The CysLT2R receptor mediates leukotriene C4-driven acute and chronic itch.

Acute and chronic itch are burdensome manifestations of skin pathologies including allergic skin diseases and atopic dermatitis, but the underlying molecular mechanisms are not well understood. Cysteinyl leukotrienes (CysLTs), comprising LTC4, LTD4, and LTE4, are produced by immune cells during type 2 inflammation. Here, we uncover a role for LTC4 and its signaling through the CysLT receptor 2 (CysLT2R) in itch. Cysltr2 transcript is highly expressed in dorsal root ganglia (DRG) neurons linked to itch in mice. We also detected CYSLTR2 in a broad population of human DRG neurons. Injection of leukotriene C4 (LTC4) or its nonhydrolyzable form NMLTC4, but neither LTD4 nor LTE4, induced dose-dependent itch but not pain behaviors in mice. LTC4-mediated itch differed in bout duration and kinetics from pruritogens histamine, compound 48/80, and chloroquine. NMLTC4-induced itch was abrogated in mice deficient for Cysltr2 or when deficiency was restricted to radioresistant cells. Itch was unaffected in mice deficient for Cysltr1, Trpv1, or mast cells (WSh mice). CysLT2R played a role in itch in the MC903 mouse model of chronic itch and dermatitis, but not in models of dry skin or compound 48/80- or Alternaria-induced itch. In MC903-treated mice, CysLT levels increased in skin over time, and Cysltr2-/- mice showed decreased itch in the chronic phase of inflammation. Collectively, our study reveals that LTC4 acts through CysLT2R as its physiological receptor to induce itch, and CysLT2R contributes to itch in a model of dermatitis. Therefore, targeting CysLT signaling may be a promising approach to treat inflammatory itch.

Cysteinyl leukotriene 2 receptor promotes endothelial permeability, tumor angiogenesis, and metastasis.

Cysteinyl leukotrienes (cys-LTs) are proinflammatory mediators that enhance vascular permeability through distinct receptors (CysLTRs). We found that CysLT2R regulates angiogenesis in isolated mouse endothelial cells (ECs) and in Matrigel implants in WT mice and enhances EC contraction and permeability via the Rho-dependent myosin light chain 2 and vascular endothelial (VE)-cadherin axis. Since solid tumors utilize aberrant angiogenesis for their growth and metastasis and their vessels exhibit vascular hyperpermeability, we hypothesized that CysLT2R, via its actions on the endothelium, might regulate tumor growth. Both tumor growth and metastases of adoptively transferred syngeneic Lewis lung carcinoma (LLC) cells are significantly reduced in CysLT2R-null mice (Cysltr2-/-) compared with WT and CysLT1R-null mice (Cysltr1-/-). In WT recipients of LLC cells, CysLT2R expression is significantly increased in the tumor vasculature, compared with CysLT1R. Further, the tumor vasculature in Cysltr2-/- recipients exhibited significantly improved integrity, as revealed by increased pericyte coverage and decreased leakage of i.v.-administered Texas Red-conjugated dextran. Administration of a selective CysLT2R antagonist significantly reduced LLC tumor volume, vessel density, dextran leakage, and metastases in WT mice, highlighting CysLT2R as a VEGF-independent regulator of the vasculature promoting risk of metastasis. Thus, both genetic and pharmacological findings establish CysLT2R as a gateway for angiogenesis and EC dysregulation in vitro and ex vivo and in an in vivo model with a mouse tumor. Our data suggest CysLT2R as a possible target for intervention.

Type 2 Cysteinyl Leukotriene Receptors Drive IL-33-Dependent Type 2 Immunopathology and Aspirin Sensitivity.

