Update on leukotriene, lipoxin and oxoeicosanoid receptors: IUPHAR Review 7

Structure–function relationships

Of the receptors addressed in the present review, the human BLT₁ receptor is the best characterized in terms of structure–function relationships. Like several rhodopsin family GPCRs, the BLT₁ receptor bears an 8th helix (H8) domain consisting of Val²⁹⁸-Gly-Phe-Val-Ala-Lys-Leu-Leu-Glu-Gly³⁰⁷ (Okuno et al., 2003). Two aromatic residues, Tyr²⁸⁵ and Phe³⁰⁰, may stabilize the inactive form of the BLT₁ receptor by holding H8 at an almost right angle from the C-terminus of the seventh transmembrane region (Okuno et al., 2003). Thus, H8-deficient BLT₁ receptor mutants exhibit a prolonged intracellular signalling after LTB₄ stimulation (Okuno et al., 2003). Hydrophobic amino acid residues in the H8, Val³⁰¹, Leu³⁰⁴ and Leu³⁰⁵, may act as anchors to the plasma membrane, whereas Thr³⁰⁸ located after the H8 is one of the ligand-induced phosphorylation sites mediated by the GPCR kinase GRK6, and involved in BLT₁ receptor inactivation (Gaudreau et al., 2002). Recently, Aratake et al. reported an inhibitory role of the H8 on the LTB₄-elicited internalization of the BLT₁ receptor (Aratake et al., 2012). The human BLT₁ receptor with the mutations of Leu³⁰⁴ and Leu³⁰⁵ in the H8 exhibited an augmentation of LTB₄-induced internalization, whereas the wild-type (WT) receptor exhibited minimal internalization. Furthermore, phosphorylations of 5 Ser and Thr residues between 308 and 319 were important for this enhanced internalization of the mutant BLT₁ receptor. Therefore, the H8 of BLT₁ may repress LTB₄-induced internalization by suppressing excessive phosphorylations.

BLT₁ receptors in inflammation

The generation of BLT₁ receptor-deficient mice confirmed the loss of responsiveness to LTB₄ in BLT₁ receptor null leukocytes (Haribabu et al., 2000; Tager et al., 2000) and the suppression of several inflammation disease models (Bäck et al., 2011). In contrast, transgenic mice overexpressing the human BLT₁ receptor exhibited enhanced responsiveness of leukocytes in acute dermal inflammation (Chiang et al., 1999). Recently, Monterio et al. reported that macrophages, but not mast cells, are involved in the migration of neutrophils, by generating LTB₄ in haem-induced neutrophilic inflammation, for example, malaria and sickle cell disease (Monteiro et al., 2011). BLT₁ receptor antagonists, CP-105696 and LY-292476, significantly impaired the haem-induced peritoneal neutrophilia, demonstrating further involvement of the BLT₁ receptor in this inflammatory response (Monteiro et al., 2011).

BLT₁ receptors in cardiovascular disease (CVD)

Recurring nocturnal episodes of airway obstruction, known as obstructive sleep apnea syndrome, causes intermittent hypoxia, which is a detrimental stimuli for the cardiovascular system associated with, for example, early atherosclerosis and an increased cardiovascular risk (Stanke-Labesque et al., 2014). Neutrophil granulocytes derived from patients with obstructive sleep apnea exhibit increased LTB₄ production in response to calcium ionophore stimulation, compared with cells derived from healthy subjects (Stanke-Labesque et al., 2012). In addition, expression of LT-synthesizing enzymes in neutrophils correlates with measures of subclinical atherosclerosis and vascular remodelling in these patients (Stanke-Labesque et al., 2012). In support of a role for the LTB₄-BLT₁ pathway in sleep apnea-associated atherosclerosis, mice deficient in both apolipoprotein E and the BLT₁ receptor are protected from the accelerated atherosclerosis observed after subjecting apolipoprotein E-deficient mice to intermittent hypoxia in vivo (Li et al., 2011).

