Antileukotriene Therapy
Future Directions

STEPHEN T. HOLGATE and ANTHONY P. SAMPSON

University Medicine, Southampton General Hospital, Southampton, United Kingdom

	INTRODUCTION

TOP INTRODUCTION HOW DO CYSTEINYL-LEUKOTRIENES... DO CYSTEINYL-LEUKOTRIENES... HOW DOES GENETIC VARIATION... FUTURE TARGETS REFERENCES

In the early 1990s, numerous clinical trials with antileukotriene drugs confirmed Brocklehurst's hypothesis that slow- reacting substance of anaphylaxis (SRS-A) is an important bronchoconstrictor agent in asthma (1). Bronchoprovocation studies showed that inhibition of the synthesis of leukotrienes (LTs) or antagonism of cysteinyl-LT receptors partly or wholly blocks constrictor responses to a wide range of asthma triggers, including allergen, platelet-activating factor (PAF), exercise, cold air, sulfur dioxide, adenosine 5'-monophosphate, and nonsteroidal antiinflammatory drugs (NSAIDs) (2). Multiple-dose studies of patients with allergic and nonallergic asthma commonly report 10-15% improvements in baseline lung function, with 25-60% improvements in secondary outcome measures, including symptom scores, night-time awakenings, use of rescue medication (₂-agonists and glucocorticoids), and days lost from work and school (3). To many researchers, the clinical improvements observed after blockade of this single family of inflammatory mediators were surprising, in view of the widely accepted model that airway narrowing is caused by numerous agents acting as components of a mediator "soup." Indeed, the efficacy of antileukotriene drugs is highlighted by the relative failure in clinical trials of other mediator antagonists such as histamine H₁ antagonists, thromboxane antagonists, and PAF antagonists (4).

The antibronchoconstrictor efficacy of antileukotriene drugs provided the main impetus behind their introduction as the first novel class of asthma therapy in more than 20 yr. However, clinical trials also provided surprising evidence for a hitherto unsuspected role of cysteinyl-leukotrienes in promoting persistent eosinophilia in the airway and blood of patients with asthma, and possibly influencing pathways involved in airway wall remodeling (5). A better understanding of these actions of antileukotriene drugs will influence their place in asthma management.

	HOW DO CYSTEINYL-LEUKOTRIENES INDUCE EOSINOPHILIA?

TOP INTRODUCTION HOW DO CYSTEINYL-LEUKOTRIENES... DO CYSTEINYL-LEUKOTRIENES... HOW DOES GENETIC VARIATION... FUTURE TARGETS REFERENCES

Chronic infiltration of the airway by eosinophils is a highly consistent finding in bronchoscopy studies both in allergic and nonallergic asthma. Eosinophilia in the airway and blood correlates with bronchial hyperresponsiveness. The factors driving eosinophilia may include enhanced eosinophilopoiesis and release from the bone marrow, priming of eosinophil activity within the circulation, upregulation of adhesion molecules on eosinophils and on the vascular endothelium, directed eosinophil migration in response to specific chemoattractant molecules, and reduced eosinophil apoptosis in the inflamed airway wall. The eosinophilopoietic cytokines interleukin 3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and chemokines including RANTES and eotaxin, are all thought to play a role in eosinophil differentiation and/ or trafficking. Of these, only IL-5 has so far been shown to induce airway eosinophilia when inhaled by patients with asthma (6), and the lack of specific cytokine antagonists at present prevents a quantitative assessment of the relative importance of each cytokine in humans. In contrast, evidence from bronchoprovocation studies and from trials of specific antileukotriene drugs suggests that cysteinyl-LTs make a significant contribution to the eosinophilia in patients with asthma.

Cysteinyl-LT Challenge Studies

The exquisite potency of cysteinyl-leukotrienes as bronchoconstrictor agents in humans was demonstrated repeatedly in challenge studies from 1981 onward (7). That inhaled LTC₄ and LTD₄ could also produce a dose-related eosinophil infiltration of the airway was first demonstrated in 1990 in the guinea pig (8). The response was blocked by the early cysteinyl-LT receptor antagonist MK-571. In the same model, eosinophil infiltration has been shown to persist up to 4 wk after a single inhaled dose of LTD₄, and to be blocked at 24 h and at 3 wk by pranlukast (9).

In a study of four subjects with asthma, 3- to 24-fold increases in eosinophil count were observed in the bronchial mucosa 4 h after inhalation of LTE₄, accompanied by much smaller increases in neutrophils, but not in mononuclear cells (10). In 12 patients with allergic asthma, a bronchoconstricting dose of inhaled LTD₄ increased the percentage of eosinophils in sputum induced 4 h later, while inhaled methacholine produced a similar degree of bronchoconstriction but had no effect on eosinophil counts (11). Our own study has shown that in mild asthma inhaled LTD₄ induced both a sputum eosinophilia and neutrophilia, but only the latter achieved significance. It is not known whether inhaled cysteinyl-LTs produce airway eosinophilia in nonatopic normal subjects, or whether the response in allergic asthmatic subjects persists beyond 4 h. Segmental allergen challenge of patients with atopic asthma also produces airway eosinophilia that remains detectable for up to 30 d, and eosinophil counts as late as 24-48 h after challenge correlate with bronchoalveolar lavage (BAL) fluid cysteinyl-LT levels (12).

Antileukotriene Drug Studies

The contribution of endogenous cysteinyl-LTs to allergen- induced eosinophilia is confirmed in animal models and in clinical trials of antileukotriene drugs. Inhaled ovalbumin challenge of sensitized guinea pigs induces a four-fold eosinophil infiltrate of the bronchial submucosa at 12 h, and this is blocked by MK-571, but not by histamine H₁ or H₂ receptor antagonists or by a cyclooxygenase inhibitor (13). In the cynomolgus monkey, airway eosinophilia and bronchial hyperresponsiveness after inhaled antigen are blocked by a cysteinyl-LT receptor antagonist (ICI- 98,615) (14) and in sheep by the 5-lipoxygenase (5-LO) inhibitor zileuton (15). In 5-LO gene-deleted mice sensitized to ovalbumin, increases in airway eosinophils after ovalbumin challenge are reduced by 50% compared with wild-type mice, while the increase in bronchial responsiveness is completely abolished (16).

