Leukotriene B4-leukotriene B4 receptor axis promotes oxazolone-induced contact dermatitis by directing skin homing of neutrophils and CD8⁺ T cells.

Leukotriene B4 (LTB4 ) is a lipid mediator that is rapidly generated in inflammatory sites, and its functional receptor, BLT1, is mostly expressed on immune cells. Contact dermatitis is a common inflammatory skin disease characterized by skin oedema and abundant inflammatory infiltrates, primarily including neutrophils and CD8(+) T cells. The role of the LTB4 -BLT1 axis in contact dermatitis remains largely unknown. In this study, we found up-regulated gene expression of 5-lipoxygenase and leukotriene A4 hydrolase, two critical enzymes for LTB4 synthesis, BLT1 and elevated LTB4 levels in skin lesions of oxazolone (OXA)-induced contact dermatitis. BLT1 deficiency or blockade of LTB4 and BLT1 by the antagonists, bestatin and U-75302, respectively, in the elicitation phase caused significant decreases in ear swelling and skin-infiltrating neutrophils and CD8(+) T cells, which was accompanied by significantly reduced skin expression of CXCL1, CXCL2, interferon-γ and interleukin-1ß. Furthermore, neutrophil depletion during the elicitation phase of OXA-induced contact dermatitis also caused significant decreases in ear swelling and CD8(+) T-cell infiltration accompanied by significantly decreased LTB4 synthesis and gene expression of CXCL2, interferon-γ and interleukin-1ß. Importantly, subcutaneous injection of exogenous LTB4 restored the skin infiltration of CD8(+) T cells in neutrophil-depleted mice following OXA challenge. Collectively, our results demonstrate that the LTB4 -BLT1 axis contributes to OXA-induced contact dermatitis by mediating skin recruitment of neutrophils, which are a major source of LTB4 that sequentially direct CD8(+) T-cell homing to OXA-challenged skin. Hence, LTB4 and BLT1 could be potential therapeutic targets for the treatment of contact dermatitis.

Immunomodulatory oligonucleotides inhibit neutrophil migration by decreasing the surface expression of interleukin-8 and leukotriene B4 receptors.

Neutrophils play important roles in many inflammatory diseases. The migration of neutrophils to the inflammatory site is tightly regulated by specific chemokines, of which interleukin-8 (IL-8) and leukotriene B4 (LTB4 ) constitute key mediators by binding to the surface receptors CXCR1/2 and BLT1, respectively. Oligonucleotides (ODN) containing CpG motifs mediate potent immunomodulatory effects through binding to Toll-like receptor 9. So far, knowledge on how ODN can affect neutrophil migration during inflammation is lacking. This study demonstrates that several novel CpG ODN significantly down-regulate the surface expression of CXCR1/2 and BLT1. In addition, the ODN significantly blocked IL-8-induced and LTB4 -induced neutrophil migration in vitro, as well as leucocyte migration in vivo demonstrated in mice by intravital microscopy and in a model of airway inflammation. The down-regulation of CXCR1 is rapid, occurring 15 min after ODN stimulation, and can be mediated through an endosomally independent mechanism. Inhibition of the IL-8 and LTB4 pathways may provide new opportunities of therapeutic intervention using ODN to reduce neutrophil infiltration during inflammation.

Expression of leukotriene B4 receptor-1 on CD8⁺ T cells is required for their migration into tumors to elicit effective antitumor immunity.

