I. Introduction

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Asthma is a complex chronic inflammatory disease of the airways that involves the activation of many inflammatory and structural cells, all of which release inflammatory mediators that result in the typical pathophysiological changes of asthma (Barnes, 1996a) (table 1). By inflammatory mediators, we mean cell products that are secreted and exert functional effects. We reviewed the mediators of asthma in 1988 (Barnes et al., 1988), but since then there have been major advances in our understanding of the mechanisms of asthma and the role of inflammatory mediators. There is now greater understanding of each mediator; in addition, novel mediators of asthma, such as the cytokines, have been identified. To date, >50 different mediators have been identified in asthma. Advances in this field have been greatly assisted by the development of potent and specific inhibitors that either block the inflammatory mediator receptors or inhibit mediator synthesis.

In writing this review, we have focused on new developments since 1988 and have emphasized studies in humans wherever possible. There is a vast and rapidly increasing body of literature on mediators of asthma; therefore, we have been forced to be somewhat selective. We have chosen to emphasize the mediators and effects that we think are most relevant to human asthma.

Many inflammatory cells are recruited to asthmatic airways or are activated in situ. These include mast cells, macrophages, eosinophils, T lymphocytes, dendritic cells, basophils, neutrophils, and platelets. It is now increasingly recognized that structural cells may also be important sources of mediators in asthma. Airway epithelial cells, smooth muscle cells, endothelial cells, and fibroblasts are all capable of synthesizing and releasing inflammatory mediators (Levine, 1995; Saunders et al., 1997; John et al., 1997). Indeed, these cells may become the major sources of inflammatory mediators in the airway, and this may explain how asthmatic inflammation persists even in the absence of activating stimuli.

There have been major advances in our understanding of the synthetic pathways involved in the synthesis of inflammatory mediators. Many of the key enzymes have now been cloned; in several cases, specific inhibitors have been developed that may have useful therapeutic effects. 5-Lipoxygenase (5-LO)^b inhibitors, which inhibit the synthesis of leukotrienes (LTs), have already been shown to have beneficial effects in the control of clinical asthma and are now available for clinical use (Israel et al., 1996).

Many inflammatory mediator receptors have now been cloned. The receptor for platelet-activating factor (PAF) was the first inflammatory mediator receptor to be cloned (Honda et al., 1991), and many inflammatory mediator receptors have been sequenced since then. The receptors for many inflammatory mediators have the typical seven-transmembrane domain structure that is expected for G protein-coupled receptors. However, receptors for cytokines and growth factors have markedly different structures, and usually two or more subunits are involved (Kishimoto et al., 1994). Receptor cloning has yielded a much better understanding of receptor function, because the receptors can be expressed in cell lines, allowing investigation of the "pure" pharmacological features of the receptor and enabling screening for drugs that interact with the receptor. This has been important in elucidating the signal transduction pathways involved in receptor function. Many signal transduction pathways have now been identified. For noncytokine mediators, inflammatory receptors are often coupled, through G proteins (G_q and G_i), to phosphoinositide (PI) hydrolysis, but it is increasingly recognized that other pathways may also be activated, including the complex mitogen-activated protein (MAP) kinase pathways that are involved in more long term effects of mediators. Cytokine receptors signal through complex pathways, including MAP kinases and other protein kinases, that result in the activation of transcription factors. Transcription factors regulate the expression of many genes, including inflammatory genes themselves.

The cloning of receptors has made it possible to study the factors regulating their expression. This may be of particular relevance in asthma, because the inflammatory state may alter the gene expression, translation, or function of receptors, thus affecting responsiveness to different mediators.

Inflammatory mediators produce many effects in the airways, including bronchoconstriction, plasma exudation, mucus secretion, neural effects, and attraction and activation of inflammatory cells. Although the acute effects of mediators have been emphasized, there is increasing recognition that mediators may result in long-lasting structural changes in the airways that are also mediated by the release of inflammatory mediators. These changes may include fibrosis resulting from the deposition of collagen, which is seen predominantly under the epithelium even in patients with mild asthma. The airway smooth muscle layer is also thickened in asthma, and this is likely the result of increases in the number of smooth muscle cells (hyperplasia) and increases in their size (hypertrophy) (Knox, 1994). There may be proliferation of airway vessels (angiogenesis) (Kuwano et al., 1993) and of mucus-secreting cells. There may also be changes in the innervation of the airways.

There are several lines of evidence that may implicate a mediator in asthma. Firstly, it may mimic features of clinical asthma. Secondly, the mediator may be produced in asthmatic patients. Thus, mediators or their metabolites may be detected in plasma (e.g., histamine), urine (e.g., LTE₄), or, more likely, the airways (in biopsies, bronchoalveolar lavage fluid, induced sputum, or exhaled air). However, this does not necessarily mean that the mediator plays any important role in asthma. The best evidence for the involvement of a mediator in asthma is obtained with the use of specific blockers. These may be drugs that block the synthesis of the mediators (e.g., 5-LO inhibitors) or drugs that block their receptors (e.g., antihistamines). Use of new and selective mediator blockers has enormously increased our understanding of the individual mediators and also of asthma itself. Although it is unlikely that blockade of a single mediator will be entirely effective in controlling asthma, there is accumulating evidence that some mediators are more important than others. PAF receptor antagonists are of no obvious clinical benefit in asthma (Kuitert et al., 1993), but cysteinyl-LT (cys-LT) receptor antagonists have considerable clinical effects (O'Byrne et al., 1997).

The role of a mediator in asthma may be difficult to assess when the mediator has a long term effect on airway function. It is easy to measure the effect of a mediator on airway smooth muscle, but it is more difficult to determine its effect on airway microvascular leakage and mucus secretion. It may be even more difficult to determine the role of a mediator on chronic inflammatory effects, such as airway smooth muscle proliferation and fibrosis, that may develop over many years. However, prevention of the long term consequences of asthmatic inflammation, such as irreversible airway narrowing, may be an important goal of asthma therapy, and it is necessary to devise methods to investigate how mediators may affect these long term consequences of asthma.

Asthma has a characteristic clinical pattern, and the histological appearance of asthma is very similar among patients, even when there are differences in asthma severity or in whether or not the asthma is allergic. However, it is likely that there are differing mechanisms of asthma among patients and that different patterns of inflammatory mediators are involved. This suggests that mediator antagonists would have different effects in different patients. This has already been observed in the use of anti-LTs, because some patients appear to have much better therapeutic responses than others. This might be related to polymorphisms of the 5-LO gene (In et al., 1997), but there might be differences that relate to the type of asthma. Patients with aspirinsensitive asthma are particularly helped by anti-LTs, consistent with a critical role for cys-LTs in this type of asthma. As more mediator antagonists become available, other patients who respond well to a particular antagonist may be identified and the heterogeneity of asthma may be revealed.

Although in the past much attention has been paid to acute inflammatory responses (such as bronchoconstriction, plasma exudation, and mucus hypersecretion) in asthma, it is being increasingly recognized that chronic inflammation is an important aspect of asthma (Redington and Howarth, 1997). This chronic inflammation may result in structural changes in the airway, such as fibrosis (particularly under the epithelium), increased thickness of the airway smooth muscle layer (hyperplasia and hypertrophy), hyperplasia of mucus-secreting cells, and new vessel formation (angiogenesis). Some of these changes may be irreversible, leading to fixed narrowing of the airways. These chronic inflammatory changes are mediated by the secretion of distinct mediators, although their role in asthma is still far from certain. These factors include cytokines and growth factors. Cytokines are a large group of protein mediators that play a critical role in determining the nature of the inflammatory response and its persistence. They play a key role in the pathophysiological changes in chronic asthma and are being increasingly recognized as important targets for treatment (Robinson et al., 1993c; Barnes, 1994a; Drazen et al., 1996).

Transcription factors are DNA-binding proteins that regulate the expression of inflammatory genes, including enzymes involved in the synthesis of inflammatory mediators and protein and peptide mediators. Transcription factors therefore play a critical role in the expression of inflammatory proteins in asthma, because many of these proteins are regulated at a transcriptional level (Barnes and Karin, 1997; Barnes and Adcock, 1998). These transcription factors include nuclear factor-B (NF-B) and activator protein-1 (AP-1), which are universal transcription factors that are involved in the expression of multiple inflammatory and immune genes and may play a key role in amplifying the inflammatory response. Other transcription factors, such as nuclear factor of activated T cells (NF-AT), are more specific and regulate the expression of a restricted set of genes in particular types of cell; NF-AT regulates the expression of interleukin (IL)-2 and IL-5 in T lymphocytes.