Cysteinyl leukotrienes (cysLTs) facilitate mucosal type 2 immunopathology by incompletely understood mechanisms. Aspirin-exacerbated respiratory disease, a severe asthma subtype, is characterized by exaggerated eosinophilic respiratory inflammation and reactions to aspirin, each involving the marked overproduction of cysLTs. Here we demonstrate that the type 2 cysLT receptor (CysLT2R), which is not targeted by available drugs, is required in two different models to amplify eosinophilic airway inflammation via induced expression of IL-33 by lung epithelial cells. Endogenously generated cysLTs induced eosinophilia and expanded group 2 innate lymphoid cells (ILC2s) in aspirin-exacerbated respiratory disease-like Ptges-/- mice. These responses were mitigated by deletions of either Cysltr2 or leukotriene C4 synthase (Ltc4s). Administrations of either LTC4 (the parent cysLT) or the selective CysLT2R agonist N-methyl LTC4 to allergen sensitized wild-type mice markedly boosted ILC2 expansion and IL-5/IL-13 generation in a CysLT2R-dependent manner. Expansion of ILC2s and IL-5/IL-13 generation reflected CysLT2R-dependent production of IL-33 by alveolar type 2 cells, which engaged in a bilateral feed-forward loop with ILC2s. Deletion of Cysltr1 blunted LTC4-induced ILC2 expansion and eosinophilia but did not alter IL-33 induction. Pharmacological blockade of CysLT2R prior to inhalation challenge of Ptges-/- mice with aspirin blocked IL-33-dependent mast cell activation, mediator release, and changes in lung function. Thus, CysLT2R signaling, IL-33-dependent ILC2 expansion, and IL-33-driven mast cell activation are necessary for induction of type 2 immunopathology and aspirin sensitivity. CysLT2R-targeted drugs may interrupt these processes.

Leukotriene E4 elicits respiratory epithelial cell mucin release through the G-protein-coupled receptor, GPR99.

Cysteinyl leukotrienes (cysLTs), leukotriene C4 (LTC4), LTD4, and LTE4 are proinflammatory lipid mediators with pathobiologic function in asthma. LTE4, the stable cysLT, is a weak agonist for the type 1 and type 2 cysLT receptors (CysLTRs), which constrict airway smooth muscle, but elicits airflow obstruction and pulmonary inflammation in patients with asthma. We recently identified GPR99 as a high-affinity receptor for LTE4 that mediates cutaneous vascular permeability. Here we demonstrate that a single intranasal exposure to extract from the respiratory pathogen Alternaria alternata elicits profound epithelial cell (EpC) mucin release and submucosal swelling in the nasal mucosa of mice that depends on cysLTs, as it is absent in mice deficient in the terminal enzyme for cysLT biosynthesis, LTC4 synthase (LTC4S). These mucosal changes are associated with mast cell (MC) activation and absent in MC-deficient mice, suggesting a role for MCs in control of EpC function. Of the three CysLTRs, only GPR99-deficient mice are fully protected from EpC mucin release and swelling elicited by Alternaria or by intranasal LTE4 GPR99 expression is detected on lung and nasal EpCs, which release mucin to doses of LTE4 one log lower than that required to elicit submucosal swelling. Finally, mice deficient in MCs, LTC4S, or GPR99 have reduced baseline numbers of goblet cells, indicating an additional function in regulating EpC homeostasis. These results demonstrate a novel role for GPR99 among CysLTRs in control of respiratory EpC function and suggest that inhibition of LTE4 and of GPR99 may have therapeutic benefits in asthma.

Phosphoinositide 3-Kinase δ Regulates Dectin-2 Signaling and the Generation of Th2 and Th17 Immunity.

