Cysteinyl leukotrienes (cys-LTs) are potent mediators of inflammation derived from arachidonic acid through the 5-lipoxygenase/leukotriene C4 synthase pathway. The derivation of their chemical structures and identification of their pharmacologic properties predated the cloning of their classical receptors and the development of drugs that modify their synthesis and actions. Recent studies have revealed unanticipated insights into the regulation of cys-LT synthesis, the function of the cys-LTs in innate and adaptive immunity and human disease, and the identification of a new receptor for the cys-LTs. This review highlights these studies and summarizes their potential pathobiologic and therapeutic implications.
Leukotrienes; 5-lipoxygenase; asthma; AERD
Antagonists of the type 1 cysteinyl leukotriene receptor (CysLT1R) are efficacious for bronchoconstriction in humans with bronchial asthma; however, the clinical response to these drugs is heterogeneous. In particular, how CysLT1R expression and function are constitutively regulated in vivo is not known. Here we show that a 7-transmembrane receptor GPR17 negatively regulates the CysLT1R-mediated inflammatory cell accumulation in the bronchoalveolar lavage fluid and lung, the levels of IgE and specific IgG1 in serum, and Th2/Th17 cytokine expression in the lung after intranasal sensitization and challenge with house dust mite (Df) in mice. Sensitization of naïve wild-type recipients with Df-pulsed bone marrow-derived dendritic cells (BMDCs) of each genotype or sensitization of each genotype with Df-pulsed wild-type BMDCs and Df challenge revealed markedly increased pulmonary inflammatory and serum IgE responses for GPR17-deficient mice as compared to wild-type mice and reduced responses in the genotypes lacking CysLT1R. These findings reveal a constitutive negative regulation of CysLT1R functions by GPR17 in both the antigen presentation and downstream phases of allergic pulmonary inflammation.
Knockout Mice; Lipid Mediators; Allergy; Inflammation; Lung
The cysteinyl leukotrienes (cys-LTs), leukotriene C4 (LTC4) and its metabolites, LTD4 and LTE4, are proinflammatory lipid mediators in asthma and other inflammatory diseases. They are generated through the 5-lipoxygenase/LTC4 synthase (LTC4S) pathway and act via at least two distinct G protein-coupled receptors. The inhibition of human LTC4S will make a simple way to treat the cys-LT relevant inflammatory diseases. Here, we show that compounds having 5-(5-methylene-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid moiety suppress LTC4 synthesis, glutathione conjugation to the precursor LTA4, in both an enzyme assay and a whole-cell assay. Hierarchical in silico screenings of 6 million compounds provided 300,000 dataset for docking, and after energy minimization based on the crystal structure of LTC4S, 111 compounds were selected as candidates for a competitive inhibitor to glutathione. One of those compounds showed significant inhibitory activity, and subsequently, its derivative 5-((Z)-5-((E)-2-methyl-3-phenylallylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid (compound 1) was found to be the most potent inhibitor. The enzyme assay showed the IC50 was 1.9 µM and the corresponding 95% confidence interval was from 1.7 to 2.2 µM. The whole-cell assay showed that compound 1 was cell permeable and inhibited LTC4 synthesis in a concentration dependent manner.
enzyme inhibitor; membrane protein; leukotriene; inflammation; in silico screening
Cysteinyl leukotrienes (cys-LTs) can mediate Th2 immunity to the house dust mite, Dermatophagoides farinae (Df), via the type 1 receptor CysLT1R on dendritic cells (DCs). However, the role of the homologous type 2 receptor CysLT2R in Th2 immunity is unknown. Df sensitization and challenge of CysLT2R-deficient mice showed a marked augmentation of eosinophilic pulmonary inflammation, serum IgE, and Th2 cytokines. Wild-type (WT) mice sensitized by adoptive transfer of Df-pulsed CysLT2R-deficient bone marrow-derived DCs (BMDCs) also had a marked increase in Df-elicited eosinophilic lung inflammation and Th2 cytokines in restimulated hilar nodes. This response was absent in mice sensitized with Df-pulsed BMDCs lacking leukotriene C4 synthase (LTC4S), CysLT1R, or both CysLT2R/LTC4S, suggesting that CysLT2R negatively regulates LTC4S- and CysLT1R-dependent DC-mediated sensitization. CysLT2R-deficient BMDCs had increased CysLT1R-dependent LTD4-induced ERK phosphorylation, whereas N-methyl LTC4 activation of CysLT2R on WT BMDCs reduced such signaling. Activation of endogenously expressed CysLT1R and CysLT2R occurred over an equimolar range of LTD4 and N-methyl LTC4, respectively. Although the baseline expression of cell surface CysLT1R was not increased on CysLT2R-deficient BMDCs, it was upregulated at 24 h by a pulse of Df, as compared to WT or CysLT2R/LTC4S-deficient BMDCs. Importantly, treatment with N-methyl LTC4 reduced Df-induced CysLT1R expression on WT BMDCs. Thus, CysLT2R negatively regulates the development of cys-LT-dependent Th2 pulmonary inflammation by inhibiting both CysLT1R signaling and Df-induced LTC4S-dependent cell surface expression of CysLT1R on DCs. Furthermore, these studies highlight how the biologic activity of cys-LTs can be tightly regulated by competition between these endogenously expressed receptors.