In 11 subjects with mild to moderate asthma, 7 d of treatment with zafirlukast reduced the influx of basophils, lymphocytes, and eosinophils measured in BAL fluid 2 d after segmental allergen challenge, and also reduced levels of inflammatory markers linked to mast cell and macrophage activation (17). One week of treatment with zileuton prevented a significant influx of eosinophils into BAL fluid 24 h after segmental allergen challenge of 10 subjects with atopic asthma (18). Zileuton reduces baseline levels of LTB₄ in BAL fluid and of LTE₄ in the urine of subjects with nocturnal asthma, accompanied by significant reductions in the BAL and peripheral blood eosinophil counts compared with placebo (19). Four weeks of treatment with montelukast resulted in an approximately 50% reduction in eosinophil counts in both induced sputum and blood of 40 adults with mild asthma (20), and slightly smaller reductions in sputum and blood eosinophils have been observed after 8 wk of treatment with pranlukast (21). In 408 adults and 201 children with moderate asthma, 20-40% of whom were using inhaled corticosteroids, treatment with montelukast for 8 and 12 wk, respectively, produced significant reductions in blood eosinophil counts compared with placebo (22, 23). Similar results have been found with zafirlukast (24).

Further work is required to show that a 30-50% reduction in blood and sputum eosinophilia also occurs in the bronchial mucosa, and to link such changes to clinical improvements seen with antileukotriene drug therapy. However, taken together, these studies suggest that eosinophil influx into the allergic asthmatic airway, and perhaps of other inflammatory cells, depends to a significant degree on cysteinyl-LT synthesis. In patients with seasonal and persistent asthma, eosinophils, rather than mast cells, are the predominant cell type in the bronchial mucosa expressing the requisite enzymes for cysteinyl-LT synthesis (25, 26). Antileukotriene drugs may therefore interrupt a cycle of cysteinyl-LT synthesis and eosinophil influx, and hence prevent the release of other eosinophil products. The blood eosinophil data also raise the suspicion that cysteinyl-LTs may promote the maturation and/or release of eosinophils from bone marrow, in an analogous manner to the endocrine activity hypothesized for eosinophilopoietic cytokines. Cysteinyl-LTs may thus modulate eosinophilia in vivo by effects on multiple stages of the eosinophil life cycle including differentiation in the bone marrow, adhesion/margination, and apoptosis (Figure 1).

View larger version (20K): [in this window] [in a new window]   Figure 1.   Possible mechanisms of eosinophilia induced by cysteinyl-LT.

Cysteinyl-LTs and Eosinophil Chemotaxis

When compared with LTB₄ and other lipid mediators such as PAF, relatively few in vitro studies have investigated the effect of cysteinyl-LTs on the directed migration of human leukocytes. However, at nanomolar concentrations, LTD₄ causes directed migration of normal human eosinophils in vitro, a response that is blocked by the Cys-LT₁ antagonist pobilukast (27). In contrast, neutrophils respond to LTD₄ only at micromolar concentrations, while LTB₄ is a moderately potent and nonselective chemoattractant for both cell types. In subjects with asthma, it is possible that the migratory responses of eosinophils to LTD₄ may be further enhanced by in vivo priming by IL-5, as has been shown for eosinophil responses to PAF and LTB₄ (28).

Cysteinyl-LT and Leukocyte Adhesion

Cysteinyl-LTs may also contribute to eosinophil infiltration of the bronchial mucosa by promoting leukocyte-endothelial interactions. Both LTC₄ and LTD₄ induce rolling of polymorphonuclear (PMN) leukocytes along vascular endothelium (29) by increasing expression of P-selectin on the surface both of the leukocyte and the endothelial cell (30, 31). The interactions between P-selectin and their sialylated protein ligands may be similar for human eosinophils and neutrophils (32), but only the eosinophil can generate cysteinyl-LT that may enhance the interaction and promote microvascular leakage. Firm adhesion of eosinophils to endothelium involves interactions between leukocyte ₂-integrins and endothelial vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), which are modulated by GM-CSF, tumor necrosis factor  (TNF-), and IL-4 (33, 34) . The process of adhesion via ICAM-1 (but not VCAM-1) primes eosinophils for enhanced LTC₄ synthesis (34), and the process of tissue migration may directly activate eosinophils to synthesize cysteinyl-LT.

Cysteinyl-LT receptors are present on endothelium in human lung blood vessels (35). However, in human umbilical cord endothelial cells (HUVECs), the Cys-LT₁ antagonists pobilukast, pranlukast, and zafirlukast fail to block cysteinyl-LT-induced expression of P-selectin, suggesting a role for Cys-LT₂ or other uncharacterized receptors (31). Since many of the vascular actions of cysteinyl-LTs are also thought to involve Cys-LT₂ receptors (36), further pharmacological and molecular characterization of Cys-LT receptors may lead to a new generation of drugs with broader antiinflammatory activity than the present compounds.

Leukotrienes, Cytokines, and Apoptosis

Eosinophilia in the asthmatic airway may depend on a reduced rate of apoptosis when compared with other leukocytes. Increasing evidence suggests a critical role of eicosanoids in leukocyte apoptosis, although information directly linking cysteinyl-LTs to eosinophil apoptosis is so far lacking. However, in human neutrophils, the constitutive rate of apoptosis is reduced by exposure to LTB₄, but not to its less potent omega metabolites or to monohydroxyeicosatetraenoic acids (mono-HETEs) (37). Neutrophils incubated with arachidonic acid experience a high rate of apoptosis when LT synthesis is blocked with the 5-LO-activating protein (FLAP) inhibitor MK-886, while blockade of prostanoid synthesis with indomethacin enhances neutrophil survival (38). Thus, a 5-LO product promotes and a cyclooxygenase (COX) product reduces human neutrophil survival. GM-CSF produces a modest increase in neutrophil survival in vitro, and this is blocked by the LTB₄ receptor antagonist SB-201146, but not by the Cys-LT₁ antagonist pobilukast (39). Therefore, GM-CSF-dependent neutrophil survival is mediated by LTB₄ acting at surface receptors in an autocrine or paracrine manner. Similar effects of LTB₄ on DNA fragmentation, apoptosis, and cell proliferation have been reported in monocytic HL-60 cells (40).