The C-type lectin receptor Dectin-2 can trigger the leukotriene C4 synthase-dependent generation of cysteinyl leukotrienes and the caspase-associated recruitment domain 9- and NF-κB-dependent generation of cytokines, such as IL-23, IL-6, and TNF-α, to promote Th2 and Th17 immunity, respectively. Dectin-2 activation also elicits the type 2 cytokine IL-33, but the mechanism by which Dectin-2 induces these diverse innate mediators is poorly understood. In this study, we identify a common upstream requirement for PI3Kδ activity for the generation of each Dectin-2-dependent mediator elicited by the house dust mite species, Dermatophagoides farinae, using both pharmacologic inhibition and small interfering RNA knockdown of PI3Kδ in bone marrow-derived dendritic cells. PI3Kδ activity depends on spleen tyrosine kinase (Syk) and regulates the activity of protein kinase Cδ, indicating that PI3Kδ is a proximal Syk-dependent signaling intermediate. Inhibition of PI3Kδ also reduces cysteinyl leukotrienes and cytokines elicited by Dectin-2 cross-linking, confirming the importance of this molecule in Dectin-2 signaling. Using an adoptive transfer model, we demonstrate that inhibition of PI3Kδ profoundly reduces the capacity of bone marrow-derived dendritic cells to sensitize recipient mice for Th2 and Th17 pulmonary inflammation in response to D. farinae Furthermore, administration of a PI3Kδ inhibitor during the sensitization of wild-type mice prevents the generation of D. farinae-induced pulmonary inflammation. These results demonstrate that PI3Kδ regulates Dectin-2 signaling and its dendritic cell function.

CysLT1 Receptor Is Protective against Oxidative Stress in a Model of Irritant-Induced Asthma.

The bronchoconstrictive and proinflammatory properties of cysteinyl leukotrienes (cysLTs) in allergic asthma mediate their effects predominantly through the cysLT1 receptor (cysLT1R). However, the role of cysLTs and cysLT1R in innate immune-triggered asthma is largely unexplored. We explored the synthesis of cysLTs and cysLT1R as determinants of airway responses in an oxidative stress-induced model of irritant asthma. Wild-type (WT) mice exposed to 100 ppm Cl2 for 5 min had airway neutrophilia, increased cysLT production, and pulmonary expression of cysLT-related biosynthetic genes. CysLT1R-deficient (CysLTr1(-/-)) mice that were exposed to Cl2 demonstrated airway hyperresponsiveness to inhaled methacholine significantly greater than in WT BALB/c mice. Compared to WT mice, airway neutrophilia and keratinocyte chemoattractant production levels were higher in CysLTr1(-/-) mice and airway hyperresponsiveness was ameliorated using a granulocyte depletion Ab. CysLTr1(-/-) mice also demonstrated prolonged bronchial epithelial cell apoptosis following Cl2 WT mice showed increased antioxidant and NF erythroid 2-related factor 2 (Nrf2) gene expression, Nrf2 nuclear translocation in bronchial epithelial cells, and increased reduced glutathione/oxidized glutathione following Cl2 exposure whereas CysLTr1(-/-) mice did not. Furthermore, CysLTr1(-/-) mice demonstrated increased pulmonary E-cadherin expression and soluble E-cadherin shedding compared with WT mice. Loss of a functional cysLT1R results in aberrant antioxidant response and increased susceptibility to oxidative injury, apparently via a cysLT1R-dependent impairment of Nrf2 function.

Aspirin-Exacerbated Respiratory Disease Involves a Cysteinyl Leukotriene-Driven IL-33-Mediated Mast Cell Activation Pathway.

Aspirin-exacerbated respiratory disease (AERD), a severe eosinophilic inflammatory disorder of the airways, involves overproduction of cysteinyl leukotrienes (cysLTs), activation of airway mast cells (MCs), and bronchoconstriction in response to nonselective cyclooxygenase inhibitors that deplete homeostatic PGE2. The mechanistic basis for MC activation in this disorder is unknown. We now demonstrate that patients with AERD have markedly increased epithelial expression of the alarmin-like cytokine IL-33 in nasal polyps, as compared with polyps from aspirin-tolerant control subjects. The murine model of AERD, generated by dust mite priming of mice lacking microsomal PGE2 synthase (ptges(-/-) mice), shows a similar upregulation of IL-33 protein in the airway epithelium, along with marked eosinophilic bronchovascular inflammation. Deletion of leukotriene C4 synthase, the terminal enzyme needed to generate cysLTs, eliminates the increased IL-33 content of the ptges(-/-) lungs and sharply reduces pulmonary eosinophilia and basal secretion of MC products. Challenges of dust mite-primed ptges(-/-) mice with lysine aspirin induce IL-33-dependent MC activation and bronchoconstriction. Thus, IL-33 is a component of a cysLT-driven innate type 2 immune response that drives pathogenic MC activation and contributes substantially to AERD pathogenesis.

