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Anti-inflammatory mechanisms of leukotriene modulators

1999; Wiley; Volume: 29; Issue: 11 Linguagem: Inglês

10.1046/j.1365-2222.1999.00711.x

1365-2222

Diamant, Anthony P. Sampson,

Inflammatory mediators and NSAID effects

Since 1996, a novel class of anti-asthma drugs has been licensed in almost 50 countries world-wide [ 1]. The mechanism of action of these compounds is based on inhibiting the synthesis of leukotrienes in inflammatory leucocytes, or blocking their activity at specific receptors on target tissues. Leukotrienes are derived from arachidonic acid by the 5-lipoxygenase (5-LO) pathway and comprise cysteinyl-leukotrienes (cys-LTs: LTC4, LTD4, and LTE4) and LTB4 [ 2]. There is ample evidence that cys-LTs are potent pro-inflammatory mediators playing an important role in the pathophysiology of asthma-producing bronchospasm, increased vascular permeability, and mucus hyper-secretion [ 2, 3]. In addition, cys-LTs have been shown to cause airway hyperresponsiveness in both asthmatic and healthy subjects [ 4]. Several classes of leukotriene modulators have been developed [ 1]. Leukotriene synthesis inhibitors (LTSI) inhibit either 5-lipoxygenase- or 5-lipoxygenase- activating protein (FLAP). Only the 5-LO inhibitor zileuton (ZyfloTM) has so far been licensed. The three cys-LT receptor antagonists (cysLTRAs) so far licensed − pranlukast (UltairTM), montelukast (SingulairTM) and zafirlukast (AccolateTM) − block the CysLT1 receptor subtype on target tissues such as airway smooth muscle. Antagonists of the LTB4 receptor (BLT) remain in clinical trials. Numerous studies in asthma have shown that leukotriene modulators are effective bronchodilators in asthmatics [ 1]. Recent studies also suggest anti-inflammatory effects on airway leucocytes that are likely to have a direct bearing on the positioning of leukotriene modulators in international guidelines for asthma management. This review will assess this evidence and discuss the possible mechanisms of the anti-eosinophilic effect of leukotriene modulators. Asthma involves abnormalities at clinical, pathophysiological and histopathological levels. Chronic airway eosinophilia [ 5], often associated with non-specific airway hyperresponsiveness (AHR) [ 6], is a typical finding which may finally result in airway remodelling, determining the chronicity and severity of the disease [ 7]. In the past decade accumulating evidence has been provided that leukotrienes are potent and specific chemoattractants for eosinophils within the airways. Increases in airway eosinophils following ovalbumin challenge of sensitized mice are reduced by 50% in 5-LO knockout mice compared with wild-type mice, while the increase in airway responsiveness is completely abolished [ 8]. In guinea-pigs, inhalation of a single dose of LTC4 and LTD4 (but not LTB4) produced a dose-related airway eosinophilia lasting at least 3 weeks, which was blocked by the cysLTRAs MK-571 and pranlukast [ 9, 10]. In sensitized cynomolgus monkeys, the allergen-induced airway eosinophilia and AHR are blocked by a specific cysLTRA (ICI 198 615) [ 11]. Similar results were obtained in sensitized sheep with the 5-LO inhibitor zileuton [ 12]. These findings have recently been confirmed in human studies in vivo. Four hours after inhalation of LTE4, marked increases in eosinophils and, to a lesser extent, in neutrophils have been demonstrated in the airway mucosa of four asthmatic patients [ 13]. Likewise, in 12 asthmatic patients, the more potent bronchoconstrictor LTD4 induced rises in eosinophils (but not in neutrophils) in hypertonic saline-induced sputum 4 hours after challenge [ 14]. Intervention with leukotriene modulators markedly blocks eosinophil influx into the airways after allergen challenge and in chronic asthma. Treatment with the 5-LO inhibitor zileuton for 8 days significantly reduced eosinophil counts and LTE4 levels in the BAL fluid of atopic asthmatic patients 24 hours after allergen challenge [ 15]. Similar results were achieved with zafirlukast (160 mg b.i.d.); 7 days of pretreatment with this cysLTRA inhibited the rises in eosinophils and basophils observed in BAL fluid 48 h after segmental allergen challenge by 45 and 57%, respectively [ 16]. These data have been confirmed in long-term studies. In patients with mild to moderate persistent asthma, 7 days of treatment with the 5-LO inhibitor zileuton (600 mg q.i.d.) significantly decreased eosinophil counts both in the bronchoalveolar lavage (BAL) fluid and in peripheral blood [ 17]. In 40 asthmatic patients, 4 weeks of treatment with the cysLTRA montelukast (10 mg once daily) reduced sputum eosinophils by 48% [ 18]. Furthermore, treatment of 17 asthmatics for 4 weeks with another cysLTRA, pranlukast (225 mg b.i.d.), decreased airway responsiveness to methacholine and reduced eosinophils, mast cells, and T-helper lymphocytes in bronchial biopsies [ 19]. The same dose of pranlukast for 8 weeks significantly