Role of Leukotrienes in Bronchial Hyperresponsiveness and Cellular Responses in Airways

ALAN R. LEFF

Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, Illinois

	INTRODUCTION

TOP INTRODUCTION MECHANISMS OF EOSINOPHIL... RELATIONSHIP BETWEEN EOSINOPHIL... PHYSIOLOGICAL CONSEQUENCES OF... LEUKOTRIENES IN CHEMOTAXIS CONCLUSIONS REFERENCES

Among constitutively present cells, bronchoactive leukotrienes are produced predominantly by mast cells (1) and macrophages (2). A unique characteristic of asthmatic inflammation is the migration of leukocytes from the peripheral blood to the conducting airways of the lung. This is especially true of the eosinophil, which is not present in the airways of normal individuals, but may be found in massive numbers during periods of airway hyperresponsiveness in asthmatic individuals [Figure 1 (3)] (4). Most experimental models indicate that eosinophils are an invariable component of asthmatic hyperresponsiveness, although some studies suggest that bronchoconstriction can occur in the relative absence of these cells.

View larger version (109K): [in this window] [in a new window]   Figure 1.   Effect of eosinophil infiltration into human airways. At later stages, a cytotoxic effect likely accounts for the epithelial denudation found in human asthma. A Histologic section from an airway of a human who had asthma (hematoxylin-eosin; original magnification, ×160). (B) The same section as in (A), stained with a fluorescent monoclonal antibody for the eosinophil major basic protein (original magnification, ×160). (C ) Higher power view of the desquamated epithelium in A (original magnification, ×400). (D) Localization of MBP in cells (arrowheads); these cells correspond directly to the cells marked in C. MBP is also localized outside cells in association with epithelial desquamation (arrow; original magnification, ×400). C and D illustrate the same area; this section was first stained for MBP by immunofluorescence and subsequently stained with hematoxylin- eosin. [Reprinted by permission from Reference 3.]

For the purposes of this discussion, the eosinophil is viewed as a leukotriene transport system capable of providing a substantial reservoir of bronchoactive leukotrienes to airways in a relatively short time. They may be delivered to conducting airways in large numbers and have the capacity to produce cysteinyl-leukotriene C₄ (Cys-LTC₄), which cannot be produced by neutrophils, but also migrate into conducting airways in some circumstances (7, 8). This discussion deals first with the mechanism by which eosinophils, a minority constituent of the circulating blood, are homed selectively to the conducting airways of the lung in human asthma. The mechanism by which the recruitment process of cellular adhesion primes migrating eosinophils for augmented leukotriene synthesis is discussed with particular reference to interactions between surface integrins on eosinophils and their counterligands, the immunoglobulin supergenes on the endothelial surface (9, 10) and fibronectin within the airway wall (11). The potential physiological significance of augmented secretion caused by adhesive interactions is suggested by experiments elucidated in this discussion. Finally, some new preliminary evidence for an autocrine mechanism of eosinophil recruitment that is mediated through the secretion of Cys-leukotrienes is considered.

	MECHANISMS OF EOSINOPHIL RECRUITMENT

TOP INTRODUCTION MECHANISMS OF EOSINOPHIL... RELATIONSHIP BETWEEN EOSINOPHIL... PHYSIOLOGICAL CONSEQUENCES OF... LEUKOTRIENES IN CHEMOTAXIS CONCLUSIONS REFERENCES

Eosinophils do not reside in human airways under normal circumstances. Normally, these cells are important in combating parasitic infections. While eosinophil morphology has a certain similarity to other granulocytes (e.g., neutrophils and basophils), this appearance is deceptive. Eosinophils have no phagocytic function as do neutrophils. The granular proteins contained in eosinophils are unique to these granulocytes and differ substantially from those of other cells. Unlike neutrophils, eosinophils represent a major defense against parasitic infections; they attach along the length of helminths and secrete their granular proteins, which are potent RNases, to cause the death of the infesting organism. Eosinophils also are capable of synthesizing Cys-leukotrienes as well as LTB₄. Neutrophils, in contrast, lack the synthase to convert LTA₄ into LTC₄ and hence are incapable of producing bronchoactive leukotrienes even if recruited to conducting airways. Both neutrophils and eosinophils are capable of synthesizing bronchoactive prostaglandins, but these do not appear to have pathogenic significance in human asthma. Leukotriene B₄ has substantial chemoattractant activity for neutrophils (12, 13) and for guinea pig eosinophils (14). However, LTB₄ has substantially less chemoattractant activity for human eosinophils (15).

