Isoflurane attenuates sepsis-associated lung injury.

Acute lung injury is one of major complications associated with sepsis, responsible for morbidity and mortality. Patients who suffer from acute lung injury often require respiratory support under sedations, and it would be important to know the role of sedatives in lung injury. We examined volatile anesthetic isoflurane, which is commonly used in surgical setting, but also used as an alternative sedative in intensive care settings in European countries and Canada. We found that isoflurane exposure attenuated neutrophil recruitment to the lungs in mice suffering from experimental polymicrobial abdominal sepsis. We found that isoflurane attenuated one of major neutrophil chemoattractants LTB4 mediated response via its receptor BLT1 in neutrophils. Furthermore, we have shown that isoflurane directly bound to BLT1 by a competition assay using newly developed labeled BLT1 antagonist, suggesting that isoflurane would be a BLT1 antagonist.

Development and Characterization of a Fluorescent Ligand for Leukotriene B4 Receptor 2 in Cells and Tissues.

The leukotriene B4 receptor 2 (BLT2) is a G-protein coupled receptor activated by 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), which has been proposed as a promising therapeutic target for diabetic wound healing and gastrointestinal lesions. In this study, the rational design of a fluorescent probe based on the synthetic BLT2 agonist CAY10583 is described. The synthesis of several derivatives of CAY10583 coupled to fluorescein resulted in a traceable ligand suitable for different fluorescence-based techniques. An HTRF-based displacement assay (Tag-lite) on stably transfected CHO-K1 cells was developed to characterize binding properties of diverse BLT2 ligands. Highly specific binding to the BLT2 receptor was demonstrated in staining experiments on mouse skin tissue, and specific modulation of BLT2-induced cAMP signaling provided further evidence for receptor binding and ligand functionality. In conclusion, the fluorescent ligands developed in this study are suitable to investigate the pharmacology of BLT2 receptor ligands in a variety of assay systems.

CAFs shape myeloid-derived suppressor cells to promote stemness of intrahepatic cholangiocarcinoma through 5-lipoxygenase.

BACKGROUND AND AIMS: We previously demonstrated that cancer-associated fibroblasts (CAFs) promote tumor growth through recruitment of myeloid-derived suppressor cells (MDSCs). 5-lipoxygenase (5-LO) is highly expressed in myeloid cells and is critical for synthesizing leukotriene B4 (LTB4), which is involved in tumor progression by activating its receptor leukotriene B4 receptor type 2 (BLT2). In this study, we investigated whether and how CAFs regulate MDSC function to enhance cancer stemness, the driving force of the cancer aggressiveness and chemotherapy refractoriness, in highly desmoplastic intrahepatic cholangiocarcinoma (ICC). APPROACH AND RESULTS: RNA-sequencing analysis revealed enriched metabolic pathways but decreased inflammatory pathways in cancer MDSCs compared with blood MDSCs from patients with ICC. Co-injection of ICC patient-derived CAFs promoted cancer stemness in an orthotopic ICC model, which was blunted by MDSC depletion. Conditioned media (CM) from CAF-educated MDSCs drastically promoted tumorsphere formation efficiency and stemness marker gene expression in ICC cells. CAF-CM stimulation increased expression and activity of 5-LO in MDSCs, while 5-LO inhibitor impaired the stemness-enhancing capacity of MDSCs in vitro and in vivo. Furthermore, IL-6 and IL-33 primarily expressed by CAFs mediated hyperactivated 5-LO metabolism in MDSCs. We identified the LTB4-BLT2 axis as the critical downstream metabolite signaling of 5-LO in promoting cancer stemness, as treatment with LTB4 was elevated in CAF-educated MDSCs, or blockade of BLT2 (which was preferentially expressed in stem-like ICC cells) significantly reduced stemness-enhancing effects of CAF-educated MDSCs. Finally, BLT2 blockade augmented chemotherapeutic efficacy in ICC patient-derived xenograft models. CONCLUSIONS: Our study reveals a role for CAFs in orchestrating the optimal cancer stemness-enhancing microenvironment by educating MDSCs, and suggests the 5-LO/LTB4-BLT2 axis as promising therapeutic targets for ICC chemoresistance by targeting cancer stemness.

