Pulmonary Pharmacology (1992) 5, 81-96

Mucus Secretion and Inflammation J . D. Lundgren*t, J. N . Baraniuk$ *Department of Infectious Diseases (144), Hvidovre Hospital, University of Copenhagen, Hvidovre DK-2650, Denmark, and National Heart and Lung Institute, Dovehouse Street, London S W3 6L Y, UK

Introduction

cystic fibrosis patients ."' High molecular weight glycoconjugates ('mucin') form a viscid, gel phase defensive barrier which floats above a .5-µm wide, watery, sol phase periciliary layer . Inhaled particles adhere to the mucus gel and are trapped before reaching the alveolar space . The presence of antimicrobial proteins in the sol and gel phases provide a biochemical and biological barrier to infection . Epithelial cell cilia are embedded in the mucus blanket, and coordinated ciliary movement results in the transport of mucus to central airways and ultimately the larynx where it is swallowed . Cough assists in mucous clearance . Mucous glycoproteins and proteoglycans are produced by goblet cells and mucosal cells of submucosal glands""' in vivo and in vitro, while some single cell cultures of serous cells may produce proteoglycans . 18,19 These glycoconjugates have high molecular weights of 105-108 Da .3,5,7,15,"6,2° The protein cores of mucous glycoproteins contain abundant threonine, serine, proline and alanine (approximately 20 :12 :12 :10, respectively) . Approximately 80% of the glycoprotein mass is carbohydrate . N-acetylgalactosamine, N-acetylglucosamine, fucose, galactose and Nacetylneuraminic acid (sialic acid) are the predominant sugar moieties in the oligosaccharide side chains . These side chains are 1600-1800 Da in size . Sialic acid and sulphate groups contribute to the uniform acidic pl of approximately 2 .6-2 .8 of mucous glycoproteins . 3,5 Human mucous glycoprotein genes have been cloned. 21 Serous cells secrete lysozyme, lactoferrin, secretory component (the transport protein of polymeric immunoglobulins) and many enzymes .", 19,22-24 Water accounts for 95-99% of the total mass of mucus . Water secretion is regulated by an osmotic effect generated by epithelial cell chloride ion transport25 and by the great water binding capabilities of the mucous glycoproteins . 26 Changes in interstitial fluid hydrostatic pressure also increase water flux under conditions of arterial dilation and enhance post-capillary venule plasma extravasation . 27

`Mucus' is the viscoelastic fluid covering the epithelial surface of the respiratory tract .' It is composed of water, proteins, glycoproteins and other macromolecules . The macromolecules originate from epithelial cells, serous and mucous cells of submucosal glands, inflammatory cells and plasma, and include glycoconjugates such as mucins, antimicrobial factors such as lysozyme, lactoferrin and immunoglobulins, and albumin . Hydration of the glycoconjugate fraction determines the rheological properties of the gel and sol phases of mucus . The secretion of mucus is highly complex in both health and disease and is regulated in part by acetylcholine and neuropeptides released from autonomic and sensory nerves . Disorders of mucus secretion contribute to airflow obstruction in asthma, chronic bronchitis and other conditions . In acute and chronic inflammatory conditions, mast cells, neutrophils, eosinophils and other inflammatory cells generate a wide spectrum of secretagogues including peptidyl-leukotrienes ('slow reacting substance of anaphylaxis'), platelet activating factor (PAF), histamine, human neutrophil elastase, and eosinophil cationic protein which may stimulate mucus secretion . Some of these neural, leukocytic, and lipid inflammatory factors may also induce metaplasia, hypertrophy and hyperplasia of secretory cells . The contributions of these inflammatory influences to the generation of mucus will be examined (Fig . 1) . Mucus `Mucus' is 95% water, 1 % dialysable solutes, and 4% non-dialysable macromolecules by weight . These components are produced by goblet, serous and Clara cells of the epithelium, the serous and mucous cells of submucosal glands, extravasated plasma, and cellular debris ." Neutrophil DNA is present in sputum of t For reprint requests . 0952-0600/92/020081 + 16 $03 .00/0

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© 1992 Academic Press Limited



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J . D . Lundgren, J . N . Baraniuk

Epithelial goblet cells

NERVES Sensory (SP, GRP ? ) Parasympathetic (acetylcholine, VIP) Sympathetic (NE, NPY)

Exocytosis Hypertrophy Hyperplosia

10

INFLAMMATORY CELLS Neutrophils (HNE, LTB4 ) Eosinophils (ECP, LTC4, PAF ) Most cells (histamine, PAF LTC4, chymase, PGD2)

Metaplasia Bradykinin

Fig. 1 Neuropeptides released from sensory and parasympathetic nerves, and inflammatory mediators from a variety of resident and recruited cells induce exocytosis from goblet cells, and submucosal gland serous and mucous cells . Some of these factors may lead to secretory cell hypertrophy, hyperplasia, and metaplasia in respiratory disease .

Sputum and increased mucus production In healthy humans, submucosal glands are found in all parts of the central airways with density decreasing progressively until they are absent from airways smaller than 1 mm in diameter . This site is usually considered part of the peripheral airways ." Goblet cells are present distally but are normally absent in the terminal bronchioli. Submucosal gland cells outnumber goblet cells 40 to 1 in human airways in toto . 29 Since normal persons do not expectorate, sputum production is an indication that greater than normal amounts of fluid are forming in the respiratory tract . Sputum production is a characteristic of acute bronchitis, asthma, and chronic bronchitis . Furthermore, in chronic emphysema, `small airways disease', and cystic fibrosis there is evidence to suggest that increased mucus production occurs in the distal airways even though this may not result in sputum production . Alterations in the number, size, and distribution of both submucosal glands and goblet cells in the central and peripheral airways of diseased subjects indicate that hypertrophy, hyperplasia and metaplasia of mucous producing cells occur.',"-" Submucosal gland cells from chronic bronchitis subjects have an increased turnover of glycoconjugates .32 Bronchial tissue from chronic bronchitic subjects produce more glycoconjugates after stimulation by parasympathomimetics . 33 The fucose and sulfate contents of sputum mucus from patients' are increased compared to sputum induced from normal subjects . These increases may be due to the secretion of more mucin molecules or increases in the size or number of carbohydrate side chains and sulfate additions per mucin molecule . Exudation of plasma proteins may also contribute to the increase in dry weight since the concentration of sialic acid (a marker of both mucins