Eosinophils share surface ligands with other myelopoeitic elements. The sequence and mechanism of activation thus determines which cells will be recruited to the airways. Recruitment occurs at the capillary level, where flow is slow enough to overcome shear forces that exist at greater perfusion pressures. Cell rolling, presumably mediated by selectins on both the endothelial and eosinophil surface, is the first phase of recruitment and this slows laminar flow further (16). Interleukin 5 (IL-5) has been found in high concentrations in the conducting airways of the lung during periods of asthmatic activity (16, 17), and this cytokine selectively causes shedding of L-selectin from the eosinophil surface and the simultaneous upregulation of ₂-integrins on the eosinophil surface. The nature of the process that causes relatively quiescent asthma to enter a phase of eosinophil recruitment is not defined. While it is presumed that the initial secretion of IL-5 is helper T cell type 2 (Th2) mediated, the trigger for this event also is unknown. Once activated, eosinophils further produce IL-5, and this suggests a possible autocrine amplification of the recruitment process.

The next phase of this recruitment process is tight adhesion of eosinophil integrins to surface ligands on the endothelium. ₁-Integrin (VLA-4) is constitutively expressed on the eosinophil surface; whether its conformation is changed to augment adhesion is unknown. ₂-Integrin expression is quantitatively upregulated by a mechanism directly related to binding at the IL-5 receptor (Figure 2 [18]). Preliminary data (unpublished results) suggest that the conformation of MAC-1, a ₂-intregrin, also is altered toward a higher affinity state by IL-5. As noted above, upregulation of these integrins by IL-5 does not occur for the neutrophil, which lacks the IL-5 receptor, and this may account for the selective migration of eosinophils, which share ₂-integrins, MAC-1 and LFA-1, with eosinophils.

View larger version (44K): [in this window] [in a new window]   Figure 2.   Adhesion of a granulocyte to the endothelial wall. Bottom: (A) "Rolling" function caused by selectin molecules is followed by tight adhesion to ICAM-1 by 2- and 1-integrin (not shown). (B) This leads to diapedesis through the endothelial cell wall and into the airway parenchyma. [Reprinted by permission from Reference 4.]

The counterligands for eosinophil surface integrins are the immunoglobulin supergenes, ICAM-1 (₂) and VCAM-1 (₁), which are endothelial surface molecules containing a five- domain transmembrane component. The specificity for binding between integrins and endothelial surface ligands is determined by the pocket formed by the  and  chains of the surface integrin (Figure 3) into which the ICAM-1 or VCAM-1 molecule inserts. As for eosinophil surface integrins, ligands of the immunoglobulin supergene family are upregulated by specific cytokines, including IL-1 and IL-4 (19, 20). The "trigger" for this process remains similarly unknown. It is not known whether ₁- or ₂-integrins further participate in the selectivity of eosinophil recruitment. ₁-Integrin is present only on the surface of eosinophils and lymphocytes, but not neutrophils. On the other hand, blockade of ₂-integrin is sufficient to prevent eosinophil migration both in vitro and in vivo (see below).

View larger version (9K): [in this window] [in a new window]   Figure 3.   Schematic characterization of the ligation between integrins on the eosinophil surface (left) and immunoglobulin supergene (e.g., ICAM-1, VCAM-1) on the endothelial surface. Specificity of binding is determined by the conformation of the  and  chains into which the terminal portion of the five-domain transmembranous portion of the immunoglobulin supergene fits.