Hypertension is associated with increased levels of LTB₄ measured in the saliva (Labat et al., 2013). In addition, cerebral LTB₄ levels are increased in spontaneously hypertensive rats compared with Wistar-Kyoto rats (Waki et al., 2013). In the latter study, microinjection of the BLT₁ receptor antagonist U75302 into the solitary nucleus of spontaneously hypertensive rats lowered arterial pressure, suggesting that LTB₄-BLT₁ receptor inflammatory circuits in the brain stem may be associated with neurogenic hypertension (Waki et al., 2013).

BLT₁ receptors in rheumatoid arthritis

Several studies have revealed that BLT₁ receptor-deficient mice are protected from the development of arthritis using different models, associated with decreased articular neutrophil recruitment (Kim et al., 2006) resulting in reduced production of IL-1 and chemokines in the joint (Chou et al., 2010). In this disease, the recruitment of neutrophils is orchestrated via two pathways, C5a receptor signalling-induced LTB₄ release followed by BLT₁ receptor activation, and Fcγ receptor signalling-elicited IL-1β release (Sadik et al., 2012).

BLT₁ receptors in other diseases

Atopic dermatitis is an inflammatory skin disease. Although LTB₄ concentration is elevated in skin lesions of patients with atopic dermatitis, little is known about the role of LTB₄ in this disease. Recently, Oyoshi et al. reported an essential role of the LTB₄-BLT₁ receptor pathway in neutrophils for allergic skin inflammation (Oyoshi et al., 2012b). In the latter study, allergic skin inflammation was significantly decreased in BLT₁-deficient mice, and it was demonstrated that both LTB₄ production and BLT₁ receptor expression in neutrophils were important for the development of this disease (Oyoshi et al., 2012b).

Recently, the role of the BLT₁ receptor signalling in tumour immunology has been investigated. Yokota et al. examined the effect of the BLT₁-deficiency on the anti-tumour memory responses elicited by s.c. administration of GM-CSF gene-transduced WEHI3B (WGM) leukaemia cells using BLT₁ receptor-deficient mice (Yokota et al., 2012). They found that the BLT₁ receptor deficiency resulted in reduced tumour-infiltrating myeloid-derived suppressor cells, increase in matured dendritic cells (DCs) in tumour tissues and augmentation of CD4⁺ T-lymphocyte stimulation capacity during GM-CSF-triggered tumour regression, demonstrating that the lack of the LTB₄-BLT₁ receptor pathway induced long-term anti-tumour memory responses after immunization with WGM leukaemia cells. In another study however, BLT₁ receptor-deficient mice exhibited accelerated tumour growth and reduced survival compared with WT mice in a cervical cancer model (Sharma et al., 2013). These findings were associated with a decreased number of CD8⁺ T-lymphocytes and NK cells in tumours derived from BLT₁ receptor-deficient mice, and decreased IFN-γ and IL-2 expression (Sharma et al., 2013). Taken together, those studies suggest that BLT₁ receptor signalling on both suppressor and effector cells of the adaptive immune system may be involved in tumour progression and regression.

CysLT₁ receptors in respiratory diseases

The role of CysLT₁ receptor signalling in bronchial asthma depends both on the bronchoconstrictive and pro-inflammatory effects of the cysteinyl-LTs (Bäck et al., 2011). Furthermore, bronchial fibroblasts derived from asthmatic subjects express more CysLT₁ receptor mRNA compared with bronchial fibroblasts derived from non-asthmatic subjects (Eap et al., 2012). These authors also showed that activation of the asthmatic bronchial fibroblast CysLT₁ receptor by cysteinyl-LTs resulted in increased TGF-β1 which in turn increased pro-collagen (Eap et al., 2012). In airway epithelial cells, IL-13 up-regulates the expression of the CysLT₁ receptor, which is associated with an increased release of CCL26 (eotaxin-3), a potent eosinophil chemoattractant (Provost et al., 2012). Taken together, those studies suggest that CysLT₁ receptor activation on bronchial fibroblasts and epithelial cells further contribute to the cysteinyl-LTs-induced bronchial narrowing and eosinophil recruitment in asthma. Indeed, cells proliferation and bronchial narrowing are hallmarks of chronic asthma. In this regard, an intriguing study (Capra and Rovati, 2014) has recently demonstrated that rosuvastatin, the latest agent of this lipid-lowering class to be introduced on the market, dose-dependently inhibited LTD₄-induced human airway smooth muscle cells growth. The letter effect was exerted by means of inhibited prenylation of signalling proteins, most likely small G proteins such as Ras that are activated in a CysLT₁ receptor-dependent manner (McMahon et al., 2002; Capra et al., 2003; 2004; Ravasi et al., 2006; Poulin et al., 2011).