and plasma proteins) is increased more relative to the fucose concentration . Albumin, transferrin and other plasma proteins are also increased in diseased conditions . In guinea-pig and bovine systems, albumin or albumin-like proteins may be synthesized and/or transported by serous cells .14,11 Mucus viscosity appears to be increased during disease . The viscosity of sputum from patients with chronic bronchitis and asthma is two to three-fold higher than that obtained from healthy patients after inhalation of various mucus secretagogues . 36,37 Mucin chemistry and concentration are the major determinants of viscosity, although albumin and DNA also contribute ."" ,", " Decreased hydration of mucus, as seen in cystic fibrosis, may predispose to the accumulation of mucus in the airways .' DNase has recently been used for treatment of viscous secretions in cystic fibrosis ." Increased production of mucus in the lower airways and perhaps a concomitant injury to the mucociliary escalator as seen in asthmatic airways may lead to decreased mucous clearance and subsequently to accumulation of mucus in the airways, mucus plugging, airflow obstruction, and atelectasis . Mucus accumulation provides a platform for bacterial or fungal growth,40 and may cause extended exposure of the surface epithelium to inhaled carcinogens such as those present in tobacco smoke ." A more detailed description of ciliary dyskinesias will not be discussed here, but may significantly contribute to the retention of pulmonary secretions .

Airflow obstruction and disease Central airways are the predominant sites of airflow resistance .28,42 Increases in thickness of the mucosa by

Mucus Secretion and Inflammation 83

vasodilation, oedema or inflammatory infiltration, increased intraluminal mucus, and smooth muscle contraction can combine to obstruct airways . 43 Mucosal thickening can result from : hyperplasia and hypertrophy of epithelial cells, vessels, glands, and smooth muscle; deposition of subepithelial basement membrane connective tissue ; oedema formation ; and infiltration by inflammatory cells .43 Peripheral airways may significantly contribute to airway resistance when the total peripheral airway diameter decreases to less than two-thirds of normal ." Goblet cells supply the mucus in these airways, and goblet cell hyperplasia and hypersecretion could contribute to the decrease in peripheral airway diameter . Intralumenal mucus may obstruct peripheral airways and impair airflow conductivity both directly by increasing the resistance to airflow in small airways 28 and indirectly due to the increase in intrathoracic pressure and consequent collapse of larger airways . Allergen exposure in allergic asthma can be followed by both immediate and late airways obstruction .' , " The early phase obstruction lasts approximately 1 h and is most likely due to activation of mast cells and nerves in the airways . Mast cell mediators such as histamine, peptidyl-leukotrienes, platelet activating factor (PAF), and others lead to submucosal gland and epithelial cell secretion, smooth muscle contraction, airway oedema, adhesion and chemotaxis of leukocytes, and sensory nerve stimulation. Sensory nerve stimulation leads to parasympathetic nerve reflexes and axon responses .23,46 The mechanisms of the late phase obstruction have not been fully determined, but neither mast cells nor smooth muscle contraction appear to be involved . Instead, oedema and infiltration by eosinophils, neutrophils and other inflammatory cells may contribute . 47"9 These inflammatory cells release many proteins and lipids which are seeretagogues . Cytokines released from T-lymphocytes and other cells may also be important inflammatory signals ." Plasma-derived factors such as bradykinin and the complement component C3a may also play roles since these can stimulate secretion in vitro .' Bradykinin's effects may be mediated by arachidonic acid metabolites ." Chronic bronchitis is defined as significant sputum production for 3 months each in two consecutive years in a patient without underlining pulmonary disease . The volume of submucosal glands in the central airways of chronic bronchitis patients is increased compared to normals . Neutrophilic infiltrates are present and probably play a pathologic role . Nearly all chronic bronchitis patients are exposed to the same pollutant (tobacco smoke), but only a fraction demonstrate profound airway obstruction . When obstruction does occur, it is usually due to irreversible destruction of lung parenchyma with the loss of elastic recoil (chronic emphysema) . In chronic bronchitis

with obstruction but without chronic emphysema, the airflow resistances of both the peripheral and central airways are markedly increased .", " The obstruction may be due to hyperplasia and hypertrophy of mucosal elements which increase mucosal thickness, and airway plugging by mucosal secretions ." In smokers with airway obstruction but without chronic bronchitis or emphysema, an inflammatory process in the peripheral airways and lung parenchyma may be present ('small airways disease') .' Increased numbers of goblet cells and mucus plugs in the peripheral airways are found ." Also, some patients with emphysema without chronic bronchitis have evidence of increased mucus production from peripheral airways . Nerve-mediated secretion Since the last century it has been known that airway secretion was neuronally regulated ." In 1912 Mollgaard 57 suggested that the airways were innervated both by the cholinergic and the adrenergic nervous systems . The concept of acetylcholine and norepinephrine as the sole autonomic neurotransmitters was dispelled when it was discovered that some physiological responses could be obtained despite the presence of cholinergic and adrenergic antagonists in animal models"' but not in human airways .61 Because these non-adrenergic, non-cholinergic ('NANC') responses were augmented by the addition of protease inhibitors, it was determined that these responses were due in large part to neuropeptides . The respiratory tract is innervated by sensory, parasympathetic, and sympathetic nerves .21,4"9,62 Sensory nerve fibres enter via the vagal nerve (cell bodies in nodose ganglion) or sympathetic nerves (thoracic efferent nerves) . These unmyelinated, type C fibres contain substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP) and other peptides ." Noxious stimuli depolarizes these nerves . Exogenous stimuli include neurotoxins such as capsaicin, while endogenous stimuli such as H + , K + , histamine, bradykinin, and arachidonic acid metabolites are generated during cell injury, mast cell degranulation, and inflammation .23 Depolarization of these sensory nerves leads to central reflexes (cough, parasympathetic reflexes) and local antidromal axon responses .23 Parasympathetic post-ganglionic fibres contain vasoactive intestinal peptide (VIP) . Neuropeptide Y (NPY) is present in adrenergic nerve fibres . Other combinations of these neuropeptides may also occur; the functions of these diverse neuropeptides and their interactions in vivo are summarized below . Arachidonic acid metabolites may also be released by some neurons and may play a role in inflammation .' To demonstrate whether a particular neurotransmitter is relevant in the physiological mucus secretion,

