ADVANCES IN IMMUNOLOGY. VOL. 51

The Pathobiology of Bronchial Asthma JONATHAN P. ARM AND TAK H. LEE Department of Allergy and Allied Respiratory Disorders, U.M.D.S., Guy's Hospital, London, SEl 9RT England

1. Introduction

Bronchial asthma is a disease that is characterized by a history of episodic wheezing, by physiologic evidence of reversible airflow obstruction, either spontaneously or following bronchodilator therapy, and by pathologic evidence of inflammatory changes in the bronchial mucosa. Asthma is a common disease and there is accumulating evidence that it may be growing in prevalence, thereby imposing an increasingly large burden on the health services and claiming ever greater numbers of lives each year. Against the epidemiologic background, it is clear that elucidation of the mechanisms for the development and perpetuation of the asthmatic diathesis is critical. In recent years there has been a major change in the conceptual basis of the pathophysiology of this disease. This has resulted from the recognition that inflammation of the airways is a characteristic feature of asthma and that obstruction of the airway lumen by smooth muscle constriction and mucus plugging may be the sequelae of the inflammatory cascades. This view has led to a significant change in the focus of research activities and in ideas about therapy of asthma. The present review will include a summary of the recent developments in research on the pathobiology of the disease. The focus will be on the cellular mechanisms of the disease as indicated by current information on the pathologic changes in the asthmatic bronchial mucosa obtained in living patients via the fiberoptic bronchoscope. Also addressed are the mediator mechanisms, with special consideration of the possible role of the sulfidopeptide leukotrienes, prostanoid metabolites, and platelet-activating factor. Bronchial asthma is not a homogeneous disease and there are welldefined subgroups of patients. Heterogeneity in asthmatic syndromes will be addressed by a consideration of two well-defined clinical subgroups, namely, aspirin-induced asthma and corticosteroid-resistant bronchial asthma. 323 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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II. Pathology of the Asthmatic Mucosa

There have been many indirect approaches to examine airway structure, especially in connection with airway inflammation and hyperresponsiveness. More recently, with the advent of fiberoptic bronchoscopy, biopsy, and lavage in asthmatic patients, pathological examinations have been extended to the living asthmatic individual. Early studies of patients dying from status asthmaticus revealed marked inflammation of the bronchial tree (1). There was plugging of the lumen with mucus, epithelial cells, and eosinophils; shedding of the epithelium; basement membrane thickening; smooth muscle hypertrophy; and an intense inflammatory cell infiltrate in the mucosa and submucosa characterized b y a predominantly eosinophilic infiltrate, but also containing mononuclear cells and neutrophils. Bronchoalveolar lavage (BAL) fluid from asthmatic subjects, in the absence of bronchial provocation, contains increased numbers of mast cells, epithelial cells, eosinophils, and neutrophils compared to that obtained from normal controls (2-7). In addition to the cellular changes in BAL fluid in asthma, elevated levels of eosinophil-derived major basic protein (MBP), histamine, prostaglandin Dz (PGDz), and the peptidoleukotrienes have been reported (4,8-10). Biopsy specimens of human bronchus taken at bronchoscopy confirm the presence of airway inflammation in subjects with mild to moderately severe asthma ( 3 , l l 15) (Fig. 1, Table I). These changes are described in detail below, with a discussion of their pathogenesis and consequences. A. EPITHELIAL CELLS AND BASEMENT MEMBRANE Accumulated evidence suggests that epithelial damage and shedding are important features of asthmatic airways and may, at least in part, be a consequence of inflammation. Laitinen and co-workers (16) found extensive damage of the epithelium and areas in which only basal cells were present. The ciliated epithelial cells were swollen and the intercellular spaces were widened. Occasionally, columnar epithelial cells were still attached to each other at the luminal side but were separated in the middle of the epithelium by homogeneous material resembling fluid. This may have been caused by edema formation and plasma exudation into the epithelium. Several studies have reported the presence of increased numbers of bronchial epithelial cells recovered by bronchoalveolar lavage from asthmatic subjects when compared with those from normal subjects and have established a positive relationship between epithelial cell counts and the extent of airway hyperresponsiveness (3,6).The mechanisms by which epithelial dam-

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FIG.1. Individual cell counts in the submucosae ofbronchial biopsies obtained from asthmatic (A) and normal (N) subjects defined by a panel of antibodies. HAM = HAM56:panmacrophage monoclonal antibody (niAb); MAC387, antimonocyte mAb; MURAM, polyclonal antimuraniidase (lysozyme); UCHL1, antimemory T cell mAb; LN3, anti-HLA-DR mAb; EG2, antieosinophil cationic protein mAb; NP57, antineutrophil elastase mAb. T h e p values given were obtained using the Mann-Witney U test. (From Ref. 77.)

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TABLE I HISTOLOGY OF THE AIRWAYMUCOSA IN BRONCHIALASTHMA^ -

Postmortem

Plugging of the lumen Epithelial desquamation Basement membrane thickening Inflammation Eosinophils Mononuclear cells

In life Epithelial fragility and shedding Subepithelial collagen (111, V); fibronectin EG2'; degranulation of eosinophils Lymphocytes; CD25'; immature macrophages; class I1 MHC on macrophages and epithelium; mast cell degranulation

" Summary of the histological findings in the airway mucosae of subjects dying from acute severe asthma (postmortem) and in bronchial biopsies obtained from individuals with mild to moderately severe asthma (in life).

age may contribute to bronchial hyperresponsiveness are exposure of nerve endings to irritant factors, augmented penetration of allergen particles into the submucosa where mediator-secreting cells reside, and decreased production of epithelial-derived relaxant factors and neutral endopeptidases. When stimulated, human airway epithelium releases a number of arachidonic acid-derived metabolites (17),and in isolated airway preparations, removal of epithelium augments airway responsiveness to other agonists ( 1 8 ~ 9 )Some . of this effect is probably related to loss ofa diffusion barrier and loss of degradative mechanisms for the tachykinins. However, it is probable that bronchodilator factors such as cyclooxygenase products are normally produced by the epithelium in health and that these are lost when the epithelium is shed (20-22). These findings support the view that the epithelium may be a major component of the regulatory processes that control bronchomotor tone. Previous studies using light microscopy have described thickening and hyalinization of the epithelial basement membrane. However, recent morphologic studies using transmission electron microscopy and the use of monoclonal antibodies to differentiate collagen subtypes have shown that the bronchial epithelial basement membrane is of normal thickness in asthma. However, there is dense deposition of collagen fibrils in the subepithelial region (23). Under light microscopy, the combination of the true basement membrane and the collagen deposition is mistakenly seen as a thickened basement membrane. Subepithelial collagen deposition also occurs in asthmatics who are asymptomatic. Immunohistochemistry indicates the presence of collagen type IV, fibronectin, and laminin in the true basement mem-

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brane, and the subepithelial collagen consists predominantly of collagen types I11 and V, together with fibronectin but not laminin. It has been proposed that the cellular source of the subepithelial collagen is the myofibroblast (23). The stimuli for collagen deposition are unknown. Mast cells present in the airway lumen and mucosa are a potential source of growth factors, as are macrophages, eosinophils, and the bronchial epithelial cell.