Dectin-2 regulates the effector phase of house dust mite-elicited pulmonary inflammation independently from its role in sensitization.

The myeloid C-type lectin receptor Dectin-2 directs the generation of Th2 and Th17 immune responses to the house dust mite Dermatophagoides farinae through the generation of cysteinyl leukotrienes and proinflammatory cytokines, respectively, but a role for Dectin-2 in effector phase responses has not been described. In this study, we demonstrate that administration of the Dectin-2 mAb solely at the time of D. farinae challenge abrogated eosinophilic and neutrophilic inflammation in the bronchoalveolar lavage fluid and Th1, Th2, and Th17 inflammation in the lung of previously sensitized mice. Furthermore, Dectin-2 null mice (Clec4n(-/-)) sensitized with the adoptive transfer of D. farinae-pulsed wild-type (WT) bone marrow-derived dendritic cells (DCs) also had less D. farinae-elicited pulmonary inflammation, supporting an effector function for Dectin-2. The protection from pulmonary inflammation seen with the Dectin-2 mAb or in Clec4n(-/-) mice was associated with little or no reduction in lung-draining lymph node cells or their cytokine production and with no reduction in serum IgE. WT and Clec4n(-/-) mice recipients, sensitized with D. farinae-pulsed WT bone marrow-derived DCs, had comparable levels of D. farinae-elicited IL-6, IL-23, TNF-α, and cysteinyl leukotrienes in the lung. By contrast, D. farinae-elicited CCL4 and CCL8 production from pulmonary CD11c(+)CD11b(+)Ly6C(+) and CD11c(+)CD11b(+)Ly6C(-)CD64(+) monocyte-derived DCs was reduced in Clec4n(-/-) recipients. Addition of CCL8 at the time of D. farinae challenge abrogated the protection from eosinophilic, neutrophilic, and Th2 pulmonary inflammation seen in Clec4n(-/-) recipients. Taken together, these results reveal that Dectin-2 regulates monocyte-derived DC function in the pulmonary microenvironment at D. farinae challenge to promote the local inflammatory response.

Cutting edge: Leukotriene C4 activates mouse platelets in plasma exclusively through the type 2 cysteinyl leukotriene receptor.

Leukotriene C4 (LTC4) and its extracellular metabolites, LTD4 and LTE4, mediate airway inflammation. They signal through three specific receptors (type 1 cys-LT receptor [CysLT1R], CysLT2R, and GPR99) with overlapping ligand preferences. In this article, we demonstrate that LTC4, but not LTD4 or LTE4, activates mouse platelets exclusively through CysLT2R. Platelets expressed CysLT1R and CysLT2R proteins. LTC4 induced surface expression of CD62P by wild-type mouse platelets in platelet-rich plasma (PRP) and caused their secretion of thromboxane A2 and CXCL4. LTC4 was fully active on PRP from mice lacking either CysLT1R or GPR99, but completely inactive on PRP from CysLT2R-null (Cysltr2(-/-)) mice. LTC4/CysLT2R signaling required an autocrine ADP-mediated response through P2Y12 receptors. LTC4 potentiated airway inflammation in a platelet- and CysLT2R-dependent manner. Thus, CysLT2R on platelets recognizes LTC4 with unexpected selectivity. Nascent LTC4 may activate platelets at a synapse with granulocytes before it is converted to LTD4, promoting mediator generation and the formation of leukocyte-platelet complexes that facilitate inflammation.