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the following questions should be addressed . (1) Does the neurotransmitter specifically stimulate mucous glycoconjugate release in vitro and in vivo? (2) Are specific neurotransmitter receptors present on secretory cells? (3) Is the neurotransmitter present in nerve fibres around the receptor containing secretory cells? (4) Does stimulation of the nerve fibre in vivo produce mucous release?

Parasympathetic nerve-induced secretion Parasympathetic nerves are important stimulants of glandular secretion production .' ,", "," Cholinergic and VIP-ergic fibres originate in tracheobronchial ganglia and extend to smooth muscle and glands .23,62 Sensory nerves can stimulate these parasympathetic ganglion cells via central pathways and through local axon responses . These ganglion cells may be important sites of neural regulation since they bear autoreceptors which can either stimulate (NK,) or inhibit (muscarinic M 2, /3Zadrenergic, azadrenergic, NPY, µopioid, others) ganglion cell activity .'" In the nose, allergen challenge, histamine, and oral capsaicin challenge stimulate parasympathetic reflexes .11.11,6' These cholinergic influences induce glandular secretion of lysozyme and lactoferrin (serous cell markers) and regulate the mass of the secretions . Vagal nerve stimulation leads to goblet cell secretion in guinea-pig airways ." Five subtypes of muscarinic receptors have been cloned. 69 '70 M, and M3 binding sites have been localized on submucosal glands, epithelium and smooth muscle using radioligand binding studies ." ," In situ hybridization has recently been introduced to determine which cells express each of the muscarinic genes. Functional studies indicate that M 3 receptors are most important for stimulating glandular secretion ." Both serous cell and mucous cell exocytosis is stimulated by muscarinic agonists, but VIP stimulates more serous than mucous secretion in human nasal mucosa in vitro ." VIP is also a potent dilator, and may induce vasodilation and vascular permeability in vivo .

receptors and mucous cells [3-adrenergic receptors' ` •75' 76 since secretions obtained from the submucosal gland ducts increase in viscosity after [3-adrenergic stimulation but decrease after a-adrenergic stimulation ." Electric field stimulation of human bronchi could not be inhibited by antagonists of the a- and (3adrenergic receptors ." Thus, the a-adrenergic and to a lesser degree the (3-adrenergic branches of the adrenergic nervous system may have physiological importance in the regulation of glycoconjugate release in the lower respiratory tract and may contribute to changes in rheology of mucus .

Non-adrenergic non-cholinergic (NA NC) secretion Vasoactive intestinal peptide (VIP) . VIP is predominantly localized with acetylcholine in postganglionic parasympathetic nerves . Using human nasal mucosa in vitro, VIP stimulated serous cell lactoferrin secretion to a greater extent than glycoconjugate secretion .73.77 VIP may inhibit glycoconjugate and lysozyme secretion in vitro from bronchial tissues obtained from non-bronchitic patients, whereas glycoconjugate secretion from the bronchi of chronic bronchitic patients was not affected by VIP ." Because VIP antagonists are not available, it is not possible to determine the relative contributions of VIP to glandular secretion in vivo . Neuropeptide Y (NPY) . NPY is predominantly colocalized with norepinephrine in adrenergic nerves and is a potent and long acting vasoconstrictor ." Nerves containing NPY innervate arterial vessels including arteriovenous anastomoses in human nasal mucosa . 80 These sites have [' 2SI]NPY-binding sites ." NPY does not stimulate glandular secretion from human respiratory tissue in vitro ." This is consistent with the lack of [' 2SI]NPY binding sites in this location ." VIP may coexist with NPY in a population of sphenopalatine nerves in porcine nasal mucosa . 81 NPY may inhibit parasympathetic effects by acting upon autoreceptors on parasympathetic ganglion cells ."

Sympathetic nerve-induced secretion Alpha-adrenergic receptor activation in vitro causes a modest but consistent increase in tracheobronchial glycoconjugate release in all species tested, whereas Radrenergic receptor activation results in a mote short lived and less remarkable increase in glycoconjugate release .7,74 Sympathetic nerve fibres have been identified around glands in the lower airways of humans and ferrets . Activation of the stellate ganglion results in increased mucus release . The distribution of adrenergic receptors on submucosal glands may differ so that serous cells contain predominantly a-adrenergic

Tachykinins The neurotransmitters of the sensory system include tachykinins, a group of neuropeptides with a common carboxy-terminal sequence (Phe-X-Gly-Leu-MetNH2 where X is an. hydrophobic amino acid) . Three different tachykinin receptor subtypes (NK,, NK2 and NK3) have been cloned ."-" They belong to the rhodopsin receptor supergene family and are associated with G-proteins and phosphoinositol hydrolysis .