B. MASTCELLS In nonasthmatic subjects, mast cells recovered by BAL constitute

0.04 to 0.6% of the total nucleated cells, whereas in asthmatics these numbers can be increased threefold to fivefold (2,5-7). Almost all the mast cells (MCs) in the bronchial epithelium and submucosa of normal airways contain secretory granules in which tryptase is the predominant neutral protease (24).These cells have been called MCTto differentiate them from those containing both tryptase and chymase (MCTC) (24). Functionally, lung mast cells differ from those of a connective tissue mast cell by their resistance to activation by non-IgE secretagogues, including compound 48180, tachykinins, basic polyamines, and opiates. They are also sensitive to the inhibitory effects of cromolyn sodium, whereas those in the skin are not (25,26). Both in uitro and in viuo studies have provided compelling evidence that IgE-mediated release of mediators from mast cells is responsible for the immediate bronchoconstriction provoked by allergen (2,2730). Direct evidence of mast cell activation has been provided by the identification of a number of individual mediators in blood and bronchoalveolar lavage fluid in uiuo. Thus, several centers have reported increases in concentrations of plasma histamine and serum highmolecular-weight neutrophil chemotactic activity during the early asthmatic response provoked by antigen and exercise (31-35). Several studies have demonstrated an increased concentration of histamine in the BAL fluid during the early reaction that follows endobronchial allergen challenge (8,36,37).These increases in histamine correlate with that of the unique mast cell protease tryptase (36),supporting the mast cell origin of these mediators. I n uitro IgE-dependent activation of mast cells leads to the production of arachidonic acid-derived metabolites (28,38).The most abundant cyclooxygenase product synthesized and released from the human lung mast cell is PGDZ (38). After local airway challenge of asthmatic airways with allergen through a bronchoscope, there is a 150-fold increase in the concentration of PGDz in BAL fluid (39,40). Following allergen provocation of the airways, concentrations of leu-

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kotriene C4 (LTC4) and its metabolite, LTE4, have been found in increased concentrations in the bronchoalveolar lavage fluid (37). LTE4 has also been found in increased concentrations in the urine in association with both the early reaction following allergen challenge and during natural exacerbations of the disease (41-43). Mast cells in the mucosae of patients with asthma show evidence of degranulation, suggesting that they are chronically activated (3).An inverse correlation between the percentage of mast cells and histamine content in the recovered bronchoalveolar lavage fluid and baseline spirometry and airway hyperresponsiveness suggests that histamine and other mast cell-derived mediators may contribute to airflow obstruction (5,6,10). It is well recognized that histamine causes contraction of bronchial smooth muscle through stimulation of H1 receptors (44). Furthermore, histamine dilates and increases the permeability of the bronchial vascular bed (45). Histamine is also capable of stimulating mucus secretion as well as altering its viscosity (46), properties that may contribute to its effect on airway caliber. Other mast cell mediators that are likely to play important roles in the mechanisms of bronchoconstriction, inflammation, and hyperresponsiveness are PGDZ, tryptase, and the sulfidopeptide leukotrienes, which will be discussed later. The emphasis on the mast cell as an essential cell in the pathogenesis of asthma has recently been tempered by the observation that albuterol, which is a very potent inhibitor of mast cell degranulation, had little effect on the allergic inflammation induced by allergen challenge (47). This has cast doubt over the hypothesis of the important effector role of the mast cell in the more prolonged and protracted phases of inflammation induced by allergic reactions.

Must Cells and C ytokines The role of mast cells in the acute asthmatic response is well documented, whereas the role of mast cells in the chronic inflammatory response within the airways is less clear. Therefore, it was of considerable interest when it was reported that rodent mast cells synthesized a wide range of cytokines upon IgE-mediated activation. Brown et aZ. (48)demonstrated that several transformed and several interleukin-3 (IL3)-dependent nontransformed mast cell lines constitutively expressed mRNA for IL-4. They also found biologically active IL-4 in the supernatants of these mast cell lines. These studies were extended to a study of cytokine production in response to cross-linking of Fc,RI by spleen cells depleted of T and B lymphocytes (49). IL-4 production was demonstrated when these cells were stimulated by

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IgE immobilized on culture dishes, and was augmented by pretreatment with IL-3 either in uiuo or in uitro. Furthermore, following treatment with IL-3 these cells also produced cytokines upon crosslinking of Fc,RII (49,50). Subsequent work showed that the cells that produced IL-4 were all Fc,RI positive. These cells were heterogeneous and the majority of IL-4 production was from cells of basophilic morphology, lacking c-Kit. Three IL-3-dependent murine mast cell lines were shown to produce IL-3, IL-4,Il-5, and IL-6 in response to cross-linking of Fc,RI or to calcium ionophores (51). Mast cells sensitized with IgE anti-2,4dinitrophenylhydrazine (anti-DNP) exhibited a dose-dependent secretion of IL-3 in response to antigen that was maximal 2 to 6 hours after activation. Secretion of IL-6, and to a lesser extent IL-4, was also observed. The optimal dose for cytokine generation was the same as that required for histamine release, although the kinetics of histamine and cytokine release were very different. mRNA for cytokines was not detected in resting cells, but was markedly up-regulated preceding cytokine secretion. This observation and the kinetics of secretion suggest that the cytokines were newly synthesized following mast cell activation. Burd et al. evaluated the generation of a large number of T cell and monocyte/macrophage-associated cytokines by growth factordependent and -independent mast cell lines following activation by DNP-HSA (human serum albumin) following passive sensitization with monoclonal IgE anti-DNP (52).The cytokines evaluated were JE, MIP-la, MIP-lP, and TCA-3 (members of a family of low-molecularweight cytokines that share significant sequence homology), interferon-? (IFN-y),granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, IL-3, IL-5, and IL-6. The pattern of cytokine transcription in unstimulated cells and following stimulation via Fc,RI varied between different cell lines. Basal levels of mRNA for J E and/or IL-6 were detected in three of the cell lines. Following activation there was increased mRNA for each of the cytokines tested in one or more cell line. The induction of TCA-3, MIP-la, and MIP-1P mRNA was completely inhibited and that of IL-6 partially inhibited by treatment with cyclosporin A. Inhibition of new protein synthesis by cyclohexamide had minimal effects. Cytokine expression was also induced by activation with concanavalin A (Con A) or phorbol ester plus ionophore, though to a lesser extent than following IgE-dependent activation. Biologically active IL-1, IL-4, and/or IL-6 were released following activation of both mast cell lines and bone marrow culture-derived mast cells (BMMCs). Further work from the same group has demon-