Macrophage MST1/2 Disruption Impairs Post-Infarction Cardiac Repair via LTB4.

[Figure: see text].

Structural insights on ligand recognition at the human leukotriene B4 receptor 1.

The leukotriene B4 receptor 1 (BLT1) regulates the recruitment and chemotaxis of different cell types and plays a role in the pathophysiology of infectious, allergic, metabolic, and tumorigenic human diseases. Here we present a crystal structure of human BLT1 (hBLT1) in complex with a selective antagonist MK-D-046, developed for the treatment of type 2 diabetes and other inflammatory conditions. Comprehensive analysis of the structure and structure-activity relationship data, reinforced by site-directed mutagenesis and docking studies, reveals molecular determinants of ligand binding and selectivity toward different BLT receptor subtypes and across species. The structure helps to identify a putative membrane-buried ligand access channel as well as potential receptor binding modes of endogenous agonists. These structural insights of hBLT1 enrich our understanding of its ligand recognition and open up future avenues in structure-based drug design.

Up-regulating microRNA-138-5p enhances the protective role of dexmedetomidine on myocardial ischemia-reperfusion injury mice via down-regulating Ltb4r1.

Both microRNAs (miRs) and dexmedetomidine (Dex) have been verified to exert functional roles in myocardial ischemia-reperfusion injury (MI/RI). Given that, we concretely aim to discuss the effects of Dex and miR-138-5p on ventricular remodeling in mice affected by MI/RI via mediating leukotriene B4 receptor 1 (Ltb4r1). MI/RI mouse model was established by ligating left anterior descending coronary artery. The cardiac function, inflammatory factors and collagen fiber contents were detected after Dex/miR-138-5p/Ltb4r1 treatment. MiR-138-5p and Ltb4r1 expression in myocardial tissues were tested by RT-qPCR and western blot assay. The target relationship between miR-138-5p and Ltb4r1 was verified by online software prediction and luciferase activity assay. MiR-138-5p was down-regulated while Ltb4r1 was up-regulated in myocardial tissues of MI/RI mice. Dex improved cardiac function, alleviated myocardial damage, reduced inflammatory factor contents, collagen fibers, and Ltb4r1 expression while increased miR-138-5p expression in myocardial tissues of mice with MI/RI. Restored miR-138-5p and depleted Ltb4r1 improved cardiac function, abated inflammatory factor contents, myocardial damage, and content of collagen fibers in MI/RI mice. MiR-138-5p directly targeted Ltb4r1. The work evidence that Dex could ameliorate ventricular remodeling of MI/RI mice by up-regulating miR-138-3p and down-regulating Ltb4r1. Thus, Dex and miR-138-3p/Ltb4r1 may serve as potential targets for the ventricular remodeling of MI/RI.

Leukotriene B4 and Its Receptor in Experimental Autoimmune Uveitis and in Human Retinal Tissues: Clinical Severity and LTB4 Dependence of Retinal Th17 Cells.

Nomacopan, a drug originally derived from tick saliva, has dual functions of sequestering leukotriene B4 (LTB4) and inhibiting complement component 5 (C5) activation. Nomacopan has been shown to provide therapeutic benefit in experimental autoimmune uveitis (EAU). Longer acting forms of nomacopan were more efficacious in mouse EAU models, and the long-acting variant that inhibited only LTB4 was at least as effective as the long-acting variant that inhibited both C5 and LTB4, preventing structural damage to the retina and a significantly reducing effector T helper 17 cells and inflammatory macrophages. Increased levels of LTB4 and C5a (produced upon C5 activation) were detected during disease progression. Activated retinal lymphocytes were shown to express LTB4 receptors (R) in vitro and in inflamed draining lymph nodes. Levels of LTB4R-expressing active/inflammatory retinal macrophages were also increased. Within the draining lymph node CD4+ T-cell population, 30% expressed LTB4R+ following activation in vitro, whereas retinal infiltrating cells expressed LTB4R and C5aR. Validation of expression of those receptors in human uveitis and healthy tissues suggests that infiltrating cells could be targeted by inhibitors of the LTB4-LTB4 receptor 1 (BLT1) pathway as a novel therapeutic approach. This study provides novel data on intraocular LTB4 and C5a in EAU, their associated receptor expression by retinal infiltrating cells in mouse and human tissues, and in attenuating EAU via the dual inhibitor nomacopan.