	RELATIONSHIP BETWEEN EOSINOPHIL RECRUITMENT AND AUGMENTED SYNTHESIS OF LEUKOTRIENES

TOP INTRODUCTION MECHANISMS OF EOSINOPHIL... RELATIONSHIP BETWEEN EOSINOPHIL... PHYSIOLOGICAL CONSEQUENCES OF... LEUKOTRIENES IN CHEMOTAXIS CONCLUSIONS REFERENCES

Another unique property of eosinophils is their ability to ligate to matrix protein within the airway wall. VLA-4, which is not present on neutrophils, binds specifically to the RGD region of fibronectin (FN) [Figure 4 (18)], and this binding also does not occur for neutrophils. Several investigations have examined the relationship between binding to FN in vitro and the stimulated synthesis of LTC₄, using isolated human eosinophils incubated with FN-coated microwell plates. The binding process is remarkably slow [60 min; Figure 5 (21)] compared with binding to cultured human umbilical vein endothelial cells (about 5 min), suggesting that the molecule(s) may need to change conformation in the process. However, once binding has occurred there is a substantial augmentation of stimulated eosinophil secretion of eosinophil peroxidase (EPO) and LTC₄. Figure 6 (21) indicates that overall secretion of EPO increases by about 40%; however, given that only about 20% of the cells are actually bound in these studies, augmentation of LTC₄ secretion in bound cells may be estimated to be about fivefold. There are preliminary data to suggest that ligation of both ₁- and ₂- integrin to the endothelial surface also augments eosinophil secretion of LTC₄ (22). Thus, the process of cellular transmigration that accompanies chemoattraction of eosinophils into the airway matrix appears to be the process by which eosinophil synthesis of leukotriene synthesis is primed. The precise mechanism by which adhesion ligation causes this priming is unknown, but currently is under active investigation

View larger version (38K): [in this window] [in a new window]   Figure 4.   Schematic characterization of the ligation between VLA-4 with its characteristic 41 chains and the RGD region on fibronectin, which causes intramural binding of eosinophils after diapedesis. [Adapted from Leff, A. (editor). Pulmonary and Critical Care Pharmacology and Therapeutics. 303. With permission from the publisher.]

View larger version (16K): [in this window] [in a new window]   Figure 5.   Time course of binding of eosinophils to FN-coated microwell plates. Significant binding requires 60 min of incubation. This is substantially longer than is required for binding to endothelial ICAM-1 by the same eosinophil surface ligand. [Reprinted by permission from Reference 21.]

View larger version (21K): [in this window] [in a new window]   Figure 6.   Augmented secretion of EPO caused by priming due to ligation of eosinophil VLA-4 and fibronectin. [From Neeley, S. P., et al. 1998. Am. J. Respir. Cell Mol. Biol. With permission from the publisher.]

	PHYSIOLOGICAL CONSEQUENCES OF AUGMENTED SECRETION

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The notion that eosinophils could exist as innocent bystanders in the process of asthmatic hyperresponsiveness has been addressed in several experimental situations. The mere presence of these cells, even if the association were invariable, does not implicate their physiological significance in the bronchoconstrictor process. As noted above, it would appear that in some circumstances eosinophil infiltration is not essential to produce a substantial bronchoconstrictor response, e.g., exercise-induced bronchocontriction. A question to be resolved is whether the transmigration of eosinophils into airways and the concurrent priming of leukotriene secretion are at least capable of causing constriction of airways. If quantities of leukotriene secreted are insufficient, or if blockade of eosinophil has no effect on airway responsiveness, the eosinophil might well be an innocent bystander.

Prior investigation in monkeys sensitized with Ascaris suum antigen has demonstrated that eosinophils migrate into sensitized airways (23). Blockade of this migration with anti-ICAM-1 antibody both reduced the number of infiltrating eosinophils and substantially reduced the airway hyperresponsiveness on bronchial challenge. This would suggest that under these experimental conditions, eosinophils are essential to airway hyperreactivity [Figure 7 (16)]. In guinea pig tracheal preparations, eosinophils isolated from normal donors, or cultured from human umbilical vein cord blood elements, cause substantial contraction [Figure 8 (24)] that is blocked completely by inhibitors of 5-lipoxygenase, the enzyme converting arachidonic acid into LTA₄, which precedes production of LTC₄. In contrast, comparable activation of isolated human neutrophils has no effect on guinea pig tracheal contraction [Figure 9 (25)].