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strated the presence of large amounts of preformed tumor necrosis factor-a (TNF-a)in murine peritoneal mast cells (53).Following crosslinking of Fc,RI there was both release of preformed TNF-a and increased levels of TNF-a mRNA. The release of IL-3 and GM-CSF was also demonstrated from BMMCs following IgE-mediated stimuli and ionophore (54). Cells were sensitized with monoclonal IgE antiphosphorylcholine (PC) and activated with PC-conjugated bovine serum albumin. Maximal mRNA for both cytokines was detected at 30 minutes postactivation and was followed by release of biologically active cytokines, with peak bioactivity for IL-3 and GM-CSF present at 30 minutes and 2 hours after activation, respectively. Other studies with BMMCs confirmed the presence of increased mRNA for GM-CSF following IgE-mediated activation, and also demonstrated a >23-fold increase in mRNA for TNF-a and IL-6 0.5 to 1 hour after activation (55).Increased secretion of biologically active TNF-a was maximal 4 hours after activation. BMMCs had a low level of basal transcription of IL-6 detected by Northern analysis. Interestingly, only about 50% of BMMCs demonstrated increased mRNA for IL-6 1hour after IgE-mediated activation, raising the possibility that only a subpopulation of BMMCs transcribe cytokines after activation. The amount of granule mediators released was only 30 to 35%. Therefore, it is possible that only a proportion of BMMCs were activated by cross-linking of Fc,RI, or, alternatively, most were activated to release a proportion of their granule constituents and there was heterogeneity in the capability of BMMCs to generate cytokines. There are as yet limited data on the generation of cytokines by human mast cells. Steffen et al. reported the presence ofTNF-a in mast cellslbasophils derived from culture of human bone marrow (56). mRNA for TNF-a was detected and localized to metachromatically staining cells by in situ hybridization. Immunoreactive TNF-a was also demonstrated in the granules of these cells. Benyon has demonstrated cytotoxicity of purified human skin mast cells toward WEHI164 fibrosarcoma cells, which was inhibited by antibody against TNF-a (57). Mast cell cytotoxicity was enhanced by cross-linking of Fc,RI. Preliminary data suggest that purified human pulmonary mast cells contain TNF-a bioactivity (58), and there are preliminary data on the localization of IL-4 to human pulmonary mast cells in bronchial biopsies from asthmatic individuals (M. K. Church, personal communication). Thus, rodent mast cells produce a wide range of cytokines in response to IgE-mediated stimuli. There are limited data on human

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mast cells, but it is likely that they will also be found to produce a range of cytokines. Nevertheless, these studies do not address the question of the relative importance of mast cell-derived cytokines compared to those derived from other cells in allergic diseases. It is tempting to postulate that the mast cell may play a pivotal role in the recruitment and priming of inflammatory cells following allergen challenge in sensitized subjects. This would be of clear relevance to seasonal and other types of allergic asthma. It is also possible that mast cells may play an important role in the recruitment and activation of T cells and eosinophils, and in the regulation of local IgE production within the airways in chronic asthma.

C. EOSINOPHILS It has been known for a long time that bronchial asthma is associated with eosinophilia of the blood and lung (59). With the finding that these cells were able to metabolize histamine, inactivate leukotrienes and platelet-activating factor (PAF), and suppress histamine release, they were attributed a protective role in allergic responses (60). Subsequent research has suggested that they may serve a proinflammatory function since they are capable of secreting preformed and newly generated mediators capable of eliciting tissue damage (61,62).Their presence in the airway and ability to express a low-affinity receptor for IgE (63)provide a mechanism whereby these cells can be activated via IgE-dependent mechanisms. With allergen challenge, there is a transient blood eosinopenia at 6 hours postchallenge that is followed by a progressive eosinophilia occurring up to 24 hours postchallenge (64).The postchallenge eosinophilia appears only in those asthmatics in whom a late asthmatic response develops and correlates not only with the magnitude of this reaction, but also with the basal airway responsiveness (64). The recent finding that circulating eosinophil precursors increase during the late asthmatic reaction (65),and that their numbers fluctuate in relation to seasonal exposure in atopic subjects (66), suggests a role for allergen in stimulating eosinophil production by the bone marrow. Factors that may be responsible for stimulating eosinophilopoiesis are granulocyte-macrophage colony-stimulating factor, IL-3, and IL-5 (67-70). In addition to supporting colony growth and maturation (71-74), these cytokines may also liberate cells from the bone marrow (67) and prime them for augmented proinflammatory functions (71-74). The observation that human eosinophils produce IL-3 and GM-CSF when stimulated by ionophore or IFN--y suggests additional mechanisms by which eosinophils may augment the local inflamma-

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tory response, including autocrine effects of IL-3 and GM-CSF upon eosinophil survival and function (75,76). An increased number of eosinophils in asthmatic airways is an almost constant finding. Several centers have identified increased numbers of eosinophils in the bronchial mucosae of asthmatic individuals (12,13,15,77). Electron microscopic and immunohistochemical studies demonstrate activation of the infiltrating eosinophils as evidenced by electron lucency of the granule matrix, loss of the central core of the granules, and positive staining with EG2, a monoclonal antibody that recognizes the cleaved form of eosinophil cationic protein (ECP) (3,12,13,15) (Fig. 2). Peripheral blood eosinophilia, levels of eosinophils and ECP in BAL fluid, and numbers of intraepithelial eosinophils correlate with the severity of asthma (12). The number of EG2-positive cells was also higher in individuals with airway hyperresponsiveness than in those without (15). Bronchial provocation with allergen elicits eosinophil influx into the airway lumen (78,79). Metzger and co-workers have shown that there is a prominent BAL eosinophilia at 4 hours and persisting for 24 hours after allergen challenge (78). The infiltrating eosinophils demonstrate

FIG.2. EG2-positive cells in the submucosa of asthmatic airway (arrows).Note the loss of epithelium (E) and subepithelial collagen deposition (C).

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appearances consistent with degranulation. DeMonchy and associates have shown that after allergen challenge there occurs an increase in the ECP/albumin ratio, thereby providing further evidence for eosinophi1 degranulation during the allergen-provoked asthmatic response

(79).

Four highly charged arginine-rich proteins have been located in the granules of the human eosinophils, namely, major basic protein, eosinophil cationic protein, eosinophil peroxidase (EPO), and the eosinophil-derived neurotoxin (EDN) (80). There is compelling evidence that MBP may be related to the inflammatory changes and tissue damage seen in the bronchial mucosa. MBP is toxic to tracheal epithelial cells in concentrations as low as 9 x M (81).This is well below the to lop5 M concentrations found in asthmatic sputum (61). Using an immunofluorescent technique, MBP deposition is seen in the bronchial wall and in the mucus in asthmatic lung tissue obtained at necropsy (62).The sites of MBP deposition coincide with the widespread epithelial damage characteristic of the airways in severe bronchial asthma. MBP levels in sputum correlate with disease activity (61) and the levels decrease after appropriate treatment. In one study, the concentration of MBP correlated with indices of airway responsiveness and the number of ciliated epithelial cells recovered by bronchoalveolar lavage (6). ECP is also located in the eosinophil granule matrix. The levels of ECP in serum rise after allergen-provoked asthma and during the pollen season in atopic individuals (82,83).ECP is neurotoxic and is toxic to guinea pig trachea (84).It is present in the submucosae of patients who have died from asthma (85). Circulating eosinophils of subjects with blood eosinophilia display a range of densities upon separation by discontinuous density centrifugation. In asthma, there is a predominance of hypodense cells. These cells are believed to be in an activated state, as shown by the increased oxygen consumption, phagocytic and cytotoxic capacity, increased spontaneous release of granule contents, and augmented generation of lipid mediators. Thus, the capacity of these activated eosinophils to effect tissue damage by releasing cytotoxic granule contents and by the generation of the newly formed mediators suggests that they are important proinflammatory cells in the asthmatic process.