	II. Amine Mediators

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1. Synthesis and metabolism. Histamine is synthesized and released by mast cells in the airway wall and by circulating and infiltrating basophils. Although airway mast cells are likely to be the major cellular source of histamine in asthma, there is increasing evidence that basophils may be recruited to asthmatic airways and may release histamine in response to cytokine histamine-releasing factors (Schroeder and MacGlashan, 1997).

2. Receptors. Histamine has multiple effects on airway function that are mediated by specific surface receptors on target cells (Barnes, 1991). Three types of histamine receptors have now been recognized pharmacologically (Hill, 1990). Histamine receptors were first differentiated into H₁ and H₂ receptors by Ash and Schild in 1966, when it was found that some responses to histamine were blocked by low doses of mepyramine (pyrilamine), whereas others were insensitive. This classification was supported by the development of H₂ receptor-selective antagonists, such as cimetidine and ranitidine. Both H₁ and H₂ receptors have been cloned. Both have the seven-transmembrane domain motif typical of G proteincoupled receptors. A third histamine receptor subtype, termed H₃, has been described more recently; this receptor acts as an inhibitory autoreceptor in the central nervous system (Schultz et al., 1991).

3. Effects on airways. a. AIRWAY SMOOTH MUSCLE. Histamine stimulates PI hydrolysis in airway smooth muscle (Grandordy and Barnes, 1987; Hall and Hill, 1988; Daykin et al., 1993), and there is a close association of receptor occupancy, PI hydrolysis, and the contractile response, indicating that there are few or no "spare" receptors (Grandordy and Barnes, 1987). Histamine also increases the concentration of inositol-1,4,5-trisphosphate (IP₃) in airway smooth muscle, although the magnitude of the increase is less than with cholinergic agonists, which may reflect lower receptor density (Chilvers et al., 1989). In cultured human airway smooth muscle cells, histamine increases [Ca²⁺]_i via an increase in IP₃ levels (Hardy et al., 1996).

d. NERVES. In many species, the bronchoconstricting effect of histamine is partially mediated by a vagal cholinergic reflex and may be modulated by muscarinic receptor antagonists. In dogs, histamine increases the discharge of "irritant" receptors in vivo (A-fibers), and these effects are abolished by H₁ antagonists. However, in vitro measurements of single afferent fibers in guinea pig trachea show no evidence for activation of either A- or C-fibers by histamine (Fox et al., 1993

4. Role in asthma. a. RELEASE. Measurement of histamine in the circulation is complicated by the spontaneous release from basophils, and measurement of stable metabolites in the urine may not reflect release from mast cells in the airways. Several previous studies demonstrated elevations of plasma histamine concentrations in patients with asthma, at rest, after exercise, at night, and after allergen challenge, but these studies are difficult to interpret because of the likelihood of contamination from basophil release in the collected blood samples (Ind et al., 1983). It is possible that basophils from patients with asthma may be more "leaky" and that this may contribute to the higher concentrations measured in asthmatic patients. Studies of histamine infusions in normal volunteers have demonstrated that doses of histamine that yield the plasma concentrations reported in patients with asthma have marked cardiovascular effects, indicating that the higher levels seen in the blood of asthmatic patients are likely to be generated in vitro during storage and preparation of the plasma samples. The histamine released from the airways may increase plasma concentrations, but this may be overwhelmed by the contribution from circulating basophils. Sampling closer to the site of histamine release may overcome these problems. Venous sampling in the arm shows an increase in plasma histamine concentrations after mast cell degranulation in the skin of the arm, induced by SP (Barnes et al., 1986), but such sampling is not feasible in the airways. Measurement of histamine in bronchoalveolar lavage fluid is likely to provide a much more direct measurement of airway histamine release. There is evidence that histamine concentrations are elevated in bronchoalveolar lavage fluid of asthmatic patients, both at rest and after allergen challenge (Liu et al., 1991; Wenzel et al., 1988). The source of histamine is presumed to be mucosal mast cells, and the contribution of infiltrating basophils is unclear.

Serotonin [5-hydroxytryptamine (5-HT)] causes bronchoconstriction in most animal species, but interest in this mediator is minimal because it is not a constrictor of human airways and its relevance in asthma seems doubtful (Barnes et al., 1988).

2. Receptors. Multiple serotonin receptors have now been recognized, based on the development of selective antagonists and molecular cloning (Saxena, 1995). There are up to seven types of 5-HT receptors, each with several subtypes. Selective antagonists have now become available for clinical use, but few have been used in investigations of human airway cells or in the treatment of patients with asthma.

3. Effects on airways. Serotonin does not constrict human airway smooth muscle in vitro and may even have bronchodilating effects, although pulmonary vessels are constricted as expected (Raffestin et al., 1985). In animals, serotonin increases acetylcholine release from airway nerves, and this has been demonstrated in human airways (Takahashi et al., 1995). The receptor mediating this response appears to be a 5-HT₃ receptor (Takahashi et al., 1995). In guinea pig airways, serotonin inhibits nonadrenergic noncholinergic (NANC), neurally induced constriction resulting from tachykinin release via a 5-HT₁-like receptor localized to sensory nerve endings (Ward et al., 1994; Dupont et al., 1996). In humans, infused serotonin has no effect on airway function but may have an inhibitory effect on cough reflexes, possibly mediated by receptors on airway sensory nerves (Stone et al., 1993). Serotonin is a potent inducer of microvascular leakage in rodent airways, but it is not certain whether serotonin has this property in human airways. Serotonin has a blocking effect on sodium channels in human airway epithelial cells, but the receptor subtype involved has not been established (Graham et al., 1992).

4. Role in asthma. Plasma serotonin levels are reported to be elevated in asthma and are significantly related to asthma severity (Lechin et al., 1996). The source of serotonin is likely to be platelets, but the clinical relevance of this observation is unclear.

C. Adenosine

1. Synthesis and metabolism. Adenosine is a purine nucleoside that is produced by dephosphorylation of 5'-AMP by the membrane-associated enzyme 5'-nucleotidase and is liberated intracellularly by cleavage of the high energy bonds of adenosine triphosphate, adenosine diphosphate, and cyclic 5'-AMP. However, during hypoxia or even excessive cell stimulation, when the utilization of energy and oxygen exceeds the supply, 5'-AMP is metabolized to adenosine (Mentzer et al., 1975). This conversion is performed by extracellular 5'-nucleotidase. Adenosine release was originally demonstrated during myocardial hypoxia (Mentzer et al., 1975), although there is now evidence that all cells are capable of producing adenosine in times of energy deficit. Adenosine can be released by lung tissue in times of hypoxia, such as after allergen-induced bronchoconstriction, when the circulating levels of adenosine have been shown to be 3 times the base-line concentrations (Mann et al., 1986). Mast cells are a likely source of adenosine in this situation, because these cells have been shown to be capable of releasing adenosine in response to IgE cross-linking and other stimuli for mast cell activation (Marquardt et al., 1986).

2. Receptors. Three distinct subtypes of receptor have been characterized to date, based on biochemical, functional, and more recent cloning studies (Linden et al., 1991; Linden, 1994). These receptors include the A₁, A_2a, A_2b, and A₃ receptor subtypes. Interaction of adenosine with these receptors leads to either inhibition of adenylyl cyclase (A₁), stimulation of adenylyl cyclase (A_2a and A_2b) (Collis and Hourani, 1993), or activation of phospholipase C (A₃) (Ali et al., 1990). The A₁ receptor is expressed in lung tissue (Ren and Stiles, 1994) and, in particular, A₁ receptors have been identified on human epithelial cells (McCoy et al., 1995). The classification of adenosine receptors into A_2a and A_2b subtypes is based on distinct rank orders of potency of a range of agonists and antagonists and distinct nucleotide sequences of the two complementary deoxyribonucleic acids (cDNAs). A_2a, A_2b, and A₃ receptors are expressed in several tissues, including lungs, and in mast cells and fibroblasts (Linden et al., 1993; Auchampach et al., 1997; Ciruela et al., 1997; Shryock and Belardinelli, 1997; Fredholm, 1997).