reduced the numbers of eosinophils in both sputum and peripheral blood in 27 asthmatic patients [ 20]. In 408 adults and 201 children with moderate asthma, 20–40% of whom were using inhaled corticosteroids, treatment with montelukast for 12 and 8 weeks, respectively, produced significant reductions in blood eosinophil counts compared with placebo [ 21, 22]. Similar results have been found with zafirlukast [ 23]. These studies with potent and specific leukotriene modulators provide definitive evidence of a major contribution of endogenous cys-LTs to airway eosinophilia in asthma, and suggest that blockade of cys-LTs also reduces airway hyperresponsiveness. Reductions in peripheral blood eosinophilia also suggest an effect of cys-LTs in promoting eosinophil differentiation from precursor cells. The specific mechanisms by which cys-LTs promote eosinophilia are unknown, but may involve direct chemotaxis of eosinophils in inflamed tissues, upregulation of adhesion molecules on eosinophils and on the vascular endothelium, effects on eosinophilopoiesis and the release of eosinophil precursors from bone marrow, and enhanced eosinophil survival. Compared with other lipid mediators, relatively few studies have investigated the chemotactic effects of cys-LTs in vitro. However, at nanomolar concentrations, LTD4 causes directed migration of normal human eosinophils in vitro, a response that is blocked by the cysLTRA pobilukast [ 24]. Neutrophils responded to LTD4 only at micromolar concentrations, while LTB4 was a non-selective chemoattractant for both cell types. A direct effect on eosinophil chemotaxis is the simplest explanation for rapid eosinophil influx into the airway mucosa and sputum after cys-LTs inhalation [ 13, 14]. Cysteinyl-LTs may also promote eosinophil–endothelial interactions. Both LTC4 and LTD4 cause rolling of PMN leucocytes along vascular endothelium [ 25] by inducing P-selectin expression on the surface of leucocytes and endothelial cells [ 26, 27]. Eosinophils overexpress a specific ligand for P-selectin that may result in selective adhesion of eosinophils relative to neutrophils [ 28]. Following P–selectin interaction, firm adhesion to endothelium occurs via ICAM-1 and VCAM-1, and the interaction primes eosinophils for enhanced LTC4 synthesis [ 29], perhaps leading to further P-selectin expression and eosinophil adhesion. Curiously, in endothelial cells from human umbilical vein (HUVEC), the induction of P-selectin by cys-LTs is not blocked by the CysLT1 receptor antagonists pobilukast, pranlukast, and zafirlukast, suggesting a role for CysLT2 or other receptor subtypes [ 27]. Novel cysLTRAs with a wider spectrum of antagonism at CysLT1, CysLT2, and possibly other receptor subtypes may thus suppress leucocyte migration more effectively than the current generation of cysLTRAs. Reduced apoptosis (programmed cell death) of eosinophils may contribute to persistent airway eosinophilia in asthma. Increasing evidence suggests a critical role of leukotrienes in suppressing leucocyte apoptosis, although specific information relating to eosinophils is so far lacking. Thus, the survival of human neutrophils, and their enhanced survival in the presence of GM-CSF, are both dependent on LTB4 synthesis [ 303132]. In asthmatic patients, the proportion of peripheral blood T cells undergoing apoptosis is lower than in normal subjects [ 33], but the proportion is increased by the cysLTRA zafirlukast. Enhanced survival of T cells in asthmatics is therefore mediated at least in part by endogenous cys-LTs. It is possible that the ability of eosinophilopoietic cytokines including IL-3, IL-5, and especially GM-CSF to enhance eosinophil survival is due to their concomitant priming of cys-LT synthesis in these cells [ 34], and leukotriene modulators may reduce eosinophil numbers in the asthmatic airway by accelerating programmed cell death. The eosinophilopoietic cytokines IL-3, IL-5 and GM-CSF and chemokines including RANTES and eotaxin are traditionally thought to regulate eosinophil differentiation and trafficking. Of the eosinophilopoietic cytokines, only IL-5 has so far been tested and shown to induce airway eosinophilia when inhaled by asthmatic patients [ 35], analogous to the effects of inhaled cys-LTs [ 13, 14]. The role of endogenous IL-5 has not yet been explored with a specific IL-5 receptor antagonist. It is therefore difficult to quantify the relative contributions of cys-LTs, IL-5, and other chemotaxins to airway eosinophilia in man, or to determine whether cys-LTs and IL-5 act independently or in series. However, in guinea-pigs, inhaled LTD4 produces immediate bronchoconstriction and a persistent airway eosinophilia lasting 3–4 weeks, both of which are inhibited by pranlukast. Intriguingly, the eosinophilia (but not the bronchoconstriction) is also blocked by a monoclonal antibody directed against IL-5 [ 9]. This indicates that LTD4-induced eosinophilia is mediated by secondary release of IL-5 from an unknown