Eosinophils and Adhesion Mechanisms The mechanisms responsible for the selective recruitment of eosinophils into the airway mucosa is not known. The endothelial cell is a major regulatory step in the passage of leukocytes into tissues. The signals generated at the site of allergic inflammation can activate both

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endothelial cells and circulating leukocytes to become adhesive for one another. Thus, it is pertinent to reflect on the adhesion molecules involved in leukocyte/endothelial cell interactions. Endothelial cells express adhesion molecules upon activation with appropriate stimuli, such as cytokines and endotoxin (86-89). The time course of expression depends upon whether the molecules are contained within intracellular stores or whether new protein synthesis is required. It is likely that surface expression of GMP-140 on endothelium may be involved in the initial phase of neutrophil migration. The mechanism does not require protein synthesis and is at least partially attributable to the redistribution of GMP-140 from the granule membrane (90).GMP-140 has been cloned and is a member of the selectin cellular adhesion molecule (LEC-CAM) family, having similarities to endothelial leukocyte adhesion molecule-1 and MEL-14 (91). Later phases of leukocyte infiltration are likely to be related to the induction or up-regulation over a matter of hours of cytokine-inducible endothelial adhesion molecules by a process involving de nouo protein synthesis. Several of these molecules have been identified; endothelial leukocyte adhesion molecule-1 (ELAM-1) (86), intercellular adhesion molecule-1 (ICAM- 1) (92), and vascular cell adhesion molecule-1 (VCAM-1) (89). ELAM-1 is a 110- to 115-kDa single-chain glycoprotein expressed on endothelial cells after stimulation with IL-1, TNF-a, or lipopolysaccharide (LPS) (86).The ligand for ELAM-1 has been identified as the sialylated Lewis x carbohydrate (93).The expression of ELAM-1 is dependent upon new protein synthesis, peaks at 4 to 6 hours, and decreases to near basal levels in cultured cells by 24 hours. ELAM-1 is not constitutively expressed in normal tissues but has been noted on the endothelium at sites of allergic inflammation and experimental delayed hypersensitivity reactions in uiuo (88,94). Incubation of skin biopsies from atopic individuals with allergen for 5 hours i n uitro also led to expression of ELAM-1 (94). Preincubation with antibodies to TNF-a alone and antibodies to IL-1 alone did not alter the allergeninduced expression of ELAM-1. However, preincubation with both anti-IL-1 and anti-TNF-a completely suppressed ELAM-1 expression. ICAM-1 is a 90-kDa single-chain glycoprotein that is basally expressed on endothelial cells and is markedly up-regulated by IL-1, TNF-a, lymphotoxin, LPS, or IFN-y (87,95). ICAM-1 has been cloned and is a member of the immunoglobulin supergene family with five immunoglobulin-like domains of C2 type (92). The increase in ICAM-1 expression on the cultured endothelial cells occurs well after ELAM-1 and plateaus at 24 hours, with expression being sustained as

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long as the cytokine is in the medium (96).Intensified ICAM-1 expression in vivo by dermal endothelial cells has been observed in skin biopsy tissue following intradermal injection of antigen that elicited a cutaneous late-phase response (88). ICAM-2 is a truncated form of ICAM-1, containing only two immunoglobulin-like domains. It is constitutively expressed in endothelial cells but is not subject to regulation by IL-1, TNF-a, or LPS (97). VCAM-1 is another member of the immunoglobulin supergene family, having six immunoglobulin-like domains of C2 type (89). Its receptor is VLA-4, a p-1 integrin, which binds VCAM at a site distinct from its fibronectin-binding domain (98). Recent work has shown that ICAM-1 and ELAM-1 bind both neutrophils and eosinophils, whereas VCAM-1 binds eosinophils preferentially (99,100). On leukocytes, the adhesion molecules that act as counterligands for ICAM-1 consist of the LEU-CAM family of p-2 integrins (101).These comprise three heterodimers with a common p chain (CD18) and different a chains designated LFA-1 (CDlla), MAC-1 (CDllb), and P150,95 (CDllc). Endothelial Iigands for C D l lalCD18 are ICAM-1 and ICAM-2 and for CDllb/CD18 may be ICAM-1. No adhesion ligand on endothelial cells for C D l l c has yet been identified. LFA-1 is found on all leukocytes, whereas MAC-1 and P150,95 are found only on phagocytes and large granular lymphocytes. LFA-1 is involved in lymphocyte adhesion and all three heterodimers are involved to varying degrees in neutrophil, eosinophil, and mononuclear cell adhesion. Leukocyte p-2 integrins are expressed on the surface of leukocytes in an inactive form (102). Stimulation of the cells with chemotaxins leads to activation of the receptors (102,103) and an increase in their cell surface expression (104-106). It has recently been shown that ELAM-1 may activate MAC-1 on the surface of neutrophils (107); recombinant soluble ELAM-1 was also shown to be a neutrophil chemoattractant. Whether ELAM-1 up-regulates MAC-1 on eosinophils was not studied; however, these experiments serve to show one level of complexity in the interaction between different endothelial adhesion molecules. The interest in the role of adhesion molecules in asthma was heightened recently when Wegner and colleagues (108)identified a relationship between eosinophilic infiltration and airway responsiveness in a primate model of asthma. They demonstrated that the presence of eosinophils in the airways following repeated administration of aerosolized ascaris extract to sensitized animals appeared to be predictive of the intensity of the early asthmatic response to inhaled antigen. In addition, antigen challenge induced a prolonged and specific eosinophil influx into the airways, which is evident at 6 hours after

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challenge and persisted for 7 days. This was associated, both in terms of magnitude and time, with increased airway responsiveness to methacholine. The administration of anti-ICAM-1 monoclonal antibody in vivo reduced eosinophil infiltration and airway hyperresponsiveness following antigen inhalation challenge. The type of response in the airways was dependent upon the model employed. When a subset of intrinsically hypereosinophilic animals was given a single inhaled allergen challenge, there was both an immediate and a delayed asthmatic response. The latter was associated with a neutrophil influx into the airways and an associated airway hyperresponsiveness, and was inhibited by anti-ELAM-1 but not anti-ICAM-1 (109). The apparently different roles of ELAM-1 and ICAM-1 in these different models of asthma are consistent with the time course of their induction. Clearly, by successfully determining the key molecular interactions that might be responsible for selective eosinophil influx in asthma and the development of bronchial hyperresponsiveness, novel targets for the development of antiinflammatory therapies in allergic disease may be elucidated.

D. NEUTROPHILS Neutrophils have been reported to be associated with the transient bronchial hyperresponsiveness induced by ozone (110).However, the evidence for the involvement of these cells in the mechanisms of bronchial asthma is much less certain. The presence of a few neutrophils in epithelium is probably a normal phenomenon because neutrophils may be found even in the lavage fluid of normal subjects (6). Neutrophil numbers were even more numerous in the control specimens in a recent biopsy study of asthmatic patients (77). A number of studies have not shown any significant difference in the neutrophil infiltration between asthma and normal individuals. Following allergen and excercise challenge, a heat-stable high-molecular-weight neutrophil chemotactic activity (NCA) has been detected in the peripheral circulation (31-35). The release ofthis molecule is associated with the increased expression of cell surface markers of neutrophil activation, namely, complement receptors (111)and IgG Fc receptors (112), and with enhanced neutrophil cytotoxicity toward opsonized schistosomulae (113).The extent of neutrophil cytotoxicity and the release of NCA was related to the magnitude of the provoked decrease in FEVl (forced expiratory volume in one second). Premedication with cromolyn sodium prevented both NCA release and the increase in neutrophi1 cytotoxicity. The significance of these findings in relation to the occurrence of airway obstruction and the increase in bronchial hyperresponsiveness has not been elucidated.