Eosinophil-derived leukotriene C4 signals via type 2 cysteinyl leukotriene receptor to promote skin fibrosis in a mouse model of atopic dermatitis.

Atopic dermatitis (AD) skin lesions exhibit epidermal and dermal thickening, eosinophil infiltration, and increased levels of the cysteinyl leukotriene (cys-LT) leukotriene C(4) (LTC(4)). Epicutaneous sensitization with ovalbumin of WT mice but not ΔdblGATA mice, the latter of which lack eosinophils, caused skin thickening, collagen deposition, and increased mRNA expression of the cys-LT generating enzyme LTC(4) synthase (LTC(4)S). Skin thickening and collagen deposition were significantly reduced in ovalbumin-sensitized skin of LTC(4)S-deficient and type 2 cys-LT receptor (CysLT(2)R)-deficient mice but not type 1 cys-LT receptor (CysLT(1)R)-deficient mice. Adoptive transfer of bone marrow-derived eosinophils from WT but not LTC(4)S-deficient mice restored skin thickening and collagen deposition in epicutaneous-sensitized skin of ΔdblGATA recipients. LTC(4) stimulation caused increased collagen synthesis by human skin fibroblasts, which was blocked by CysLT(2)R antagonism but not CysLT(1)R antagonism. Furthermore, LTC(4) stimulated skin fibroblasts to secrete factors that elicit keratinocyte proliferation. These findings establish a role for eosinophil-derived cys-LTs and the CysLT(2)R in the hyperkeratosis and fibrosis of allergic skin inflammation. Strategies that block eosinophil infiltration, cys-LT production, or the CysLT(2)R might be useful in the treatment of AD.

Identification of GPR99 protein as a potential third cysteinyl leukotriene receptor with a preference for leukotriene E4 ligand.

The cysteinyl leukotrienes (cys-LTs), leukotriene C4 (LTC4), a conjugation product of glutathione and eicosatetraenoic acid, and its metabolites, LTD4 and LTE4, are lipid mediators of smooth muscle constriction and inflammation in asthma. LTD4 is the most potent ligand for the type 1 cys-LT receptor (CysLT1R), and LTC4 and LTD4 have similar lesser potency for CysLT2R, whereas LTE4 has little potency for either receptor. Cysltr1/Cysltr2(-/-) mice, lacking the two defined receptors, exhibited a comparable dose-dependent vascular leak to intradermal injection of LTC4 or LTD4 and an augmented response to LTE4 as compared with WT mice. As LTE4 retains a cysteine residue and might provide recognition via a dicarboxylic acid structure, we screened cDNAs within the P2Y nucleotide receptor family containing CysLTRs and dicarboxylic acid receptors with trans-activator reporter gene assays. GPR99, previously described as an oxoglutarate receptor (Oxgr1), showed both a functional and a binding response to LTE4 in these transfectants. We generated Gpr99(-/-) and Gpr99/Cysltr1/Cysltr2(-/-) mice for comparison with WT and Cysltr1/Cysltr2(-/-) mice. Strikingly, GPR99 deficiency in the Cysltr1/Cysltr2(-/-) mice virtually eliminated the vascular leak in response to the cys-LT ligands, indicating GPR99 as a potential CysLT3R active in the Cysltr1/Cysltr2(-/-) mice. Importantly, the Gpr99(-/-) mice showed a dose-dependent loss of LTE4-mediated vascular permeability, but not to LTC4 or LTD4, revealing a preference of GPR99 for LTE4 even when CysLT1R is present. As LTE4 is the predominant cys-LT species in inflamed tissues, GPR99 may provide a new therapeutic target.

Cysteinyl leukotriene 2 receptor on dendritic cells negatively regulates ligand-dependent allergic pulmonary inflammation.