In patients with asthma, the proportion of peripheral blood T cells undergoing apoptosis is less than in normal subjects (41). In contrast to neutrophils, the proportion of T cells undergoing apoptosis in the patients with asthma was increased by the Cys-LT₁ antagonist zafirlukast. Apoptosis in different leukocyte types may therefore be regulated by different leukotriene subfamilies. In eosinophils, survival is greatly enhanced by cytokines including IL-3, IL-5, and especially GM-CSF, which also prime eosinophils for enhanced cysteinyl-LT synthesis (42). It is a reasonable hypothesis that the effects of these cytokines on eosinophil survival may be mediated by cysteinyl-LT, and that antileukotriene drugs may reduce eosinophil numbers in the airway by accelerating their programmed cell death.

Eicosanoids may also be important in mediating the transcription of cytokine genes in response to proinflammatory stimuli. LTB₄ is recognized to enhance transcription of a number of cytokines including IL-6 and IL-8, possibly by activating the nuclear transcription factor NF-B (43), and to promote the synthesis of IL-5 by T cells (44). There is little information on the effect of cysteinyl-LTs or Cys-LT₁ receptor antagonists on cytokine synthesis by human cells. However, in guinea pigs, inhaled LTD₄ produces immediate bronchoconstriction and a persistent airway eosinophilia lasting 3-4 wk, both of which are inhibited by pranlukast. The eosinophilia (but not the bronchoconstriction) is blocked by a monoclonal antibody directed against IL-5 (9), showing that in guinea pigs LTD₄-induced eosinophilia is mediated by secondary release of IL-5 from an unknown cell type.

	DO CYSTEINYL-LEUKOTRIENES CONTRIBUTE TO AIRWAY REMODELING?

TOP INTRODUCTION HOW DO CYSTEINYL-LEUKOTRIENES... DO CYSTEINYL-LEUKOTRIENES... HOW DOES GENETIC VARIATION... FUTURE TARGETS REFERENCES

Chronic overproduction of cysteinyl-LT in asthma is indicated by multiple-dose studies with antileukotriene drugs, which show rapid improvements of 10-20% in baseline lung function maintained over several weeks of treatment (2). Excess cysteinyl-LT release in mild stable asthma has been confirmed directly by urinary LTE₄ assays, while histamine production is not significantly elevated (45). The resting tone of normal human bronchi in vitro is uniquely dependent on cysteinyl-LTs, as it is markedly inhibited by a cysteinyl-LT antagonist and by a 5-LO inhibitor, but not by cyclooxygenase or thromboxane synthetase inhibitors, a histamine H₁ antagonist, or atropine (46). It follows that the asthmatic airway may be under excessive "leukotriene tone" when compared with normal bronchi. Since human airway smooth muscle cells do not express 5-LO or FLAP, the endogenous cysteinyl-LTs in human bronchi may be generated by infiltrating mast cells and/or eosinophils. Mast cells are capable of continuous cysteinyl-LT synthesis in response to low levels of physiological stimulation (47), such as environmental allergen. Blood eosinophils from patients with asthma have an inherently increased capacity to generate cysteinyl-LTs (48), which may lead to cysteinyl-LT overproduction in the asthmatic airway even in the absence of a marked airway eosinophilia.

Chronic overproduction of relatively low levels of cysteinyl-LTs by mast cells or eosinophils may have subtle effects on structural cells of the airway, leading to bronchial hyperresponsiveness and airway remodeling. Inhaled cysteinyl-leukotrienes cause bronchial hyperresponsiveness in patients with asthma (49), and reductions in bronchial responsiveness are observed after multiple-dose treatment with antileukotriene drugs including pranlukast and zafirlukast (50, 51). The reduction in cold air responsiveness obtained after 13 wk of treatment with zileuton is comparable to that provided by high-dose inhaled budesonide (52), and persists for 10 d after washout of the drug, suggesting that leukotrienes may increase responsiveness by altering the characteristics of structural airway cells.

No bronchial biopsy studies have been published reporting the effects of antileukotriene drugs on airway pathology. However, in Brown Norway rats, the increase in bronchial responsiveness induced by inhaled antigen is directly related to an increase in the mass of bronchial smooth muscle, and both these increases are blocked by the cysteinyl-LT antagonist MK-571, suggesting a mitogenic effect of cysteinyl-LTs on smooth muscle (53). In vitro, LTD₄ has no direct effect on the proliferation of human airway smooth muscle (HASM) cells, but greatly augments that produced by epidermal growth factor (EGF) (54). The secretion of EGF and expression of EGF receptors are increased in asthma (55). The effect of LTD₄ on HASM proliferation is blocked by pranlukast and pobilukast, but not by zafirlukast (54), suggesting that it is mediated by a receptor subtype other than that which mediates bronchial smooth muscle contraction (Cys-LT₁). In contrast, induction of COX-2 expression by proinflammatory cytokines inhibits serum-induced proliferation of HASM, an effect that is attributable to PGE₂ synthesis (56). The rate of HASM proliferation in an inflammatory environment may thus reflect the balance between upregulation induced by leukocyte-derived cysteinyl-LTs and downregulation induced by PGE₂ from HASM cells themselves. Micromolar concentrations of LTD₄ do not alter the expression of extracellular matrix components including collagen, elastin, and fibronectin by HASM cells (54). However, at nanomolar concentrations, LTC₄ strongly induces the expression and activity of collagenase in human lung fibroblast cell lines and in primary fibroblasts from patients with idiopathic pulmonary fibrosis (57), providing a mechanism for contribution of LTC₄ to extracellular matrix remodeling in chronic airway inflammation.

Epithelial denudation, proliferation, and hyperplasia are typical pathological features of chronic inflammation in the asthmatic airway. Loss of epithelium may contribute to bronchial hyperresponsiveness by increasing access of stimuli to the bronchial mucosa and afferent nerve fibers, and by reducing the availability of bronchodilator and antiinflammatory mediators such as PGE₂. The cysteinyl-LTs are potent mitogens for human airway epithelial cells in culture, with LTC₄ being effective even at sub-picomolar concentrations (58). Cysteinyl-LT could contribute to abnormal epithelial proliferation, and as with smooth muscle, may interact with enhanced expression of EGF receptors on the asthmatic epithelium (55) to modulate epithelial repair processes. Cysteinyl-LT may exacerbate the sensitizing effects of epithelial denudation by directly stimulating local afferent nerves to release tachykinins, leading to bronchoconstriction and plasma exudation, as shown in a guinea pig model with a 5-LO inhibitor and with Cys-LT₁ receptor antagonists (59). At the time of writing, it is not known whether treatment with antileukotriene drugs can protect the epithelium from damage by inhibiting eosinophil trafficking and hence reducing the impact of eosinophil basic proteins on epithelial structure.