[A Research on Drug Discovery for Intracerebral Hemorrhage Focusing on Leukotriene B4 and Its Receptor].

Intracerebral hemorrhage (ICH) results from blood vessels rupture in the brain, forming a blood clot in the brain parenchyma. Leakage of blood constituents causes detrimental tissue damages, ensuing long-lasting neurological deficits; however, effective therapeutic approaches are not yet developed to date. In this study, leukotriene B4 (LTB4) and its receptor leukotriene B4 receptor 1 (BLT1) are proposed as novel therapeutic targets for ICH therapy. After the onset of ICH, the LTB4 content in the brain transiently elevated. Microglia are considered as the source of LTB4 production. Thrombin, a blood constituent, activated the BV-2 microglia and increased the LTB4 secretion from the BV-2 cells. Microglia-released LTB4 promoted its own microglial activation and neutrophil-like differentiated HL-60 cell migration activity. LTB4 receptors comprised of two types: BLT1 and BLT2, with BLT1 known to be a high-affinity receptor associated with chemotaxis. BLT1 knockout mice showed decreased neutrophil invasion, attenuating sensorimotor dysfunction after ICH. Furthermore, therapeutic administration of ONO-4057, an orally active LTB4 receptor antagonist, attenuated neutrophil invasion, microglial activation, axonal fragmentation, and sensorimotor deficits induced by ICH. These results suggest that LTB4 and its receptor BLT1 can be potential promising therapeutic targets that prevent tissue damages following ICH.

Leukotriene B4 Receptor Type 2 Accelerates the Healing of Intestinal Lesions by Promoting Epithelial Cell Proliferation.

Leukotriene B4 receptor type 2 (BLT2) is a low-affinity leukotriene B4 receptor that is highly expressed in intestinal epithelial cells. Previous studies demonstrated the protective role of BLT2 in experimentally induced colitis. However, its role in intestinal lesion repair is not fully understood. We investigated the role of BLT2 in the healing of indomethacin-induced intestinal lesions in mice. There was no significant different between wild-type (WT) and BLT2-deficient (BLT2KO) mice in terms of the development of indomethacin-induced intestinal lesions. However, healing of these lesions was significantly impaired in BLT2KO mice compared with WT mice. In contrast, transgenic mice with intestinal epithelium-specific BLT2 overexpression presented with superior ileal lesion healing relative to WT mice. An immunohistochemical study showed that the number of Ki-67-proliferative cells was markedly increased during the healing of intestinal lesions in WT mice but significantly attenuated in BLT2KO mice. Exposure of cultured mouse intestinal epithelial cells to CAY10583, a BLT2 agonist, promoted wound healing and cell proliferation in a concentration-dependent manner. Nevertheless, these responses were abolished under serum-free conditions. The CAY10583-induced proliferative effect was also negated by Go6983, a protein kinase C (PKC) inhibitor, U-73122, a phospholipase C (PLC) inhibitor, LY255283, a BLT2 antagonist, and pertussis toxin that inhibits G protein-coupled receptor signaling via Gi/o proteins. Thus, BLT2 plays an important role in intestinal wound repair. Moreover, this effect is mediated by the promotion of epithelial cell proliferation via the Gi/o protein-dependent and PLC/PKC signaling pathways. The BLT2 agonists are potential therapeutic agents for the treatment of intestinal lesions. SIGNIFICANCE STATEMENT: The healing of indomethacin-induced Crohn's disease-like intestinal lesions was impaired in mice deficient in low-affinity leukotriene B4 receptor type 2 (BLT2). They presented with reduced epithelial cell proliferation during the healing. In contrast, healing was promoted in mice overexpressing intestinal epithelial BLT2. In cultured intestinal epithelial cells, the BLT2 agonist CAY10583 substantially accelerated wound repair by enhancing cell proliferation rather than migration. Thus, BLT2 plays an important role in the intestinal lesions via acceleration of epithelial cell proliferation.

Therapeutic potential of BLT1 antagonist for COPD: involvement of inducing autophagy and ameliorating inflammation.

