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Takehiko Yokomizo - One of the best experts on this subject based on the ideXlab platform.

  • role of the high affinity Leukotriene B4 Receptor signaling in fibrosis after unilateral ureteral obstruction in mice
    PLOS ONE, 2019
    Co-Authors: Mariko Kamata, Takehiko Yokomizo, Takao Shimizu, Hideki Amano, Tomoe Fujita, Kanako Hosono, Kouju Kamata, Yasuo Takeuchi, Fumisato Otaka, Masataka Majima
    Abstract:

    Leukotriene B4 (LTB4) is a lipid mediator that acts as a potent chemoattractant for inflammatory leukocytes. Kidney fibrosis is caused by migrating inflammatory cells and kidney-resident cells. Here, we examined the role of the high-affinity LTB4 Receptor BLT1 during development of kidney fibrosis induced by unilateral ureteral obstruction (UUO) in wild-type (WT) mice and BLT1 knockout (BLT1-/-) mice. We found elevated expression of 5-lipoxygenase (5-LOX), which generates LTB4, in the renal tubules of UUO kidneys from WT mice and BLT1-/- mice. Accumulation of immunoreactive type I collagen in WT UUO kidneys increased over time; however, the increase was less prominent in BLT1-/- UUO kidneys. Accumulation of S100A4-positive fibroblasts increased temporally in WT UUO kidneys, but was again less pronounced in-BLT1-/- UUO kidneys. The same was true of mRNA encoding transforming growth factor-β (TGF)-β and fibroblast growth factor (FGF)-2. Finally, accumulation of F4/80-positive macrophages, which secrete TGF-β, increased temporally in WT UUO and BLT1-/- UUO kidneys, but to a lesser extent in the latter. Following LTB4 stimulation in vitro, macrophages showed increased expression of mRNA encoding TGF-β/FGF-2 and Col1a1, whereas L929 fibroblasts showed increased expression of mRNA encoding α smooth muscle actin (SMA). Bone marrow (BM) transplantation studies revealed that the area positive for type I collagen was significantly smaller in BLT1-/—BM→WT than in WT-BM→WT. Thus, LTB4-BLT1 signaling plays a critical role in fibrosis in UUO kidneys by increasing accumulation of macrophages and fibroblasts. Therefore, blocking BLT1 may prevent renal fibrosis.

  • role of the high affinity Leukotriene B4 Receptor signaling in fibrosis after unilateral ureteral obstruction in mice
    bioRxiv, 2018
    Co-Authors: Mariko Kamata, Takehiko Yokomizo, Takao Shimizu, Hideki Amano, Yoshiya Ito, Tomoe Fujita, Kanako Hosono, Kouju Kamata, Yasuo Takeuchi, Masataka Majima
    Abstract:

    Leukotriene B4 (LTB4) is a lipid mediator that acts as a potent chemoattractant for inflammatory leukocytes. Kidney fibrosis is caused by migrating inflammatory cells and kidney-resident cells. Here, we examined the role of the high-affinity LTB4 Receptor BLT1 during development of kidney fibrosis in wild-type (WT) mice and BLT1 knockout (BLT1-/-) mice with unilateral ureteral obstruction (UUO). We found elevated expression of 5-lipoxygenase (5-LOX), which generates LTB4, in the renal tubules of WT and BLT1-/- UUO mice. Accumulation of immunoreactive type I collagen in UUO kidneys of WT mice increased over time; however, the increase was less prominent in BLT1-/- mice. Accumulation of S100A4-positive fibroblasts also increased temporally in WT UUO kidneys, but was again less pronounced in those of BLT1-/- mice. The same was true of mRNA encoding transforming growth factor-β (TGF)-β and fibroblast growth factor (FGF)-2. Finally, accumulation of F4/80-positive macrophages, which secrete TGF-β, also increased temporally in WT UUO and BLT1-/- kidneys, but to a lesser extent in the latter. Following LTB4 stimulation in vitro, macrophages showed increased expression of mRNA encoding TGF-β/FGF-2 and Col1a1, whereas L929 fibroblasts showed increased expression of mRNA encoding α smooth muscle actin (SMA). Bone marrow (BM) transplantation studies revealed that the area positive for type I collagen was significantly smaller in BLT1-/--BM→WT UUO kidneys than in WT-BM→WT kidneys. Thus, LTB4-BLT1 signaling plays a critical role in fibrosis in UUO kidneys by increasing accumulation of macrophages and fibroblasts. Therefore, blocking BLT1 may prevent renal fibrosis.