3. Effects on airways. a. AIRWAY SMOOTH MUSCLE. Adenosine elicits little or no contraction of human bronchi from nonasthmatic subjects but potently constricts asthmatic airways in vitro (Björck et al., 1992). This constriction is blocked by histamine and LT antagonists and is therefore likely to be attributable to the release of mediators from mast cells in asthmatic airways. It is likely that the bronchoconstricting effects of adenosine are indirect, resulting from the activation of mast cell degranulation, because adenosine causes histamine release from mast cells (Church et al., 1986). Comparable results have been observed in vivo, where adenosine and AMP are able to elicit bronchoconstricting effects in atopic and asthmatic subjects but have no effect in normal subjects (Cushley et al., 1983). Furthermore, dipyridamole (an inhibitor of adenosine uptake into tissues) enhances adenosine-induced bronchospasm in asthmatic subjects (Crimi et al., 1988), an effect that can be inhibited by theophylline (a nonselective adenosine antagonist) (Cushley et al., 1984). The receptor mediating the bronchoconstricting effect of adenosine in asthma is not yet known. In rabbits, the A₁ receptor is a likely candidate, because tracheal strips from rabbits immunized with house dust mites are more responsive to adenosine and the adenosine A₁-selective agonist cyclopentyladenosine than are tracheal strips isolated from naive animals (Ali et al., 1994a). Furthermore, immunized animals are considerably more responsive to the bronchoconstricting effects of adenosine (Thorne and Broadley, 1994) and cyclopentyladenosine in vivo (Ali et al., 1994b; el Hashim et al., 1996). No bronchoconstricting effects of the A₃-selective agonist aminophenylethyladenosine have been found in rabbits (el Hashim et al., 1996) or guinea pigs (Hannon et al., 1995), although studies in rats have shown that aminophenylethyladenosine can elicit bronchoconstriction (Meade et al., 1996). The histamine-releasing effect of adenosine may involve the A_2b receptor, because this effect is sensitive to enprofylline (an A_2b receptor antagonist) (Feoktistov and Biaggioni, 1995). Certainly, there is clinical evidence showing that elevated levels of histamine can be demonstrated in plasma after the inhalation of AMP by atopic subjects (Phillips et al., 1990), and increased levels of histamine have been detected after the instillation of AMP directly into the airways (Polosa et al., 1995). Furthermore, the H₁ receptor antagonist terfenadine has a protective effect against adenosine-induced bronchoconstriction in asthmatic subjects (Rafferty et al., 1987).

4. Role in asthma. a. RELEASE. Increased levels of adenosine have been found in bronchoalveolar lavage fluid obtained from asthmatic subjects, compared with normal subjects (Driver et al., 1993), and, as discussed above, adenosine concentrations in plasma are higher in allergic patients minutes after allergen provocation (Mann et al., 1986). A₃ receptor expression is increased in asthmatic lungs, compared with lungs of normal subjects, although, because the A₃ receptor is expressed predominantly in eosinophils, this may be a reflection of eosinophilic infiltration (Walker et al., 1997).

	III. Lipid-Derived Mediators

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1. Synthesis and metabolism. Prostanoids are generated from arachidonic acid by two forms of COX (Mitchell et al., 1995). COX-1 is constitutive and is responsible for basal release of prostanoids, whereas COX-2 is inducible by inflammatory stimuli, such as endotoxin and proinflammatory cytokines, and its induction is inhibited by glucocorticoids. Both COX-1 and COX-2 are expressed in human lung (Demoly et al., 1997). Human airway epithelial cells basally express COX-1, whereas COX-2 is induced by IL-1 and TNF- (Mitchell et al., 1994; Newton et al., 1997b; Asano et al., 1997) and is enhanced by NO (Watkins et al., 1997). COX-2 is also induced in cultured human airway smooth muscle cells by proinflammatory cytokines and bradykinin (Belvisi et al., 1997; Pang and Knox, 1997a,b), and the formation of prostanoids is blocked by the selective COX-2 inhibitor L745,337 (Saunders et al., 1998). COX-2 expression is inhibited by dexamethasone in both epithelial cells and smooth muscle cells. The induction of COX-2 is regulated in part by NF-B, and this may also account for the inhibitory action of glucocorticoids (Newton et al., 1997a). There is no difference in the profiles of prostanoids formed by COX-1 and COX-2. In epithelial cells and airway smooth muscle cells, the predominant prostanoids are PGE₂ and 6-keto-PGF₁ (metabolite of PGI₂), whereas there is relatively little formation of Tx (Mitchell et al., 1994; Belvisi et al., 1997). Tx is formed from the intermediate PGH₂ by a distinct enzyme, Tx synthase, which has been cloned (Ohashi et al., 1992).

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2. Receptors. Several prostanoid receptors have now been cloned (Ushikubi et al., 1995; Pierce et al., 1995). Pharmacologically, prostanoid receptors are classified according to the prostanoid that causes selective activation; PGE₂ preferentially activates EP receptors, PGI₂ (prostacyclin) activates IP receptors, PGF₂ activates FP receptors, PGD₂ activates DP receptors, and Tx activates TP receptors (Coleman et al., 1994). Within each receptor type there may be distinct subtypes, many of which have been identified using selective ligands and cloning; the EP receptor has at least four subtypes, which are differentially expressed in different cell types. EP₁ receptors mediate activation responses and are involved in hyperalgesic responses, whereas EP₂ and EP₄ receptors mediate smooth muscle relaxation responses and EP₃ receptors modulate neurotransmitter release. In airway smooth muscle, several constrictor PGs (PGD₂, PGF₂, and 8-epi-PGF₂) appear to work through activation of TP receptors (Coleman and Sheldrick, 1989; Kawikova et al., 1996).

3. Effects on airways. a. AIRWAY SMOOTH MUSCLE. PGE₂ relaxes human airway smooth muscle in vitro via EP receptors (Knight et al., 1995). The relaxation response to PGE₂ in human airways is mediated by EP₂ receptors (McKenniff et al., 1988), but in animal airways an EP₁ receptor subtype is also involved (Ndukwu et al., 1997). Inhaled PGE₂ causes bronchodilation in normal subjects (Walters and Davies, 1982) but may cause constriction in patients with asthma because of activation of reflex cholinergic bronchoconstriction. Inhaled PGE₂ protects against exercise-, metabisulfite-, and allergen-induced bronchoconstriction in asthmatic patients, however (Melillo et al., 1994; Pavord et al., 1992, 1993). PGI₂ is less potent than PGE₂ in relaxing human airways in vitro (Tamaoki et al., 1993) and, in contrast to PGE₂, does not protect against histamine-induced contraction (Knight et al., 1995). Inhaled PGI₂ has little effect on airway function (Hardy et al., 1985).

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4. Role in asthma. a. RELEASE. Bronchoalveolar lavage studies have demonstrated increased concentrations of PGF₂, PGD₂, and TxB₂ in patients with asthma (Liu et al., 1990; Oosterhoff et al., 1995; Dworski et al., 1994; Smith et al., 1992). PGD₂ is the prostanoid present in highest concentration, and this is correlated with an increase in mast cell tryptase, indicating the likely mast cell origin of the mediator. After allergen challenge, there is an increase in PGD₂ and TxB₂ levels (Dworski et al., 1994). A urinary metabolite of Tx (11-dehydro-TxB₂) is increased in asthmatic subjects after challenge with allergen (Kumlin et al., 1992). COX-2 shows increased expression in the airways of asthmatic patients and is presumably induced by proinflammatory cytokines (Demoly et al., 1997). In peripheral leukocytes of asthmatic patients, there is increased expression of COX-1 and COX-2 mRNA (Kuitert et al., 1996).

b. EFFECTS OF INHIBITORS. Nonselective COX inhibitors, including aspirin and flurbiprofen, have little or no beneficial effect in challenge studies or in the treatment of clinical asthma, but this may be because they block production of both bronchoconstricting (PGD₂, PGF₂, and TxA₂) and bronchodilating (PGE₂ and PGI₂) mediators. Specific Tx synthase inhibitors have been developed for use in asthma. Ozagrel (ONO-046), a moderately potent orally active Tx synthase inhibitor, reduces airway hyperresponsiveness to cholinergic agonists when given orally or by aerosol, but the effect is very small and unlikely to be of clinical significance (Fujimura et al., 1990a

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c. CONCLUSIONS. Prostanoids are produced in asthmatic airways and appear to have several effects on the airways, including bronchoconstriction, plasma exudation, sensitization of nerve endings, and effects on inflammatory cells, which are mediated by prostanoid receptors. However, inhibition of their formation with COX or Tx synthase inhibitors or inhibition of TP receptors does not appear to benefit asthmatic patients. One possibility is that COX inhibitors, while blocking the formation of bronchoconstricting prostanoids (PGD₂, PGF₂, and TxA₂), also inhibit the formation of the bronchodilating PGs (PGE₂ and PGI₂), which may counteract these effects. Furthermore, isoprostanes may be formed in response to oxidative stress in asthma, and their formation occurs independently of COX function.