Leukotriene B4 (LTB4) receptor (BLT)1 is expressed on variety of immune cells and has been implicated as a mediator of diverse inflammatory diseases. However, whether biological responses initiated via this receptor generate tumor-promoting inflammation or antitumor immunity remains unexplored. In this study, we investigated the role of BLT1 in antitumor immunity using syngeneic TC-1 cervical cancer model, and observed accelerated tumor growth and reduced survival in BLT1⁻/⁻ mice compared with BLT1⁺/⁺ mice. Analysis of the tumor infiltrates by flow cytometry and confocal microscopy revealed a significant decrease in effector immune cells, most notably, CD8⁺ T cells and NK cells in the tumors of the BLT1⁻/⁻ mice. Gene expression profiling confirmed the dramatic decrease of IFN-γ, granzyme B, and IL-2 in tumors growing in BLT1⁻/⁻ mice. Furthermore, depletion of CD8⁺ T cells enhanced the tumor growth in BLT1⁺/⁺ but not in BLT1⁻/⁻ mice. However, similar levels of Ag-dependent CD8⁺ T cell-mediated killing activity were observed in spleens of BLT1⁺/⁺ and BLT1⁻/⁻ mice. Adoptive transfer of CD8⁺ T cells from tumor-bearing BLT1⁺/⁺ but not BLT1⁻/⁻ mice significantly reduced tumor growth and increased the survival of Rag2⁻/⁻ mice. Although the homeostatic proliferation and expression profiles of other chemokine receptors of adoptively transferred BLT1⁺/⁺ and BLT1⁻/⁻ CD8⁺ T cells appears to be similar, BLT1⁺/⁺ T lymphocytes entered the tumors in greater numbers. These results suggest that BLT1 expression on CD8⁺ T cells plays an important role in their trafficking to tumors.

Nonredundant roles for leukotriene B4 receptors BLT1 and BLT2 in inflammatory arthritis.

Lipid mediators derived from arachidonic acid through the cyclooxygenase and lipoxygenase pathways are known to be important mediators of inflammation. Studies in mouse models demonstrated an important role for the high-affinity leukotriene B(4) receptor BLT1 in arthritis, atherosclerosis, and asthma. BLT2, a low-affinity leukotriene B(4) receptor, was also shown to be a high-affinity receptor for cyclooxygenase-1 derived 12(S)-hydroxyheptadeca-5Z, 8E, 10E-trienoic acid. However, its biochemical activities and physiological roles remain unknown. In this study, we developed mice deficient in BLT2 by targeted disruption. The BLT2(-/-) mice developed normally, and analysis of immune cells showed that disruption of BLT2 did not alter BLT1 expression or function. Mast cells from the C57BL/6 mice but not from the BLT2(-/-) mice showed intracellular calcium mobilization in response to 12(S)-hydroxyheptadeca-5Z, 8E, 10E-trienoic acid. In an autoantibody-induced inflammatory arthritis model, the BLT2(-/-) mice showed reduced incidence and severity of disease, including protection from bone and cartilage loss. Reciprocal bone marrow transplant experiments identified that loss of BLT2 expression on a bone marrow-derived cell lineage offers protection against severe disease. Thus, BLT2, a unique receptor for 5-lipoxygenase- and cyclooxygenase-1-derived lipid mediators, represents a novel target for therapies directed at treating inflammation associated with arthritis.

[Prognostic implications and functional enrichment analysis of LTB4R in patients with acute myeloid leukemia].

OBJECTIVE: To explore the expression patterns, prognostic implications, and biological role of leukotriene B4 receptor (LTB4R) in patients with acute myeloid leukemia (AML). METHODS: We collected the data of mRNA expression levels and clinical information of patients with AML from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database for mRNA expression analyses, survival analyses, Cox regression analyses and correlation analyses using R studio to assess the expression patterns and prognostic value of LTB4R. The correlation of LTB4R expression levels with clinical characteristics of the patients were analyzed using UALCAN. The co-expressed genes LTB4R were screened from Linkedomics and subjected to functional enrichment analysis. A protein-protein interaction network was constructed using STRING. GSEA analyses of the differentially expressed genes (DEGs) were performed based on datasets from TCGA-LAML stratified by LTB4R expression level. We also collected peripheral blood mononuclear cells (PBMCs) from AML patients and healthy donors for examination of the mRNA expression levels of LTB4R and immune checkpoint genes using qRT-PCR. We also examined serum LTB4R protein levels in the patients using ELISA. RESULTS: The mRNA expression level of LTB4R was significantly increased in AML patients (4.898±1.220 vs 2.252±0.215, P < 0.001), and an elevated LTB4R expression level was correlated with a poor overall survival (OS) of the patients (P=0.004, HR=1.74). LTB4R was identified as an independent prognostic factor for OS (P=0.019, HR=1.66) and was associated with FAB subtypes, cytogenetic risk, karyotype abnormalities and NPM1 mutations. The co- expressed genes of LTB4R were enriched in the functional pathways closely associated with AML leukemogenesis, including neutrophil inflammation, lymphocyte activation, signal transduction, and metabolism. The DEGs were enriched in differentiation, activation of immune cells, and cytokine signaling. Examination of the clinical serum samples also demonstrated significantly increased expressions of LTB4R mRNA (P=0.044) and protein (P=0.008) in AML patients, and LTB4R mRNA expression was positively correlated with the expression of the immune checkpoint HAVCR2 (r= 0.466, P=0.040). CONCLUSION: LTB4R can serve as a novel biomarker and independent prognostic indicator of AML and its expression patterns provide insights into the crosstalk of leukemogenesis signaling pathways involving tumor immunity and metabolism.