  • Biological functions of 12(S)-hydroxyheptadecatrienoic acid as a ligand of Leukotriene B4 Receptor 2
    BMC, 2018
    Co-Authors: Toshiaki Okuno, Takehiko Yokomizo
    Abstract:

    Abstract Although 12(S)-hydroxyheptadecatrienoic acid (12-HHT) is an abundant fatty acid, it is long considered a byproduct of thromboxane A2 production. We identified a Leukotriene B4 Receptor 2 (BLT2)-specific agonistic activity in lipid extracts from rat small intestine, and mass spectrometric analysis of partially purified lipids containing BLT2 agonistic activity revealed that 12-HHT is an endogenous ligand of BLT2. In a dextran sulfate sodium (DSS)-induced inflammatory colitis model, BLT2-deficient mice exhibited enhanced intestinal inflammation, possibly due to impaired epithelial barrier function. In a skin wound healing model, BLT2-deficient mice exhibited delayed wound healing via dampened keratinocyte migration. BLT2 also accelerates corneal wound healing, and eye drops containing a non-steroidal anti-inflammatory drug (NSAID) inhibit the production of 12-HHT, resulting in delayed corneal wound healing. Furthermore, BLT2 is expressed in pulmonary epithelial type II cells and vascular endothelial cells in the mouse lung, and BLT2-deficient mice are more susceptible to lung damage by pneumolysin. In this review, we summarize the identification and characterization of 12-HHT as a ligand for BLT2 and discuss recent research on the physiological and pathophysiological roles of the 12-HHT-BLT2 axis. Some side effects of NSAIDs such as delayed wound healing may be caused by reduced 12-HHT production rather than diminished production of prostaglandins

  • a synthetic Leukotriene B4 Receptor type 2 agonist accelerates the cutaneous wound healing process in diabetic rats by indirect stimulation of fibroblasts and direct stimulation of keratinocytes
    Journal of Diabetes and Its Complications, 2017
    Co-Authors: Takehiko Yokomizo, Lin Luo, Rica Tanaka, Shigeyuki Kanazawa, Ayato Hayashi, Hiroshi Mizuno
    Abstract:

    Abstract Aims The synthetic Leukotriene B 4 Receptor type 2 (BLT2) agonist CAY10583 (CAY) accelerates wound healing in diabetic mice by promoting keratinocyte migration. However, its effects on fibroblast activity and granulation are unknown. We investigated the mechanisms by which CAY promotes wound healing. Methods CAY was applied to wounds on streptozotocin-induced diabetic rats, and wound closure, granulation thickness, and epithelialization gaps were analyzed. BLT2 expression was examined by RT-PCR. Migration and proliferation were studied by scratch assays and MTS assays. Keratinocyte supernatants with CAY were applied to fibroblasts, and cytokines were measured by enzyme-linked immunosorbent assays. Results CAY significantly accelerated wound healing in diabetic rats (CAY, 78.05±12.22% vs. control, 59.84±11.09%; p =0.0222), with increased re-epithelialization and granulation compared to controls. BLT2 was expressed in keratinocytes, but not in fibroblasts. Keratinocyte treatment with the CAY supernatant enhanced fibroblast proliferation and migration (fibroblast scratch closure: CAY, 75.95±4.09% vs. control, 49.69±4.49%; p Conclusions CAY directly promotes keratinocyte migration and indirectly enhances fibroblast proliferation by increasing keratinocyte production of TGF-β1 and bFGF, accelerating wound closure. CAY is a promising pharmaceutical agent for diabetic wounds.