	HOW DOES GENETIC VARIATION IN LEUKOTRIENE SYNTHESIS CONTRIBUTE TO ASTHMA PHENOTYPES?

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At least eight proteins are sequentially involved in the synthesis, release, conversion, and activity of cysteinyl-LTs (Figure 2), and genetic variation in the activity of any or all of these may contribute to cysteinyl-LT overproduction in specific asthma phenotypes. Most information is available on the molecular biology of 5-LO, FLAP, and LTC₄ synthase (60), which act sequentially to convert arachidonic acid to LTC₄. Mutations in the number of GC-rich recognition sites for the transcription factor Sp1 have been described in the human 5-LO gene (63). One or two additions or deletions of the Sp1 recognition sites are associated with reduced Sp1 binding and with 20-40% reductions in transcription efficiency compared with the wild type. In 236 subjects with asthma, homozygotes for mutant 5-LO alleles were relatively resistant to treatment with the 5-LO inhibitor ABT-761, with mean FEV₁ improving by approximately 5% compared with 15% improvements in heterozygotes and in homozygotes for the wild-type allele (64). The mutant 5-LO alleles may therefore help to indicate individuals with a phenotype of asthma that is relatively independent of LTs.

View larger version (25K): [in this window] [in a new window]   Figure 2.   Principal enzymes, cofactors, carrier proteins, and receptors involved in the synthesis, export, interconversion, and activity of leukotrienes (LTs). sPLA2 = secretory phospholipase A2; cPLA2 = cytosolic PLA2; PLAP = phospholipase activating protein; LC = lipocortin; 5-LO = 5-lipoxygenase; FLAP = 5-lipoxygenase activating protein; 5-HPETE = 5-hydroperoxyeicosatetraenoic acid; 5-HETE = 5-hydroxyeicosatetraenoic acid; GPX = glutathione perioxidase; LTA4H = leukotriene A4 hydrolase; LTC4S = LTC4 synthase; PKC = protein kinase C; MRP = multidrug resistance-associated protein (an LTC4 export carrier protein); BC = LTB4 export carrier protein; gGT = -glutamyl-transpeptidase; DP = dipeptidase; CysLT1, CysLT2 = cysteinyl-leukotriene receptor subtypes 1 and 2; BLT = LTB4 receptor; MGST2 = microsomal glutathione transferase 2.

In contrast, aspirin-sensitive asthma (AIA), a chronic severe form of the disease that affects 10-20% of the asthmatic population, is closely associated with cysteinyl-LT overproduction and with excellent clinical responses to antileukotriene drugs. Acute adverse responses to aspirin and other COX inhibitors are caused by a surge in cysteinyl-LT release, which is superimposed on persistently elevated cysteinyl-LT production, as judged by cysteinyl-LT levels in BAL fluid and in urine when compared with subjects with aspirin-tolerant asthma (ATA) (65, 66). A profound overexpression of LTC₄ synthase has been described in the bronchial biopsies of patients with AIA, while expression of 5-LO, FLAP, COX-1, and COX-2 did not differ between ATA and normal biopsies (25, 67). Overrepresentation of LTC₄ synthase in the bronchial mucosa was the only factor to correlate with elevated cysteinyl-LT levels in the BAL fluid of patients with AIA, and with bronchial responsiveness to inhaled lysine-aspirin. By immunohistochemistry, most LTC₄ synthase localized to eosinophils, and the proportion of eosinophils overexpressing LTC₄ synthase was significantly higher in patients with AIA than in patients with ATA or in normal subjects. A polymorphism in the LTC₄ synthase gene promoter has been reported to be significantly more prevalent in the AIA population than in patients with ATA or normal subjects (68). The A₄₄₄C transversion creates an extra recognition site for the transcription factor AP-2, which may lead to its overexpression in eosinophils or other leukocytes. Some 40% of ATA and normal subjects are heterozygotic for the variant LTC₄ synthase allele, but it is not known whether this predisposes to enhanced cysteinyl-LT synthesis or to good clinical responses of patients with aspirin-tolerant asthma to antileukotriene drugs.

Arachidonic acid for LT synthesis is thought to be generated by phospholipase A₂ (PLA₂), but it is unclear which of the isoenzymes of cytosolic (85-kD) or secretory (14-kD) PLA₂ are most important in mast cells and eosinophils. In human monocytes, cPLA₂ is not required for LT synthesis (69), but in alveolar macrophages, both cPLA₂ and sPLA₂ are involved, with cPLA₂ playing a greater role in cells from patients with asthma than in normal subjects (70). Intriguingly, human blood eosinophils express large amounts of sPLA₂ (71).

After synthesis by 5-LO, FLAP, and LTC₄ synthase, the release of LTC₄ from human eosinophils requires a specific carrier protein (72) that has been identified as the multidrug resistance-associated protein (MRP1), which exports diverse cytotoxic drugs in a glutathione-conjugated form (73). The contribution of other glutathione carrier proteins to LTC₄ release is unknown. LTC₄ is then converted sequentially to receptor-active LTD₄ and LTE₄ by isozymes of -glutamyl-transpeptidase (-GT) and dipeptidase found on the surface of many cells and in plasma. The genes of -GT and dipeptidase have not been characterized. The three cysteinyl-LTs act with varying affinities at two or more receptors in human lung (74).

	FUTURE TARGETS

TOP INTRODUCTION HOW DO CYSTEINYL-LEUKOTRIENES... DO CYSTEINYL-LEUKOTRIENES... HOW DOES GENETIC VARIATION... FUTURE TARGETS REFERENCES

Molecular characterization of Cys-LT receptor sub-types may have the most profound effect on the development of novel antagonists which may be more efficacious or have a broader spectrum of action than current drugs by interacting with Cys-LT₁ and Cys-LT₂ receptor subtypes. In particular, these may help create a better understanding of the vascular activities of cysteinyl-LTs. Studies have suggested a significant contribution to various asthma phenotypes of genetic variation in 5-LO pathway enzymes. These studies need to be extended to include enzymes proximal to 5-LO in the pathway, such as PLA₂, and distal proteins such as those involved in cysteinyl-LT release and signaling. Improved targeting of antileukotriene drugs may result, perhaps involving genetic testing for responder subgroups. The role of 5-LO within the euchromatin of the nucleus in activated leukocytes remains unclear (75).