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.

Molecular and biological characterization of the murine leukotriene B4 receptor expressed on eosinophils.

The movement of leukocytes into tissues is regulated by the local production of chemical mediators collectively referred to as chemoattractants. Although chemoattractants constitute a diverse array of molecules, including proteins, peptides, and lipids, they all appear to signal leukocytes through a related family of seven transmembrane-spanning G protein-coupled receptors. The eosinophil is a potent proinflammatory cell that is attracted into tissues during allergic inflammation, parasitic infection, and certain malignancies. Since the molecular mechanisms controlling eosinophil recruitment are incompletely understood, we performed a degenerate polymerase chain reaction on cDNA isolated from murine eosinophils to identify novel chemoattractant receptors. We report the isolation of a cDNA that encodes a 351-amino acid glycoprotein that is 78% identical to a human gene that has been reported to be a purinoceptor (P2Y7) and a leukotriene B4 (LTB4) receptor (BLTR). Chinese hamster ovary (CHO) cells transfected with this cDNA specifically bound [3H]LTB4 with a dissociation constant of 0.6 +/- 0.1 nM. Furthermore, LTB4 induced a dose-dependent intracellular calcium flux in transfected CHO cells. In contrast, [35S]dATP did not specifically bind to these transfectants. This mRNA was expressed at high levels in interleukin 5-exposed eosinophils, elicited peritoneal macrophages and neutrophils, and to a lesser extent interferon gamma stimulated macrophages. Low levels of expression were detected in the lung, lymph node, and spleen of unchallenged mice. Western blot analysis detected the mBLTR protein in murine eosinophils and alveolar macrophages as well as human eosinophils. In addition, elevated levels of mBLTR mRNA were found in the lungs of mice in a murine model of allergic pulmonary inflammation in a time course consistent with the influx of eosinophils. Our findings indicate that this murine receptor is an LTB4 receptor that is highly expressed on activated leukocytes, including eosinophils, and may play an important role in mediating eosinophil recruitment into inflammatory foci.

Pleural mesothelial cells express both BLT2 and PPARalpha and mount an integrated response to pleural leukotriene B4.

Leukotriene B(4) (LTB(4)) plays a crucial role in the recruitment of neutrophils into the pleural space. We identified for the first time the mechanisms by which LTB(4) interacts with mesothelial cells and recruits neutrophils in the pleural compartment. Primary pleural mesothelial cells express both the proinflammatory receptor for LTB(4) BLT2, and the anti-inflammatory receptor for LTB(4), PPARalpha. Parapneumonic pleural effusions highly increase BLT2 expression and, via BLT2 activation, increase the adhesion between mesothelial cells and neutrophils and the expression of ICAM-1 on mesothelial cells. The block of PPARalpha further increases both cell adhesion and ICAM-1 expression. BLT2 activation promotes the activation, on mesothelial cells, of STAT-1 but not the activation of NF-kappaB transcription factor. The increase of ICAM-1 expression is achieved via increased tyrosine phosphorylation activity since herbimycin, a tyrosine kinase inhibitor, reduces and since Na orthovanadate, a tyrosine phosphatase inhibitor, further increases ICAM-1 expression. This study demonstrates that pleural mesothelial cells, expressing both proinflammatory and anti-inflammatory LTB(4) receptors, are able to mount an integrated response to LTB(4) with a prevalence of BLT2 activities in the presence of an inflammatory milieu within the pleura.

Corticosteroids enhance CD8+ T cell-mediated airway hyperresponsiveness and allergic inflammation by upregulating leukotriene B4 receptor 1.