1. Synthesis and metabolism. LTs are potent lipid mediators produced by arachidonic acid metabolism in cell or nuclear membranes. They are derived from arachidonic acid, which is released from membrane phospholipids via the activation of phospholipase A₂. Arachidonic acid is subsequently metabolized by the enzyme 5-LO, to produce LTs. The free 5-LO enzyme is found in the cytoplasm and cannot metabolize arachidonic acid. However, after the free 5-LO has been activated, it is translocated to the nuclear membrane, where a membrane-bound protein termed 5-LO-activating protein stabilizes the translocated 5-LO, thus allowing the transformation of arachidonic acid into LTA₄ (Evans et al., 1991). Recently, a family of mutations of 5-LO genes have been reported in asthmatics. These are characterized by a variable number of tandem repeat segments in the promoter region, and they modify reporter gene transcription. This may account for differences in the susceptibility of patients to drugs modifying 5-LO activity (In et al., 1997). LTA₄ is further metabolized to LTC₄ (via the activation of LTC₄ synthase) or to LTB₄ (by LTA₄ hydrolase). After release into the extracellular environment, LTC₄ can be further metabolized to LTD₄ and LTE₄ by cleavage of the peptide side chain of LTC₄. Several types of airway cells, including mast cells, eosinophils, macrophages, neutrophils, and epithelial cells, can synthesize LTs in response to a variety of stimuli. LTB₄, synthesized predominantly by LTA₄ hydrolase in neutrophils, is an extremely potent activator of neutrophils, causing aggregation, chemotaxis, and degranulation (Ford-Hutchinson, 1991; Brain and Williams, 1990). LTC₄, LTD₄, and LTE₄ are the active constituents of what was once termed "slow reacting substance of anaphylaxis."

2. Receptors. The biological effects of LTs occur through their ability to stimulate specific receptors, which have been identified on several cell types. There are probably multiple receptors, although two major classes have been well characterized. The BLT receptors are activated by LTB₄ and to a lesser extent by 20-OH-LTB₄ and 12-(R)-hydroxyeicosatetraenoic acid (HETE). The BLT receptor is a 60-kDa plasma membrane protein (Miki et al., 1990) and has recently been cloned (Yokomizo et al., 1997). Cys-LTs act via cys-LT receptors, of which two types have been pharmacologically characterized. Cys-LT₁ receptors mediate all of the known airway effects of cys-LTs in human cells (Coleman et al., 1995). A second receptor type, the cys-LT₂ receptor, has been described on pulmonary veins, on the basis of responses to certain LT antagonists (Metters, 1995; Gorenne et al., 1996). To date, none of these LT receptors has been cloned.

3. Effects on airways. a. AIRWAY SMOOTH MUSCLE. Cys-LTs are very potent contractile agents for human bronchi in vitro, being approximately 1000 times more potent than histamine, and they elicit this effect via activation of cys-LT₁ receptors (Krell et al., 1990). There is a certain degree of tone in human airways in vitro, and this is partly mediated by cys-LTs, because it can be reduced by 5-LO inhibitors and by cys-LT₁ receptor antagonists (Ellis and Undem, 1994). The ability of cys-LTs to act as potent bronchoconstricting agents has also been demonstrated in vivo, both in normal subjects and in patients with asthma (Drazen, 1988). Inhaled LTD₄ also increases the maximal airway narrowing induced by inhaled methacholine (Bel et al., 1987), and LTE₄ induces airway hyperresponsiveness to inhaled histamine, an effect that may persist for several days (Arm et al., 1988; O'Hickey et al., 1991). LTB₄ has no direct effect on human airway smooth muscle and does not cause bronchodilation after inhalation in asthmatic patients, even when combined with PGD₂ (Black et al., 1989a). Cys-LTs may also stimulate airway smooth muscle proliferation (Cohen et al., 1995), although this has not yet been shown for human airway smooth muscle and may be secondary to release of Tx.

4. Role in asthma. a. RELEASE. In humans, elevated levels of cys-LTs have been detected in plasma, bronchoalveolar lavage fluid, and sputum samples obtained from asthmatics during spontaneous exacerbations of their asthma or after allergen exposure (Taylor et al., 1989; Wenzel et al., 1995). Furthermore, several groups have shown elevated levels of LTE₄ in the urine of allergic patients undergoing allergen exposure (Taylor et al., 1989; Drazen et al., 1992) and exhibiting nocturnal asthma (Bellia et al., 1996). In another study, the increase in urinary LTE₄ levels in allergic asthmatics parallels the bronchoconstriction and subsides with resolution of the airway response (Kumlin et al., 1992). Urinary LTE₄ levels are increased in aspirin-sensitive asthmatic patients (Kumlin et al., 1992), supporting the view that in these patients aspirin produces its effect by increasing cys-LT production. This is consistent with the recent demonstration of increased LTC₄ synthase expression in bronchial biopsies of aspirin-sensitive asthmatics (Sampson et al., 1997), and this may be linked to a polymorphism of the LTC₄ synthase gene (Sanak et al., 1997).

1. Synthesis and metabolism. PAF is an ether-linked phospholipid (1-O-alkyl-sn-glycero-3-phosphocholine) that was first described as a substance released from IgE-stimulated basophils. The synthesis of PAF occurs in a wide variety of inflammatory cells, including platelets, neutrophils, basophils, macrophages, and eosinophils (Barnes et al., 1989; Chung, 1992). The synthesis of PAF in inflammatory cells is generally via a two-step enzymatic pathway involving first the activation of phospholipase A₂, which cleaves a free fatty acid from ether-linked phospholipids (called plasmalogens) to yield lyso-PAF; under appropriate conditions, lyso-PAF can be acetylated, to form the biologically active PAF, by a rate-limiting enzyme that is termed acetyl transferase and is found in the cytoplasm of inflammatory cells (Barnes et al., 1989). Large amounts of PAF can be synthesized by several inflammatory cell types in the lung, including resident cells such as mast cells (Triggiani et al., 1991) and alveolar macrophages (Bratton et al., 1994).

2. Receptors. A PAF receptor has been cloned from human platelets and leukocytes and shown to be a typical G protein-linked receptor with seven transmembrane domains (Nakamura et al., 1993; Shimizu and Izumi, 1995). PAF receptors are expressed in animal and human lung (Shirasaki et al., 1994b). Recent evidence has shown that substitution of the Cys90, Cys95, or Cys173 residues in the PAF receptor with alanine or serine yields mutant receptors that do not bind PAF and are not expressed on the surface of cells but are found intracellularly (Le Gouill et al., 1997). The cell signaling pathways initiated by PAF interactions with its receptor are well characterized and include increases in [Ca²⁺]_i (Mazer et al., 1991), increases in IP₃ and diacylglycerol levels, and induction of cell cycle-active genes, such as fos, jun, and egr-1 (Mazer et al., 1991; Schulam et al., 1991). PAF also activates the transcription factor AP-1 in bronchial epithelial cells (Le Van et al., 1998). The PAF receptor undergoes homologous desensitization by phosphorylation of cytoplasmic tail sites in the receptor molecule (Takano et al., 1994), and related lipids such as PAGPC can also desensitize the classical PAF receptor (Mazer et al., 1998). PAF exposure, however, leads to an increase in PAF receptor mRNA levels, suggesting increased turnover of the receptor (Shirasaki et al., 1994a). Overexpression of the PAF receptor in transgenic mice results in airway hyperresponsiveness, which is attenuated by Tx, LT, and muscarinic antagonists (Nagase et al., 1997).

3. Effects on airways. a. AIRWAY SMOOTH MUSCLE. PAF has little direct effect on human airway smooth muscle contraction in vitro but may elicit constriction through the release of other mediators (Johnson et al., 1992). PAF produces acute bronchoconstriction when inhaled by patients with asthma (Barnes et al., 1989). PAF-induced bronchoconstriction is not inhibited by the H₁ receptor antagonist ketotifen (Chung et al., 1988) or the Tx antagonist GR32191B (Stenton et al., 1990b). However, PAF-induced bronchoconstriction can be inhibited by LT antagonists, including SKF 104353-Z (Spencer et al., 1991) and ICI 204,219 (Kidney et al., 1993), suggesting the involvement of LTD₄ in this response.

4. Role in asthma. a. RELEASE. Several groups have attempted to quantify the release of PAF in plasma or bronchoalveolar lavage fluid from asthmatic and allergic subjects, with conflicting results (Nakamura et al., 1987; Miadonna et al., 1989; Stenton et al., 1990a; Tsukioka et al., 1996). However, high levels of lyso-PAF were found in these studies and, because lyso-PAF is the precursor as well as the metabolite of PAF, this complicates the interpretation of these data. After segmental allergen challenge in asthmatic patients, high levels of lyso-PAF were correlated with increased acetylhydrolase and phospholipase A₂ activity (Chilton et al., 1996). PAF has also been detected in the plasma of patients exhibiting a late asthmatic response (Chan Yeung et al., 1991).

