Leukotriene receptor antagonists in the treatment of asthma : Implications for eosinophilic inflammation

Correspondence and reprints: Dr R Moqbel, 574 Heritage Medical Research Centre, Pulmonary Research Group, Department of Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2S2. Telephone 780-492-1909, fax 780-492-5329, e-mail redwan.moqbel@ualberta.ca R Moqbel. Leukotriene receptor antagonists in the treatment of asthma: Implications for eosinophilic inflammation. Can Respir J 1999;6(5):453-457.

A sthma is a heterogeneous and complex condition af- flicting a wide range of population of varying age and sex.It is now generally recognized that the disease is caused mainly by inflammation in the airways (Figure 1).This results in reversible airway obstruction and bronchial hyperresponsiveness, which at times can be fatal.Until recently, the treatment of asthma was confined to symptom relieving (beta 2 -agonists) and/or preventive therapy, ie, corticosteroids (inhaled and/or systemic).
Activated T cells and eosinophils are thought to play a major role in asthma, and the numbers of these cells correlate broadly with disease severity (1).Mucosal damage in chronic asthma is believed to be the consequence of cytotoxic and proinflammatory mediator release from activated eosinophils (2,3).These include cytotoxic granule proteins (major basic protein, eosinophil cationic protein, eosinophil peroxidase, and eosinophil-derived neurotoxin) together with phospholipidderived, pharmacologically active mediators.Cytokines derived from T helper (Th) 2-type cells, particularly interleukin (IL)-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF), are thought to regulate eosinophil activation and promote their survival (4,5).IL-5 is an obligatory terminal differentiator of eosinophil precursors (6).

SULPHIDOPEPTIDE (CYSTEINYL)
LEUKOTRIENES Eosinophils are a rich source of the sulphidopeptide leukotrienes (2).These are de novo-synthesized lipid mediators derived from arachidonic acid (AA) by the action of phospholipase A 2 (PLA 2 ) (7).AA is found in association with the plasma membrane as well as cytoplasmic lipid bodies in these cells (8), and is metabolized through one of two pathways, cyclo-oxygenase and lipoxygenase (9).The former leads to the generation of prostaglandins, thromboxanes and prostacyclins.The second pathway, via the 5-lipoxygenase (5-LO) enzyme acting in concert with a cofactor, 5-LO activating protein (FLAP), generates leukotrienes (LTs), including LTB 4 , LTC 4 , LTD 4 and LTE 4 (10).
When LTC 4 is formed, it is transported actively out of the cell.LTD 4 and LTE 4 are produced from LTC 4 by the removal of glutamic acid (via the action of -glutamyl transpeptidase) and glycine (via the action of dipeptidase), respectively (7,9).LTC 4 , D 4 and E 4 collectively form the activity previously recognized as 'slow reacting substance of anaphylaxis', due to their prolonged in time spasmogenic effects on smooth muscles.Although LTD 4 and LTE 4 are rapidly degraded in the body through oxidative metabolism, small amounts of LTE 4 can be measured in the urine (21).Interestingly, in humans, the leukotriene pathway, via 5-LO, is observed only in myeloid cells, ie, mast cells, basophils, neutrophils, eosinophils and alveolar macrophages (22).Recent studies have suggested that cysteinyl LTs may also have selective eosinophilotactic activity (23,24).

LT RECEPTORS
Two receptors for cysteinyl LTs have been identified on smooth muscle cells, namely, Cys-LT1 and Cys-LT2.Cys-LT1 is now recognized as the probable regulating receptor for bronchial smooth muscle contraction and, thus, may be directly relevant to asthma treatment (25).Cys-LT2, on the other hand, appears to be mainly involved in pulmonary vein contraction (26).In addition to LTD 4 , both LTC 4 and LTE 4 bind to Cys-LT1, although LTE 4 exhibits a greatly reduced binding capacity (25).There is, however, no evidence available in the literature to suggest that eosinophils express either of these two receptors for LTs.One exception is the observation that HL-60 cells, which were differentiated into eosinophils, in vitro, expressed a very low affinity LTD 4 receptor (Kd 41.91 nM) (27).However, these cells may not reflect fully all the properties and the biological profile of circulating mature eosinophils.