Little is presently known about the effects of cysteinyl-LTs and of treatment with antileukotriene drugs on the inflammatory cell profile in the bronchial wall and on airway remodeling. It is in this arena that maintenance therapy with oral antileukotriene drugs compared with long-term inhaled corticosteroids must be judged. Possible differences in activity between cysteinyl-LT receptor antagonists and the leukotriene synthesis inhibitors, which block LTB₄ synthesis as well as that of the cysteinyl-LT, are also most likely to emerge in longer-term studies of airway inflammation and repair.

	Footnotes

Correspondence and requests for reprints should be addressed to Tony Sampson, Ph.D., Immunopharmacology Group (825), Level F, Centre Block, Southampton General Hospital, Southampton SO16 6YD, UK. E-mail: aps{at}soton.ac.uk.

	References

TOP INTRODUCTION HOW DO CYSTEINYL-LEUKOTRIENES... DO CYSTEINYL-LEUKOTRIENES... HOW DOES GENETIC VARIATION... FUTURE TARGETS REFERENCES

1. Brocklehurst, W. E.. 1953. Occurrence of an unidentified substance during anaphylactic shock in cavy lung. J. Physiol. 120: 16-17 .

2. Holgate, S. T., P. Bradding, and A. P. Sampson. 1996. Leukotriene antagonists and synthesis inhibitors: new directions in asthma therapy. J. Allergy Clin. Immunol. 98: 1-13 [Medline].

3. Spector, S. L., L. J. Smith, and M. Glass. 1994. Effects of 6 weeks of therapy with oral doses of ICI 204,219, a leukotriene D₄ receptor antagonist, in subjects with bronchial asthma. Am. J. Respir. Crit. Care Med. 150: 618-623 [Abstract].

4. Kuitert, L., and N. C. Barnes. 1995. PAF and asthmatime for an appraisal? Clin. Exp. Allergy 25: 1159-1162 [Medline].

5. Hay, D. W. P., T. J. Torphy, and B. J. Undem. 1995. Cysteinyl leukotrienes in asthma: old mediators up to new tricks. Trends Pharmacol. Sci. 16: 304-309 [Medline].

6. Shi, H. Z., C. Q. Xiao, D. Zhong, S. M. Qin, Y. Liu, G. R. Liang, H. Xu, Y. Q. Chen, X. M. Long, and Z. F. Xie. 1998. Effect of inhaled interleukin-5 on airway hyperreactivity and eosinophilia in asthmatics. Am. J. Respir. Crit. Care Med. 157: 204-209 [Abstract/Free Full Text].

7. Holroyde, M. C., R. E. C. Altounyan, M. Cole, M. Dixon, and E. V. Elliott. 1981. Bronchoconstriction produced in man by leukotrienes C and D.  Lancet ii: 17-18 .

8. Chan, C. C., K. McKee, P. Tagari, P. Chee, and A. W. Ford-Hutchinson. 1990. Eosinophil-eicosanoid interactions: inhibition of eosinophil chemotaxis in vivo by a LTD₄-receptor antagonist. Eur. J. Pharmacol. 191: 273-280 [Medline].

9. Underwood, D. C., R. R. Osborn, S. J. Newsholme, T. J. Torphy, and D. W. P. Hay. 1996. Persistent airway eosinophilia after leukotriene (LT) D₄ administration in the guinea pig: modulation by the LTD₄ receptor antagonist, pranlukast, or an interleukin-5 monoclonal antibody. Am. J. Respir. Crit. Care Med. 154: 850-857 [Abstract].

10. Laitinen, L. A., A. Laitinen, T. Haahtela, V. Vilkka, B. W. Spur, and T. H. Lee. 1993. Leukotriene E₄ and granulocytic infiltration into asthmatic airways. Lancet 341: 989-990 [Medline].

11. Diamant, Z., J. T. Hiltermann, E. L. Van Rensen, P. M. Callenbach, M. Veselic-Charvat, H. Van Der Veen, J. K. Sont, and P. J. Sterk. 1997. The effect of inhaled leukotriene D₄ and methacholine on sputum cell differentials in asthma. Am. J. Respir. Crit. Care Med. 155: 1247-1253 [Abstract].

12. Kane, G. C., M. Tollino, M. Pollice, C. J. Kim, J. Cohn, J. J. Murray, R. Dworski, J. Sheller, J. E. Fish, and S. P. Peters. 1995. Insights into IgE-mediated lung inflammation derived from a study employing a 5-lipoxygenase inhibitor. Prostaglandins 50: 1-18 [Medline].

13. Foster, A., and C. C. Chan. 1991. Peptide leukotriene involvement in pulmonary eosinophil migration upon antigen challenge in the actively-sensitised guinea pig. Int. Arch. Allergy Appl. Immunol. 96: 279-284 [Medline].

14. Turner, C. R., W. B. Smith, C. J. Andresen, A. C. Swindell, and J. W. Watson. 1994. Leukotriene D₄ receptor antagonism reduces airway hyperresponsiveness in monkeys. Pulm. Pharmacol. 7: 49-58 [Medline].

15. Abraham, W. M., A. Ahmed, A. Cortes, M. W. Sielczak, W. Hinz, J. Bouska, C. Lanni, and R. L. Bell. 1992. The 5-lipoxygenase inhibitor zileuton blocks antigen-induced late airway responses, inflammation and airway hyperresponsiveness in allergic sheep. Eur. J. Pharmacol. 217: 119-126 [Medline].

16. Irvin, C. G., Y.P. Tu, J. R. Sheller, and C. D. Funk. 1997. 5-Lipoxygenase products are necessary for ovalbumin-induced airway responsiveness in mice. Am. J. Physiol. 272: L1053-L1058 [Abstract/Free Full Text].