Mucus Secretion and Inflammation

These receptors have been defined pharmacologically ." SP has the highest affinity for the NK, receptor, NKA for the NK 2 receptor, and NKB for the NK3 receptor. SP is more potent than NKA at stimulating glycoconjugate release from cat and human airways (Table 1) .87-89 [125I]SP but not [t25 I]NKA bound to submucosal glands and epithelium in human nasal mucosa90 and human tracheobronchial glands" suggesting that NK, receptors are involved . Goblet cell secretion is stimulated in guinea-pig trachea by capsaicin-sensitive sensory nerve axon responses via NK, receptors ." Tachykinins may be degraded by neutral endopeptidase 24 .11 ('enkephalinase'), neutral endopeptidase 24 .15, angiotensin converting enzyme, aminopeptidase M and other proteases . Alterations of these proteases may regulate the effects of neuropeptides . 93,94 The effects of SP on glandular secretion, bronchial smooth muscle contraction, vascular permeability, and neurogenic inflammation are potentiated by inhibitors of neutral enkephalinase (thiorphan, phosphoramidon) 89,95,96 and serine esterase inhibitors (aprotinin).89 Reduction of neutral endopeptidase activity by viral infections," ," cigarette smoke, 99 ozone, 10° and toluene diisocyanate (TDI) 101 also accentuate peptide-induced events and neurogenic inflammation . It is hypothesized that release of peptides into areas with reduced NEP activity leads to enhanced bronchoconstriction, glandular secretion and vascular permeability .", ' Calcitonin gene-related peptide (CGRP) . Calcitonin gene-related peptide (CGRP) is commonly colocalized with tachykinins in sensory nerves . CGRP binding sites were found on arterial vessels but not on glands or epithelium . 102 a o3 CGRP does not stimulate glandular secretion from human tissues in vitro .

85

peptides share a common C-terminal sequence (GlyAsn-X-Trp-Ala-Val-Gly-His-Leu-Met) . This sequence contains the receptor binding and biological activities ." Only a single receptor type has been identified . 105 Specific [125I]GRP binding sites are found on submucosal glands and the surface epithelium in human 10' and guinea-pig nasal turbinates, 107 cat 104 and human bronchi (Fig . 2) . In human nasal mucosa, GRP nerve fibres were seen around submucosal glands .'o6 GRP appears to be a neurotransmitter of the efferent vagal system, 108 and the sensory nervous system.63,'09 GRP is also present in neuroendocrine cells,' os,no nerve fibres, 102,106,111 in some extratracheal ganglion cells, 104 and can be recovered from bronchoalveolar lavage fluid from humans ."' GRP causes mucous glycoconjugate release from feline tracheal explants 104 and glycoconjugate and serous cell lactoferrin secretion by human nasal turbinates in vitro .' 06 In guinea-pig nasal mucosa in vivo, GRP causes submucosal gland alkaline phosphatase and total

Gastrin releasing peptide (GRP)

GRP is a 27 amino acid long mammalian peptide, which has functional similarities with the amphibian peptide bombesin (14 amino acids) . These and other

C

Table 1 Minimal concentrations of substance P reported to cause glandular secretion (modified from reference no . 7) Species

Parameter

Dog

Mucin secretion

Ferret

Mucin secretion Serous cell degranulation

Cat

Mucin secretion in vitro Mucin secretion in vitro Submucosal gland contraction

Human

Fucose secretion Hexose secretion Protein secretion

nM 0 .1 10 1000 1 100 0 .001 1 100 1000

Fig. 2 ["SI]GRP binding sites on human tracheobronchial submucosal glands . A, Bright-field image of submucosal glands (G) . Bar= 100 gm . B, Dark-field autoradiographic image of the same field showing silver grains which represent ['ZSI]GRP binding sites . Silver grains are seen over submucosal glands . C, Dark-field autoradiographic image in the presence of excess GRP . Excess GRP reduced the density of the silver grains over submucosal glands (G) .

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protein secretion, while vascular permeability is unaffected . 107 Bombesin stimulates serous and mucous cell secretion from human nasal mucosa in vivo . 13 Bombesin acts as a vasoconstrictor on canine trachea, 79 and a bronchoconstrictor in guinea-pigs."' Cigarette smoke increases bombesin content in hamster pulmonary neuroendocrine cells ."' GRP may regulate the growth of normal fetal and adult surface epithelium and small cell lung carcinoma cell lines 10 ' suggesting that these peptides may play a role in mucosal growth and homeostasis . These results indicate that GRP and GRP-like peptides are present in neuroendocrine cells, ganglion cells, and nerve fibres in various species, and that the GRP or C-terminal derivatives can act upon GRP/bombesin receptors on the epithelium and submucosal glands to induce mucosal secretion.

Endogenous opioids The endogenous opioids include the enkephalins, the endorphins, and the dynorphins . They are derived from genetically distinct precursors and interact with unique receptors."' Nerves containing endogenous opioid-immunoreactive material have been found in guinea-pig airways around submucosal glands ."' The anatomical origins of these neurons and their functions remain ambiguous . Dynorphin (kappa-receptor) stimulates feline glycoconjugate release ."' Rogers and Barnes1' found that morphine inhibited capsaicin induced glandular secretion, suggesting that g-opioid receptors inhibited sensory nerve axon responses or parasympathetically mediated gland secretion . Modulation of these neural sites by µ-opioid receptors has been confirmed in vitro . 120