EVIDENCE FOR A BIOLOGICAL ROLE FOR LTS
IN ASTHMA Evidence has accumulated to suggest that, in vivo, LTs are among the most potent constrictors of airway smooth muscle and are, thus, major players in the complex picture of asthmatic inflammation.A substantial amount of the published literature shows that LTs are critical elements in the development, progression and chronicity of the inflammatory response associated with various clinical presentations of asthma (28)(29)(30).For instance, both LTC 4 and LTD 4 , when inhaled, are an order of magnitude greater in pharmacological potency than histamine in inducing airflow obstruction in normal subjects, and their effects lasted longer.In these studies, asthmatic airways appeared to be more sensitive (100 to 1000 times) to inhaled cysteinyl LTs than those of nonasthmatic subjects.These patients also demonstrated increased bronchial hyperresponsiveness to methacholine or histamine following inhalation of LTC 4 and LTD 4 (31).

LT MODIFIERS
The potential contribution of the sulphidopeptide LTs in bronchoconstriction has been identified by recent developments in the field of LT-modifying therapies (30,32).Earlier studies and trials with LT inhibitors and receptor antagonists were disappointing, mainly due to toxicity, lack of specificity and limited potency of the agents (33).
Two new strategies aimed at blocking the effects of the LT pathway have recently gained rapid momentum and provided hope for better and more efficient treatment for asthma.The first area relates to the development of LT synthesis inhibitors, such as zileuton and BAYx1005.These were shown to produce significant reductions in LT generation levels, including both LTB 4 and the cysteinyl LTs (34,35).Zileuton inhibits the 5-LO and, thus, prevents the synthesis of LTA 4 (34).In contrast, BAYx1005 is an antagonist of FLAP, which prevents the translocation of 5-LO and blocks the formation of LTA 4 (35).This area of FLAP inhibition is currently under intensive development because it will determine the role of other products of 5-LO metabolic pathway in asthmatic inflammation.
More recently, second generation LTD 4 receptor antagonists have been introduced.These new agents have the potential to inhibit the biological activities of LTD 4 and the other members of the cysteinyl LT family by competing for their receptors on smooth muscle cells (36).These include monte-lukast (Singulair [Merck Frosst Canada Inc, Kirkland, Quebec]), zafirlukast (Accolate [Zeneca Pharma Inc, Mississauga, Ontario]), and pranlukast (Ultair, SmithKline Beecham, United Kingdom), which, when used in inhalation challenge studies, in vivo, have demonstrated a greater potency in blocking LT effects and a better safety record (37)(38)(39)(40).

LT MODIFIERS AND THE EOSINOPHIL IN ASTHMA
The clinical effects of LT receptor antagonists (including montelukast and zafirlukast) on dampening the pathophysiological sequelae of inflammation in asthma have been amply reviewed previously (37)(38)(39)(40)(41).These reports show clearly that these agents have significant efficacy in controlling asthma symptoms in a large percentage of patients.The present paper concentrates on the as yet unanswered question of whether LT receptor antagonists are anti-inflammatory drugs, potent bronchodilators or both.In particular, the issue of the biological relevance and efficacy of LT receptor antagonists from the perspective of their effect on the eosinophil is addressed.As stated earlier, the latter is a highly relevant inflammatory cell type in asthmatic airway mucosal tissue (Figure 1) (3).
Recent observations have shown that both montelukast and zafirlukast suppress eosinophil numbers in the peripheral