Knockout mouse; Lipid mediator; G protein-coupled receptor; Asthma; Lung
Lipid signaling is dysregulated in many diseases with vascular pathology, including cancer, diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration. We have previously demonstrated that diets enriched in ω-3 polyunsaturated fatty acids (PUFAs) effectively reduce pathological retinal neovascularization in a mouse model of oxygen-induced retinopathy, in part through metabolic products that suppress microglialderived tumor necrosis factor–α. To better understand the protective effects of ω-3 PUFAs, we examined the relative importance of major lipid metabolic pathways and their products in contributing to this effect. ω-3 PUFA diets were fed to four lines of mice deficient in each key lipid-processing enzyme (cyclooxygenase 1 or 2, or lipoxygenase 5 or 12/15), retinopathy was induced by oxygen exposure; only loss of 5-lipoxygenase (5-LOX) abrogated the protection against retinopathy of dietary ω-3 PUFAs. This protective effect was due to 5-LOX oxidation of the ω-3 PUFA lipid docosahexaenoic acid to 4-hydroxy-docosahexaenoic acid (4-HDHA). 4-HDHA directly inhibited endothelial cell proliferation and sprouting angiogenesis via peroxisome proliferator–activated receptor γ (PPARγ), independent of 4-HDHA’s anti-inflammatory effects. Our study suggests that ω-3 PUFAs may be profitably used as an alternative or supplement to current anti–vascular endothelial growth factor (VEGF) treatment for proliferative retinopathy and points to the therapeutic potential of ω-3 PUFAs and metabolites in other diseases of vasoproliferation. It also suggests that cyclooxygenase inhibitors such as aspirin and ibuprofen (but not lipoxygenase inhibitors such as zileuton) might be used without losing the beneficial effect of dietary ω-3 PUFA.
House dust mites are a significant source of airborne allergen worldwide, but there is little understanding of how they so potently generate allergic inflammation. We found that extracts from the house dust mites Dermatophagoides farinae (Df) and Dermatophagoides pteronyssinus and from the mold Aspergillus fumigatus stimulated a rapid and robust production of cysteinyl leukotrienes (cys-LTs), proinflammatory lipid mediators, from mouse bone marrow-derived dendritic cells (BMDCs). Con A affinity chromatography of the Df extract revealed that the relevant ligand is a glycan(s), suggesting stimulation via a dendritic cell (DC) lectin receptor. Cys-LT production in BMDCs from wild-type mice was inhibited by spleen tyrosine kinase (Syk) inhibitors and was abolished in BMDCs from FcRγ−/− mice, implicating either Dectin-2 or DC immunoactivating receptor. Transfection of each receptor in bone marrow-derived mast cells revealed that only Dectin-2 mediates cys-LT production by Df, Dermatophagoides pteronyssinus, and Aspergillus fumigatus. Lentiviral knockdown of Dectin-2 in BMDCs attenuated Df extract-elicited cys-LT generation, thereby identifying Dectin-2 as the receptor. Lung CD11c+ cells, but not peritoneal or alveolar macrophages, also generated cys-LTs in response to Df. These findings place Dectin-2 among the C-type lectin receptors that activate arachidonic acid metabolism and identify the Dectin-2/FcRγ/Syk/cys-LT axis as a novel mechanism by which three potent indoor allergens may activate innate immune cells to promote allergic inflammation.
Sepsis impairs hypoxic pulmonary vasoconstriction (HPV) in patients and animal models contributing to systemic hypoxemia. Levels of cysteinyl leukotrienes are elevated in the bronchoalveolar lavage fluid of patients with sepsis, but the contribution of cysteinyl leukotrienes to the impairment of HPV is uncertain.