D. Other Lipid Mediators

1. Synthesis and metabolism. Several other lipid mediators, including hydroperoxyeicosatetraenoic acid (HPETEs), mono- and di-HETEs, and lipoxins (LXs), have been shown to have effects in the airways that are of potential relevance to asthma (Sigal and Nadel, 1991). Most of these substances are metabolic products of the 15-LO enzyme, which catalyzes the insertion of molecular oxygen at the carbon atom at position 15 in the arachidonic acid molecule (Samuelsson et al., 1987). 15-LO has been demonstrated in human tracheal epithelium (Hunter et al., 1985), eosinophils (Turk et al., 1982), endothelial cells (Hopkins et al., 1984), and monocytes (Conrad et al., 1992). Furthermore, immunohistochemical studies have revealed that 15-LO is expressed in airway epithelium and eosinophils (Sigal et al., 1992; Bradding et al., 1995). LXs (LO interaction products), of which the most prevalent is LXA₄, are produced by interactions between 15-LO and 5-LO or between 12-LO and 5-LO.

2. Receptors. Little is known regarding receptors for 15-LO products, and it is not clear whether there are distinct receptors for these HETEs and HPETEs. Specific LXA₄ receptors have been identified in murine and human cells (Takano et al., 1997; Fiore et al., 1994).

3. Effects on airways. Both mono- and di-HETEs are chemotactic for neutrophils and eosinophils (Johnson et al., 1985; Kirsch et al., 1988; Morita et al., 1990; Schwenk et al., 1992). In addition, 15-HETE has been demonstrated to induce LTC₄ release from mastocytoma cells (Goetzl et al., 1983) and mucus secretion from dog trachea (Johnson et al., 1985). LXs have been demonstrated to contract airway smooth muscle (Dahlen et al., 1987; Meini et al., 1992) and to activate PKC (Hansson et al., 1986). LXA₄ inhibits neutrophil and eosinophil activation by N-formyl-methionyl-leucyl-phenylalanine and PAF, respectively (Lee et al., 1991; Soyombo et al., 1994), and inhibits adhesion of leukocytes (Scalia et al., 1997), suggesting that it has an anti-inflammatory role. LXA₄ also inhibits cholinergic neurotransmission in airways, an effect that may be mediated by release of NO (Tamaoki et al., 1995).

4. Role in asthma. Immunoreactive LXA₄ has been detected in increased concentrations in bronchoalveolar lavage fluid from asthmatic patients (Lee et al., 1990). Inhaled LXA₄ has little effect on airway function but antagonizes the bronchoconstricting effect of inhaled LTC₄ (Christie et al., 1992), supporting the view that LXs may function as endogenous antagonists of cys-LTs (Lee, 1995). Stable LXA₄ analogues have anti-inflammatory effects and inhibit neutrophil chemotaxis and activation, suggesting that these endogenous substances are anti-inflammatory (Scalia et al., 1997). 15-LO may therefore function as an anti-inflammatory regulator in asthma by controlling the formation of LXs in response to cys-LT formation in the airways. There is an increase in levels of mRNA for 15-LO in circulating leukocytes of asthmatic patients (Kuitert et al., 1996) and increased expression of 15-LO in epithelial cells of asthmatic patients (Bradding et al., 1995). IL-4 selectively increases the expression of 15-LO in epithelial cells, and this may account for the increase in expression in asthma (Sigal et al., 1993).

	IV. Peptide Mediators

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Several peptides, including bradykinin, tachykinins, CGRP, endothelins (ETs), and complement, are involved in asthma. They are usually cleaved from larger precursors and are released in an active form. They are subject to degradation by peptidases (such as NEP) both in the circulation and in the airways.

Bradykinin has long been considered to be a mediator involved in asthma, since the first demonstration of bronchoconstriction in asthmatic patients after bradykinin inhalation. The development of potent and long-lasting bradykinin receptor antagonists has focused attention on the role of bradykinin and other kinins in the pathophysiological mechanisms of asthma, as well as on the potential uses of bradykinin antagonists in asthma therapy (Barnes, 1992b).

1. Synthesis and metabolism. Kinins are vasoactive peptides that are formed, during the inflammatory response, from the ₂-globulins high molecular weight (HMW) and low molecular weight (LMW) kininogens, by the action of kininogenases (Bhoola et al., 1992). Kininogenases include plasma kallikrein and tissue kallikrein. HMW and LMW kininogens are produced from the same gene (containing 11 exons and 10 introns) as a consequence of alternative splicing (Nakanishi, 1987). Both kininogens are synthesized in the liver. HMW kininogen is present only in plasma, whereas LMW kininogen also occurs in tissues. Two kinins are formed in humans, i.e., the nonapeptide bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg), which is generated from HMW kininogen, and the decapeptide lysyl-bradykinin (kallidin), which is generated from LMW kininogen. Kallidin is rapidly converted to bradykinin by the enzyme aminopeptidase-N (Proud and Kaplan, 1988). There is evidence for kinin activity in bronchoalveolar lavage fluid from asthmatic patients (Christiansen et al., 1987, 1992), and it is likely that bradykinin is formed, by the action of plasma and tissue kallikreins, in plasma that has been exuded from the inflamed airways. The concentrations of kallikrein and kinins in bronchoalveolar lavage fluid increase after allergen challenge (Christiansen et al., 1992). HMW kininogen is the preferred substrate for plasma kallikrein, which is generated from inactive prekallikrein by contact with certain negatively charged surfaces, including basement membrane components and proteoglycans, such as heparin released from mast cells. Tissue kallikreins are produced in glandular secretions and release kinins from both HMW and LMW kininogens. Tissue kallikrein has been localized immunocytochemically to serous cells in the submucosal glands of human airways (Proud and Vio, 1993). Serine proteases, such as ₁-antitrypsin, are effective inhibitors of kallikrein in the circulation, but in tissues kallikrein may remain activated for prolonged periods. Kallistatin is a kallikrein inhibitor that is present in some tissues, but its role in airways is not yet known (Chao et al., 1996).

2. Receptors. Bradykinin exerts several effects on the airways that are mediated by specific surface receptors. At least two subtypes of bradykinin receptors are recognized, based on the rank order of potency of kinin agonists (Regoli and Barabé, 1980), as follows: B₁, [des-Arg10]-lysyl-bradykinin > [des-Arg9]-bradykinin = lysyl-bradykinin bradykinin; B₂, bradykinin = lysyl-bradykinin [des-Arg10]-lysyl-bradykinin > [des-Arg9]-bradykinin. B₁ receptors are selectively activated by lysyl-bradykinin (kallidin) and [des-Arg9]-bradykinin and are inducible by inflammatory signals. B₁ receptors are expressed in chronic inflammation induced by IL-1 and IL-6 in rats and may play an important role in hyperalgesia. The effects of bradykinin on airways are mediated by B₂ receptors, and there is no evidence for functional B₁ receptors in the airways. A B₃ receptor has also been proposed in airway smooth muscle of sheep (Farmer et al., 1991), but there are doubts regarding its existence, because it has been defined with weak antagonists.

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4. Role in asthma. a. RELEASE. Although the role of bradykinin in asthma is still not clear, the development of potent, stable, B₂ receptor antagonists offers the possibility of soon clarifying the role of bradykinin in airway disease (Burch et al., 1990). Bradykinin is generated in asthmatic airways by the action of various kininogenases (generated in the inflammatory response) on HMW kininogen present in the exuded plasma and on LMW kininogens secreted in the airways. Bradykinin has been detected in bronchoalveolar lavage fluid from asthmatic patients (Christiansen et al., 1992). The degradation of bradykinin in the airways may be impaired when NEP is down-regulated in asthmatic airways or epithelial shedding occurs (Nadel, 1991). In experimental animals, aerosol exposure to IL-1 markedly increases the bronchoconstriction response to bradykinin (Tsukagoshi et al., 1994a), and this may be the result of reduced expression of NEP in the airways (Tsukagoshi et al., 1995).

Airway sensory nerves have the capacity to release neuropeptides, particularly the tachykinins SP and NKA, as well as CGRP, which may have proinflammatory effects in the airway. Because airway sensory nerves are activated in asthma, this has suggested that the release of sensory neuropeptides may contribute to the inflammatory response in asthma (Barnes, 1995a).