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E. MACROPHAGES AND MONOCYTES

The classification of lung macrophages has traditionally divided them into two categories based upon their anatomic distribution: alveolar or interstitial. The term “alveolar” referred to the macrophages retrieved by bronchoalveolar lavage. However, considerable proof now exists that macrophages also reside at the air-surface interface of conducting airways in the lower respiratory tract of humans. The presence of macrophages in asthmatic lungs has been evaluated in uivo using bronchoalveolar lavage from patients with mild disease, and bronchial biopsies taken from patients with moderate to severe disease. Most studies of BAL fluid have found that the numbers of mononuclear phagocytes present were not increased, compared to the control subjects (6,36,39,78,79,114,115).One exception to this was the study by Metzger and colleagues (116). In contrast, immunohistochemistry of the bronchial biopsy specimens showed that the submucosa had a significantly increased macrophage population in asthmatic patients (77). The macrophage population had phenotypic characteristics of peripheral blood monocytes, suggesting that they had migrated recently into the lung. HLA class I1 antigen was expressed on the infiltrating cells of the airway mucosa to a greater extent in asthmatic subjects than in normal individuals (77). Metzger has shown that the number of monocytes in the airways increases at 48 hours after antigen provocation in asthmatic patients (116).The mononuclear cells of patients with bronchial asthma demonstrate increased complement receptor expression, as compared with those of normal subjects, and also greater enhancement of receptor expression following stimulation with casein. Capron and co-workers were the first to discover that rat macrophages could be activated by IgE-dependent mechanisms and that they possess a low-affinity IgE receptor (Fc,RII), as opposed to the IgE receptor on basophils and mast cells, which is high affinity and is referred to as Fc,RI. From sequence analysis ofthe cloned cDNAs, it is now clear that the FcR,II on B cells (Fc,RII,) and on U937 cells, a human monocytic cell line (Fc,RIIb), differ at the N-terminal cytoplasmic region but share C-terminal extracellular regions (117). In normal subjects, the percentage of Fc,RII-positive lung macrophages and peripheral blood monocytes is low, approximately 5-10 and 10-15%, respectively (118-120). In atopic individuals, the numbers of IgE Fc,RII-positive lung macrophages and peripheral blood monocytes are increased (120).Patients with severe asthma and atopic dermatitis treated with corticosteroids had the !owest percentage of IgE Fc,RII-positive peripheral blood monocytes, suggesting that the

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expression of this receptor can be modulated by therapy (120). Lung macrophages retrieved by bronchoalveolar lavage from mild atopic asthmatic subjects showed that the percentage of Fc,RII-positive lung macrophages was increased in these individuals to approximately 20%, compared with nonatopic controls (119). Speigelberg and colleagues found that peripheral blood monocytes from severely atopic individuals induced significantly more 51Cr release from IgE-coated red blood cells than did monocytes from nonatopic or mildly atopic asthmatic subjects (118). The number of Fc,RII present on mononuclear phagocytes can be reegulated by a variety of cytokines. Using the U937 cell, it has been shown that IL-4 or IFN-7 enhance Fc,RII gene expression and the production of protein (121). IgE is also able to augment the number of Fc,RII on these cells (122).These observations suggest that cytokines and other molecules, such as IgE, may be important regulators of mononuclear phagocyte Fc,RII in uiuo. Dessaint et al. demonstrated that IgE-antigen complexes activated rat peritoneal macrophages to release lysosomal enzymes and superoxide anion (123). Macrophage products of oxygen metabolism have proinflammatory effects and may contribute to the inflammatory airway reaction observed in patients with asthma. Bach et at. (124) observed that rat peritoneal macrophages were capable of releasing leukotriene C4. Rankin and co-workers (125,126) demonstrated that normal rat macrophages could be activated by monoclonal IgE and its specific antigen to release both leukotriene B4 (LTB4) and LTC4. Several laboratories have now demonstrated that mononuclear phagocytes could have an important role in IgE-mediated diseases. Ferreri et al. (127) challenged peripheral blood mononuclear cells in uitro with chemically aggregated IgE and found that these cells release small quantities of LTB4, LTC4, and PGE2. Fuller et al. (128) observed the release of LTB4, PGF2,, thromboxane Bz, and P-glucuronidase from macrophages obtained from patients with a variety of lung disease when these cells were challenged with anti-IgE. Analysis of bronchoalveolar lavage fluid of patients with asthma after antigen challenge revealed increased amounts of p-glucuronidase, whereas macrophage intracellular levels were decreased (129). These results suggest that macrophage secretory processes can be activated by allergen, acting through Fc,RII. In view of the evidence that alveolar macrophages (AMs) secrete molecules that can influence the functions of inflammatory granulocytes, and the compelling evidence for the participation of eosinophils in airway inflammation, Howell and colleagues have studied the interactions of AMs and eosinophils in asthmatic subjects (130). Eosino-

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phils incubated with AM supernatants from asthmatic patients, followed by stimulation with A23187, demonstrated an enhanced capacity to secrete LTC4. AM supernatants derived from normal individuals had no enhancing effects when compared with culture medium alone. The activity derived from asthmatic AMs could be neutralized by incubation with specific antibodies to human GM-CSF, suggesting that the major active component is identical or closely related to GM-CSF. This is supported by the observation that pretreatment of eosinophils with recombinant GM-CSF primed the cells for enhanced LTC4 generation following stimulation with A23187. GMCSF is an acidic glycoprotein with a PI of 4.5 and a molecular weight of 22,000. It elutes from size exclusion columns with an apparent molecular weight of between 15,000and 40,000 due to variations in its glycosylation. It stimulates the proliferation and differentiation of normal granulocytes and monocytic stem cells. It also modifies the function of mature granulocytes, leading to enhancement of expression of granulocyte functional antigens 1 and 2, Mol, Leu-M5, and C3bi. GM-CSF induces histamine release from basophils and enhances eosinophil survival in culture. Thus, the presence of GM-CSF in the lung may precondition eosinophils for enhanced proinflammatory functions upon subsequent stimulation, and either alone or in concert with other cytokines, lead to eosinophil colony formation from bone marrow progenitors. GM-CSF may therefore play an important role in the amplification of the eosinophilic inflammation, which is characteristic of asthmatic airways. F. LYMPHOCYTES An area of considerable current interest is the role of the T lymphocyte in the regulation of the inflammation associated with allergy and asthma. T cell-derived lymphokines, IL-4 and IFN-y, are involved in the regulation of IgE production (131,132). Other lymphokines (IL-5, GM-CSF, and 1L-3) control eosinophil production and function (see above) and regulate mast cell differentiation. Necropsy examination of the airways of asthmatic patients showed large numbers of lymphocytes (1).Increased natural killer cell activity has been described in the peripheral blood of asthmatic patients (133). Since natural killer cell activity is an inducible property of T cells and of non-T, non-B lymphocytes, this is a nonspecific indicator of lymphocyte activation. Lymphocytes from the peripheral blood of patients with status asthmaticus demonstrate significant elevations of the expression of T