17. Calhoun, W. J., B. J. Lavins, M. C. Minkwitz, R. Evans, G. J. Gleich, and J. Cohn. 1998. Effect of zafirlukast (Accolate) on cellular mediators of inflammation: bronchoalveolar lavage fluid findings after segmental antigen challenge. Am. J. Respir. Crit. Care Med. 157: 1381-1389 [Abstract/Free Full Text].

18. Kane, G. C., M. Pollice, C. Kim, J. Cohn, R. T. Dworski, J. J. Murray, J. R. Sheller, J. E. Fish, and S. P. Peters. 1996. A controlled trial of the effect of the 5-lipoxygenase inhibitor, zileuton, on lung inflammation produced by segmental antigen challenge in human beings. J. Allergy Clin. Immunol. 97: 646-654 [Medline].

19. Wenzel, S. E., J. B. Trudeau, D. A. Kaminsky, J. Cohn, R. J. Martin, and J. Y. Westcott. 1995. Effect of 5-lipoxygenase inhibition on bronchoconstriction and airway inflammation in nocturnal asthma. Am. J. Respir. Crit. Care Med. 152: 897-905 [Abstract].

20. Leff, J. A., E. Pizzichini, A. Efthimiadis, L. P. Boulet, L. X. Wei, D. E. Weinland, L. Hendeles, and F. E. Hargreave. 1997. Effect of montelukast (MK-0476) on airway eosinophilic inflammation in mildly uncontrolled asthma: a randomised placebo-controlled trial (abstract). Am. J. Respir. Crit. Care Med. 155: A977 .

21. Isogai, S., M. Taniguchi, K. Anzai, C. Nakagawa, K. Matsui, I. Tanaka, K. Kako, M. Sato, M. Okazawa, H. Sakakibara, and S. Suetsugu. 1998. Effects of peptide-leukotriene receptor antagonist pranlukast on eosinophil counts in peripheral blood and sputum in patients with chronic asthma (abstract). Am. J. Respir. Crit. Care Med. 157: A412 .

22. Knorr, B., J. Matz, J. A. Bernstein, H. Nguyen, B. C. Seidenberg, T. F. Reiss, and A. Becker. 1998. Montelukast for chronic asthma in 6- to 14-year-old children: a randomized, double-blind trial. J.A.M.A. 279: 1181-1186 [Abstract/Free Full Text].

23. Reiss, T. F., P. Chervinsky, R. J. Dockhorn, S. Shingo, B. Seidenberg, and M. D. Edwards. 1998. Montelukast, a once-daily leukotriene receptor antagonist, in the treatment of chronic asthma: a multicenter, randomized, double-blind trial. Arch. Intern. Med. 158: 1213-1220 [Abstract/Free Full Text].

24. Kylstra, J. W., D. E. Sweitzer, C. J. Miller, and C. M. Bonucelli. 1998. Zafirlukast (Accolate) in moderate asthma: patient-reported outcomes and peripheral eosinophil data from a 13-week trial (abstract). Am. J. Respir. Crit. Care Med. 157: A410 .

25. Cowburn, A., K. Sladek, J. Soja, L. Adamek, E. Nizankowska, A. Szczeklik, B. K. Lam, J. F. Penrose, K. F. Austen, S. T. Holgate, and A. P. Sampson. 1998. Over-expression of leukotriene C₄ synthase in bronchial biopsies of aspirin-intolerant asthmatics. J. Clin. Invest. 101: 834-846 [Medline].

26. Seymour, M. L., D. Aberg, G. Riise, S. Rak, S. T. Holgate, and A. P. Sampson. 1998. Seasonal allergen exposure increases expression of leukotriene pathway enzymes and induces eosinophil influx in bronchial mucosa of atopic asthmatics (abstract). J. Allergy Clin. Immunol. 101: 711 .

27. Spada, C. S., A. L. Nieves, A. H. Krauss, and D. F. Woodward. 1994. Comparison of leukotriene B₄ and D₄ effects on human eosinophil and neutrophil motility in vitro. J. Leukocyte Biol. 55: 183-191 [Abstract].

28. Sehmi, R., A. J. Wardlaw, O. Cromwell, K. Kurihara, P. Waltmann, and A. B. Kay. 1992. Interleukin-5 selectively enhances the chemotactic response of eosinophils obtained from normal but not eosinophilic subjects. Blood 79: 2952-2959 [Abstract/Free Full Text].

29. Heimburger, M., and J. E. Palmblad. 1996. Effects of leukotrienes C₄ and D₄, histamine, and bradykinin on cytosolic calcium concentrations and adhesiveness of endothelial cells and neutrophils. Clin. Exp. Immunol. 103: 454-460 [Medline].

30. Kanwar, S., B. Johnston, and P. Kubes. 1995. Leukotriene C₄/D₄ induces P-selectin and sialyl-Lewis-X-dependent alterations in leukocyte kinetics in vivo. Circ. Res. 77: 879-887 [Abstract/Free Full Text].

31. Pedersen, K. E., B. S. Bochner, and B. J. Undem. 1997. Cysteinyl leukotrienes induce P-selectin expression in human endothelial cells via a non-CysLT₁ receptor-mediated mechanism. J. Pharmacol. Exp. Ther. 281: 655-662 [Abstract/Free Full Text].

32. Wein, M., S. A. Sterbinsky, C. A. Bickel, R. P. Schleimer, and B. S. Bochner. 1995. Comparison of human eosinophil and neutrophil ligands for P-selectin: ligands for P-selectin differ from those for E-selectin. Am. J. Respir. Cell Mol. Biol. 12: 315-319 [Abstract].

33. Sano, H., N. Nakagawa, H. Nakajima, S. Yoshida, and I. Iwamoto. 1995. Role of vascular cell adhesion molecule-1 and platelet-activating factor in selective eosinophil migration across vascular endothelial cells. Int. Arch. Allergy Immunol. 107: 533-540 [Medline].

34. Nagata, M., J. B. Sedgwick, H. Kita, and W. W. Busse. 1998. Granulocyte macrophage colony-stimulating factor augments ICAM-1 and VCAM-1 activation of eosinophil function. Am. J. Respir. Cell Mol. Biol. 19: 158-166 [Abstract/Free Full Text].