1. Synthesis and metabolism. SP and NKA, but not NKB, are localized to sensory nerves in the airways of several species (Barnes et al., 1991; Joos et al., 1994; Uddman et al., 1997). SP-immunoreactive nerves are abundant in rodent airways but are sparse in human airways (Martling et al., 1987; Laitinen et al., 1992; Komatsu et al., 1991). Rapid enzymatic degradation of SP in airways, and the fact that SP concentrations may decrease with age and possibly with cigarette smoking, could explain the difficulty in demonstrating this peptide in some studies. SP-immunoreactive nerves in the airway are found beneath and within the airway epithelium, around blood vessels, and, to a lesser extent, within airway smooth muscle. SP-immunoreactive nerves fibers also innervate parasympathetic ganglia, suggesting a sensory input that may modulate ganglionic transmission and thus result in local reflexes. SP in the airways is localized predominantly to capsaicin-sensitive unmyelinated nerves, but chronic administration of capsaicin only partially depletes the lung of tachykinins, indicating the presence of a population of capsaicin-resistant SP-immunoreactive nerves, as in the gastrointestinal tract (Dey et al., 1991). Similar capsaicin denervation studies are not possible in human airways, but after extrinsic denervation during heart-lung transplantation there appears to be a loss of SP-immunoreactive nerves in the submucosa (Springall et al., 1990). Tachykinins are derived from preprotachykinins (PPTs) that are expressed in nodose and jugular ganglia. There are three PPT genes; -PPT codes for SP alone, -PPT codes for SP and NKA, and -PPT codes for SP, NKA, and a novel, amino-terminally extended form of NKA termed NP-. Synthesis may be partly determined by local inflammation in the airways, because allergen exposure increases the expression of PPT mRNA in nodose ganglia of guinea pigs (Fischer et al., 1996). There is some evidence that tachykinins may be synthesized in nonneuronal cells, such as macrophages. Human macrophages express -PPT, and SP is released from these cells by capsaicin (Ho et al., 1997). In rat alveolar macrophages, -PPT mRNA and SP-like immunoreactivity are expressed in response to inflammatory stimuli, suggesting that this may result in increased SP release in inflammatory diseases (Killingsworth et al., 1997).

2. Receptors. At least three subtypes of tachykinin receptors have been characterized pharmacologically by the rank order of potency of agonists, by the development of selective antagonists, and by molecular cloning (Nakanishi, 1991). SP acts preferentially at NK₁ receptors, NKA at NK₂ receptors, and NKB at NK₃ receptors. Tachykinin receptors are differentially expressed and are also subject to differential regulation, for example by inflammatory stimuli. Tachykinins are typical G protein-coupled receptors and lead to increased PI hydrolysis, with an increase in the release of intracellular Ca²⁺, IP₃, and diacylglycerol. Tachykinin receptors in the airways have been mapped using autoradiographic techniques and labeled tachykinins (Carstairs and Barnes, 1986; Walsh et al., 1994; Strigas and Burcher, 1996; Miyayasu et al., 1993; Zhang et al., 1995). NK₁ receptors are localized to bronchial vessels, epithelial cells, and submucosal glands, whereas NK₂ receptors are predominantly localized to airway smooth muscle.

4. Role in asthma. In rodents, there is now considerable evidence for neurogenic inflammation in airways resulting from antidromic release of neuropeptides from nociceptive nerves or C-fibers, via an axon reflex, and this process may contribute to the inflammatory response in asthma (Barnes, 1986).

b. RELEVANCE IN ASTHMA. Sensory nerves may be activated in airway disease. In asthmatic airways the epithelium is often shed, thereby exposing sensory nerve endings. Sensory nerves in asthmatic airways may be "hyperalgesic" as a result of exposure to inflammatory mediators such as PGs and certain cytokines (such as IL-1, TNF-, and nerve growth factor) and may then be activated more readily by other mediators, such as kinins. In animals, capsaicin has been used as a tool to explore the release of sensory neuropeptides. In humans, capsaicin inhalation causes cough and transient bronchoconstriction, which is inhibited by cholinergic blockade and is probably attributable to a laryngeal reflex (Fuller et al., 1985

C. Calcitonin Gene-Related Peptide

1. Synthesis and metabolism. CGRP-immunoreactive nerves are abundant in the respiratory tract of several species, and CGRP is stored and localized with SP in afferent nerves. CGRP has been extracted from and is localized to human airways (Palmer et al., 1987; Komatsu et al., 1991). CGRP is found in trigeminal, nodose-jugular, and dorsal root ganglia and has also been detected in neuroendocrine cells of the lower airways (Uddman et al., 1997).

2. Receptors. CGRP acts on specific receptors that are coupled (via G_s) to adenylyl cyclase, resulting in an increase in intracellular cyclic AMP concentrations. Subtypes of CGRP receptors have been proposed, based on the selectivity of different CGRP analogues and the related peptide amylin (Poyner, 1992). CGRP receptors have been mapped autoradiographically in human airways and are predominantly located in bronchial vascular smooth muscle, rather than airway epithelium (Mak and Barnes, 1988).

3. Effects on airways. a. AIRWAY SMOOTH MUSCLE. CGRP causes constriction of human bronchi in vitro (Palmer et al., 1987). This is surprising, because CGRP increases cyclic AMP levels. There are few, if any, CGRP receptors in airway smooth muscle in human or guinea pig airways, and this suggests that the paradoxical bronchoconstriction response reported in human airways may be mediated indirectly. In guinea pig airways, CGRP has no consistent effect on tone (Martling et al., 1988). The variable effects of CGRP on airways may be explained by the fact that CGRP may release other mediators that have effects on tone. CGRP may release both NO and ET in airways, so that its effects would depend on the balance between these bronchodilating and bronchoconstricting mediators (Ninomiya et al., 1996).

ETs are potent constrictor peptides that were originally described as vasoconstrictors released from endothelial cells. There is now considerable circumstantial evidence that they are involved in the pathophysiological mechanisms of asthma (Barnes, 1994b; Hay et al., 1996).

1. Synthesis and metabolism. There are three ET peptides, and each is encoded by a distinct gene (Inoue et al., 1989), which codes for the precursor peptide. Prepro-ET-1 is cleaved to a 38-amino acid intermediate form termed big ET-1 or pro-ET-1. Pro-ET-1 is rapidly cleaved by a specific enzyme, termed ET-converting enzyme (ECE), to form mature ET-1. ECE is a neutral metalloendopeptidase and is inhibited by phosphoramidon (Ikegawa et al., 1990). Mast cell chymase may also cleave pro-ET-1 (Wypij et al., 1992). The human prepro-ET-1 gene is on chromosome 6, and its upstream regulatory region reveals multiple regulatory elements, indicating that several factors may regulate its expression (Masaki et al., 1992). Several proinflammatory cytokines, including transforming growth factor (TGF)-, TNF-, and IL-1, may increase expression of ET-1. Less is known regarding the synthetic pathways and regulation of ET-2 and ET-3.

2. Receptors. Pharmacological responses to ETs are mediated by at least two receptor subtypes. Two distinct receptors, with structures typical of G protein-coupled receptors, have been cloned; they exhibit approximately 60% homology (Masaki et al., 1992). For the ET_A receptor, the rank order of potency is ET-1 > ET-2 ET-3 and the binding affinity for ET-1 is approximately 100 times greater than that for ET-3. ET_B receptors show similar affinities for all three ETs and for the related sarafotoxins. The distinction between ET_A and ET_B receptors has been confirmed with the development of selective agonists and antagonists. Although the existence of a third ET receptor, which is selective for ET-3 (ET_C receptor), has been proposed (Masaki et al., 1992), there is little conclusive evidence for this in human tissues. Radioligand binding studies and in situ hybridization studies with receptor cDNA probes have demonstrated that ET receptors are widely distributed, in keeping with the multiple actions of these peptides. ET_A and ET_B receptors are expressed in lung and are differentially distributed (Nakamichi et al., 1992). Selective ET_A and ET_B agonists and antagonists have greatly aided the study of receptor subtype expression. BQ-123, FR-139317, and PD 145065 are selective ET_A receptor antagonists, whereas IRL 1038 is a selective antagonist of ET_B receptors.