PURPOSE: Leukotriene B4 (LTB4) is a major pro-inflammatory mediator that leads to the persistence of chronic inflammation in chronic obstructive pulmonary disease (COPD). The purpose of this study was to evaluate therapeutic potential of BLT1 antagonist for cigarette smoke (CS)-induced COPD and to explore the underlying mechanism. MATERIALS AND METHODS: In vitro, autophagy proteins were determined by Western blotting in RAW264.7 macrophages treated with U75302 (BLT1 antagonist) or autophagy inhibitor in cigarette smoke extract-induced inflammation. In vivo, C57BL/6J mice were randomly divided into three groups: Control group, CS group and CS+U75302 group. After 12-week exposure, histological analysis and lung function tests were performed to evaluate the inflammatory infiltration and emphysema. The expression of inflammatory cytokines was measured by real-time PCR and enzyme-linked immunosorbent assay. Immunohistochemical analysis and Western blotting detected the expression of autophagy-related proteins. Transmission electron microscopy (TEM) showed the alterations of autophagosomes and lysosomes. RESULTS: Lower levels of inflammatory factors and autophagy markers were detected in U75302-treated cells and mice after CS exposure than control. In vitro, LC3 mRNA expression was elevated when treated with U75302. Autophagy inhibition resulted in augmented inflammatory response and autophagy proteins even with U75302 treatment. Furthermore, BLT1 antagonist decreased the number of lysosomes and autophagosomes in alveolar macrophages of mice and potentially enhanced the expression of transcriptional activation of transcription factor-EB (TFEB) in vitro and vivo. CONCLUSION: Insufficient autophagy of macrophages was associated with LTB4-mediated inflammation in CS-exposure models. BLT1 antagonist ameliorated inflammatory response through inducing autophagy.

20-Hydroxy- and 20-carboxy-leukotriene (LT) B4 downregulate LTB4 -mediated responses of human neutrophils and eosinophils.

Leukotriene B4 (LTB4 ) plays a prominent role in innate immunity as it induces phagocyte recruitment, the release of antimicrobial effectors, and as it potentiates the ingestion and killing of pathogens. In humans, LTB4 has a short half-life and is rapidly metabolized by leukocytes, notably into 20-OH- and 20-COOH-LTB4 by neutrophils. Although these LTB4 metabolites bind to the BLT1 receptor with high affinity, they activate neutrophils to a much lower extent than LTB4 . We thus postulated that LTB4 metabolites could dampen BLT1 -mediated responses, therefore limiting the impact of LTB4 on human neutrophil functions. We found that 20-OH-LTB4 and 20-COOH-LTB4 inhibited all of the LTB4 -mediated neutrophil responses we tested (migration, degranulation, leukotriene biosynthesis). The potencies of the different compounds at inhibiting LTB4 -mediated responses were 20-OH-LTB4  = CP 105,696 (BLT1 antagonist) > > 20-COOH-LTB4 ≥ resolvin E1 (RVE1 ). In contrast, the fMLP- and IL-8-mediated responses we tested were not affected by the LTB4 metabolites or RVE1 . 20-OH-LTB4 and 20-COOH-LTB4 also inhibited the LTB4 -mediated migration of human eosinophils but not that induced by 5-KETE. Moreover, using 20-COOH-LTB4 , LTB4 , and LTB4 -alkyne, we show that LTB4 is a chemotactic, rather than a chemokinetic factor for both human neutrophils and eosinophils. In conclusion, our data indicate that LTB4 metabolites and RVE1 act as natural inhibitors of LTB4 -mediated responses. Thus, preventing LTB4 ω-oxidation might result in increased innate immunity and granulocyte functions.

Mediatory role of BLT2 in the proliferation of KRAS mutant colorectal cancer cells.