View larger version (18K): [in this window] [in a new window]   Figure 7.   Relationship between inhibition of eosinophil migration caused by ICAM-1 and airway hyperresponsiveness in monkeys sensitized with Ascaris suum antigen. Blockade of eosinophil migration (A) is associated with a return of airway hyperresponsiveness to near normal levels (B). [From Wegner, C., et al., as reprinted in Leff, A. R., K. Hamann, and C. Wegner. 1991. Am. J. Physiol. (Lung Cell. Mol. Physiol.) 260:L189-L206. With permission from the publisher.]

View larger version (15K): [in this window] [in a new window]   Figure 8.   Effect of activated eosinophils cultured from human cord blood on isometric tone of guinea pig trachealis muscle in vivo. Human cells were activated first with fMLP plus cytochalasin B and then applied directly to the trachea. Force is expressed as isometric tension per unit area of the tracheal surface. [Reprinted by permission from Reference 24.]

View larger version (15K): [in this window] [in a new window]   Figure 9.   Effect of human neutrophils activated as in Figure 8 on isometric tone of guinea pig trachealis. There is no contractile effect caused by the activating substance. fMLP, given alone (A) or by fMLP-activated neutrophils (C ) (compared with Figure 8 for eosinophils). Neutrophils lack the enzyme to produce Cys-leukotrienes. Positive controls are HL-60 cells (B), which produce bronchoconstriction prostaglandins, but not leukotrienes. [Reprinted by permission from Reference 25.]

To test further whether activated human eosinophils could cause contraction of human airway smooth muscle, our laboratory developed a system for videomicrometry of small sections of human airways. Fifth- to seventh-generation airways can be incubated in 96-well microplates. The airways are photographed through an overhead microscope, and the images are stored in real time on a computer. Changes in airway diameter are measured by determining lumenal pixel fitting in a manner described in another article in this supplement (26). Exposure to progressively larger numbers of human eosinophils causes progressive contraction as measured by lumenal narrowing (27, 28). This narrowing was blocked completely by pretreatment with the 5-lipoxygenase inhibitor, A63162, a congener of the drug, zileuton, which is now marketed in the United States for the treatment of asthma.

To determine if the degree of augmentation of secretion of leukotriene caused by adhesion of isolated human eosinophils to FN also caused augmented contraction of human airways, the effect on airway narrowing in human airway sections, using isolated human eosinophils, was examined. Cells exposed to FN caused twofold greater narrowing of lumenal area than cells exposed to bovine serum albumin (BSA) as a control [Figure 10 (29)]. Contraction was blocked completely for both BSA- and FN-treated cells activated with formyl-methionyl-leucyl-phenylalanine (fMLP) plus cytochalasin B by pretreatment with the 5-lipoxygenase inhibitor, A63162 (29). Contraction of airways in all studies was elicited with 10⁵ cells. While these data do not predict the in vivo situation, they do indicate that a physiologically meaningful augmentation of human airway contraction is elicited by adhesive ligation. However, given the dramatic nature of the blockade of contractile effects caused by eosinophils in this system, it is perhaps somewhat disappointing that antileukotriene therapies are less efficacious than would be predicted from these models. These data indicate that the process of asthmatic bronchoconstriction is substantially more complicated than that which can be predicted by a single in vitro model in an isolated cell system.

View larger version (13K): [in this window] [in a new window]   Figure 10.   Effect of fibronectin ligation of human eosinophils on contraction of human bronchial explants in vitro. There is substantial augmentation of lumenal narrowing in fMLP-activated human eosinophils first exposed to FN versus controls exposed to bovine serum albumin (BSA). Exposure of eosinophils to FN alone had no contractile effects. Hence, adhesion molecule ligation has a priming rather than a direct stimulatory effect on airway smooth muscle contraction. [Reprinted by permission from Reference 29.]