3. Effects on airways. a. AIRWAY SMOOTH MUSCLE. ET-1 and ET-2 are potent constrictors of human airway smooth muscle in vitro, being even more potent than LTD₄ (Advenier et al., 1990; Henry et al., 1990; McKay et al., 1991b; Takahashi et al., 1997; Goldie et al., 1995). The contractile response is slow in onset and sustained, and ET-1 appears to cause a maximal contractile response. The contractile response in human airways is unaffected by calcium antagonists or (in contrast to other species) COX inhibitors or LT antagonists (McKay et al., 1991a; Nally et al., 1994), suggesting a direct effect on airway smooth muscle. This is consistent with the demonstration of ET binding sites on human airway smooth muscle, using autoradiography (Henry et al., 1990; McKay et al., 1991b; Brink et al., 1991; Goldie et al., 1995; Knott et al., 1995). ET-1 may produce a prolonged contractile response in human airway smooth muscle by activating PKC, because the PKC inhibitor staurosporine reduces the constricting effect of ET-1 (McKay et al., 1996). ET-3 is less potent that ET-1 or ET-2 (Advenier et al., 1990; Hay et al., 1993), but the potency differences are complicated by differential metabolism. Mechanical removal of airway epithelium potentiates the constricting effects of ETs, but the effect is greater for ET-3 than for ET-1 (Candenas et al., 1992; McKay et al., 1992). After epithelium removal or phosphoramidon treatment, the potencies of ET-1, ET-2, and ET-3 are similar, suggesting that any differences in previous studies were the result of more rapid degradation of ET-3 by epithelial NEP. ET-3-mediated contraction of human airways is partly reduced by COX inhibition (Nally et al., 1994).

4. Role in asthma. a. RELEASE. There is increased formation of ETs in asthma. Elevated concentrations of ET-1 have been detected in bronchoalveolar lavage fluid from asthmatic patients (Mattoli et al., 1991; Sofia et al., 1993; Redington et al., 1995), and these are reduced after treatment with steroids (Vittori et al., 1992). ET-1 is present in induced sputum, but the levels are not elevated in asthmatic patients, compared with normal subjects (Chalmers et al., 1997b). An increase in the concentration of plasma ET-1 has been reported in asthmatic children and adults and is related to asthma severity (Aoki et al., 1994; Chen et al., 1995), although another study showed no increase in plasma ET-1 levels in patients with mild asthma (Chalmers et al., 1997b). Furthermore, in patients with nocturnal asthma, there is a significantly lower level of ET-1 in bronchoalveolar lavage fluid at night than during the day (Kraft et al., 1994). There is a significant increase in the expression of ET-1 immunoreactivity in the epithelial layer in fiber-optic bronchial biopsies from asthmatic patients (Springall et al., 1991). It is tempting to speculate that this is the result of the action of proinflammatory cytokines (IL-1, TNF-, and IL-6) released from activated macrophages in asthmatic airways. Anti-CD23 also induces release of ET-1 in epithelial cells from asthmatic patients, suggesting that allergen acting via a low affinity IgE receptor (FcRII) may be a mechanism for releasing ET-1 in asthma (Campbell et al., 1994). There is also an increase in the ET-1 content of alveolar macrophages from asthmatic patients, compared with normal subjects, although there is no increase in the release of ET-1 after stimulation with lipopolysaccharide (Chanez et al., 1996).

E. Complement

1. Synthesis and metabolism. The complement system contains a series of 30 distinct circulating proteins, including proteolytic proenzymes, nonenzymatic components that form functional enzymes when activated, and receptors (Ember and Hugli, 1997). The proenzymes become sequentially activated in a cascade that finally leads to the formation of the so-called terminal attack sequence, which can promote cell lysis and is central to our defense against invading microorganisms. However, there are several by-products generated during the activation of the complement cascade that have proinflammatory activity and therefore have the potential to be involved in asthma. The larger fragments of C3 and C4 (i.e., C3b and C4b) are involved in a range of biological activities, including opsonization, phagocytosis, and immunomodulation. There are also several smaller fragments generated during the activation of C5, such as C3a and C5a, which have been referred to as anaphylatoxins and which have several airway effects (Ember and Hugli, 1997).

2. Receptors. There are distinct receptors for C3a and C5a, which have been cloned (Ember and Hugli, 1997). Both are members of the G protein-coupled receptor superfamily.

3. Effects on airways. C3a and C5a induce airway smooth muscle contraction and chemotaxis of leukocytes, including eosinophils (Daffern et al., 1995; Regal, 1997). Aerosolization of C5a into the airways induces transient hyperresponsiveness to inhaled histamine (Irvin et al., 1986; Armour et al., 1987), an effect that is partially inhibited by pretreatment with indomethacin (Berend et al., 1986). Both C3a and C5a are potent stimulants of eosinophil degranulation (Takafuji et al., 1996), and the response of circulating eosinophils to C5a is enhanced after the late response to inhaled allergen in asthmatic patients (Evans et al., 1996b). C5a is also a potent chemoattractant of human monocytes and may therefore be involved in recruitment of macrophages into asthmatic airways (Pieters et al., 1995).

4. Role in asthma. There have been conflicting reports regarding changes in the complement cascade in asthmatic patients (Barnes et al., 1988). An increased amount of C3a has been demonstrated in the circulation of asthmatics during exercise-induced bronchoconstriction (Smith et al., 1990), and increased levels of C3a have been demonstrated in bronchoalveolar lavage fluid obtained from some, but not all, asthmatics (Van de Graaf et al., 1992). Furthermore, patients with severe asthma have been reported to show increased serum levels of C3a and to exhibit a different pattern of complement activation, compared with patients with bronchial infections (Lin et al., 1992). In asthmatic patients, the neutrophil chemotactic activity of bronchoalveolar lavage fluid is largely explained by C5a (Teran et al., 1997), suggesting that this is an important mediator of neutrophilic infiltration in asthmatic airways.

	V. Small Molecules

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There is increasing evidence that oxidative stress and reactive oxygen species (ROS) are involved in inflammatory airway diseases, including asthma (Barnes, 1990; Repine et al., 1997), although relatively few studies have been undertaken in humans. This is partly because of the difficulties of measuring oxidative stress in the airways in vivo and partly because of the relative inefficacy of currently available antioxidants. However, new noninvasive techniques have been developed to assess oxidative stress in the airways, making it possible to reassess the role of oxidative stress in asthma.

1. Synthesis and metabolism. Many inflammatory and structural cells that are activated in asthmatic airways, including eosinophils, macrophages, mast cells, and epithelial cells, produce ROS (Barnes, 1990). Superoxide anions are generated by NADPH oxidase and then are converted to hydrogen peroxide by superoxide dismutases (SODs). Hydrogen peroxide is then degraded to water by catalases. Superoxide and hydrogen peroxide may interact in the presence of free iron to form the highly reactive hydroxyl radical. Superoxide may also combine with NO to form peroxynitrite, which also generates hydroxyl radicals (Beckman and Koppenol, 1996). Oxidative stress describes an imbalance between ROS and antioxidants. The normal production of oxidants is counteracted by several antioxidant mechanisms in the human respiratory tract (Cantin et al., 1990). The major intracellular antioxidants in the airways are catalase, SOD, and glutathione, which is formed by the selenium-dependent enzyme glutathione peroxidase. Extracellular antioxidants include the dietary antioxidants vitamin C (ascorbic acid) and vitamin E (-tocopherol), uric acid, and lactoferrin. Oxidant stress activates the inducible enzyme heme oxygenase-1, which converts heme and hemin to biliverdin, with the formation of carbon monoxide (Wong and Wispe, 1997; Choi and Alam, 1996). Biliverdin is converted, by bilirubin reductase, to bilirubin, which is a potent antioxidant.

2. Effects on airways. a. AIRWAY SMOOTH MUSCLE. Hydrogen peroxide directly constricts airway smooth muscle in vitro, and this effect is mediated partly via the release of prostanoids (Rhoden and Barnes, 1989). ROS may damage airway epithelium, resulting in increased epithelial shedding and increased bronchoconstriction responses (Yukawa et al., 1990). In vitro, hydrogen peroxide induces an increase in the responsiveness of human airways (Hulsmann et al., 1994a). Formation of peroxynitrite also increases airway responsiveness in guinea pigs in vitro and in vivo (Sadeghi-Hashjin et al., 1996), but its effect in human airways is not yet known.

e. INFLAMMATORY CELLS. Oxidants also activate NF-B (which orchestrates the expression of multiple inflammatory genes that undergo increased expression in asthma), thereby amplifying the inflammatory response (Barnes and Karin, 1997

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3. Role in asthma. a. RELEASE. Bronchoalveolar lavage fluid cells from asthmatic patients show increased production of superoxide anions, compared with cells from normal individuals (Jarjour and Calhoun, 1994), and this production is increased further after allergen challenge (Calhoun and Bush, 1990). Increased generation of superoxide has also been reported for circulating monocytes and neutrophils from asthmatic patients (Vachier et al., 1994), and there is evidence for increased oxidative stress in the circulation (Rahman et al., 1996). Circulating eosinophils from asthmatic patients produce excessive superoxide after activation (Chanez et al., 1990), and this is increased even further after allergen challenge (Evans et al., 1996b). In experimental animals, certain viral infections (e.g., influenza) induce various indices of oxidative stress in the lungs (Choi and Alam, 1996), and this may be relevant to exacerbations of asthma.