Wild-type mice, mice deficient in leukotriene C4 synthase, the enzyme responsible for cysteinyl leukotriene synthesis, and mice deficient in cysteinyl leukotriene receptor 1 were studied at 18 h after challenge with either saline or endotoxin. HPV was measured by the increase in left pulmonary vascular resistance induced by left mainstem bronchus occlusion. Levels of cysteinyl leukotrienes were determined in the bronchoalveolar lavage fluid.
In the bronchoalveolar lavage fluid of all three strains, cysteinyl leukotrienes were not detectable after saline challenge; whereas endotoxin challenge increased cysteinyl leukotriene levels in wild-type mice and mice deficient of cysteinyl leukotriene receptor 1, but not in mice deficient of leukotriene C4 synthase. HPV did not differ between the three mouse strains after saline challenge (120±26, 114±16, and 115±24%, respectively; mean±SD). Endotoxin challenge markedly impaired HPV in wild-type mice (41±20%) but only marginally in mice deficient in leukotriene C4 synthase (96±16%, P<0.05 vs. wild-type mice), thereby preserving systemic oxygenation. While endotoxin modestly decreased HPV in mice deficient in cysteinyl leukotriene receptor 1 (80±29%, P<0.05 vs. saline challenge), the magnitude of impairment was markedly less than in endotoxin-challenged wild-type mice.
Cysteinyl leukotrienes importantly contribute to endotoxin-induced impairment of HPV in part via a cysteinyl leukotriene receptor 1-dependent mechanisms.
Dectin-2 expression on GM-CSF–cultured bone marrow cells is required for the generation of cysteinyl leukotrienes and Th2 cytokines in response to the house dust mite Dermatophagoides farinae in vivo.
The innate signaling pathways for Th2 immunity activated by inhaled antigens are not well defined. We previously identified Dectin-2 as a receptor for glycans in allergen extracts from the house dust mite Dermatophagoides farinae (Df) that mediates cysteinyl leukotriene (cys-LT) generation from pulmonary CD11c+ cells and from GM-CSF–cultured bone marrow cells (BMCsGM-CSF). Using lentiviral knockdown of Dectin-2 in BMCsGM-CSF and adoptive transfer of Df-pulsed BMCsGM-CSF to sensitize naive mice, we now report that Dectin-2 is critical for the development of Df-elicited eosinophilic and neutrophilic pulmonary inflammation and Th2 cytokine generation in the lungs and restimulated lymph nodes. Sensitization with Df-pulsed BMCsGM-CSF from LTC4 synthase (LTC4S)–deficient mice or type 1 cys-LT receptor (CysLT1R)–deficient mice demonstrated that both proteins were required for Df-elicited eosinophilic pulmonary inflammation and Th2 cytokine generation in the lungs and restimulated lymph nodes. Direct sensitization and challenge of Ltc4s−/− and Cysltr1−/− mice confirmed that cys-LTs mediate these parameters of Df-elicited Th2 pulmonary inflammation. Thus, the Dectin-2–cys-LT pathway is critical for the induction of Th2 immunity to a major allergen, in part through CysLT1R. These findings identify a previously unrecognized link between a myeloid C-type lectin receptor and Th2 immunity.
Leukotriene (LT) C4 and its metabolites, LTD4 and
LTE4, are involved in the pathobiology of bronchial asthma.
LTC4 synthase is the nuclear membrane-embedded enzyme responsible
for LTC4 biosynthesis, catalyzing the conjugation of two substrates
that have considerably different water solubility; that amphipathic
LTA4 as a derivative of arachidonic acid and a water-soluble
glutathione (GSH). A previous crystal structure revealed important details of
GSH binding and implied a GSH activating function for Arg-104. In addition,
Arg-31 was also proposed to participate in the catalysis based on the putative
LTA4 binding model. In this study enzymatic assay with mutant
enzymes demonstrates that Arg-104 is required for the binding and activation of
GSH and that Arg-31 is needed for catalysis probably by activating the epoxide
group of LTA4.