35. Ortiz, J. L., I. Gorenne, J. Cortijo, A. Seller, C. Labat, B. Sarria, T. S. Abram, P. J. Gardiner, E. Morcillo, and C. Brink. 1995. Leukotriene receptors on human pulmonary vascular endothelium. Br. J. Pharmacol. 115: 1382-1386 [Medline].

36. Gardiner, P. J., T. S. Abram, S. R. Tudhope, N. J. Cuthbert, P. Norman, and C. Brink. 1994. Leukotriene receptors and their selective antagonists. Adv. Prostaglandin Thromboxane Leukotriene Res. 22: 49-61 [Medline].

37. Hebert, M. J., T. Takano, H. Holthofer, and H. R. Brady. 1996. Sequential morphologic events during apoptosis of human neutrophils: modulation by lipoxygenase-derived eicosanoids. J. Immunol. 157: 3105-3115 [Abstract].

38. Koller, M., P. Wachtler, A. David, G. Muhr, and W. Konig. 1997. Arachidonic acid induces DNA-fragmentation in human polymorphonuclear neutrophil granulocytes. Inflammation 21: 463-474 [Medline].

39. Lee, E., K. Ryan, N. Vosper, N. Jackson, M. Haswell, L. Robertson, A. A. Hill, and S. A. Kilfeather. 1998. Leukotriene B₄ (LTB₄) mediates LPS and GM-CSF-induced neutrophil survival: reversal by LTB₄ receptor blockade (abstract). Am. J. Respir. Crit. Care Med. 157: A142 .

40. Dittmann, K. H., C. Mayer, H. P. Rodemann, P. E. Petrides, and C. Denzlinger. 1998. MK-886, a leukotriene biosynthesis inhibitor, induces antiproliferative effects and apoptosis in HL-60 cells. Leukocyte Res. 22: 49-53 .

41. Melis, M., L. Siena, E. Pace, A. M. Vignola, G. Garinoff, F. Cibella, M. Gjomarkaj, and G. Bonsignore. 1998. Effect of zafirlukast on peripheral blood T-lymphocytes (PBT) isolated from normal and asthmatic patients (abstract). Am. J. Respir. Crit. Care Med. 157: A394 .

42. Takafuji, S., S. C. Bischoff, A. L. De Weck, and C. A. Dahinden. 1991. IL-3 and IL-5 prime normal human eosinophils to produce leukotriene C₄ in response to soluble agonists. J. Immunol. 147: 3855-3861 [Abstract].

43. Brach, M. A., S. de Vos, C. Arnold, H. J. Gruss, R. Mertelsmann, and F. Herrmann. 1992. Leukotriene B₄ transcriptionally activates interleukin-6 expression involving NK-B and NF-IL6. Eur. J. Immunol. 22: 2705-2711 [Medline].

44. Yamaoka, K. A., and J. P. Kolb. 1993. Leukotriene B₄ induces interleukin 5 generation from human T lymphocytes. Eur. J. Immunol. 23: 2392-2398 [Medline].

45. Asano, K., C. M. Lilly, W. J. O'Donnell, E. Israel, A. Fischer, B. J. Ransil, and J. M. Drazen. 1995. Diurnal variation of urinary leukotriene E₄ and histamine excretion rates in normal subjects and patients with mild-to-moderate asthma. J. Allergy Clin. Immunol. 96: 643-651 [Medline].

46. Kohno, S., N. Tsuzuike, H. Yamamura, T. Nabe, M. Horiba, and K. Ohata. 1993. Important role of peptide leukotrienes (p-LTs) in the resting tonus of isolated human bronchi. Jpn. J. Pharmacol. 62: 351-355 [Medline].

47. Malaviya, R., and B. A. Jakschik. 1993. Reversible translocation of 5-lipoxygenase in mast cells upon IgE/antigen stimulation. J. Biol. Chem. 268: 4939-4944 [Abstract/Free Full Text].

48. Laviolette, M., C. Ferland, J.-F. Comtois, K. Champagne, M. Bosse, and L. P. Boulet. 1995. Blood eosinophil leukotriene C₄ production in asthma of different severities. Eur. Respir. J. 8: 1465-1472 [Abstract].

49. Arm, J. P., B. W. Spur, and T. H. Lee. 1988. The effects of inhaled leukotriene E₄ on the airway responsiveness to histamine in subjects with asthma and normal subjects. J. Allergy Clin. Immunol. 82: 654-660 [Medline].

50. Taki, F., R. Suzuki, K. Torii, S. Matsumoto, H. Taniguchi, and K. Takagi. 1994. Reduction of the severity of bronchial hyperresponsiveness by the novel leukotriene antagonist 4-oxo-8-[4-(4-phenyl-butoxy)benzoylamino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate. Arzneimittelforschung. 44: 330-333 [Medline].

51. Rosenthal, R. R., B. J. Lavins, and L. A. Hanby. 1996. Effect of treatment with zafirlukast (Accolate) on bronchial hyperresponsiveness in patients with mild-to-moderate asthma (abstract). J. Allergy Clin. Immunol. 97: 250 .

52. Fischer, A. R., C. A. McFadden, R. Frantz, W. M. Awni, J. Cohn, J. M. Drazen, and E. Israel. 1995. Effect of chronic 5-lipoxygenase inhibition on airway hyperresponsiveness in asthmatic subjects. Am. J. Respir. Crit. Care Med. 152: 1203-1207 [Abstract].

53. Wang, C. G., T. Du, L. J. Xu, and J. G. Martin. 1993. Role of leukotriene D₄ in allergen-induced increases in airway smooth muscle in the rat. Am. Rev. Respir. Dis. 148: 413-417 [Medline].

54. Panettieri, R. A., E. M. L. Tan, V. Ciocca, M. A. Luttmann, T. B. Leonard, and D. W. P. Hay. 1998. Effects of LTD₄ on human airway smooth muscle cell proliferation, matrix expression, and contraction in vitro: differential sensitivity to cysteinyl leukotriene receptor antagonists. Am. J. Respir. Cell Mol. Biol. 19: 453-461 [Abstract/Free Full Text].

55. Amishima, M., M. Munakata, Y. Nasuhara, A. Sato, T. Takahashi, Y. Homma, and Y. Kawakami. 1998. Expression of epidermal growth factor and epidermal growth factor receptor immunoreactivity in the asthmatic human airway. Am. J. Respir. Crit. Care Med. 157: 1907-1912 [Abstract/Free Full Text].