BACKGROUND: Leukotriene B4 (LTB4) is a potent inflammatory lipid mediator that binds to LTB4 receptor 1 (BLT1). Ligation of BLT1 by LTB4 plays an important role in the recruitment of effector memory CD8+ T cells into the airways of sensitized and challenged mice. OBJECTIVES: The effects of the corticosteroid dexamethasone (DEX) on BLT1-expressing effector memory CD8+ T cells and effector memory CD8+ T cell-mediated airway hyperresponsiveness (AHR) and allergic inflammation were determined. METHODS: Effector memory CD8+ T cells were generated from ovalbumin(257-264)-primed mononuclear cells from OT-1 mice in the presence of IL-2. In some cultures DEX was added. The effects of DEX on BLT1 expression, LTB4-induced Ca2+ influx, phosphorylation of extracellular signal-regulated kinase 1/2, chemotaxis, and effector memory CD8+ T cell-mediated AHR were examined. RESULTS: DEX-treated effector memory CD8+ T cells showed significant increases in surface expression of BLT1, LTB4-induced intracellular Ca2+ influx, phosphorylation of extracellular signal-regulated kinase 1/2, and chemotaxis. Upregulation of BLT1 by DEX was accompanied by increased IL-2 receptor expression. Adoptive transfer of DEX-treated effector memory CD8+ T cells into ovalbumin-sensitized and ovalbumin-challenged CD8-/- mice resulted in significant increases in AHR, allergic inflammation, goblet cell metaplasia, and numbers of both CD8+ and CD4+ T cells in the bronchoalveolar lavage fluid and lungs. CONCLUSIONS: Corticosteroids upregulate BLT1 on effector memory CD8+ T cells and related signaling pathways and potentiate allergic airway inflammation and AHR induced by these cells.

Leukotriene B4 receptor transgenic mice reveal novel protective roles for lipoxins and aspirin-triggered lipoxins in reperfusion.

Polymorphonuclear neutrophil (PMN) activation is pivotal in acute inflammation and injury from reperfusion. To elucidate components controlling PMNs in vivo, we prepared novel transgenic mice with the human leukotriene (LT) B4 receptor (BLTR) for functional characterization. Overexpression of BLTR in leukocytes dramatically increased PMN trafficking to skin microabscesses and lungs after ischemia-reperfusion, whereas mice deficient in 5-lipoxygenase (5-LO) showed diminished PMN accumulation in reperfused lungs. Hence, both BLTR expression and LT biosynthesis are critical for PMN infiltration in reperfusion-initiated second-organ injury. Also, in BLTR transgenic mice, 5-LO expression and product formation were selectively increased in exudates, demonstrating that receptor overexpression amplifies proinflammatory circuits. Endogenous lipoxin (LX) A4 was produced in ischemic lungs and elevated by reperfusion. Because LXA4 and aspirin-triggered 15-epimeric LXA4 (ATL) selectively regulate leukocyte responses, they were tested in BLTR transgenic mice. Despite excessive PMN recruitment in BLTR transgenic mice, intravenous injection of ATL sharply diminished reperfusion-initiated PMN trafficking to remote organs, and topical application of LX was protective in acute dermal inflammation. These results demonstrate a direct role for BLTR with positive feedback, involving BLTR and 5-LO signaling in controlling PMNs. Moreover, LXA4 and ATL counter BLTR-amplified networks, revealing a novel protective role for LX and ATL in stress responses that has applications in perioperative medicine.

Role of leukotriene B4 receptors in the development of atherosclerosis: potential mechanisms.