Inflammatory lipid mediators play various roles in colorectal cancer progression through complex pathways. However, the mechanism by which lipoxygenase-derived inflammatory lipid mediators contribute to colorectal cancer progression remains elusive. In this study, we found that BLT2, a cell surface GPCR for leukotriene B4 and 12­hydroxyeicosatetraenoic acid, is highly upregulated in KRAS mutant LOVO and SW480 colorectal cancer cells and plays critical roles in mediating proliferation through activation of phosphatidylinositol 3­kinase (PI3K)/protein kinase B (Akt) and subsequent upregulation of cyclin D1. Exposure to BLT2 siRNA or LY255283, a specific BLT2 inhibitor, clearly suppressed the proliferation of KRAS mutant colorectal cancer cells and markedly increased cell cycle arrest by downregulating the PI3K/Akt-cyclin D1 cascade. Xenograft tumor formation by LOVO and SW480 cells in athymic mice was also substantially reduced by treatment with the BLT2 inhibitor in vivo. Together, our study demonstrates that BLT2 is necessary for the proliferation of LOVO and SW480 cells and thus may be a potential therapeutic target for the treatment of KRAS mutant colorectal cancer.

Comparative Modeling and Evaluation of Leukotriene B4 Receptors for Selective Drug Discovery Towards the Treatment of Inflammatory Diseases.

Leukotriene B4 (LTB4) exerts its biological effects through stimulation of specific G protein-coupled receptors (GPCRs)-namely BLT1 and BLT2. Due to the absence of human BLT1 and BLT2 crystal structures, the current study was set to predict the 3D structures of these two receptors for structure-based anti-inflammatory drug discovery. Homology modeling of the BLT1 receptor was first constructed, based on various X-ray and NMR GPCR templates, followed by molecular dynamics (MD) refinement. Using a single-template approach, nine well-established alignment methods and ten secondary structure prediction methods during the backbone generation were implemented and assessed. The binding sites of the BLT1 receptor were then mapped using fifteen chemical probes with the help of FTMAP and AutoDock Vina 4.2 software. Model validation was performed through the docking of eight specific antagonists that have experimental inhibition constants (ki) towards BLT1. The antagonists-BLT1 docked structures were then subjected to AMBER-based molecular mechanical minimization and the corresponding binding energies were calculated using molecular mechanics-generalized Born surface area (MM/GBSA) approach. According to the results, the most energetically stable models were constructed using SAlign method for the alignment process and PSIPRED for secondary structure prediction. In comparison, the refined BLT1 model built on 2KS9 as an NMR template has the lowest DOPE energy compared to those built on 4EA3 and 4XT1 as X-ray templates. According to the mapping results, two main binding sites were identified: one was among TMs II, III and VII and the other was among TMs III, IV and V. For the antagonists, correlation between binding energies and experimental data was in a good agreement, with a correlation coefficient (R2 value) of 0.91. Due to the great amino acid sequence similarity between BLT1 and BLT2 receptors (calculated as 45.2%), BLT2 model was constructed based on the predicted BLT1 model.

BLTR1 in Monocytes Emerges as a Therapeutic Target For Vascular Inflammation With a Subsequent Intimal Hyperplasia in a Murine Wire-Injured Femoral Artery.

Given the importance of high-mobility group box 1 (HMGB1) and 5-lipoxygenase (5-LO) signaling in vascular inflammation, we investigated the role of leukotriene signaling in monocytes on monocyte-to-macrophage differentiation (MMD) induced by HMGB1, and on vascular inflammation and subsequent intimal hyperplasia in a mouse model of wire-injured femoral artery. In cultured primary bone marrow-derived cells (BMDCs) stimulated with HMGB1, the number of cells with macrophage-like morphology was markedly increased in association with an increased expression of CD11b/Mac-1, which were attenuated in cells pre-treated with Zileuton, a 5-LO inhibitor as well as in 5-LO-deficient BMDCs. Of various leukotriene receptor inhibitors examined, which included leukotriene B4 receptors (BLTRs) and cysteinyl leukotriene receptors (cysLTRs), the BLTR1 inhibitor (U75302) exclusively suppressed MMD induction by HMGB1. The importance of BLTR1 in HMGB1-induced MMD was also observed in BMDCs isolated from BLTR1-deficient mice and BMDCs transfected with BLTR1 siRNA. Although leukotriene B4 (LTB4) had minimal direct effects on MMD in control and 5-LO-deficient BMDCs, MMD attenuation by HMGB1 in 5-LO-deficient BMDCs was significantly reversed by exogenous LTB4, but not in BLTR1-deficient BMDCs, suggesting that LTB4/BLTR1-mediated priming of monocytes is a prerequisite of HMGB1-induced MMD. In vivo, both macrophage infiltration and intimal hyperplasia in our wire-injured femoral artery were markedly attenuated in BLTR1-deficient mice as compared with wild-type controls, but these effects were reversed in BLTR1-deficient mice transplanted with monocytes from control mice. These results suggest that BLTR1 in monocytes is a pivotal player in MMD with subsequent macrophage infiltration into neointima, leading to vascular remodeling after vascular injury.