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lymphocyte activation markers, namely interleukin-2 receptor (IL-BR), class I1 HLA-DR, and “very late activation” antigen (VLA-l), compared with control subjects (134). Phenotypic analysis of the IL-2Rpositive T cells showed that these cells were exclusively of the CD4 helper-inducer phenotype. Percentages of IL-2R-positive and HLADR-positive but not VLA-l-positive lymphocytes tended to decrease as the patients were treated and improved clinically. The serum concentrations of IFN-.)I and soluble IL-2R were also significantly elevated in patients with acute severe asthma (135).Concentrations decreased as the patients improved clinically during the first 7-day period of hospital treatment. A significant correlation was observed between the degree of airway obstruction as measured by the peak expiratory flow rate and the percentages of peripheral blood T cells expressing IL-2R and the serum concentrations of soluble IL-2R. These observations provide evidence that CD4 T cell activation is associated with acute severe asthma. Biopsies of asthmatic airways reveal a tendency for increased numbers of T cells compared to biopsies from normal controls (15,77),with an increase in the number of cells expressing receptors for IL-2R (CD25),reflecting lymphocyte activation (15,136).CD25-positive cells were greater in airways of asthmatic subjects with bronchial hyperresponsiveness than in those without (15).Hamid et al. used the technique of in situ hybridization to examine the expression of IL-5 in bronchial biopsies from normal and asthmatic subjects (136).Using an antisense cRNA probe for IL-5, they found IL-5 mRNA in the bronchial mucosa of 6 of 10 asthmatic subjects, and in none ofthe 9 controls. No signal was obtained with sense cRNA probes, nor in tissue pretreated with RNase A, demonstrating the specificity of the hybridization. Although the number of patients studied was small, there was a trend for the six IL-5-positive asthmatics to have more severe asthma than those in whom no signal for IL-5 was observed. Further, biopsies positive for IL-5 mRNA also had a greater number of CD25-positive cells, a greater number of eosinophils, and a greater number of EG2positive cells. These data are consistent with the suggestion that IL-5, secreted by activated T lymphocytes, contributes to the recruitment and activation of eosinophils in the bronchial mucosa in asthma. 111. Eicosanoids and the Pathophysiology of Asthma

Several observations suggest that leukotrienes, prostaglandins, and thromboxane are important mediators in bronchial asthma. They are potent proinflammatory and spasmogenic mediators; they are present

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TABLE I1 PEPTIDOLEUKOTR~ENES AND ASTHMA"

Properties Potent bronchoconstrictor agonists Increase mucus secretion Increase vascular permeability Selective hyperresponsiveness to LTE4 in asthma Increase airway hyperresponsiveness Presence in the airways and release in asthma In asthmatic airways at rest Release following allergen challenge, aspirin-induced asthma, isocapnic hyperventilation, and acute severe asthma Leukotriene antagonists Decrease resting airway tone in asthma Attenuate early asthmatic response to exercise, allergen, and isocapnic hyperventilation Attenuate late asthmatic response to allergen and accompanying hyperresponsiveness Efficacy in chronic asthma Evidence for the role of leukotrienes in bronchial asthma. LTE4, Leukotriene E+

in asthmatic airways at rest and during an acute attack of asthma; and the acute asthmatic response to various stimiili is attenuated b y potent and selective receptor antagonists and inhibitors of the relevant enzymes (Table 11). A. LEUKOTRIENES

1. Synthesis and Metabolism The synthesis and metabolism of eicosanoids has been reviewed extensively (137). Metabolism of arachidonic acid by 5-lipoxygenase generates the unstable intermediate 5-hydroperoxyeicosatetraenoic acid (5-HPETE) (138), which is reduced to 5-hydroxyeicosatetraenoic acid (5-HETE)or is converted to an epoxide, leukotriene A4 (139-141) (Fig. 3 ) . LTAQis processed by an epoxide hydrolase to LTB4 (142) or, by a glutathione-S-transferase, to LTC4 (124,143,144).LTCl is cleaved by y-glutamyl-transpeptidase to LTD4, which is cleaved by a dipeptidase to LTE4 (124,143,145-148). LTA4 also undergoes nonenzymatic diastereoihydrolysis to 5S,12R- and 5S,12S-dihydroxy-6-trans-LTB4 somers and to minor products, 5,6-dihydroxyeicosatetraenoicacid diastereoisomers (149). LTC4, LTD4 and LTE4 comprise the activity previously recognized as slow-reacting substance of anaphylaxis (SRS-A), and are collectively known as the sulfidopeptide leukotrienes.

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JONATHAN P. ARM AND TAK H . LEE Ether Phospholipids

I

I

Acetyl transferase

2-lyso-PAF

1

PAF

Acetyl hydro lase

ARACHl DON IC

5-HETE

+

+5-HPETE

PGG2

PGD2 Hydrolase

LTB4

I

PGH

l

1

PGE 2

PG'2

LTC4

-+

LTD4 +LTE

2

PGF

2a

T X A2

4

FIG.3. Generation of platelet-activating factor (PAF) from ether phospholipids and metabolism of arachidonic acid by 5-lipoxygenase and cyclooxygenase pathways to leukotrienes (LT) and prostaglandins (PG), respectively; 5-HETE, 5-hydroxyeicosatetraenoic acid; 5-HPETE, 5-hydroperoxyeicosatetraenoicacid.

With the molecular cloning of 5-lipoxygenase (5-LO) it became apparent that cellular 5-LO activity was dependent upon an additional factor. Osteosarcoma cells transfected with the cDNA for 5-LO were unable to generate leukotrienes upon stimulation with the calcium ionophore A23187, although cell lysates expressed active enzyme (150). Furthermore, a class of compounds, of which MK-886 is an example, inhibit the generation of leukotrienes by intact cells but have no inhibitory effect on soluble 5-LO (151).The target of MK-886 was identified as a membrane protein of M,18,000, termed 5-LO-activating protein (FLAP) (152). Osteosarcoma cells transfected with 5-LO or FLAP alone did not generate leukotrienes upon activation with A23187. Transfection with cDNAs for both 5-LO and FLAP was required for significant generation of leukotrienes.

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LTB4 is converted intracellularly by a hydroxylase to 20-hydroxy LTB4, and by further oxidation to a biologically inactive molecule, 20-aldehyde LTB4 (153- 155). The sulfidopeptide leukotrienes may be metabolized by granulocytes, upon the triggering of the respiratory burst (156), through an extracellular hydrogen peroxide-peroxidase chloride-dependent reaction. In addition to their generation by inflammatory cells, leukotrienes may be synthesized and metabolized b y lung tissue. Thus, the conversion of LTA4 to LTB4, LTC4, LTD4, and LTE4 has been demonstrated in guinea pig lung (157),and of LTC4 to LTD4 and LTE4 in human lung parenchyma (158).