OBJECTIVE: Leukotriene B4 (LTB4), a potent leukocyte chemoattractant, is known to promote several inflammatory diseases, including atherosclerosis. We sought to determine mechanisms through which LTB4 modulates atherosclerosis in cell lines expressing LTB4 receptors, BLT-1, and in mice deficient in BLT-1 as well as macrophage cell lines derived from BLT-1+/+ and BLT-1-/- mice. METHODS AND RESULTS: Analysis of global changes in gene expression induced by LTB4 in rat basophilic leukemia cells (RBL-2H3) expressing the human BLT-1 showed highest-fold increase in expression of fatty acid translocase/CD36 and the chemokine MCP1/JE/CCL2, which are critical in atherogenesis. To determine the importance of BLT-1 in atherogenesis, we crossed BLT-1-null mice with apolipoprotein (apo)-E-deficient mice, which develop severe atherosclerosis. Deletion of BLT-1 significantly reduced the lesion formation in apo-E-/- mice only during initiating stages (4 and 8 weeks) but had no effect on the lesion size in mice fed atherogenic diet for 19 weeks. Macrophage cell lines from BLT-1-deficient mice expressed the low-affinity LTB4 receptor, BLT-2, and exhibited chemotaxis to LTB4. CONCLUSIONS: The effects of LTB4 in atherosclerosis are likely mediated through the high-affinity BLT-1 and the low-affinity BLT-2 receptors. LTB4 promotes atherosclerosis by chemo-attracting monocytes, by providing an amplification loop of monocyte chemotaxis via CCL2 production, and by converting monocytes to foam cells by enhanced expression of CD36 and fatty acid accumulation.

Two distinct leukotriene B4 receptors, BLT1 and BLT2.

Leukotriene B4 (LTB4) is a potent inflammatory mediator derived from arachidonic acid. Two G protein-coupled receptors for LTB4 have been identified: a high-affinity receptor, BLT1, and a low-affinity receptor, BLT2. Both receptors mainly couple to pertussis toxin-sensitive Gi-like G proteins and induce cell migration. 12(S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid (12-HHT) was identified to bind BLT2 with higher affinity than LTB4. Expression of BLT1 was confirmed in type 1 helper T cells, type 2 helper T cells, type 17 helper T cells, effector CD8(+) T cells, dendritic cells and osteoclasts in addition to granulocytes, eosinophils and macrophages, and BLT1-deficient mice showed greatly reduced phenotypes in models of various inflammatory diseases, such as peritonitis, bronchial asthma, rheumatoid arthritis, atherosclerosis and osteoporosis. In mice, BLT2 expression is restricted to intestinal epithelial cells and epidermal keratinocytes. BLT2-deficient mice showed enhanced colitis after administration of dextran sulfate, possibly due to reduced intestinal barrier function. An aspirin-dependent reduction in 12-HHT production was responsible for delayed skin wound healing, showing that the 12-HHT/BLT2 axis also plays an important role in skin biology. BLT1 and BLT2 are therefore potential targets for the development of novel drugs.

Myeloid differentiation primary response gene 88-leukotriene B4 receptor 2 cascade mediates lipopolysaccharide-potentiated invasiveness of breast cancer cells.

Inflammation and local inflammatory mediators are inextricably linked to tumor progression through complex pathways in the tumor microenvironment. Lipopolysaccharide (LPS) exposure to tumor cells has been suggested to promote tumor invasiveness and metastasis. However, the detailed signaling mechanism involved has not been elucidated. In this study, we showed that LPS upregulated the expression of leukotriene B4 receptor-2 (BLT2) and the synthesis of BLT2 ligands in MDA-MB-231 and MDA-MB-435 breast cancer cells, thereby promoting invasiveness. BLT2 depletion with siRNA clearly attenuated LPS-induced invasiveness. In addition, we demonstrated that myeloid differentiation primary response gene 88 (MyD88) lies upstream of BLT2 in LPS-potentiated invasiveness and that this 'MyD88-BLT2' cascade mediates activation of NF-κB and the synthesis of IL-6 and IL-8, which are critical for the invasiveness and aggression of breast cancer cells. LPS-driven metastasis of MDA-MB-231 cells was also markedly suppressed by the inhibition of BLT2. Together, our results demonstrate, for the first time, that LPS potentiates the invasiveness and metastasis of breast cancer cells via a 'MyD88-BLT2'-linked signaling cascade.

Flow cytometric mapping of the leukotriene B4 receptor, BLT1, in human bone marrow and peripheral blood using specific monoclonal antibodies.