Cysteinyl leukotrienes (cys-LTs) can mediate Th2 immunity to the house dust mite, Dermatophagoides farinae, via the type 1 receptor CysLT(1)R on dendritic cells (DCs). However, the role of the homologous type 2 receptor CysLT(2)R in Th2 immunity is unknown. D. farinae sensitization and challenge of CysLT(2)R-deficient mice showed a marked augmentation of eosinophilic pulmonary inflammation, serum IgE, and Th2 cytokines. Wild-type (WT) mice sensitized by adoptive transfer of D. farinae-pulsed CysLT(2)R-deficient bone marrow-derived DCs (BMDCs) also had a marked increase in D. farinae-elicited eosinophilic lung inflammation and Th2 cytokines in restimulated hilar nodes. This response was absent in mice sensitized with D. farinae-pulsed BMDCs lacking leukotriene C(4) synthase (LTC(4)S), CysLT(1)R, or both CysLT(2)R/LTC(4)S, suggesting that CysLT(2)R negatively regulates LTC(4)S- and CysLT(1)R-dependent DC-mediated sensitization. CysLT(2)R-deficient BMDCs had increased CysLT(1)R-dependent LTD(4)-induced ERK phosphorylation, whereas N-methyl LTC(4) activation of CysLT(2)R on WT BMDCs reduced such signaling. Activation of endogenously expressed CysLT(1)R and CysLT(2)R occurred over an equimolar range of LTD(4) and N-methyl LTC(4), respectively. Although the baseline expression of cell surface CysLT(1)R was not increased on CysLT(2)R-deficient BMDCs, it was upregulated at 24 h by a pulse of D. farinae, compared with WT or CysLT(2)R/LTC(4)S-deficient BMDCs. Importantly, treatment with N-methyl LTC(4) reduced D. farinae-induced CysLT(1)R expression on WT BMDCs. Thus, CysLT(2)R negatively regulates the development of cys-LT-dependent Th2 pulmonary inflammation by inhibiting both CysLT(1)R signaling and D. farinae-induced LTC(4)S-dependent cell surface expression of CysLT(1)R on DCs. Furthermore, these studies highlight how the biologic activity of cys-LTs can be tightly regulated by competition between these endogenously expressed receptors.

Crystal structure of a human membrane protein involved in cysteinyl leukotriene biosynthesis.

The cysteinyl leukotrienes, namely leukotriene (LT)C4 and its metabolites LTD4 and LTE4, the components of slow-reacting substance of anaphylaxis, are lipid mediators of smooth muscle constriction and inflammation, particularly implicated in bronchial asthma. LTC4 synthase (LTC4S), the pivotal enzyme for the biosynthesis of LTC4 (ref. 10), is an 18-kDa integral nuclear membrane protein that belongs to a superfamily of membrane-associated proteins in eicosanoid and glutathione metabolism that includes 5-lipoxygenase-activating protein, microsomal glutathione S-transferases (MGSTs), and microsomal prostaglandin E synthase 1 (ref. 13). LTC4S conjugates glutathione to LTA4, the endogenous substrate derived from arachidonic acid through the 5-lipoxygenase pathway. In contrast with MGST2 and MGST3 (refs 15, 16), LTC4S does not conjugate glutathione to xenobiotics. Here we show the atomic structure of human LTC4S in a complex with glutathione at 3.3 A resolution by X-ray crystallography and provide insights into the high substrate specificity for glutathione and LTA4 that distinguishes LTC4S from other MGSTs. The LTC4S monomer has four transmembrane alpha-helices and forms a threefold symmetric trimer as a unit with functional domains across each interface. Glutathione resides in a U-shaped conformation within an interface between adjacent monomers, and this binding is stabilized by a loop structure at the top of the interface. LTA4 would fit into the interface so that Arg 104 of one monomer activates glutathione to provide the thiolate anion that attacks C6 of LTA4 to form a thioether bond, and Arg 31 in the neighbouring monomer donates a proton to form a hydroxyl group at C5, resulting in 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC4). These findings provide a structural basis for the development of LTC4S inhibitors for a proinflammatory pathway mediated by three cysteinyl leukotriene ligands whose stability and potency are different and by multiple cysteinyl leukotriene receptors whose functions may be non-redundant.