56. Belvisi, M. G., M. Saunders, M. Yacoub, and J. A. Mitchell. 1998. Expression of cyclooxygenase-2 in human airway smooth muscle is associated with profound reductions in cell growth. Br. J. Pharmacol. 125: 1102-1108 [Medline].

57. Medina, L., J. Perez, Ramos, R. Ramirez, M. Selman, and A. Pardo. 1994. Leukotriene C₄ upregulates collagenase expression and synthesis in human lung fibroblasts. Biochim. Biophys. Acta 1224: 168-174 [Medline].

58. Leikauf, G. D., H. E. Claesson, C. A. Doupnik, S. Hybbinette, and R. C. Grafstrom. 1990. Cysteinyl leukotrienes enhance growth of human airway epithelial cells. Am. J. Physiol. 259: L255-L261 [Abstract/Free Full Text].

59. McAlexander, M. A., A. C. Myers, and B. J. Undem. 1998. Inhibition of 5-lipoxygenase diminishes neurally evoked tachykinergic contraction of guinea pig isolated airway. J. Pharmacol. Exp. Ther. 285: 602-607 [Abstract/Free Full Text].

60. Funk, C. D., S. Hoshiko, T. Matsumoto, O. Rådmark, and B. Samuelsson. 1989. Characterization of the human 5-lipoxygenase gene. Proc. Natl. Acad. Sci. U.S.A. 86: 2587-2591 [Abstract/Free Full Text].

61. Hoshiko, S., O. Rådmark, and B. Samuelsson. 1990. Characterization of the human 5-lipoxygenase promoter. Proc. Natl. Acad. Sci. U.S.A. 87: 9073-9077 [Abstract/Free Full Text].

62. Kennedy, B. P., R. E. Diehl, Y. Boie, M. Adam, and R. A. Dixon. 1991. Gene characterization and promoter analysis of the human 5-lipoxygenase-activating protein (FLAP). J. Biol. Chem. 266: 8511-8516 [Abstract/Free Full Text].

63. In, K., K. Asano, D. Beler, J. Grobholz, P. W. Finn, E. K. Solverman, E. S. Silverman, T. Collins, A. R. Fischer, T. P. Keith, K. Serino, S. W. Kim, G. W. De Sanctis, C. Yandava, A. Pillari, P. Rubin, J. Kemp, E. Israel, W. Busse, and J. M. Drazen. 1997. Naturally-ocurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription. J. Clin. Invest. 99: 1130-1137 [Medline].

64. Drazen, J. M. 22nd Symposium of Collegium Internationale Allergologicum, Corfu, September 1998, Abstract 39.

65. Szczeklik, A., K. Sladek, R. Dworski, E. Nizankowska, J. Soja, J. Sheller, and J. Oates. 1996. Bronchial aspirin challenge causes specific eicosanoid response in aspirin-sensitive asthmatics. Am. J. Respir. Crit. Care Med. 154: 1608-1614 [Abstract].

66. Kumlin, M., B. Dahlén, T. Björck, O. Zetterström, E. Granstrom, and S. E. Dahlén. 1992. Urinary excretion of leukotriene E₄ and 11-dehydro-thromboxane B₂ in response to bronchial provocations with allergen, aspirin, leukotriene D₄, and histamine in asthmatics. Am. Rev. Respir. Dis. 146: 96-103 [Medline].

67. Sampson, A. P., A. S. Cowburn, K. Sladek, L. Adamek, E. Nizankowska, A. Szczeklik, B. K. Lam, J. F. Penrose, K. F. Austen, and S. T. Holgate. 1997. Profound overexpression of leukotriene C₄ synthase in bronchial biopsies from aspirin-intolerant asthmatic patients. Int. Arch. Allergy Immunol. 113: 355-357 [Medline].

68. Sanak, M., H. U. Simon, and A. Szczeklik. 1997. Leukotriene C₄ synthase promoter polymorphism and risk of aspirin-induced asthma. Lancet 350: 1599-1600 [Medline].

69. Marshall, L. A., B. Bolognese, J. D. Winkler, and A. Roshak. 1997. Depletion of human monocyte 85-kDa phospholipase A₂ does not alter leukotriene formation. J. Biol. Chem. 272: 759-765 [Abstract/Free Full Text].

70. Shamsuddin, M., E. Chen, J. Anderson, and L. J. Smith. 1997. Regulation of leukotriene and platelet-activating factor synthesis in human alveolar macrophages. J. Lab. Clin. Med. 130: 615-626 [Medline].

71. Blom, M., A. T. Tool, P. C. Wever, G. J. Wolbink, M. C. Brouwer, J. Calafat, A. Egesten, E. F. Knol, C. E. Hack, D. Roos, and A. J. Verhoeven. 1998. Human eosinophils express, relative to other circulating leukocytes, large amounts of secretory 14-kD phospholipase A₂. Blood 91: 3037-3043 [Abstract/Free Full Text].

72. Lam, B. K., K. Xu, M. B. Atkins, and K. F. Austen. 1992. Leukotriene C₄ uses a probenecid-sensitive export carrier that does not recognise leukotriene B₄. Proc. Natl. Acad. Sci. U.S.A. 89: 11598-11602 [Abstract/Free Full Text].

73. Leier, I., G. Jedlitschky, U. Buchholz, S. P. Cole, R. G. Deeley, and D. Keppler. 1994. The MRP gene encodes an ATP-dependent export pump for leukotriene C₄ and structurally related conjugates. J. Biol. Chem. 269: 27807-27810 [Abstract/Free Full Text].

74. Capra, V., S. Nicosia, D. Ragnini, M. Mezzetti, D. Keppler, and G. E. Rovati. 1998. Identification and characterization of two cysteinyl-leukotriene high affinity binding sites with receptor characteristics in human lung parenchyma. Mol. Pharmacol. 53: 750-758 [Abstract/Free Full Text].

75. Woods, J. W., M. J. Coffey, T. G. Brock, I. I. Singer, and M. Peters-Golden. 1995. 5-Lipoxygenase is located in the euchromatin of the nucleus in resting human alveolar macrophages and translocates to the nuclear envelope upon cell activation. J. Clin. Invest. 95: 2035-2046 .