	LEUKOTRIENES IN CHEMOTAXIS

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Leukotrienes have variable chemotactic properties that are highly cell dependent. There also is a strong species dependence, which makes data from other species unreliable for prediction of the human condition. The guinea pig eosinophil is rich in LTB₄ receptors and hence migrates well by chemotaxis in a variety of experimental systems (14). However, the human eosinophil (as noted above) is weakly attracted by LTB₄, implying a weaker receptor population. In contrast, human neutrophils migrate strongly, and this was an initial concern in the development of antileukotriene therapies directed against 5-lipoxygenase. The specific concern was whether the concomitant blockade of LTB₄as well as Cys-leukotriene synthesisthat results as a consequence of the blockade of 5-lipoxygenase might also prevent neutrophil chemotaxis. The system has proven more robust than feared, and there is no increase in significant respiratory infection when a 5-lipoxygenase agent is used versus a leukotriene receptor antagonist that has no effect on LTB₄. Unfortunately, there also is no evidence that eosinophil chemotaxis is selectively diminished by LTB₄ inhibition that results from synthesis inhibitors. Hence, there is no obvious therapeutic benefit to drugs that block the entire leukotriene synthetic pathway relative to receptor antagonists that block specifically the cysteinyl-leukotriene receptor.

One phenomenon is the ability of the antileukotriene agents to block eosinophil migration at least partially, even with short-term use. In guinea pig tracheal explants, the chemotactic agent fMLP causes substantial and selective migration of eosinophils that reside naturally (and quiescently) in the lamina propria [Figure 11 (30)]. Administration of zileuton in concentrations > 10¹⁰ M caused substantial inhibition of eosinophil migration, and full blockade was achieved with greater concentrations (14). Because LTB₄ receptors are significantly involved in the chemotactic process, it was postulated that the action of zileuton resulted from the unique ability of this antileukotriene to block synthesis of this leukotriene as well as the SP1 analogs. The selective LTB₄ antagonist, LTB₄-diethylamide, caused even more potent blockade of eosinophil migration in this model [Figure 12 (30)]. However, administration of the highly selective leukotriene D₄ receptor antagonist, zafirlukast, caused equipotent blockade. Significant inhibition of eosinophil chemotaxis was observed at 10¹² M zafirlukast. Comparable trials have been conducted in humans. Calhoun and coworkers have reported in a preliminary study that large doses of zafirlukast given over 24 h caused an approximate 50% decrease in the migration of eosinophils into the airways of challenged subjects with asthma (31), and Sterk and colleagues have demonstrated independently comparable findings (32).

View larger version (15K): [in this window] [in a new window]   Figure 11.   Effect of pretreatment with the 5-lipoxygenase inhibitor, zileuton, on eosinophil migration from the lamina propria to the lumen in guinea pig tracheal explants. Chemotaxis was initiated by lumenal instillation of fMLP. [Reprinted by permission from Reference 30.]

View larger version (11K): [in this window] [in a new window]   Figure 12.   Effect of pretreatment with inhibitors of LTB4 (left panel ) and LTD4 (right panel ) on eosinophil chemotaxis, using the same preparation as for Figure 11. LTB4 causes inhibition of chemotaxis at even lesser concentrations than zileuton, suggesting that there is a chemotactic effect of this Cys-leukotriene compound. The mechanisms by which LTD4 causes chemoattraction in this model are undefined, but similar findings are suggested from human studies (see text). [Reprinted by permission from Reference 30.]