Oxidative stress suppresses cysteinyl leukotriene generation by mouse bone marrow-derived mast cells.

Cysteinyl leukotrienes and oxidative stress have both been implicated in bronchial asthma; however, there is no previous study that focused on the ability of oxidative stress to alter cysteinyl leukotriene generation. In this study, treatment of bone marrow-derived mast cells with prostaglandin D(2) reduced their ability to generate leukotriene (LT) C(4) upon calcium ionophore stimulation but had little effect on LTB(4) generation. This effect could be reproduced by a selective agonist of the DP(2) receptor, 15R-methyl prostaglandin D(2) (15R-D(2)). 15R-D(2) dose-dependently inhibited LTC(4) generation with an IC(50) of 2 µM, and the effect was not altered by a DP(2)/thromboxane antagonist or by a peroxisome proliferator-activated receptor-γ antagonist. 15R-D(2) exerted its suppressive effect via a reduction in intracellular GSH, a mechanism that involved the conjugation of its non-enzymatic breakdown product to GSH. At 10 µM, 15R-D(2) reduced LTC(4) generation to 10%, intracellular GSH to 50%, and LTC(4) synthase (LTC(4)S) activity to 33.5% of untreated cells without altering immunoreactive LTC(4)S protein expression or 5-lipoxygenase activity. The effects of 15R-D(2) on LTC(4)S activity could be partially reversed by reducing reagent. The sulfhydryl-reactive oxidative agent diamide suppressed LTC(4)S activity and induced a reversible formation of covalent dimer LTC(4)S. LTC(4)S bearing a C56S mutation was resistant to the effect of diamide. Covalent dimer LTC(4)S was observed in nasal polyp biopsies, indicating that dimerization and inactivation of LTC(4)S can occur at the site of inflammation. These results suggest a cellular redox regulation of LTC(4)S function through a post-translational mechanism.

The catalytic architecture of leukotriene C4 synthase with two arginine residues.

Leukotriene (LT) C(4) and its metabolites, LTD(4) and LTE(4), are involved in the pathobiology of bronchial asthma. LTC(4) synthase is the nuclear membrane-embedded enzyme responsible for LTC(4) biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA(4) 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 LTA(4) 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 LTA(4).

Neutrophil-derived leukotriene B4 is required for inflammatory arthritis.

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.

GPR17 regulates immune pulmonary inflammation induced by house dust mites.

Antagonists of the type 1 cysteinyl leukotriene receptor (CysLT(1)R) are efficacious for bronchoconstriction in humans with bronchial asthma; however, the clinical response to these drugs is heterogeneous. In particular, how CysLT(1)R expression and function are constitutively regulated in vivo is not known. In this study, we show that a seven-transmembrane receptor, GPR17, negatively regulates the CysLT(1)R-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 the house dust mite (extract of Dermatophagoides farinae [Df]) in mice. Sensitization of naive wild-type recipients with Df-pulsed bone marrow-derived dendritic cells of each genotype or sensitization of each genotype with Df-pulsed wild-type bone marrow-derived dendritic cells and Df challenge revealed markedly increased pulmonary inflammatory and serum IgE responses for GPR17-deficient mice as compared with wild-type mice and reduced responses in the genotypes lacking CysLT(1)R. These findings reveal a constitutive negative regulation of CysLT(1)R functions by GPR17 in both the Ag presentation and downstream phases of allergic pulmonary inflammation.

GPR17 is a negative regulator of the cysteinyl leukotriene 1 receptor response to leukotriene D4.