We have previously raised two monoclonal antibodies (7B1, 14F11) recognizing the high-affinity leukotriene B4 receptor, BLT1. They were presently used to determine receptor surface expression in the hematopoietic system. In peripheral blood, BLT1 was primarily recognized in granulocytes, monocytes and, to a lower extent, in certain lymphocytes except the CD4 subpopulation. The expression pattern was similar in bone marrow cells. In vitro differentiation of CD34+ progenitor cells induced BLT1 expression within 7 days, which remained constant up to day 17 when a further increase was measured and maintained up to day 20. BLT1 expression was modified by inflammatory mediators: LPS, TNFalpha, fMLP, as well as LTB4 itself, caused a slight down-regulation at 30 min, an effect that was particularly marked with PMA, whereas the effect was least pronounced with IL-8. The antibodies have proved to be useful in an extensive mapping of BLT1 in both peripheral blood and bone marrow and as a tool to elucidate changes in the receptor expression.

Differential inhibition of receptor activation by two mouse monoclonal antibodies specific for the human leukotriene B4 receptor, BLT1.

The inflammatory mediator leukotriene B(4) (LTB(4)) binds to and activates a G-protein-coupled receptor named BLT(1). We have previously produced two monoclonal antibodies, named 7B1 and 14F11, that bind specifically to this receptor. Using a HeLa cell line expressing human BLT(1), we find that both antibodies inhibit LTB(4)-induced calcium release, and activation of a MAP-kinase-sensitive luciferase reporter system. The normal chemotactic movement of polymorphonuclear cells towards higher LTB(4) concentrations was also strongly inhibited by both antibodies. Neither antibody was found to activate BLT(1), and experiments using cyclic peptide fragments of the BLT(1) n-terminal and extracellular loops showed that these antibodies bind only to complex epitopes in the tertiary, membrane bound, conformation of the receptor protein. In ligand binding experiments, 7B1 was found to be a competitive antagonist, while 14F11 was a noncompetitive antagonist that inhibited receptor activation, but not agonist (LTB(4)) binding. 14F11 will be a useful tool for studying the mechanisms of receptor activation.

Enhancing antitumor effects in pancreatic cancer cells by combined use of COX-2 and 5-LOX inhibitors.

Cyclooxygenase (COX)-2 and lipoxygenase (LOX)-5 are involved in carcinogenesis of pancreatic cancer. COX-2 inhibitor celecoxib displays inhibitory effects in pancreatic cancer cell growth. Recently, it has been reported that COX-2 inhibitor may not be able to suppress pancreatic tumor growth in vivo and its application is further limited by untoward side effects. The present study provides evidence that combined use of celecoxib and 5-LOX inhibitor MK886 markedly suppresses pancreatic tumor cell growth in vitro. Compared to the single inhibitor treatment, dual treatment with celecoxib and MK886 exerted additive antitumor effects in pancreatic tumor cells. We found that MK886 reversed celecoxib-induced increases in 5-LOX gene expression and Erk1/2 activation in pancreatic tumor cells. Moreover, Dual treatment of pancreatic tumor cells with celecoxib and MK886 inhibited the levels of LBT4 receptor BLT1 and vascular endothelial growth factor. Our results imply that combined use of celecoxib and MK886 might be an effective way to treat clinical patients with pancreatic cancer.

Leukotriene B4 regulates proliferation and differentiation of cultured rat myoblasts via the BLT1 pathway.

Skeletal muscle regeneration is a highly orchestrated process initiated by activation of adult muscle satellite cells. Upon muscle injury, the inflammatory process is always accompanied by muscle regeneration. Leukotriene B(4) is one of the essential inflammatory mediators. We isolated and cultured primary satellite cells. RT-PCR showed that myoblasts expressed mRNA for LTB(4) receptors BLT1 and BLT2, and LTB4 promoted myoblast proliferation and fusion. Quantitative real-time PCR and immunoblotting showed that LTB(4) treatment expedited the expression process of differentiation markers MyoD and M-cadherin. U-75302, a specific BLT1 inhibitor, but not LY2552833, a specific BLT2 inhibitor, blocked proliferation and differentiation of myoblasts induced by LTB(4), which implies the involvement of the BLT1 pathway. Overall, the data suggest that LTB(4) contributes to muscle regeneration by accelerating proliferation and differentiation of satellite cells.

Preparation of 2-, 3-, 4- and 7-(2-alkylcarbamoyl-1-alkylvinyl)benzo[b]furans and their BLT1 and/or BLT2 inhibitory activities.