BLT1-mediated O-GlcNAcylation is required for NOX2-dependent migration, exocytotic degranulation and IL-8 release of human mast cell induced by Trichomonas vaginalis-secreted LTB4.

Trichomonas vaginalis is a sexually-transmitted protozoan parasite that causes vaginitis and cervicitis. Although mast cell activation is important for provoking tissue inflammation during infection with parasites, information regarding the signaling mechanisms in mast cell activation and T. vaginalis infection is limited. O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of serine and threonine residues that functions as a critical regulator of intracellular signaling, regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). We investigated if O-GlcNAcylation was associated with mast cell activation induced by T. vaginalis-derived secretory products (TvSP). Modified TvSP collected from live trichomonads treated with the 5-lipooxygenase inhibitor AA861 inhibited migration of mast cells. This result suggested that mast cell migration was caused by stimulation of T. vaginalis-secreted leukotrienes. Using the BLT1 antagonist U75302 or BLT1 siRNA, we found that migration of mast cells was evoked via LTB4 receptor (BLT1). Furthermore, TvSP induced protein O-GlcNAcylation and OGT expression in HMC-1 cells, which was prevented by transfection with BLT1 siRNA. TvSP-induced migration, ROS generation, CD63 expression and IL-8 release were significantly suppressed by pretreatment with OGT inhibitor ST045849 or OGT siRNA. These results suggested that BLT1-mediated OGlcNAcylation was important for mast cell activation during trichomoniasis.

Leukotriene receptors as potential therapeutic targets.

Leukotrienes, a class of arachidonic acid-derived bioactive molecules, are known as mediators of allergic and inflammatory reactions and considered to be important drug targets. Although an inhibitor of leukotriene biosynthesis and antagonists of the cysteinyl leukotriene receptor are clinically used for bronchial asthma and allergic rhinitis, these medications were developed before the molecular identification of leukotriene receptors. Numerous studies using cloned leukotriene receptors and genetically engineered mice have unveiled new pathophysiological roles for leukotrienes. This Review covers the recent findings on leukotriene receptors to revisit them as new drug targets.

Substance P-regulated leukotriene B4 production promotes acute pancreatitis-associated lung injury through neutrophil reverse migration.

Leukotriene B4 (LTB4) is a potent chemoattractant and inflammatory mediator involved in multiple inflammatory diseases. Substance P (SP) has been reported to promote production of LTB4 in itch-associated response in vivo and in some immune cells in vitro. Here, we investigated the role of LTB4 in acute pancreatitis (AP), AP-associated acute lung injury (ALI) and the related mechanisms of LTB4 production in AP. In vivo, murine AP model was induced by caerulein and lipopolysaccharide or L-arginine. The levels of LTB4 and its specific receptor BLT1 were markedly upregulated in both AP models. Blockade of BLT1 by LY293111 attenuated the severity of AP, decreased neutrophil reverse transendothelial cell migration (rTEM) into the circulation and alleviated the severity of ALI. In vitro, treatment of pancreatic acinar cells with SP increased LTB4 production. Furthermore, SP treatment increased phosphorylation of protein kinase C (PKC) α and mitogen activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), p-38 MAPK and c-Jun NH2-terminal kinase (JNK). Finally, blockade of neurokinin-1 receptor by CP96345 significantly attenuated the severity of AP and decreased the level of LTB4 when compared to AP group. In summary, these results show that SP regulates the production of LTB4 via PKCα/MAPK pathway, which further promotes AP-associated ALI through neutrophil rTEM.

Novel mechanism of regulation of the 5-lipoxygenase/leukotriene B4 pathway by high-density lipoprotein in macrophages.