Cysteinyl-LTs are increased in children following infection with respiratory syncytial virus (RSV), associated with a potential subsequent development of asthma-like symptoms. In a mouse model of primary and secondary RSV-infection of newborn mice, pretreatment with montelukast, a selective CysLT₁ receptor antagonist, decreased RSV-induced airway hyperresponsiveness, airway inflammation and increased IFN-γ production in primary, but not secondary, infected neonate mice (Han et al., 2010).

In addition to asthma, CysLT₁ receptor signalling has also been implicated in chronic obstructive pulmonary disease (COPD). Bronchial mucosa samples from patients with COPD exacerbations of increased CysLT₁ receptor protein and mRNA expression was demonstrated on inflammatory cells, particularly mast cells and monocytes/macrophages (Zhu et al., 2012).

In line with an activation of the 5-lipoxygenase pathway, urinary LTE₄ has been associated with the degree of obstructive sleep apnea in adults (Stanke-Labesque et al., 2009) and children (Shen et al., 2011). In addition, increased cysteinyl-LTs and CysLT₁ receptor expression have been shown in tonsillar tissues derived from children with sleep apnea in China (Shen et al., 2012) and in Greece (Tsaoussoglou et al., 2012), suggesting that CysLT₁ receptor signalling may contribute to local proliferative and inflammatory pathways within tonsils in paediatric sleep-disordered breathing (Stanke-Labesque et al., 2014). In support of the latter notion, a recent randomized double-blind study of 46 children showed that a 12 week treatment with daily, oral montelukast reduced the severity of obstructive sleep apnea and the underlying adenoidal hypertrophy (Goldbart et al., 2012).

A role for activation of CysLT₁ receptors by cysteinyl-LTs in experimental pulmonary fibrosis has been supported by montelukast treatment in several mouse models. In a bleomycin-induced pulmonary fibrosis model in mice, montelukast decreased expression of IL-6, IL-13, IL-10 and TGF-β and attenuated lung fibrosis and hydroxyproline content (Shimbori et al., 2011). In a GATA-3 transcription factor overexpression model, montelukast-treated mice exhibited less airway inflammation in response to ovalbumin challenge, as demonstrated by decreased levels of T_H2 cytokines and TGF-β and decreased smooth muscle cell hyperplasia (Kiwamoto et al., 2011).

CysLT₁ receptor expression increases during T_H2 cell differentiation (Parmentier et al., 2012). Human T_H2 cells selectively express the CysLT₁ receptor, whereas CysLT₂ receptor, GPR17 and P2Y₁₂ (other receptors that can respond to cysteinyl-LT; see below) are undetectable. The T_H2 cell CysLT₁ receptor couples through both Gα_q and Gα_I to transduce a chemotactic response. In addition, the dectin-2-cysteinyl-LT pathway is essential for T_H2 predominant immunity in mice in response to house dust mite, in part through the CysLT₁ receptor (Barrett et al., 2011).