2 . Biological Activities

LTB4 is a potent proinflammatory mediator. Its in uitro activities are apparent at concentrations as low as lo-” M and include chemokinesis and chemotaxis of human neutrophils and eosinophils (104,159),chemokinesis of monocytes (16O), aggregation of neutrophils (159), enhanced expression of complement receptors on granulocytes (105), release of lysosomal enzymes from neutrophils (161), and augmentation of neutrophil adherence to endothelial cell monolayers (162).In uiuo, intradermal injection of LTB4 promotes a prolonged neutrophil infiltration into human skin, with induration and tenderness 4-6 hours after injection (163). LTB4 also contracts smooth muscle through the biosynthesis of cyclooxygenase products (164). Sulfidopeptide leukotrienes constrict nonvascular smooth muscle, enhance mucus secretion, constrict arterioles, and enhance venopermeability (163,165-167). The activity and binding of the sulfidopeptide leukotrienes in various tissues and cells have been characterized. Stereospecific, reversible, and saturable binding of LTC4, LTD4, and LTE4 have been demonstrated in guinea pig and human lung. The existence of receptor heterogeneity for these agonists in guinea pig lung is suggested by differences in the contractile properties and kinetics of action of the separate leukotrienes, the effects of leukotriene receptor antagonists, and radioligand binding studies (145,168-177). In contrast to the results in guinea pig tissues, a study conducted in the presence of bioconversion inhibitors on intralobar airways isolated from human subjects undergoing surgery for carcinoma of the bronchus did not reveal evidence for multiple leukotriene receptors (178). However, it should be emphasized that data from human tissue are limited; the effects of underlying disease on the expression of the different leukotriene receptors have not been studied and data are not available for asthmatic lung.

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3. Potency LTC4 and LTD4 are potent constrictors of human airways both in vitro and in vivo (168,179,180). LTC4 and LTD4 are approximately 1000-fold more potent than histamine, on a molar basis, in contracting isolated human bronchi in vitro (180). In normal subjects the concentrations of LTC4 and histamine required to produce a 30% decrease in V30 were 2-20 pg/ml and 2-10 mg/ml, respectively (181).LTC4 was 600- to 9500-fold more potent than histamine and LTD4 was 6000-fold more potent than histamine on a molar basis (182). By comparison, in asthmatic subjects, LTD4 was 140-fold more potent than histamine in eliciting a 30% decrease in V3o (183). Asthmatic subjects were only one-third more responsive to LTD4 than the normal subjects, despite an approximate 100-fold hyperresponsiveness to inhaled histamine. The relative lack of hyperresponsiveness to LTC4 and LTD4 in asthmatic subjects was confirmed in other studies (184,185).In addition, correlation was observed between airway responsiveness to methacholine and the relative responsiveness to LTC4 and LTD4; subjects with the most responsive airways demonstrated the lowest relative responsiveness to LTC4 and LTD4 as compared to methacholine (185).In contrast, the relative potency of LTE4 compared with histamine and methacholine was two to three times greater in asthmatics than in normal subjects (186). Because of the inherent difficulties in comparing studies performed in different subjects using different methodologies, the potencies of LTC4, LTD4, and LTE4 relative to one another and to both histamine and methacholine were compared in normal and asthmatic subjects (187). The airways of asthmatic subjects were 14-fold, 15-fold, 6-fold, 9-fold, and 219-fold more responsive than the airways of normal subjects to histamine, methacholine, LTC4, LTD4,and LTE4, respectively. The cumulative data therefore suggest that the airways of asthmatic subjects are relatively unresponsive to LTC4 and LTD4, but have a marked hyperresponsiveness to LTE4. 4 . Leukotrienes and Airway Hyperresponsiveness Brocklehurst demonstrated that slow-reacting substance of anaphylaxis enhanced the contractile response of guinea pig ileum to histamine in vitro (188). It was subsequently shown that pretreatment of guinea pig tracheal spirals with 10-23 nM LTE4, but not LTC4, or LTD4, enhanced the subsequent contractile response to histamine (168). This effect was not observed when parenchymal strips were contracted with LTE4. A detailed in vitro investigation of LTE4-

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induced hyperresponsiveness suggested that LTE4 augments the contractile response of guinea pig tracheal spirals to histamine by facilitating cholinergic neurotransmission, and is mediated via the secondary generation of cyclooxygenase products acting at the thromboxane A2 (TP) receptor (189). A similar mechanism may operate in human airways (189). In vivo studies support a role for the sulfidopeptide leukotrienes in enhancing airway hyperresponsiveness in asthma. Inhalation of a bronchoconstricting dose of LTD4 in normal subjects produced an approximate twofold increase in airway methacholine responsiveness (190),which was maximal at day 7 and persisted for up to 2 to 3 weeks (191). In normal subjects inhalation of LTD4 did not significantly enhance the airway response to exercise (192) or histamine (184,193), although it increased the sensitivity of the airways to inhaled PGF2, by approximately sevenfold (184). Normal and asthmatic airway responses in vivo differ not only in their sensitivity to a wide range of pharmacological and nonpharmacological stimuli (194),but also by the presence of maximal airway narrowing to histamine and methacholine in nonasthmatic subjects. Asthmatic subjects show a leftward shift of the dose-response curve and progressive airway narrowing with increasing dose of agonist, whereas the airway response in normal subjects reaches a plateau at mild degrees of airway narrowing (195,196). The degree of maximal airway narrowing is greater in response to LTD4 than to methacholine. Prior inhalation of LTD4 did not change the position of the methacholine dose-response curve, although the maximal airway response to methacholine increased (197).The maximal airway narrowing response to LTD4 was diminished and the LTD4-induced augmentation of maximal airway narrowing in response to methacholine was prevented by pretreatment with inhaled budesonide for 1 week (198). Studies in asthmatic subjects have been more limited. The inhalation of bronchoconstricting doses of LTC4 did not enhance the airway response to ultrasonically nebulized distilled water (199). In contrast, preinhalation of a bronchoconstricting dose of LTC4, LTD4, or LTE4 in asthmatic subjects increased histamine responsiveness by approximately threefold, 4 to 7 hours after inhalation of the leukotriene (193,200). Neither LTC4, LTD4, nor LTE4 elicited any change in airway responses to histamine in normal subjects, although each mediator was administered in a dose that elicited a mean 35% fall in SGaw (airways specific conductance) (193,200).The lack of effect in normal individuals is in contrast to the studies of Kaye (191) and Kern (190) (see above), and may be due to a selective effect of LTD4 on normal

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airway responses to methacholine (as opposed to histamine), to the timing of measurements of airway responsiveness, or to individual variability. In addition to the capacity of inhaled leukotrienes to enhance subsequent airway responses to histamine in subjects with asthma, LTC4 may interact synergistically with histamine and PGDz in the acute bronchoconstrictor response (201).

5. Release of Leukotrienes in Asthma Using various physicochemical techniques, leukotrienes have been detected in bronchoalveolar lavage fluid of asthmatic subjects, both at rest and following bronchial challenge. Lam found LTE4 in the BAL fluid of 15 out of 17 asthmatic subjects (9); LTD4 was detected in 2 subjects and 20-hydroxy-LTB4 was found in 12 subjects. Other studies have confirmed the presence of significant quantities of LTC4 and LTB4 in the BAL fluid of asthmatic subjects compared to normal controls (202-204). Following allergen challenge, mean LTC4 levels rose from 64 pg/ml of lavage fluid to 616 pg/ml (203). Following asthma provoked by isocapnic hyperventilation, BAL concentrations of LTB4 and immunoreactive sulfidopeptide leukotrienes rose from baseline levels of 10 and 46 pg/ml, respectively, to 121 and 251 pglml, respectively

(204).

Measurement of urinary LTE4 has been used as a marker of sulfidopeptide leukotriene generation (205-207). Increased urinary LTE4 levels have been reported during acute severe asthma and at 3 hours following antigen challenge of asthmatic subjects (41-43), but not following exercise-induced asthma (42).