Several 2-alkylcarbamoyl-1-alkylvinylbenzo[b]furans were designed to find a selective leukotriene B4 (LTB4) receptor antagonist. 2-(2-Alkylcarbamoyl-1-alkylvinyl)benzo[b]furans having a substituent group at the 3-position, 4-(2-alkylcarbamoyl-1-methylvinyl)benzo[b]furans having a substituent group at the 3-position, and 7-(2-alkylcarbamoyl-1-methylvinyl)benzo[b]furans and 3-(2-alkylcarbamoyl-1-alkylvinyl)benzo[b]furans were prepared and evaluated for LTB4 receptor (BLT1 and BLT2) inhibitory activities. (E)-3-Amino-4-[2-[2-(3,4-dimethoxyphenyl)ethylcarbamoyl]-1-methylvinyl]benzo[b]furan ((E)-17c) showed potent and selective inhibitory activity for BLT2. On the other hand, (E)-7-(2-diethylcarbamoyl-1-methylvinyl)benzo[b]furan ((E)-27a) showed potent inhibitory activity for both BLT1 and BLT2.

Regulation of dendritic cell migration and adaptive immune response by leukotriene B4 receptors: a role for LTB4 in up-regulation of CCR7 expression and function.

Trafficking of dendritic cells (DCs) to peripheral tissues and to secondary lymphoid organs depends on chemokines and lipid mediators. Here, we show that bone marrow-derived DCs (BM-DCs) express functional leukotriene B4 (LTB4) receptors as observed in dose-dependent chemotaxis and calcium mobilization responses. LTB4, at low concentrations, promoted the migration of immature and mature DCs to CCL19 and CCL21, which was associated with a rapid (30-minute) increase of CCR7 expression at the membrane level. At longer incubation times (6 hours), gene array analysis revealed a promoting role of LTB4, showing a significant increase of CCR7 and CCL19 mRNA levels. BM-DCs cultured from BLT1-/- or BLT1/2-/- mice showed a normal phenotype, but in vivo BLT1/2-/-DCs showed dramatic decrease in migration to the draining lymph nodes relative to wild-type (WT) DCs. Consistent with these observations, BLT1/2-/- mice showed a reduced response in a model of 2,4-dinitro-fluorobenzene (DNFB)-induced contact hypersensitivity. Adoptive transfer of 2,4-dinitrobenzene sulfonic acid (DNBS)-pulsed DCs directly implicated the defect in DC migration to lymph node with the defect in contact hypersensitivity. These results provide strong evidence for a role of LTB4 in regulating DC migration and the induction of adaptive immune responses.

Eicosapentaenoic acid modulates neutrophil leukotriene B4 receptor expression in cystic fibrosis.

In patients with cystic fibrosis (CF), high intrapulmonary concentrations of the neutrophil chemotaxin leukotriene B4 (LTB4) are associated with specific reduction of LTB4-induced chemotaxis of circulating neutrophils. The chemotactic abnormality is partially corrected by dietary supplementation with eicosapentaenoic acid (EPA). LTB4-induced neutrophil chemotaxis is mediated by specific, high-affinity, cell surface LTB4 receptors. The hypotheses that neutrophil LTB4 receptors are down-regulated in CF, and that EPA normalizes receptor expression, were tested by measuring the number (Rmax) and affinity (Kd) of LTB4 receptors on neutrophils from eight CF patients before and after EPA (6 weeks of 2.7 g/day), and from nine normal individuals. High-affinity receptor Rmax was depressed in CF patients (0.6 +/- 0.2 x 10(4)/cell (mean +/- s.d.) versus 1.8 +/- 0.7 x 10(4)/cell in normals), but corrected to normal (2.0 +/- 1.9 x 10(4)/cell) after EPA. High-affinity receptor Kd was depressed in CF patients (0.4 +/- 0.3 nM versus 1.4 +/- 0.5 nM in normals), and also corrected to normal with EPA (1.4 +/- 1.2 nM). Low-affinity receptors were depressed, but did not change significantly with EPA. These results indicate that neutrophil responses in chronic inflammatory lung disease can be influenced directly by LTB4 receptor modulation, and that this effect of EPA predominates over alterations in neutrophil signal transduction in situations of chronic exposure to LTB4.