High-density lipoprotein (HDL) interacts with various cells, particularly macrophages, in functional cell-HDL interactions. Here, we found that HDL protein quality and lipid quality play critical roles in HDL functions. HDL fractions from healthy volunteers (HDLHealthy) and patients with recurrent coronary atherosclerotic disease (HDLCAD) were prepared. To analyse functional HDL-macrophage interactions, macrophages were co-incubated with each HDL, and lipid mediator production was assessed by liquid chromatography/mass spectrometry-based metabololipidomics. HDLHealthy treatment attenuated the pro-inflammatory lipid mediator production, particularly that of leukotriene (LT) B4, and this treatment enhanced lipoxin (LX) B4 and resolvin (Rv) E2 production. HDLHealthy treatment enhanced the proteasome-mediated degradation of the LTB4-producing enzyme 5-lipoxygenase (LO) in activated macrophages; however, HDLCAD did not show these anti-inflammatory effects. HDLHealthy was engulfed by macrophages via clathrin-mediated endocytosis, which was a critical step in 5-LO/LTB4 regulation. We also found that HDLCAD showed higher levels of the LTB4-producing enzymes and thus promoted LTB4 production from HDLCAD. In addition, LTB4 attenuated HDL endocytosis, HDL-mediated 5-LO degradation in macrophages, and HDL-derived augmentation of macrophage phagocytosis. These results indicated that local LTB4 produced de novo from HDLCAD regulates HDL-macrophage functional interactions and plays critical roles in dysfunctional, inflammatory HDL characteristics.

Biochemical and immunological characterization of a novel monoclonal antibody against mouse leukotriene B4 receptor 1.

Leukotriene B4 (LTB4) receptor 1 (BLT1) is a G protein-coupled receptor expressed in various leukocyte subsets; however, the precise expression of mouse BLT1 (mBLT1) has not been reported because a mBLT1 monoclonal antibody (mAb) has not been available. In this study, we present the successful establishment of a hybridoma cell line (clone 7A8) that produces a high-affinity mAb for mBLT1 by direct immunization of BLT1-deficient mice with mBLT1-overexpressing cells. The specificity of clone 7A8 was confirmed using mBLT1-overexpressing cells and mouse peripheral blood leukocytes that endogenously express BLT1. Clone 7A8 did not cross-react with human BLT1 or other G protein-coupled receptors, including human chemokine (C-X-C motif) receptor 4. The 7A8 mAb binds to the second extracellular loop of mBLT1 and did not affect LTB4 binding or intracellular calcium mobilization by LTB4. The 7A8 mAb positively stained Gr-1-positive granulocytes, CD11b-positive granulocytes/monocytes, F4/80-positive monocytes, CCR2-high and CCR2-low monocyte subsets in the peripheral blood and a CD4-positive T cell subset, Th1 cells differentiated in vitro from naïve CD4-positive T cells. This mAb was able to detect Gr-1-positive granulocytes and monocytes in the spleens of naïve mice by immunohistochemistry. Finally, intraperitoneal administration of 7A8 mAb depleted granulocytes and monocytes in the peripheral blood. We have therefore succeeded in generating a high-affinity anti-mBLT1 mAb that is useful for analyzing mBLT1 expression in vitro and in vivo.

Involvement of Leukotriene B4 Released from Keratinocytes in Itch-associated Response to Intradermal Interleukin-31 in Mice.

A recent study suggests that interleukin-31 (IL-31) exerts its effect via indirect mechanisms rather than through direct stimulation of cutaneous nerves. However, the underlying peripheral mechanisms of IL-31-induced itch in the skin remain unclear. Therefore, the present study investigated the peripheral mechanisms underlying IL-31-induced itch in mice. IL-31-induced itch-related response was inhibited by anti-allergic drugs (tranilast and azelastine), but not by an H1 histamine receptor antagonist (terfenadine). Furthermore, a 5-lipoxygenase inhibitor (zileuton), but not a cyclooxygenase inhibitor (indomethacin), and a leuko-triene B4 (LTB4) receptor antagonist (CMHVA) attenuated the action of IL-31. IL-31 receptor-immunoreactivity was observed in the epidermis and primary sensory neurones. IL-31 receptor mRNA was expressed in mouse keratinocytes and dorsal root ganglia neurones. IL-31 increased the production of LTB4 in mouse keratinocytes. These results suggest that IL-31 elicits itch not only through direct action on primary sensory neurones, but also by inducing LTB4 production in keratinocytes.