The mechanism by which LTD₄ receptor blockade inhibits eosinophil migration remains elusive. There are no Cys-LT receptors on the eosinophil surface in either rodents or humans. Hence, the effect is either nonspecific, i.e., nonreceptor related, or the result of complex interactions within the airway involving other cells and/or tissues. On the basis of the guinea pig model, the airway itself appears to contain all elements necessary to inhibit eosinophil migration through blockade of the Cys-LT receptor. One investigation using the leukotriene receptor antagonist pranlukast has shown that the ability of this compound to block eosinophil migration into the airways of challenged guinea pigs is blocked with the monoclonal antibody TRFK-5, which is directed against IL-5 (33). This implies, but does not by any means establish, that blockade of the LTD₄ receptor on some element contained within the airway wall could have an inhibitory effect on IL-5 secretion, which is an important component of adhesion model upregulation in eosinophil chemotaxis (see above). However, definitive studies still are required to elucidate the mechanism whereby LTD₄ receptor blockade affects eosinophil migration. It also remains to be determined whether the magnitude of inhibition of eosinophil migration in humans is clinically and pathophysiologically significant. If so, antileukotriene therapies could properly be viewed as being broadly antiinflammatory in a manner (if not degree) comparable to that conferred by corticosteroid treatment. The implications of this for subsequent airway remodeling are considered elsewhere in this supplement.

	CONCLUSIONS

TOP INTRODUCTION MECHANISMS OF EOSINOPHIL... RELATIONSHIP BETWEEN EOSINOPHIL... PHYSIOLOGICAL CONSEQUENCES OF... LEUKOTRIENES IN CHEMOTAXIS CONCLUSIONS REFERENCES

Asthma is viewed as an inflammatory process mediated at least in part by leukotrienes; eosinophils are the major transport systems for these compounds to the airway smooth muscle, where they cause contraction, and to the airway vasculature, where they cause edema. Leukotrienes are synthesized de novo in eosinophils directly from membrane phospholipids after activation by phospholipase A₂ (PLA₂). The process of selective chemoattraction is a fascinating one, as eosinophils are but a minor component of the circulating granulocytes. Even though eosinophils share common surface ligands with neutrophils, they are capable of selective migration into the airway wall. It is likely that cytokine-specific processes regulate this selective migration, e.g., IL-5.

It is also of considerable interest that the process of molecular adhesion and transmigration is intimately linked to the priming of eosinophil secretion of leukotrienes. The mechanism by which this occurs is unclear, but appears from some preliminary studies to be related to the direct phosphorylation of PLA₂-IV, which may occur as a consequence of adhesion. Another property of leukotrienes that remains unexplained is the apparent ability of these compounds to cause, by a mechanism yet to be defined, substantial chemotaxis of eosinophils in both animal models and in humans.

While eosinophils are the unique inflammatory cell of asthmatic inflammation, it still is unclear if they are essential to all manifestations of the asthma syndrome. Furthermore, it is unclear whether leukotriene synthesis alone accounts for the bioactivity of these cells in causing airway narrowing in asthma. Blockade of leukotriene activity in human asthma does not cause improvement in airflow obstruction in a manner comparable to that obtained with corticosteroids or high-efficacy ₂-adrenoceptor drugs. The invariable presence of eosinophils in human asthma does not itself imply a role for these cells in the pathogenesis of the disease. However, the demonstration that adhesion-primed eosinophils are capable of causing massive augmentation of leukotriene secretion and that this secretion is of a magnitude sufficient to cause contraction of human airway explants suggests a contributory role of eosinophils as the source of leukotrienes in human asthma. Nonetheless, the role of eosinophils as well as the role of leukotrienes in human asthma may vary among the various asthma phenotypes that are only now being defined.

	Footnotes

Correspondence and requests for reprints should be addressed to Alan R. Leff, M.D., Section of Pulmonary and Critical Care Medicine, Department of Medicine MC6076, University of Chicago, Chicago, IL 60637. E-mail: aleff{at}medicine.bsd.uchicago.edu

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TOP INTRODUCTION MECHANISMS OF EOSINOPHIL... RELATIONSHIP BETWEEN EOSINOPHIL... PHYSIOLOGICAL CONSEQUENCES OF... LEUKOTRIENES IN CHEMOTAXIS CONCLUSIONS REFERENCES

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