CysLT₁ receptors in CVDs

Cysteinyl-LTs are locally produced in coronary atherosclerotic plaques and contribute to vascular inflammation. Although previous studies have shown a dominant CysLT₂ receptor expression in vascular smooth muscle cells, lipopolysaccharide stimulation induces CysLT₁ receptor expression in human coronary artery vascular smooth muscle cells (Eaton et al., 2012). Interestingly, the CysLT₁ receptor exhibited a perinuclear expression in those cells, and its activation was coupled to a predominant nuclear calcium signalling and up-regulation of pro-atherosclerotic genes such as PAI-2 (Eaton et al., 2012). A nuclear membrane localization of the CysLT₁ receptor expression was initially reported in intestinal epithelial cells (Nielsen et al., 2005), and perinuclear expression of the CysLT₁ receptor has subsequently been demonstrated in human aortic valve myofibroblasts (Nagy et al., 2011). In the latter cells, the LTC₄-induced rise in intracellular calcium was most pronounced in the nuclear and perinuclear region of the cell, associated with mitochondrial permeability transition, changes in cell morphology, increased ROS production and an up-regulation of mRNA encoding bone morphogenic proteins (Nagy et al., 2011), all important pathophysiological processes in the calcification of the aortic valve observed in patients with calcific aortic stenosis.

The clinical use of the CysLT₁ receptor antagonists has allowed testing the hypothesis of their beneficial role in CVD in observational studies. In a pharmacoepidemiological cohort of approximately 7 million subjects, montelukast exposure was associated with a lower risk for recurrent stroke and with a lower risk for recurrent myocardial infarction in male subjects (Ingelsson et al., 2012).

CysLT₁ receptors in other diseases

In a mouse model of experimental autoimmune encephalitis (EAE) expression of the CysLT₁ receptor mRNA was up-regulated in spleen and lymph node tissue and in the spinal cord, and cysteinyl-LT concentrations in the blood and CSF were higher than in normal mice (Wang et al., 2011). In this EAE mouse model, treatment with CysLT₁ receptor-selective antagonists, either montelukast or zafirlukast, reduced CNS CD4⁺ T-lymphocyte influx and demyelination, associated with decreased EAE disease scores.

A role for CysLT₁ receptor signalling in various cancers has also been demonstrated. LTD₄-induced CysLT₁ receptor activation on chronic lymphocytic leukaemia cells and normal B-lymphocytes increases calcium and promote chemotaxis. CysLT₁ receptor antagonists induced apoptosis and reduced viability suggesting a potential therapeutic treatment (Drost et al., 2012). In line with the latter suggestion, high CysLT₁ and low CysLT₂ receptor protein expression in breast cancer tissue is associated with increased cancer-related mortality (Magnusson et al., 2011).

Clinical use of CysLT₁ receptor antagonists

It is worth noting here that LT modifiers (including LT receptor antagonists and 5-lipoxygenase inhibitors) are a class of drugs that may be used as an add-on treatment for adult patient with mild persistent asthma not satisfactorily controlled with inhaled glucocorticosteroids, or as alternative treatment particularly in patients suffering from aspirin-sensitive asthma or concomitant allergic rhinitis (Scott and Peters-Golden, 2013). They are generally well tolerated and present few, if any, class-related side effects. LT modifiers can also be safely used in children at all level of asthma severity, particularly against exercise-induced bronchoconstriction, considering virtually the absence of safety concerns (from the Global Strategy for Asthma Management and Prevention, Global Initiative for Asthma (GINA) 2012. Available from http://www.ginasthma.org/). However, LT modifiers are generally less effective than inhaled glucocorticosteroids when used alone as controller (Chauhan and Ducharme, 2012), while long-acting β₂-adrenoceptor agonists are modestly superior to LT receptor antagonists in reducing oral corticosteroid-treated exacerbations (Chauhan and Ducharme, 2014).

A number of clinical studies have been published from 2011 to 2013 using the selective CysLT₁ receptor antagonists montelukast or pranlukast in asthmatics. For example, montelukast pretreatment decreased hypertonic saline induced bronchoconstriction in asthmatics (Kazani et al., 2011), and provided disease control, similar to that of fluticasone. in actively smoking asthmatics (Price et al., 2013). Furthermore, in an allergen challenge model, pranlukast pretreatment inhibited nasal obstruction and nasal eosinophil cationic protein in allergic Japanese children (Gotoh et al., 2012). Finally, a pilot study indicated that montelukast prevented inflammatory cell responses in patients with persistent rhinitis, with particular emphasis on macrophages and neutrophils (Braido et al., 2012).