6 . Leukotriene Antagonists and Inhibitors If leukotrienes play a significant role in the pathogenesis of asthma, then attempts to inhibit their generation or to antagonize their action at specific receptors should be of some benefit. Several studies have found that administration of peptidoleukotriene antagonists leads to bronchodilatation in asthmatic but not in normal individuals (208210), suggesting that leukotrienes may contribute to the resting airway tone in asthma. This effect is additive to that of albuterol (208-210). These agents have also been shown to inhibit the acute asthmatic response to exercise (211,212), allergen (213-215), and isocapnic hyperventilation (216). In addition ICI 204,219 inhibited the allergeninduced late asthmatic response and the increased airway hyperresponsiveness that followed allergen challenge (213).Preliminary data also suggest that administration of leukotriene antagonists leads to an improvement in the severity of clinical asthma (218,219).Compared to placebo, treatment with MK-571 led to a mean 8-14% improvement in

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FEVI, 30% decrease in morning and evening symptom scores, and an approximate 30% decrease in usage of albuterol (218). There are few data yet on the effects of 5-LO inhibitors in asthma. A significant inhibition of the asthmatic response to cold, dry air was demonstrated (217), and a small inhibition of the early asthmatic response to allergen (220).Although the latter effect was not statistically significant, there was a correlation between the inhibition of urinary LTE4 excretion and the attenuation of the early asthmatic response, suggesting that the lack of clinical effect may have been related to insufficient inhibition of 5-LO in the lung.

B. PROSTAGLANDINS AND THROMBOXANE

1 . Synthesis und Metabolism Arachidonic acid may be metabolized by cyclooxygenase to the cyclic endoperoxides, PGGz and PGHZ, which are then converted b y specific synthesis to thromboxane (TX) Az, or to various prostaglandins, PGDZ, PGFZ,, PGE2, and PGIz (221,222) (Fig. 3). PGEz is the predominant cyclooxygenase product of a number of different types of cells, including epithelial cells and macrophages. PGD2 is the major cyclooxygenase product of the mast cell (223,224)and is metabolized to 9m,ll/3-PGFz, which contracts airway smooth muscle both in vitro and in vivo (225).

2 . Biological Activities

The cyclic endoperoxides, PGGz and PGHz, and TXAz are labile molecules with short half-lives and appear to act at a common receptor (226).They constrict vascular and bronchial smooth muscle and aggregate platelets (227,228). PGIz is active in many tissues, producing vasodilatation, inhibiting platelet aggregation, and relaxing bronchial smooth muscle (229,230).PGEZ has diverse properties, including inhibition of platelet aggregation and contraction or relaxation of vascular and nonvascular smooth muscle (231,232).In human airways it acts as a bronchodilator (233). PGFZ,, PGDz, and its stable metabolite 9a, 11P-PGFZ are potent bronchoconstrictors (225). In addition, PGDz stimulates neutrophil chemokinesis (234), causes vasodilatation, and increases postcapillary venular permeability (163),and may act synergistically with LTB4 in promoting neutrophil infiltration (163). 3 . Potency Initial studies of the bronchoconstrictor effects of prostaglandins were directed to the properties of PGFz,. PGFz, was shown to contract human airways in vivo, and asthmatic subjects were shown to be more

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sensitive to PGF2, than were normal controls. The airway responsiveness to PGFz, correlates with that to methacholine (235).Furthermore, the airway hyperresponsiveness that is characteristic of asthma is markedly greater toward PGF2, than toward histamine (233,235,236). However, subsequent studies suggested that the airway response to PGF2, in uiuo might not be as simple as originally described. Both Fish (237)and Beasley (238)have reported complex biphasic or triphasic responses to inhaled PGFz,, possibly due to the action of PGFz, on separate receptors mediating bronchodilatation and bronchoconstriction. Studies of the effects of cholinergic blockade on airway responses to PGFz, have yielded conflicting results (236-241), but the cumulative data suggest that the contribution of cholinergic pathways to PGFz,-induced bronchoconstriction is small. Neither a-adrenergic blockade (239) nor pretreatment with cromolyn (239,240) inhibited airway responses to PGFz,. In normal and asthmatic subjects PGDz is a potent contractile agent when inhaled (225,238,242), being approximately 3.5 and 10 times more potent than PGFz, and histamine, respectively. The major metabolite of PGD2, 9a,llP-PGF2, is a potent contractile agonist for human airways both in uitro and in uiuo (225). It is approximately 4 times more potent than PGD2 in contracting human bronchial smooth muscle in uitro, but is equipotent in eliciting bronchoconstriction in uiuo, suggesting that some of the contractile activity of PGD2 may be mediated through its metabolite. Prostaglandins may constrict human airways directly via TP receptors and indirectly through cholinergic pathways (238). The inhalation of 55 pg of PGEl and PGE2 in normal human subjects led to a mean increase in SGaw of 10 and 18%, respectively (233). In asthmatic subjects the same doses of these agonists led to a mean increase of 41 and 39%, respectively. These increases in airway caliber were comparable to those induced by 550 pg of inhaled isoprenaline. PGEz was also noted to speed the recovery from PGFz,-induced bronchoconstriction. However, both PGEl and PGE2 were highly irritating when inhaled, making them unsuitable for therapeutic use. PGIz has complex effects on the airways in humans. Precontracted human bronchus relaxes in response to PGI2 in uitro (229). However, in uiuo, inhaled PGIz had no consistent effect on airway caliber as measured by changes in SGaw in normal and asthmatic subjects (243). In contrast, concentration-related decreases in both FEVl and Vmax30 were observed in allergic asthmatic subjects. Inhaled PGIz protected the airways against the bronchoconstrictor effects of PGD2 and methacholine. The paradoxical effects of PGI2 on the airways of asthmatic

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subjects might be explained by its effect on the vasculature within the airways. Increased mucosal blood flow might lead to engorgement of the mucosa with a significant reduction in caliber of the small airways. Increased mucosal blood flow might also lead to a more rapid clearance of inhaled bronchoconstrictor agonists from the airways, providing a degree of functional antagonism (243). 4 . Prostanoids and Airway Hyperresponsiveness

Inhalation of a subthreshold dose of PGF2, enhanced airway responsiveness to histamine by approximately fourfold in asthmatic subjects but had no effect on the airway response to methacholine (244). Inhalation of a noncontractile dose of PGDZ, but not saline, bradykinin, or histamine, enhanced airway responses to subsequent histamine and methacholine in asthmatic individuals by approximately twofold (245). Hardy et al. (246) confirmed the potentiating effect of PGDz on airway histamine responsiveness in three asthmatic subjects, but suggested that the results may represent a physiological rather than a pharmacological effect of PGDZ. They showed that histamine and PGDz were additive and not synergistic in their bronchoconstrictor effects on the airways of asthmatic subjects.

5 . Measurements of Prostanoids in Biological Fluids There have been various attempts to measure prostanoids in the lungs, blood, and urine of asthmatic subjects in both stable asthma and asthma provoked by a number of stimuli. Liu and colleagues found that levels of PGDz, 9a,l lP-PGF2, and PGF2, in bronchoalveolar lavage fluid were elevated 10- to 20-fold in subjects with atopic asthma, compared to levels in the controls (8).There was an inverse correlation between levels of these mediators and the responsiveness of the airways to methacholine. PGD2 is the major cyclooxygenase product released by activated mast cells (38,223), and has therefore been measured in the BAL fluid of asthmatic subjects after allergen challenge. PGDz levels rose from basal levels of