cell-type in guinea-pigs. Although enhanced synthesis of cytokines including IL-5, IL-6, and IL-8 has been observed in lymphocytes and other cells in response to LTB4 [ 36, 37], the effects of cys-LTs on cytokine synthesis have been little studied. However, an interesting study by Tohda and colleagues published in this issue [ 38] throws some light on the problem. Peripheral blood mononuclear cells from mite-sensitive asthmatic patients were incubated for 72 h in vitro with mite allergen in the presence or absence of the cysLTRA pranlukast (ONO-1078). Assessed by ELISA, the production of GM-CSF, IL-3, and IL-4 was significantly suppressed at the highest concentration of pranlukast (10 μg/mL), with a trend towards reduced IL-5 synthesis [ 38]. IL-2 synthesis was not inhibited. Although cys-LT release was not quantified directly, the data suggest that endogenous cys-LTs released by allergen-specific blood mononuclear cells can stimulate the synthesis of cytokines implicated in eosinophilia and IgE synthesis, and that this can be inhibited by physiological concentrations of a specific cysLTRA. The work of Tohda and colleagues raises as many questions as it answers. The cellular source of the putative endogenous cys-LTs was not determined. Monocytes are the most likely source, as lymphocytes appear to synthesize LTB4 but not cys-LTs [ 39]. The cellular provenance of the cytokines is also unclear; both monocytes and lymphocytes might contribute, but the profile of cytokines inhibited by pranlukast (IL-4 but not IL-2) is consistent with activity against TH2-like lymphocytes. The authors further argue that the lack of proven CysLT receptors on lymphocytes suggests that pranlukast acts independently of cys-LT antagonism. However, this cannot be sustained unless other cysLTRAs fail to block cytokine synthesis in this system, and until cys-LT receptors are characterized sufficiently for their cellular localization to be properly elucidated. Nevertheless, the study suggests a scenario in which cys-LTs, perhaps released from degranulating mast cells, may initiate airway eosinophilia by direct chemotaxis, and that this may be sustained indirectly by cys-LT induction of eosinophilopoietic cytokine synthesis in T cells. Chronic over-production of cys-LTs by mast cells or infiltrating eosinophils may promote airway remodelling by effects on structural cells of the airway, including smooth muscle, fibroblasts, and epithelium, leading to airway hyperresponsiveness. The work of Tohda and colleagues [ 38] suggests that such effects of cys-LTs might even be mediated by secondary release of cytokines and growth factors from leucocytes or even from structural cells themselves. Leukotriene modulators may thus combat airway remodelling by inhibiting the activity of cys-LTs from a variety of cell types, leading to suppression of cytokine/growth factor synthesis and leucocyte influx. Epidermal growth factor (EGF) is a potent mitogen for human airway smooth muscle (HASM) cells, and expression of EGF and its receptors is increased in the asthmatic airway [ 40]. In vitro, LTD4 profoundly augments the proliferation of HASM cells produced by EGF, and this is blocked by specific cysLTRAs [ 41]. In rats, the cysLTRA MK-571 suppresses the increase in bronchial responsiveness and the increased airway smooth muscle mass induced by inhaled antigen [ 42], suggesting a mitogenic effect of endogenous cysteinyl-LTs on airway smooth muscle in vivo. Epithelial damage and epithelial cell proliferation are also typical pathological features of airway remodelling in asthma. The cys-LTs are potent mitogens for human airway epithelial cells in vitro, with LTC4 being effective even at sub-picomolar concentrations [ 43]. Cysteinyl-LTs may exacerbate the sensitizing effects of epithelial denudation by 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 CysLT1 receptor antagonists [ 44]. In addition, 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 [ 45]. This provides a mechanism for LTC4 to cause extracellular matrix remodelling in the chronically inflamed airway that might also be blocked by leukotriene modulators. Models to explain the cytokine and chemokine mechanisms of airway eosinophilia in asthma must now take into account the definitive evidence from clinical trials of specific leukotriene modulators that cysteinyl-LTs make a substantial, perhaps even the predominant, contribution to eosinophil migration in vivo. Indeed, until the advent of clinical trials of relevant cytokine antagonists, the leukotriene modulators may remain the only family of drugs with a known and specific mechanism of action that are capable of suppressing airway and blood eosinophilia. Recent evidence that at least part of the eosinophilic activity of cys-LTs may depend on secondary release of eosinophilopoietic cytokines such as IL-5 by T lymphocytes and other cells [ 9, 38] should promote further study of the effects of cys-LTs on cytokine expression in leucocytes and airway cells.