There is increasing evidence that endogenous NO plays a key role in physiological regulation of airway functions and is implicated in airway diseases, including asthma (Barnes and Belvisi, 1993; Gaston et al., 1994; Barnes, 1995b).

1. Synthesis and metabolism. NO is a gas that is derived from the amino acid L-arginine by the enzyme NOS, of which at least three isoforms exist (Nathan and Xie, 1994). There are two cNOS forms; one was first described in brain and is localized to neural tissue [neuronal NOS (nNOS) or NOSI], and the other is localized to endothelial cells [endothelial NOS (eNOS) or NOSIII], although it has become apparent that both enzymes are also expressed in other cells, such as epithelial cells. Both enzymes are activated by increases in [Ca²⁺]_i and produce small amounts of NO, which serve a local regulatory function. In contrast, iNOS (NOSII) is not normally expressed but is induced by inflammatory cytokines and endotoxin. This enzyme form is less dependent on increases in [Ca²⁺]_i, because calmodulin is tightly bound to the enzyme; when the enzyme is induced it is activated and produces much larger amounts of NO than do cNOS isoforms. NO produced by cNOS is involved in physiological regulation of airway function, whereas NO produced by iNOS is involved in inflammatory diseases of the airways and in host defenses against infection.

2. Receptors. NO does not have conventional receptors but, rather, diffuses into cells and activates soluble guanylyl cyclase, resulting in an increase in the formation of cyclic GMP. In airway smooth muscle, cyclic GMP causes relaxation (Ward et al., 1995a). Some of the effects of NO are mediated by the formation of peroxynitrite, as discussed above.

3. Effects on airways. NO has many effects on airway function, although the effects of endogenous NO depend on the site of production and the amount produced (Barnes, 1996b).

4. Role in asthma. a. RELEASE. There is evidence for increased expression of iNOS in asthmatic airways, particularly in epithelial cells and macrophages (Hamid et al., 1993; Giaid et al., 1998). It is likely that this arises from the effects of proinflammatory cytokines, oxidants, and perhaps other inflammatory mediators. Because NO is a gas, it diffuses into the airway lumen and may be detected in exhaled air (Barnes and Kharitonov, 1996). There is an increase in NO levels in the exhaled air from asthmatic patients (Alving et al., 1993; Kharitonov et al., 1994; Persson et al., 1994), which is derived from the lower airways (Kharitonov et al., 1996b; Massaro et al., 1996). The increased exhaled NO in asthma is related to airway inflammation (Jatakanon et al., 1998), is increased during the late response to allergen (Kharitonov et al., 1995) and during exacerbations (Massaro et al., 1995), and is decreased by treatment with inhaled corticosteroids (Kharitonov et al., 1996a).

	VI. Cytokines

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A. General Overview

1. The cytokine network in chronic inflammation. Cytokines are small protein mediators that play an integral role in the coordination and persistence of inflammation in asthma, although the precise role of each cytokine remains to be determined. The chronic airway inflammation of asthma is unique, in that the airway wall is infiltrated by T lymphocytes of the Th2 phenotype, eosinophils, macrophages/monocytes, and mast cells. In addition, "acute-on-chronic" inflammation may be observed in acute exacerbations, with increases in eosinophils and neutrophils and release of mediators such as histamine and cys-LTs from eosinophils and mast cells, to induce bronchoconstriction, airway edema, and mucus secretion.

Th2 lymphocytes produce a panel of cytokines, including IL-3, IL-4, IL-5, IL-9, IL-10, IL-13, and GM-CSF. The primary signals that activate Th2 cells are unknown but may be related to the presentation of a restricted panel of antigens in the presence of appropriate cytokines. Dendritic cells are ideally suited to act as the primary contacts between the immune system and external allergens. Interaction of co-stimulatory molecules on the surface of antigen-presenting cells (in particular, the B7.2/CD28 interaction) may lead to proliferation of Th2 cells, thus perpetuating mast cell activation and eosinophilic inflammation. This may lead to the production of specific IgE by B lymphocytes under the influence of IL-4, which plays a critical role in the isotype switching of B lymphocytes from IgG to IgE production. Other cytokines, including TNF- and IL-6, may also be important. The IgE produced in asthmatic airways binds to FcRI on mast cells, priming them for activation by antigen. The development of mast cells from bone marrow cells represents a process of maturation and expansion, involving growth factors and cytokines [such as stem cell factor (SCF) and IL-3] produced by structural cells. Mast cells recovered from asthmatic patients by bronchoalveolar lavage show increased release of mediators such as histamine.

IL-4 also increases the expression of an inducible form of the low affinity receptor for IgE (FcRII or CD23) on B lymphocytes and macrophages. This may account for the increased expression of CD23 on alveolar macrophages from asthmatic patients, which in turn could account for the increased release of cytokines from these macrophages. In addition, IL-4 is very important in driving the differentiation of CD4⁺ Th precursors into Th2-like cells.

The differentiation, migration, and pathobiological effects of eosinophils may occur through the effects of GM-CSF, IL-3, and IL-5. Once recruited from the circulation, mature eosinophils in the presence of these cytokines change phenotype into hypodense eosinophils, which show increased survival in bronchial tissue. These eosinophils are primed for ligand-initiated generation of increased amounts of cys-LTs and for cytotoxicity to other cells, such as those of the airway epithelium. Eosinophils themselves may also generate other cytokines.

Cytokines may also play an important role in antigen presentation and may enhance or suppress the ability of macrophages to act as antigen-presenting cells. Airway macrophages are normally poor at antigen presentation and suppress T cell proliferative responses [possibly via release of cytokines such as IL-1 receptor antagonist (IL-1ra)], but in asthma there is evidence for reduced suppression after exposure to allergen (Spiteri et al., 1994

2. Cytokine receptors. The receptors for many cytokines have now been cloned and, based on common homology regions, these have been grouped into superfamilies (Kishimoto et al., 1994).

a. CYTOKINE RECEPTOR SUPERFAMILY. This largest receptor superfamily includes IL-2 receptor - and -chains, IL-4 receptor, IL-3 receptor - and -chains, IL-5 - and -chains, IL-6 receptor, gp130, IL-12 receptor, and GM-CSF receptor. The extracellular regions of the cytokine receptor family contain combinations of cytokine receptor domains, fibronectin type III domains, and usually C2 Ig constant region-like domains. Some members are composed of a single polypeptide chain that binds its ligand with high affinity. For other receptors, there may be more than one binding site for the ligand (typically sites with high and low binding affinities). For these receptors, additional subunits that are required for high affinity receptor expression have been identified. Some of these subunits are shared by more than one cytokine receptor, giving rise to heterodimeric structures. Such examples include (a) receptors sharing the GM-CSF receptor -chain (IL-3, IL-5, and GM-CSF); (b) receptors sharing the IL-6 receptor -chain, gp130 (IL-6, leukemia inhibitory factor, and oncostatin M); and (c) receptors sharing the IL-2 receptor -chain (IL-2, IL-4, IL-7, and IL-15).

Many proteins of the cytokine receptor superfamily are secreted as soluble forms, which are produced by alternative splicing of their mRNA transcripts to yield proteins lacking the transmembrane region and the cytoplasmic proximal charged residues that anchor the protein into the membrane. They may act as antagonists, as transport proteins to carry cytokines to other sites, or as agonists.

d. INTERFERON RECEPTOR SUPERFAMILY. This group includes the IFN-/ receptor, IFN- receptor, and IL-10 receptor. They are single-transmembrane domain glycoproteins that are characterized by either one (IFN- and IL-10 receptors) or two (IFN-/ receptors) homologous extracellular regions. Signal transduction involves phosphorylation and activation of Janus protein kinase and tyrosine kinase 2 protein tyrosine kinases.

1. Interleukin-2. a. SYNTHESIS AND RELEASE. Activated T cells, particularly Th0 and Th1 T cells, are major sources of IL-2 (Morgan et al., 1976), whereas B lymphocytes can be induced under certain conditions to secrete IL-2 in vitro. IL-2 is secreted by antigen-activated T cells 4 to 12 h after activation, accompanied later by up-regulation of high affinity IL-2 receptors on the same cells. Binding of IL-2 to IL-2 receptors induces proliferation of T cells, secretion of cytokines, and enhanced expression of receptors for other growth factors, such as insulin. The IL-2-receptor complex is then removed from the T cell surface by internalization. IL-2 can also be produced by eosinophils (Levi Schaffer et al., 1996) and by airway epithelial cells (Aoki et al., 1997).

b. RECEPTORS. The IL-2 receptor complex is composed of three chains (, , and ) and belongs to the family of hematopoietic cytokine receptors (Taniguchi and Minami, 1993