Crystal Structure; Eicosanoid-specific Enzymes; Enzyme Mechanisms; Enzyme Structure; Membrane Proteins; LTC4S; Leukotriene C4 Synthase
Ischemia with subsequent reperfusion (IR) injury is a significant clinical problem that occurs after physical and surgical trauma, myocardial infarction, and organ transplantation. IR injury of mouse skeletal muscle depends on the presence of both natural IgM and an intact C pathway. Disruption of the skeletal muscle architecture and permeability also requires mast cell (MC) participation, as revealed by the fact that IR injury is markedly reduced in c-kit defective, MC-deficient mouse strains. In this study, we sought to identify the pathobiologic MC products expressed in IR injury using transgenic mouse strains with normal MC development, except for the lack of a particular MC-derived mediator. Histologic analysis of skeletal muscle from BALB/c and C57BL/6 mice revealed a strong positive correlation (R2 = 0.85) between the extent of IR injury and the level of MC degranulation. Linkage between C activation and MC degranulation was demonstrated in mice lacking C4, in which only limited MC degranulation and muscle injury were apparent. No reduction in injury was observed in transgenic mice lacking leukotriene C4 synthase, hemopoietic PGD2 synthase, N-deacetylase/N-sulfotransferase-2 (enzyme involved in heparin biosynthesis), or mouse MC protease (mMCP) 1. In contrast, muscle injury was significantly attenuated in mMCP-5-null mice. The MCs that reside in skeletal muscle contain abundant amounts of mMCP-5 which is the serine protease that is most similar in sequence to human MC chymase. We now report a cytotoxic activity associated with a MC-specific protease and demonstrate that mMCP-5 is critical for irreversible IR injury of skeletal muscle.
The intracellular parent of the cysteinyl leukotrienes (cys-LTs), leukotriene (LT) C4, is formed by conjugation of LTA4 and reduced glutathione by LTC4 synthase in mast cells, eosinophils, basophils, and macrophages. After extracellular export, LTC4 is converted to LTD4 and LTE4 by sequential enzymatic removal of glutamic acid and then glycine. Only LTE4 is sufficiently stable to be prominent in biologic fluids, such as urine or bronchoalveolar lavage fluid of asthmatic individuals and at sites of inflammation in animal models. LTE4 has received little attention because it binds poorly to the classical receptors, CysLT1R and CysLT2R, and was much less active on normal airways than LTC4 or LTD4. However, early studies indicated that LTE4 caused skin swelling in humans as potently as LTC4 and LTD4, that airways of asthmatic subjects (particularly those that were aspirin-sensitive) were selectively hyperresponsive to LTE4, and that a potential distinct LTE4 receptor was present in guinea pig trachea. Recent studies have begun to uncover receptors selective for LTE4; P2Y12, an ADP receptor, and CysLTER, observed functionally in skin of mice lacking CysLT1R and CysLT2R. These findings prompt a renewed focus on LTE4 receptors as therapeutic targets that are not currently addressed by available receptor antagonists.
Leukotriene E4; G protein-coupled receptor; bronchial asthma; inflammation; knockout mouse
Of the potent lipid inflammatory mediators comprising the cysteinyl leukotrienes (LTs; LTC4, LTD4, and LTE4), only LTE4 is stable and abundant in vivo. Although LTE4 shows negligible activity at the type 1 and 2 receptors for cys-LTs (CysLT1R and CysLT2R), it is a powerful inducer of mucosal eosinophilia and airway hyperresponsiveness in humans with asthma. We show that the adenosine diphosphate (ADP)–reactive purinergic (P2Y12) receptor is required for LTE4-mediated pulmonary inflammation. P2Y12 receptor expression permits LTE4 -induced activation of extracellular signal-regulated kinase in Chinese hamster ovary cells and permits chemokine and prostaglandin D2 production by LAD2 cells, a human mast cell line. P2Y12 receptor expression by LAD2 cells is required for competition between radiolabeled ADP and unlabeled LTE4 but not for direct binding of LTE4, suggesting that P2Y12 complexes with another receptor to recognize LTE4. Administration of LTE4 to the airways of sensitized mice potentiates eosinophilia, goblet cell metaplasia, and expression of interleukin-13 in response to low-dose aerosolized allergen. These responses persist in mice lacking both CysLT1R and CysLT2R but not in mice lacking P2Y12 receptors. The effects of LTE4 on P2Y12 in the airway were abrogated by platelet depletion. Thus, the P2Y12 receptor is required for proinflammatory actions of the stable abundant mediator LTE4 and is a novel potential therapeutic target for asthma.