LT receptor antagonists have also been studied outside their respiratory indications. A prospective study with zafirlukast in female patients with long-standing mild/severe capsular contracture after surgical procedure for breast prosthesis. The results show a significant decrease in breast compliance values after 6 months of treatment, followed by a substantial increase 1 year after the end of drug intake, suggesting that the control of inflammation is crucial to prevent this multifactorial process (Mazzocchi et al., 2012). Finally, a retrospective study in children with food allergies suggested that montelukast can prevent food-induced adverse allergic reactions such as abdominal pain, which occur during oral immunotherapy (Takahashi et al., 2014).

CysLT₂ receptors in CVDs

A number of studies have reported the involvement of the CysLT₂ receptor in the inflammatory process subsequent to brain vascular insults, such as vascular ischaemia or oxygen-glucose deprivation (OGD; Bäck et al., 2011). In a model of focal cerebral ischaemia in the rat, a spatiotemporal up-regulation of the CysLT₂ receptor was associated with neuronal and glial cell activation (Zhao et al., 2011). The same authors also demonstrated that the mechanism underlying CysLT₂-mediated ischaemic astrocyte injury induced by OGD involves increased expression of the CysLT₂ receptor and of the water channel aquaporin 4 (AQP4). The latter was supported by reduced cell injury and AQP4 up-regulation by the CysLT₂ receptor antagonist BayCysLT2, (also known as CysLT2cpd; Carnini et al., 2011), but not by the highly selective CysLT₁ receptor antagonist montelukast (Qi et al., 2011). Furthermore, intracerebroventricular injection of HAMI3379, another even more selective CysLT₂ receptor antagonist (Wunder et al., 2010), before focal cerebral ischaemia in rats protects against acute brain injury attenuating the neurological deficits and reducing infarct volume, brain oedema, IgG exudation, neuronal degeneration and neuronal loss (Shi et al., 2012). Of note, the protective effect induced by CysLT₂ receptor antagonism was similar to that of pranlukast. In the latter context, it should be considered that pranlukast, zafirlukast and pobilukast have been found to be partially active also at the CysLT₂ receptor (Heise et al., 2000; Wunder et al., 2010; Capra et al., 2011).

In further support of neuronal effects of cysteinyl-LTs mainly being CysLT₂ receptor-dependent, HAMI3379 inhibited OGD/recovery, as well as LTD₄ and N-methyl-LTC₄-induced cell injury and neuronal loss in mixed cultures of cortical cells (Zhang et al., 2013). Although no effect of HAMI3379 was observed on OGD/recovery-induced neuronal injury in primary neurons, this antagonist inhibited neuronal loss and necrosis in neuron-microglial co-cultures, indicating that microglial activation may be crucial in this signalling. Similar effects were obtained by CysLT₂ receptor knock-down by shRNA, further supporting that these neuronal effects might indeed be CysLT₂ receptor-dependent (Zhang et al., 2013)

As mentioned above, the CysLT₂ receptor is highly expressed in endothelial cells of some vascular beds, and has been implicated in a variety of cardiovascular functions. BayCysLT2 administered either before or after ischaemia/reperfusion in transgenic mice overexpressing endothelium specific human CysLT₂ receptors attenuated the increased myocardial infarction damage, while this CysLT₂ receptor antagonist decreased neutrophil infiltration and leukocyte adhesion molecule (L-selectin) expression (Ni et al., 2011).