Inflammatory cells Inflammatory cells accumulate in the airways in a variety of diseases . Macrophages and neutrophils are important in fighting infections . Mast cells and eosinophils appear to play pathological roles in asthma . These inflammatory cells and the many mediators they release are important not only for host defence, but also tissue damage which may occur during an exuberant inflammatory response . They may also set in progress mechanisms of tissue repair and healing . Mast cells Activation of mast cells results in the production of a variety of mediators capable of stimulating smooth muscle, mucus secreting cells and vessels . These mast cell mediators induce and amplify local inflammatory responses 12 ' and play key roles in the early phase of allergen-induced bronchospasm . Cross-linking of surface IgE molecules or stimulation by opiates, basically charged molecules, and other non-specific stimuli leads to the release of a large number of preformed (histamine, tryptase, chymase, heparin) and newly synthesized (PAF, PGD 2, LTC4/ D4, LTB4, free radicals, bradykinin) mediators . Human mast cell degranulation leads to glandular secretion in vivo and in vitro .','," Chymase, but not tryptase, stimulates secretion from cultured bovine airway serous cells."' Histamine induces vascular permeability, smooth muscle contraction, and sensory nerve stimulation . In the human trachea in vitro, histamine is a secretagogue . 2s, "23 However, it does not stimulate secretion from human nasal turbinates in vitro ." In the nose, histamine may induce glandular secretion via cholinergic parasympathetic reflexes . 22,66,67

Bradykinin

Neutrophils

Bradykinin is generated from plasma kininogens by activated Hageman Factor, kallikrein and mast cell kininogenase during inflammation and is present in secretions after allergen challenge and during viral infections ." Bradykinin stimulates vascular permeability by directly stimulating B2 receptors on vessels,", " and also depolarizes nociceptive sensory nerves ." in vitro, bradykinin stimulates mucoglycoconjugate secretion from human nasal mucosal fragments, but does not affect serous cell secretion ." These effects on secretion may be secondary to the generation of arachidonic acid metabolites ." , "' The ubiquitous nature of bradykinin's generation, and its ability to stimulate further inflammatory pathways indicate that it may play a major role in generating vascular responses and amplifying inflammation after tissue injury .

Acute inflammation is characterized by accumulation of neutrophils in the tissue . Neutrophils can be detected in the mucosa or bronchial washings from patients with sputum production including asthmatics, chronic bronchitics, cystic fibrosics, and bacterial bronchitics . 4°,111,126 Thompson et al 117 found a positive correlation of percent neutrophils in bronchoalveolar lavage fluid with sputum production and abnormalities in pulmonary function tests among chronic bronchitic subjects . The routes leading to accumulation of neutrophils in these diseases are not completely understood, but the airway epithelium, macrophages and mast cells are capable of releasing LTB 4 and 15hydroxyeicosatetraenoic acid (15-HETE) . 12" 0 Both are neutrophil chemoattractants (Table 2) . Bronchoalveolar lavage fluid from patients with bacterial pneumonia contains LTB4, activated complement

Mucus Secretion and Inflammation Table 2 Arachidonic acid metabolites produced by airway cells (ng from 10 6 cells) (modified from reference no . 7) PGD, Mast cells Alveolar macrophages Neutrophils Eosinophils Airway epithelial cells

60 2

5-HETE 15 2.6

1

0 .5

LTB,

LTC,

< 4 36 50 2-6 1

20 1 7 40-70 1

(C5a), and an additional unidentified neutrophil chemoattractant . LTB4 concentrations in the BAL were nearly seven-fold higher than normal controls ."' Neutrophils generate LTB 4 in preference to other arachidonic acid metabolites. The azurophilic granules of neutrophils contain enzymes such as elastase, cathepsin G and myeloperoxidase . The specific granules contain lactoferrin and a vitamin B 12 binding protein . Lysed human neutrophils and zymosan-activated neutrophils release various factors which cause increased glycoconjugate release from human bronchi ."' Human neutrophil elastase (HNE) and cathepsin G which may be involved in stimulating glycoconjugate release and increase the number of mucous producing cells in the airways . HNE increases the release of glycoconjugates from human nasal and bronchial explants (personal observations), feline tracheal explants (personal observations), rodent tracheal explants, 133 a bovine serous cell line, 114 and cultures of canine,' 35 feline,' 36 and rodent surface cells." HNE releases glycoconjugates attached to the surface epithelium 133 and stimulates exocytosis from submucosal serous cells, 114 suggesting that HNE has different effects on the surface and the submucosal secretory cells . Cathepsin G stimulates glycoconjugate release from feline tracheal epithelial cell lines 136 and bovine submucosal serous cell cultures ."' The effect of HNE can be blocked by inhibitors of HNE activity,"'-"' and by blocking endogenous eicosanoid production in the airways (J . Lundgren, personal observation) . HNE causes the release of eicosanoids from airway epithelial cultures ."' Thus, HNE stimulate mucus secretion indirectly by stimulating the production of eicosanoids in the airways which are secretagogues . Experimental intratracheal instillation of supernatants from lysed neutrophils causes an increase in the number of goblet cells in the peripheral 138 and central 139 airways of hamsters and in the tracheas of rats . 140 HNE causes an increase in goblet cell number in both hamster' 38, ' 39 and rat 14° airways . Pretreatment of rats with dexamethasone before instillation of neutrophil products prevents the accumulation of inflammatory cells and the increase in goblet cell number . 140 HNE inhibitors such as eglin-C and chloromethyl ketone prevent the increase in goblet cell number in the peripheral airways of hamsters . 141