Cysteinyl leukotrienes (cys-LTs) induce inflammatory responses through type 1 (CysLT1R) and type 2 (CysLT2R) cys-LT receptors, and activate mast cells (MCs) in vitro. We previously demonstrated that cys-LTs cross-desensitized interleukin (IL)-4-primed primary human MCs (hMCs) to stimulation with the nucleotide uridine diphosphate (UDP). We now report that hMCs, mouse bone marrow-derived mast cells (mBMMCs), and the human MC line LAD2 all express UDP-selective P2Y6 receptors that cooperate with CysLT1R to promote cell survival and chemokine generation by a pathway involving reciprocal ligand-mediated cross-talk. LTD4, the most potent CysLT1R ligand, and UDP both induced phosphorylation of extracellular signal regulated kinase (ERK) and prolonged the survival of cytokine-starved hMCs and mBMMCs. ERK activation and cytoprotection in response to either ligand were attenuated by treatment of the cells with a selective P2Y6 receptor antagonist (MRS2578), which did not interfere with signaling through recombinant CysLT1R. Surprisingly, both UDP and LTD4-mediated ERK activation and cytoprotection were absent in mBMMCs lacking CysLT1R and the biosynthetic enzyme LTC4 synthase (LTC4S), implying a requirement for a cys-LT-mediated autocrine loop. In IL-4-primed LAD2 cells, LTD4 induced the generation of macrophage inflammatory protein 1β (MIP-1β) by IL-4-primed LAD2 cells, a response blocked by short hairpin RNA (shRNA)-mediated knockdown of CysLT1R or of P2Y6 receptors, but not of CysLT2R. Thus, CysLT1R and P2Y6 receptors, which are co-expressed with on many cell types of innate immunity, reciprocally amplify one another’s function in MCs through endogenous ligands.
Mast Cells/Basophils; Lipid Mediators; Allergy; inflammation
Conclusions regarding the contribution of low molecular weight secretory phospholipase A2 (sPLA2) enzymes in eicosanoid generation have relied on data obtained from transfected cells or the use of inhibitors that fail to discriminate between individual members of the large family of mammalian sPLA2 enzymes. To elucidate the role of group V sPLA2, we used targeted gene disruption to generate mice lacking this enzyme. Zymosan-induced generation of leukotriene C4 and prostaglandin E2 was attenuated ~50% in peritoneal macrophages from group V sPLA2-null mice compared to macrophages from wild-type littermates. Furthermore, the early phase of plasma exudation in response to intraperitoneal injection of zymosan and the accompanying in vivo generation of cysteinyl leukotrienes were markedly attenuated in group V sPLA2-null mice compared to wild-type controls. These data provide clear evidence of a role for group V sPLA2 in regulating eicosanoid generation in response to an acute innate stimulus of the immune response both in vitro and in vivo, suggesting a role for this enzyme in innate immunity.
Neutrophils serve as a vanguard of the acute innate immune response to invading pathogens. Neutrophils are also abundant at sites of autoimmune inflammation, such as the rheumatoid joint, although their pathophysiologic role is incompletely defined and relevant effector functions remain obscure. Using genetic and pharmacologic approaches in the K/BxN serum transfer model of arthritis, we find that autoantibody-driven erosive synovitis is critically reliant on the generation of leukotrienes, and more specifically on leukotriene B4 (LTB4), for disease induction as well as perpetuation. Pursuing the cellular source for this mediator, we find via reconstitution experiments that mast cells are a dispensable source of leukotrienes, whereas arthritis susceptibility can be restored to leukotriene-deficient mice by intravenous administration of wild-type neutrophils. These experiments demonstrate a nonredundant role for LTB4 in inflammatory arthritis and define a neutrophil mediator involved in orchestrating the synovial eruption.
In the fission yeast Schizosaccharomyces pombe the nrd1+ gene encoding an RNA binding protein negatively regulates the onset of differentiation. Its biological role is to block differentiation by repressing a subset of the Ste11-regulated genes essential for conjugation and meiosis until the cells reach a critical level of nutrient starvation. By using the phenotypic suppression of the S. pombe temperature-sensitive pat1 mutant that commits lethal haploid meiosis at the restrictive temperature, we have cloned ROD1, a functional homologue of nrd1+, from rat and human cDNA libraries. Like nrd1+, ROD1 encodes a protein with four repeats of typical RNA binding domains, though its amino acid homology to Nrd1 is limited. When expressed in the fission yeast, ROD1 behaves in a way that is functionally similar to nrd1+, being able to repress Ste11-regulated genes and to inhibit conjugation upon overexpression. ROD1 is predominantly expressed in hematopoietic cells or organs of adult and embryonic rat. Like nrd1+ for fission yeast differentiation, overexpressed ROD1 effectively blocks both 12-O-tetradecanoyl phorbol-13-acetate-induced megakaryocytic and sodium butyrate-induced erythroid differentiation of the K562 human leukemia cells without affecting their proliferative ability. These results suggest a role for ROD1 in differentiation control in mammalian cells. We discuss the possibility that a differentiation control system found in the fission yeast might well be conserved in more complex organisms, including mammals.