CysLT₂ receptors in other diseases

Using a loss-of-function murine model (CysLT₂R-LacZ), CysLT₂ receptor expression has been identified in neurons of the myenteric and submucosal plexus in the small intestine, colonic myenteric plexus, dorsal root ganglia and inferior ganglion of the vagal nerve (Barajas-Espinosa et al., 2011). In this model, LTC₄/D₄ stimulation of colonic submucosal venules elicited a reduced permeability response in CysLT₂ receptor knockout (CysLT₂^−/−) mice compared with WT mice, while basal neuronal activity of colonic-projecting nociceptive neurons from dorsal root ganglia showed significantly higher excitability. These data suggest that CysLT₂ receptor signalling in the murine colonic myenteric plexus may be involved in colitis disease progression, controlling inflammation-associated tissue oedema, and in the increased neuronal sensitivity to nociceptive stimuli (Barajas-Espinosa et al., 2011).

Sensitization and challenge using the dust mite Dermatophagoides farinae induces a marked augmentation of eosinophilic pulmonary inflammation, serum IgE, and T_H2 cytokines in CysLT₂ receptor-deficient compared with WT mice (Barrett et al., 2012). These observations could be replicated in WT mice sensitized by adoptive transfer of D. farina-pulsed CysLT₂ receptor-deficient bone marrow-derived DCs. Those results, taken together with a previous observation of a counter-regulatory role of CysLT₂ with respect to CysLT₁ receptor activity by dimerization (Jiang et al., 2007), suggest that the CysLT₂ receptor negatively regulates the development of cysteinyl-LT–dependent T_H2 pulmonary inflammation by inhibiting both CysLT₁ receptor signalling and D. farinae-induced LTC₄ synthase-dependent cell surface expression of CysLT₁ receptors on DCs (Barrett et al., 2012).

Proliferative diabetic retinopathy is associated with an increased synthesis of LTs (Talahalli et al., 2010) and oxygen-induced retinopathy (OIR) in mice induces CysLT₂ receptor up-regulation. CysLT₂ receptor knockout mice exhibit decreased vascular leakage and retinal oedema, but, surprisingly, increased tissue damage (vaso-obliteration/vasoproliferation) compared with WT mice. In addition, only PGs and hydroxyeicosatetraenoic acids, but not LTs, were detected in A23187-treated retina preparations (Barajas-Espinosa et al., 2012). Taken together, these data point to a confusing role of CysLT₂ receptor signalling in OIR progression that could be interpreted as either beneficial or detrimental to retinal health.

As mentioned above, atopic dermatitis is a chronic, relapsing, inflammatory skin disease characterized by dermal thickening, eosinophil infiltration and increased levels of LTE₄ in the urine. Although the role of cysteinyl-LTs in the inflammation associated with atopic dermatitis is unclear, there are reports suggesting improvements in atopic dermatitis with the use of LT receptor antagonists at the doses generally recommended for asthma treatment (see Capra et al., 2006 and Bäck et al., 2011 for more details). Recently, it has been reported that skin thickening and collagen deposition were significantly reduced in ovalbumin-sensitized skin of CysLT₂ receptor knockout mice. In addition, LTC₄ stimulation caused increased collagen synthesis by human skin fibroblasts, which, in turn, secreted factors that elicited keratinocyte proliferation. These effects were blocked by the dual CysLT₁/CysLT₂ receptor antagonist BAY u9773 (Oyoshi et al., 2012a).

Since the discovery that activation of CysLT₁ receptors could induce MAPK phosphorylation and thus, proliferation, survival and migration in a variety of cells, a number of papers have reported CysLT₁ receptor expression in brain tumours, as well as in prostate and breast cancer cells (Bäck et al., 2011). Interestingly, while low expression of CysLT₁ and high expression of CysLT₂ receptors correlated with high differentiation in epithelial colon cancer cells (Magnusson et al., 2007) and mediate good prognosis in colon (Magnusson et al., 2010) and breast cancer patients (Magnusson et al., 2011), very recently, the same group also demonstrated that all-trans retinoic acid (ATRA) induced CysLT₂ receptor and LTC₄ synthase mRNA expression (without affecting CysLT₁ receptor expression) and differentiation of colorectal cancer cells. This latter effect was inhibited by the CysLT₂ receptor-specific antagonist BayCysLT2, suggesting that ATRA can have anti-tumorigenic effects through the cysteinyl-LT pathway (Bengtsson et al., 2013).