87

Eosinophils Eosinophils are involved in the host defence against some types of parasites, and are characteristically present in allergic diseases . Eosinophils have specific granules which contain four distinct proteins : eosinophil peroxidase (EPO) ; eosinophil cationic protein (ECP) ; eosinophil derived neurotoxin (EDN); and major basic protein (MBP) . 142 Eosinophil protein X may be a fifth protein of the granules or may be EDN . One-million eosinophils contain approximately 15 µg of EPO, 5 pg of MBP and 80-800 ng of ECP . `Small' granules contain arylsulfatase and acid phosphatase . The eosinophil membrane contains lysophospholipase, the Charcot-Leyden crystal protein . Eosinophils are recruited during the asthmatic late phase response . 45 There is a selective increase in the number of eosinophils and solubilized eosinophil granule protein in bronchoalveolar lavage fluid during the late phase reaction ."' The number of eosinophils in bronchoalveolar lavage fluid correlates with the severity of the asthmatic attack .144 Glucocorticoids reduce ECP levels in nasal lavage fluid during allergic late phase reactions .' 45 Eosinophil products may destroy or otherwise influence the surface epithelium . 14' EPO causes ciliostasis and damages the surface epithelium of guineapig tracheal epithelium . The toxic effects of EPO are markedly potentiated by the addition of H2O, and halide . MBP (10-100 gg/ml) causes ciliostasis and exfoliation of the surface epithelium, and epithelial chloride secretion and passive water secretion . 141 MBP and, to a much lower degree, ECP, cause histamine release from mast cells and human basophils by an IgE-independent mechanism ." EDN is ineffective . ECP does not affect cilia function but causes exfoliation of the tracheal epithelium after a lag period of 6 h . ECP stimulates the release of glycoconjugates from feline and human lower airway explant cultures, and serous cell lactoferrin release from human bronchi .149 MBP, however, inhibits glycoconjugate release from feline airways . 14' Eosinophils generate large amounts of LTC 4 and PAF 7, ' 43 which can also influence secretion .

Monocytes and macrophages Accumulation of mononuclear cells in the airways are usually the predominant cell in chronic inflamed airways. Macrophages are able to kill certain microorganisms and act as antigen presenting cells in the specific cellular and humoral immunological system . Furthermore, both monocytes and macrophages release a molecule with a molecular weight of 2000 which stimulates glycoconjugate release .150 "5' This socalled monocyte/macrophage mucus secretagogue (MMS) is produced and released upon activation of



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J . D. Lundgren, J. N . Baraniuk

the cell, whereas mucus secretagogues from neutrophils (e .g . HNE) and eosinophils (ECP) are stored intracellularly . Alveolar macrophages generate LTB 4 (Table 2) . Arachidonic acid metabolites Arachidonic acid metabolites are intimately involved in many cellular functions including secretion production . Arachidonic acid is released from membrane phospholipids by the actions of phospholipase A 2 (Fig . 3) . Arachidonic acid is metabolized by cyclooxygenase, 5-lipoxygenase or other enzymes . The spectrum of arachidonic acid metabolites formed by any given cell is a reflection of the restricted number of metabolizing enzymes expressed by that cell . Thus, a cell may be capable of generating the products of either only a single, or multiple pathways . The cyclooxygenase pathway gives rise to prostaglandins and thromboxanes . 5-Lipoxygenase is produced by bone marrow derived cells and not epithelial or other resident respiratory cells . Therefore, the products of 5-lipoxygenase should be limited to infiltrating leukocytes . However, transcellular transfer of LTA, or other intermediates may occur . The lipoxygenase pathway leads to production of mono- and di-hydroxyeicosatetraenoic acids (HETEs), lipoxins and leukotrienes . Eicosanoids may mediate some of the effects of PAF,' 23 .152 neutrophil elastase, protein kinase C activators, oxygen metabolites, 153 prostaglandin generating factor of anaphylaxis, 154 cotton bract extract . . . and bradykinin . 51,156 Lipoxygenase products appear to play a key role in regulating both baseline and mediator stimulated mucous glycoconjugate release (Table 3) . The peptidyl leukotrienes are the most potent eicosanoids, being active in picomolar concentrations . Mono-hydroxyeicosatetraenoids are active in the

Glucocorticoids

Receptors

Ir PLA2

V

C

Lipocortin

Arachidonic Acid

PAF

WOOO~ Cyclooxygenase

Lipoxygenase

Prostaglandins Thromboxanes

Leukotrienes HETES Lipoxins

Fig . 3 Phospholipase A2 activity releases arachidonic acid, the substrate for cyclooxygenase and lipoxygenase enzymes which produce potent secretagogues and inflammatory agents . Glucocorticoids induce lipocortin which can inhibit PLA2 activity .

nanomolar range . Prostaglandins stimulate glycoconjugate release in micromolar concentrations ."' Prostaglandins may regulate other epithelial functions such as the chloride secretion induced by bradykinin . 156 Inhibitors of the lipoxygenase pathway and inhibitors of the whole eicosanoid metabolism [eicosatetraynoic acid (ETYA) and nordihydroguaiaretic acid (NDGA)] inhibit baseline glycoconjugate release ."' Inhibition of the cyclooxygenase pathway by either indomethacin in human bronchi 158 or ibuprofen in feline trachea 152 leads to increased baseline glycoconjugate release . The quantities of HETEs produced in unstimulated human bronchial explants are sufficient to cause an enhancement of glycoconjugate release when added exogenously . 159 Baseline secretion of glycoconjugates is likely induced by lipoxygenase products .", "' Glucocorticoids block eicosanoid generation and reduce baseline glycoconjugate secretion ." 165

Platelet activating factor Platelet activating factor (PAF) is derived from membrane phosphatidylcholine after the sn-2 fatty acid (usually arachidonic acid) is cleaved by a phospholipase A, isoenzyme and substituted with acetate .' 66 PAF is a potent molecule with a wide range of actions and may play a key role in the generation of the late phase response ." Inhalation of PAF in healthy men causes prolonged airway hyperreactivity . 161 Intratracheal instillation or intravenous injection of PAF causes inflammation in airway tissue, including oedema and accumulation of eosinophilic granulocytes, as well as bronchoconstriction, mucus secretion and airway hyperreactivity .' 68-17o A selective PAF receptor antagonist (WEB-2086) attenuates the late phase response and the accumulation of eosinophils after allergen challenge of sheep and guinea-pigs .", "' PAF causes release of glycoconjugates in vivo and in vitro in rodents, 170,171 cats,"' and humans . 111,152 The inactive analog lyso-PAF did not stimulate secretion 123 •' 7 'and the PAF receptor antagonist Ro 19-3704 partially blocked PAF-induced glycoconjugate release in human airways, 123,112 and totally blocked the response using feline 112 and rodent airways . '7 ' PAF may cause an increase in glycoconjugate release due to endogenous stimulation of lipoxygenase metabolites . 121,111,111 PAF stimulates both the cyclooxygenase and the lipoxygenase pathway of the eicosanoid metabolism in feline tracheal explants, and Ro 19-3704 inhibits the PAF-induced release of the peptidyl leukotrienes . 172 Adler et al"' noted that the combined cyclooxygenase and lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) was able to inhibit the PAF-induced increase in glycoconjugate release, and this observation has subsequently been