The cysteinyl leukotrienes (cys-LTs) are proinflammatory lipid mediators acting on the type 1 cys-LT receptor (CysLT(1)R) to mediate smooth muscle constriction and vascular permeability. GPR17, a G protein-coupled orphan receptor with homology to the P2Y and cys-LT receptors, failed to mediate calcium flux in response to leukotriene (LT) D(4) with stable transfectants. However, in stable cotransfections of 6xHis-tagged GPR17 with Myc-tagged CysLT(1)R, the robust CysLT(1)R-mediated calcium response to LTD(4) was abolished. The membrane expression of the CysLT(1)R analyzed by FACS with anti-Myc Ab was not reduced by the cotransfection, yet both LTD(4)-elicited ERK phosphorylation and the specific binding of [(3)H]LTD(4) to microsomal membranes were fully inhibited. CysLT(1)R and GPR17 expressed in transfected cells were coimmunoprecipitated and identified by Western blots, and confocal immunofluorescence microscopy revealed that GPR17 and CysLT(1)R colocalize on the cell surface of human peripheral blood monocytes. Lentiviral knockdown of GPR17 in mouse bone marrow-derived macrophages (BMMPhis) increased both the membrane expression of CysLT(1)R protein by FACS analysis and the LTD(4)-elicited calcium flux in a dose-dependent manner as compared with control BMMPhis, indicating a negative regulatory function of GPR17 for CysLT(1)R in a primary cell. In IgE-dependent passive cutaneous anaphylaxis, GPR17-deficient mice showed a marked and significant increase in vascular permeability as compared with WT littermates, and this vascular leak was significantly blocked by pretreatment of the mice with the CysLT(1)R antagonist, MK-571. Taken together, our findings suggest that GPR17 is a ligand-independent, constitutive negative regulator for the CysLT(1)R that suppresses CysLT(1)R-mediated function at the cell membrane.

Cysteinyl leukotrienes impair hypoxic pulmonary vasoconstriction in endotoxemic mice.

BACKGROUND: Sepsis impairs hypoxic pulmonary vasoconstriction (HPV) in patients and animal models, contributing to systemic hypoxemia. Concentrations of cysteinyl leukotrienes are increased in the bronchoalveolar lavage fluid of patients with sepsis, but the contribution of cysteinyl leukotrienes to the impairment of HPV is unknown. METHODS: Wild-type mice, mice deficient in leukotriene C(4) synthase, the enzyme responsible for cysteinyl leukotriene synthesis, and mice deficient in cysteinyl leukotriene receptor 1 were studied 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. Concentrations of cysteinyl leukotrienes were determined in the bronchoalveolar lavage fluid. RESULTS: In the bronchoalveolar lavage fluid of all three strains, cysteinyl leukotrienes were not detectable after saline challenge; whereas endotoxin challenge increased cysteinyl leukotriene concentrations in wild-type mice and mice deficient in cysteinyl leukotriene receptor 1, but not in mice deficient in leukotriene C(4) synthase. HPV did not differ among 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 C(4) synthase (96 ± 16%, P < 0.05 vs. wild-type mice), thereby preserving systemic oxygenation. Although 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. CONCLUSION: Cysteinyl leukotrienes importantly contribute to endotoxin-induced impairment of HPV in part via a cysteinyl leukotriene receptor 1-dependent mechanism.

P2Y6 receptors require an intact cysteinyl leukotriene synthetic and signaling system to induce survival and activation of mast cells.

Cysteinyl leukotrienes (cys-LTs) induce inflammatory responses through type 1 (CysLT1R) and type 2 (CysLT2R) cys-LT receptors and activate mast cells in vitro. We previously demonstrated that cys-LTs cross-desensitized IL-4-primed primary human mast cells (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. Leukotriene (LT) D4, the most potent CysLT1R ligand, and UDP both induced phosphorylation of 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, implying a requirement for a cys-LT-mediated autocrine loop. In IL-4-primed LAD2 cells, LTD4 induced the generation of MIP-1beta, a response blocked by short hairpin RNA-mediated knockdown of CysLT1R or P2Y6 receptors, but not of CysLT2R. Thus, CysLT1R and P2Y6 receptors, which are coexpressed on many cell types of innate immunity, reciprocally amplify one another's function in mast cells through endogenous ligands.