Figure 1) A schematic representation of the current understanding of putative immune and inflammatory mechanisms regulating the early late phase asthmatic response. Cytokines and chemokines have the potential to exert regulatory influences on the inflammatory cascade involving the eosinophil. Leukotriene (LTs) modifiers are thought to modulate the response ( ) at the level of the smooth muscle cells. Little is known about the potential of these agents to influence eosinophil or epithelial cell responses ( ) ). APC Antigen presenting cells; ECM Extracellular matrix proteins; ECP Eosinophil cationic protein; EPO Eosinophil peroxidase; FEV 1 Forced expiratory volume in 1 s; GM-CSF Granulocyte-macrophage colony-stimulating factor; Ig Immunoglobulin; IL Interleukin; MBP Major basic protein; MHC Major histocompatibility complex; PAF Platelet activiating factor; PG Prostaglandin; Th T helper cells; Thp T helper precursor; TCR T cell receptor
blood and airways (42,43).Using hypertonic saline-induced sputum as an indicator of cellular infiltration associated with asthmatic inflammation, researchers demonstrated significant reductions in the numbers of sputum eosinophils following treatment with montelukast (44).A consequence of such studies has been to provide further fuel to the hypothesis, and possibly a subtle conclusion, that cysteinyl LTs possess eosinophilotactic properties (23,24,42).
There are many areas that have not yet been studied in relation to the effect of LT receptor antagonists on eosinophil biology and its natural history in asthma (Figure 1).In order to reach a better conclusion about the biological effects of leukotriene modifiers on eosinophils in asthma, further supportive evidence is needed.LT activity blockers, such as montelukast and zafirlukast, may indeed influence the eosinophilic response but through various and potentially complex paths.
The first missing evidence relates to the potential effects of LT modifiers on bone marrow or in situ eosinophil progenitors and committed precursors (45).It is possible, if not likely, that these drugs exert anti-inflammatory properties at the level of the bone marrow.Studies aimed at examining the mode of action of these agents in cultures of CD34 + cells obtained either from bone marrow or other hemopoietic tissue (including umbilical cord blood) in vitro (46) are needed.In addition, the effects of these agents on bone marrow levels of eosinophil-sensitive chemokines (such as RANTES and eotaxin) require investigation.This would help determine whether there is a potential blocking action of these agents on the proximal arm controlling the egress of eosinophils from hemopoietic sites (Figure 1).
Second, there is a strong likelihood that the pharmacological effects of montelukast on eosinophil numbers may be an indirect one.I prefer this hypothesis in favour of the notion that cysteinyl LTs exhibit eosinophilotactic properties.Previous and carefully conducted studies concluded that LTC 4 , D 4 and E 4 had negligible chemotactic activity for eosinophils (13,47).Furthermore, these sulphidopeptide LTs showed no upregulatory effects on eosinophil effector function (including cytotoxicity), at least compared with LTB 4 (48).Thus, it seems more likely that this indirect effect on eosinophil recruitment to the airway, as shown by reduced eosinophil counts in sputum, may be mediated via LT receptor-mediated effects on epithelial cells.This may in turn influence the synthesis, storage and release of eosinophilotactic chemokines, such as RANTES and eotaxin (49,50), both of which have been shown to be present in the bronchial tissue in asthma (51,52).In addition to chemokines, epithelial cell-induced eosinophil chemoattraction may also be influenced by cytokines, particularly IL-16 (Figure 1).IL-16, a potent T-cell and eosinophilotactic cytokine and a major product of bronchial epithelial cells, as well as other inflammatory cells including eosinophils, uses CD4 receptors on eosinophils in its chemotactic activity (53,54).IL-16 expression has recently been shown to be a pathological feature of human bronchial asthma (55).
Third, it seems that we know very little about the influence of montelukast treatment on IL-5 bioactivity both locally and systemically.This cytokine is a critical factor in eosinophil terminal differentiation and, together with IL-3 and GM-CSF, prolongs eosinophil survival in the tissue.However, data from a recent study (42) have suggested that IL-5 levels may be reduced in the sputum.This may provide further support to the notion that LT modifiers may exert their influence on the eosinophilic response via IL-5 protein synthesis and turnover in asthmatic airways (Figure 1).
Finally, montelukast-induced reduction in the number of peripheral blood and airway eosinophils may be due to the ability of this agent to induce eosinophil death, particularly apoptosis.Furthermore, whether engaging putative Cys-LT1 on eosinophils may be important in prolonging the survival of these cells is another worthwhile area of investigation.

CONCLUSIONS
The eosinophil may be an important target for the pharmacological activities of LT receptor antagonists.It is likely that the anti-asthma efficacy of these antagonists may relate partly to their direct and/or indirect anti-eosinophilic properties.However, much remains to be unveiled in regard to their anti-eosinophilic effects.Our aim should be to obtain a better understanding of the mode of action of leukotriene modifiers with a view to clarify their potential 'anti-inflammatory' and "steroid-sparing" properties.Such data should contribute to a more efficacious administration of these therapies in asthma.