Mucus Secretion and Inflammation

Table 3

Lowest effective doses for eicosanoids in vitro and in vivo

Agent

Dose

Species

System

PGF2a

100 µM 200 nM 10 breaths 1 mm 100 µM 100 µM 1 nM 6 pmol by aerosol I nM I nM I µM 40 pM InM No effect 600 nM 14 nmol i .a . 40 pM 1nM

Human Human Human Human Cat Human Dog Human Human Cat Human Human Cat Cat Dog Human Human

In In In In In In In In In In In In In In In In In

PGAZ 15-HETE 12-HETE 5-HETE LTB, LTC,

LTD,

89

vitro vitro vivo vitro vitro vitro vivoa vitro vitro vitro vitro' vitro vitro vivob vivob.0 vitrob vitro

Reference no . 158 190 36 158 163 158 191 158 158 163 192 193 194 194 195 192 193

'Inhibited by atropine. :Inhibited by the leukotriene antagonist FPL55712 . Inhibited by vagus nerve section, hexamethonium, or atropine .

reproduced in feline and human airways . 123,152 Indomethacin 123 and ibuprofen'S 2 enhanced PAF-induced glycoconjugate release . On the other hand, an inhibitor (L-651,392) 152 of the 5-lipoxygenase pathway and two different specific LTD, receptor antagonists (L660,711) 152 and (LY 171883) 123 inhibited the PAFinduced enhancement of glycoconjugate release . Thus, PAF may cause glycoconjugate release from the lower airways by stimulation of endogenous lipoxygenase product formation including the peptidyl leukotrienes . Phospholipase A 2 Phospholipase A2 acts upon phospholipids to release arachidonic acid and the precursor of PAF .' Therefore, this activity plays a key role in cell activation . Knowledge about the influence of PLA 2 isoenzymes on various physiological and pathophysiological functions in the airways has been limited by the lack of specific and potent inhibitors . The products of PLA 2 s actions have been described- above . Glucocorticoids induce lipocortin which inhibits PLA 2.' 63,165,173 A nonapeptide of lipocortin, `antiflammin', 14 contains the active sequence . Lipocortin is present in alveolar macrohages and in bronchoalveolar lavage from patients on glucocorticoid therapy . "5,1'6 Incubation of feline tracheal explants with dexamethasone produces a slow induction of lipocortin over a 24-h period ."' This is followed by a subsequent reduction in glycoconjugate secretion .162,163,165,177,178 The addition of monoclonal antilipocortin antibodies can totally prevent the decrease in glycoconjugate release after dexamethasone incubation with feline airways . 161 Inhibition of PLA 2 in feline airway cultures with 4-bromophenacyl bromide causes inhibition of baseline glycoconjugate release

(R. D . Rieves, personal communication) . Additional mechanisms to lipocortin induction may contribute to the antiinflammatory effects of glucocorticoids .' 1¢181 Protein kinase C Protein kinase C (PKC) plays an important role in intracellular signal transduction . Activation of many receptors leads to activation of phospholipase C which hydrolyses phosphatidylinositol to generate inositol phosphates (1P3 and derivatives) and diacylglycerol (DAG) generation . IP3 release leads to increases in cytosolic Ca" concentration and Cal'_ calmodulin mediated secretion . DAG and its analogues such as phorbol myristate acetate (PMA) and mezerine activate PKC . PKC stimulation activates cells in exocrine and endocrine glands in the gastrointestinal tract, the saliva system and the thyroid gland."' In respiratory tissues, incubation with PMA enhances the release of chloride ions, stimulates airway smooth muscle contraction, 183 and leads to dosedependent chronic glycoconjugate secretion . 114 The PKC receptor antagonists H-7 and sphingosine partly inhibit the PMA effect . PKC activation may cause chronically increased glycoconjugate release from airways in vitro due to stimulation of the eicosanoid metabolism ." The chronicity of these changes may have relevance in pathological states and secretory cell hypertrophy and hyperplasia . Management of mucus hypersecretion This review has concentrated on the modes of action of increase in mucus production and it is not the intention here to describe the management of bronchorrhea since little clinical information is available . In general, management of patients with sputum

