaved by NEP mediates vascular extravasation induced by hypertonic saline aerosols. A selective NK- 1 receptor inhibitor completely inhibited the response (20). Together, these studies indicate that inhalation of hypertonic saline aerosols in rats causes plasma extravasation due to the release of tachykinins from sensory nerves and acting on NK-1 receptors, presumably on postcapillary venules. Bradykinin (BK) is a 9-amino-acid peptide formed from a plasma precursor, plays a role in inflammatory responses,30 and has been implicated in various respiratory.di~eases.~' BK can act directly through its own receptors. However, BK also has apotent stimulatory effect on bronchial C-fiber~'~ and releases sensory neuropeptides in various tissues." In fact, the vascular extravasation caused by local application of BK to the rat nasal mucosa is prevented in a dose-dependent manner by a selective antagonist of the NK-1 tachykinin receptor, indicating that this vascular response of BK is indirect, acting by the release of neuropeptides (e.g., SP) which increase vascular permeability by an action on NK-1 receptors.34 Thus, sensory nerve-induced release of neuropeptides may be a final pathway for responses by multiple mediators. The extravasation of plasma and edematous swelling of the airways as a result
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of increased vascular permeability may be important in the pathophysiology of asthma, by increasing resistance to airflow and by the recruitment of mediators and inflammatory cells from the blood. Thus, inhibitors of NK-1 receptors, by preventing edema formation and inflammation, may contribute to the reduction of airway narrowing and to airway hyperresponsiveness. Irritants such as cigarette smoke also stimulate the sensory nerves and cause neurogenic inflammatory responses.35 Various damaging inhaled materials also act by inhibiting NEP activity. Thus, respiratory viral infections and pollutants such as toluene diisocyanate exaggerate neurogenic inflammatory responses, and they decrease NEP activity (reviewed in 1). Respiratory viruses infect and damage epithelial cells, so it is not difficult to understand how this might decrease NEP activity. It is of interest that respiratory viral infections not only increase airway smooth muscle responses, but they also potentiate neurogenic extravasation. The exact mechanisms of these effects remain unknown. One can speculate that many inhaled irritants may stimulate the sensory nerves to release neuropeptides. In the presence of normal NEP activity, substantial cleavage of the released peptides results in only a low concentration of the peptides reaching the target receptors, so neurogenic inflammatory responses are limited. However, when NEP activity is decreased (e.g., by infection, air pollutants o r cigarette smoke), neurogenic inflammatory responses may become exaggerated because of less inactivation and may contribute to the pathogenesis of disease.
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Effects of Increased Neutral Endopeptidase on Neurogenic Inflammation
Decreased NEP causes exaggerated responses, so it seems reasonable that increased NEP should depress neurogenic inflammatory responses. Two approaches have been used: delivery of exogenous recombinant NEP and upregulation of endogenous NEP. Human recombinant NEP (rNEP) has been used successfully to prevent neurogenic inflammatory responses. Thus, aerosolized rNEP inhibits cough produced by both endogenous and exogenous t a c h y k i n i n ~In . ~the ~ skin, various tachykinins cause increased plasma extravasation, and rNEP inhibits these rsep~nses.~’ Recombinant human NEP also prevents substance P-induced contraction of the iris in the eyes of rabbitsa3*These findings suggest that rNEP might be useful in the treatment of diseases involving inflammatory peptides such as SP and other peptides such as bradykinid’ and endotheha that are cleaved by NEP. Glucocorticoids are known to suppress inflammation, largely through unknown mechanisms. Glucocorticoids induce the expression of NEP41 and ACE42in cultured cells, and this upregulation could suppress neurogenic inflammatory responses by increased cleavage and inactivation of released neuropeptides. Studies of plasma extravasation induced by neuropeptides in rats suggest that this could be an important mechanism for suppressing acute inflammatory responses. Thus, dexamethasone suppressed neurogenic extravasation in rat airways in a dosedependent manner,43and glucocorticoids also inhibited SP-induced plasma extravasation but did not affect PAF-induced extravasation.4 The suppressive effect of dexamethasone on SP-induced extravasation was completely reversed by simultaneously inhibiting NEP and ACE activities, but did not affect PAF-induced extravasation. These studies provide evidence that NEP and ACE mediate a selective inhibitory effect of glucocorticoids on neurogenic plasma extravasation.
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