90

J . D . Lundgren, J . N . Baraniuk

production involves : (1) accurate diagnosis ; (2) avoidance of precipitating causes; and (3) therapies directed at the sources and clearance of the secretions .' While the first two points will not be discussed further . Therapeutic approaches to reduce mucus release include antagonism of the effects of neurotransmitters and inflammatory mediators, and alterations of mucus viscoelastic properties . Inhibition of the muscarinic receptor by atropine may decrease sputum production in patients with chronic bronchitis ."' Ipratropium bromide, which is non-absorbable, may be more beneficial since it does not decrease the clearance of mucus, a side-effect of atropine . Beta-adrenergic agonists may increase the clearance of mucus from the airways, 18 ' by dilating the bronchi or stimulating ciliary function . However, J3agonists may stimulate the secretion of more viscous mucus from the submucosal glands ." Receptor antagonists for neuropeptides are not yet available for clinical use . The most effective route for blocking the effects of inflammatory mediators is to prevent the accumulation of inflammatory cells . For infections this is most effectively done by specific antimicrobial chemotherapy . Erythromycin inhibits the release of glycoconjugates from human bronchi in vitro in a dosedependent manner in concentrations from 10 -5 to 10 - 'm, and the inhibition of glycoconjugate release was further enhanced by exposure of the explants to glucocorticoid . 187 In asthma, glucocorticoids may inhibit the accumulation of inflammatory cells in airways 16 ' and may directly inhibit the release of mucus from airway cells . Glucocorticoids may inhibit the stimulatory effect of mucus secretagogues which act via receptors coupled to PLA 2 which generated endogenous eicosanoids and PAF . 165 However, no clinical study is available demonstrating a beneficial effect of glucocorticoids on mucus release in vivo . The effect of cromoglycate, an inhibitor of mast cell degranulation, on bronchorrhea is minor . Specific receptor antagonists will allow better definition of the neural and cellular pathways that lead to sputum production . Potential pathways to target include : (1) sensory nerve axon responses of neurogenic inflammation using NK„ NK2 , NK3 , CGRP, and other neuropeptide antagonists ; (2) cholinergic and non-cholinergic parasympathetic reflex pathways using specific M„ M 2 , M 3 , and VIP antagonists ; (3) subsets of tracheobronchial ganglion cells using drugs active upon inhibitory autoreceptors ; (4) central sensory-parasympathetic reflex pathways ; (5) non-adrenergic sympathetic fibres and NPY ; (6) lipid mediators released by the actions of PLA 2 and other enzymes including PAF, prostaglandins, peptidyl leukotrienes, various HETEs, lipoxins, and others through the use of specific antagonists or enzyme inhibitors ; (7) bradykinin generation and action upon

B 2 receptors ; (8) modulation of intracellular enzymes and regulatory proteins ; (9) inhibition of proinflammatory cytokine production, release or action by Tlymphocytes, macrophages, and other cells ; (10) identification of leukocyte chemoattractants ; (11) antagonism, inhibition, or inactivation of leukocytic proteins such as HNE and ECP ; (12) free radical scavenging; and (13) administration of neutral endopeptidase to reduce the actions of inflammatory peptides (Table 4) . In short, it will be necessary to : (1) understand the basic mechanisms leading to mucus generation in acute infectious bronchitis, chronic bronchitis due to cigarette smoke and other pollutants, acute allergic asthma, non-allergic asthma, and other clinicopathologic syndromes; (2) identify the relevant secretagogues for each pathologic syndrome ; (3) administer specific therapy directed at secretory cells, inflammatory cells and their mediators ; and (4) remove or modify the inciting factors . The clearance of mucus from the lower airways may be enhanced by (3-agonists as mentioned above . Aerosolized saline, chest physiotherapy and/or postural drainage are effective adjunctive therapies ."' Decreasing the viscosity of mucus by mucolytic agents such as N-acetylcysteine may be useful ; however, this drug has been shown to be effective only in patients with cystic fibrosis . DNase may be helpful in decreasing the viscosity of mucus in cystic fibrosis patients ." Reductions in the production and release of mucoglycoconjugates may be possible with specific inhibitors of glycosyl transferases .' 89 Of these treatment options, glucocorticoids and other antiinflammatory agents will be expected to have the most dramatic effects since the basic, underlying pathological processes will be addressed . Combination therapy with, for example, leukotriene and neurotransmitter antagonists, may be necessary . Novel therapeutic advances may be conceived as more is learned about the neural control of secretion, the roles of inflammatory leukocytes and their mediators, and the regulation of secretory cell differentiation and proliferation . However, relevant models of human mucus production in vivo are required to effectively study these issues ." Conclusion Mucus is a complex mix of proteins, water and ions . It is the product of a complex integrated mucosal system . In health, nerves and arachidonic acid metabolites probably play key roles in maintaining baseline levels of secretion which is a crucial component of the non-immunological host defence system . In contrast, increased mucus production may be harmful to the host . After acute mucosal injury or chronic inflammation, nerves, infiltrating cells and activated resident cells release a host of secretagogues ranging from

Mucus Secretion and Inflammation Table 4

91

Sources of secretagogues

Source

Secretagogue

Potential therapy

Sensory nerve Axon responses Parasympathetic reflexes

Substance P Gastrin releasing peptide Acetylcholine

?

Sympathetic nerves

Neuroendocrine cells Mast cells

Neutrophils

Eosinophil

Macrophages

Epithelial cells T-lymphocytes Macrophages Mast cells Epithelium Fibroblasts Inflammation

VIP Norepinephrine a-receptors (i-receptors NPY GRP, bombesin SP LTC 4 PAF PGD, Histamine Chymase Kininase LTB, Elastase Cathepsin G LTC, PAF ECP LTB, Macrophage mucus secretagogue 15-HETE Cytokines' 96

Bradykinin

simple chemicals, to lipids, peptides and enzymes . These secretagogues may act via specific receptors on secretory cells . The wide variety of secretagogues suggests that secretory cells have many types of receptors so that they can respond to neural, humoral, inflammatory, and other signals of injury . Which secretagogues are most important in causing mucus hypersecretion is unknown at present, since our knowledge of the factors causing increases in mucus release are largely derived from in vitro models . Drugs designed to inhibit the action of these secretagogues are needed for this purpose . However, it appears that mucus hypersecretion in asthma is due to the production of eicosanoids, PAF, histamine, and eosinophil cationic protein, and the release of neurotransmitters . In chronic bronchitis, small airways disease and acute bronchitis, neutrophil products including elastase, and macrophage products are involved . Although our current therapeutic options for reducing mucus production are limited, research to identify which of the myriad of secretagogues are most important in causing hypersecretion should lead to the development of practical pharmacological agents which will interfere with secretion from mucous, serous and goblet cells .

Non-selective muscarinic antagonists Receptor specific muscarinic antagonists ? Serous secretagogue? Mucous secretagogue?

Antihistamines ? Bradykinin antagonist

7

v 7

Antagonist

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Date received : 25 February 1991 Date revised : 17 April 1991 Date accepted : 15 July 1991