  • Biochemical and immunological characterization of a novel monoclonal antibody against mouse Leukotriene B4 Receptor 1
    2017
    Co-Authors: Fumiyuki Sasaki, Tomoaki Koga, Kazuko Saeki, Toshiaki Okuno, Yasuyuki Ohkawa, Saiko Kazuno, Tsutomu Fujimura, Takehiko Yokomizo
    Abstract:

    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.

Toshiaki Okuno - One of the best experts on this subject based on the ideXlab platform.

  • na mimicking ligands stabilize the inactive state of Leukotriene B4 Receptor blt1
    Nature Chemical Biology, 2018
    Co-Authors: Toshiaki Okuno, Yoshiaki Kawano, Motonao Nakamura, Keitaro Yamashita, Kunio Hirata, Masakatsu Hato, Masaki Yamamoto
    Abstract:

    A structure of Leukotriene B4 Receptor BLT1 bound with a benzamidine-containing compound, BIIL260, reveals an inverse-agonist mechanism involving ligand binding in the sodium ion-centered water cluster adjacent to the conserved orthosteric site of class A GPCRs.

  • Biological functions of 12(S)-hydroxyheptadecatrienoic acid as a ligand of Leukotriene B4 Receptor 2
    BMC, 2018
    Co-Authors: Toshiaki Okuno, Takehiko Yokomizo
    Abstract:

    Abstract Although 12(S)-hydroxyheptadecatrienoic acid (12-HHT) is an abundant fatty acid, it is long considered a byproduct of thromboxane A2 production. We identified a Leukotriene B4 Receptor 2 (BLT2)-specific agonistic activity in lipid extracts from rat small intestine, and mass spectrometric analysis of partially purified lipids containing BLT2 agonistic activity revealed that 12-HHT is an endogenous ligand of BLT2. In a dextran sulfate sodium (DSS)-induced inflammatory colitis model, BLT2-deficient mice exhibited enhanced intestinal inflammation, possibly due to impaired epithelial barrier function. In a skin wound healing model, BLT2-deficient mice exhibited delayed wound healing via dampened keratinocyte migration. BLT2 also accelerates corneal wound healing, and eye drops containing a non-steroidal anti-inflammatory drug (NSAID) inhibit the production of 12-HHT, resulting in delayed corneal wound healing. Furthermore, BLT2 is expressed in pulmonary epithelial type II cells and vascular endothelial cells in the mouse lung, and BLT2-deficient mice are more susceptible to lung damage by pneumolysin. In this review, we summarize the identification and characterization of 12-HHT as a ligand for BLT2 and discuss recent research on the physiological and pathophysiological roles of the 12-HHT-BLT2 axis. Some side effects of NSAIDs such as delayed wound healing may be caused by reduced 12-HHT production rather than diminished production of prostaglandins

  • Biochemical and immunological characterization of a novel monoclonal antibody against mouse Leukotriene B4 Receptor 1
    2017
    Co-Authors: Fumiyuki Sasaki, Tomoaki Koga, Kazuko Saeki, Toshiaki Okuno, Yasuyuki Ohkawa, Saiko Kazuno, Tsutomu Fujimura, Takehiko Yokomizo
    Abstract:

    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.

  • Leukotriene B4 Receptor type 2 protects against pneumolysin dependent acute lung injury
    Scientific Reports, 2016
    Co-Authors: Tomoaki Koga, Kazuko Saeki, Misako Shigematsu, Ayako Ishimori, Yumiko Ishii, Yoshitaka Taketomi, Mai Ohba, Airi Jowatanabe, Toshiaki Okuno
    Abstract:

    Although pneumococcal infection is a serious problem worldwide and has a high mortality rate, the molecular mechanisms underlying the lethality caused by pneumococcus remain elusive. Here, we show that BLT2, a G protein-coupled Receptor for Leukotriene B4 and 12(S)-hydroxyheptadecatrienoic acid (12-HHT), protects mice from lung injury caused by a pneumococcal toxin, pneumolysin (PLY). Intratracheal injection of PLY caused lethal acute lung injury (ALI) in BLT2-deficient mice, with evident vascular leakage and bronchoconstriction. Large amounts of cysteinyl Leukotrienes (cysLTs), classically known as a slow reactive substance of anaphylaxis, were detected in PLY-treated lungs. PLY-dependent vascular leakage, bronchoconstriction, and death were markedly ameliorated by treatment with a CysLT1 Receptor antagonist. Upon stimulation by PLY, mast cells produced cysLTs that activated CysLT1 expressed in vascular endothelial cells and bronchial smooth muscle cells, leading to lethal vascular leakage and bronchoconstriction. Treatment of mice with aspirin or loxoprofen inhibited the production of 12-HHT and increased the sensitivity toward PLY, which was also ameliorated by the CysLT1 antagonist. Thus, the present study identifies the molecular mechanism underlying PLY-dependent ALI and suggests the possible use of CysLT1 antagonists as a therapeutic tool to protect against ALI caused by pneumococcal infection.

  • Leukotriene B4 Receptor type 2 blt2 enhances skin barrier function by regulating tight junction proteins
    The FASEB Journal, 2016
    Co-Authors: Yumiko Ishii, Tomoaki Koga, Kazuko Saeki, Fumiyuki Sasaki, Keiko Kitajima, Chikara Meno, Toshiaki Okuno
    Abstract:

    GPCRs are involved in numerous physiologic functions and are important drug targets. Although the epithelial barrier is important for protection from invading pathogens, the correlation between GPC...

Takao Shimizu - One of the best experts on this subject based on the ideXlab platform.

  • role of the high affinity Leukotriene B4 Receptor signaling in fibrosis after unilateral ureteral obstruction in mice
    PLOS ONE, 2019
    Co-Authors: Mariko Kamata, Takehiko Yokomizo, Takao Shimizu, Hideki Amano, Tomoe Fujita, Kanako Hosono, Kouju Kamata, Yasuo Takeuchi, Fumisato Otaka, Masataka Majima
    Abstract:

    Leukotriene B4 (LTB4) is a lipid mediator that acts as a potent chemoattractant for inflammatory leukocytes. Kidney fibrosis is caused by migrating inflammatory cells and kidney-resident cells. Here, we examined the role of the high-affinity LTB4 Receptor BLT1 during development of kidney fibrosis induced by unilateral ureteral obstruction (UUO) in wild-type (WT) mice and BLT1 knockout (BLT1-/-) mice. We found elevated expression of 5-lipoxygenase (5-LOX), which generates LTB4, in the renal tubules of UUO kidneys from WT mice and BLT1-/- mice. Accumulation of immunoreactive type I collagen in WT UUO kidneys increased over time; however, the increase was less prominent in BLT1-/- UUO kidneys. Accumulation of S100A4-positive fibroblasts increased temporally in WT UUO kidneys, but was again less pronounced in-BLT1-/- UUO kidneys. The same was true of mRNA encoding transforming growth factor-β (TGF)-β and fibroblast growth factor (FGF)-2. Finally, accumulation of F4/80-positive macrophages, which secrete TGF-β, increased temporally in WT UUO and BLT1-/- UUO kidneys, but to a lesser extent in the latter. Following LTB4 stimulation in vitro, macrophages showed increased expression of mRNA encoding TGF-β/FGF-2 and Col1a1, whereas L929 fibroblasts showed increased expression of mRNA encoding α smooth muscle actin (SMA). Bone marrow (BM) transplantation studies revealed that the area positive for type I collagen was significantly smaller in BLT1-/—BM→WT than in WT-BM→WT. Thus, LTB4-BLT1 signaling plays a critical role in fibrosis in UUO kidneys by increasing accumulation of macrophages and fibroblasts. Therefore, blocking BLT1 may prevent renal fibrosis.

  • role of the high affinity Leukotriene B4 Receptor signaling in fibrosis after unilateral ureteral obstruction in mice
    bioRxiv, 2018
    Co-Authors: Mariko Kamata, Takehiko Yokomizo, Takao Shimizu, Hideki Amano, Yoshiya Ito, Tomoe Fujita, Kanako Hosono, Kouju Kamata, Yasuo Takeuchi, Masataka Majima
    Abstract:

    Leukotriene B4 (LTB4) is a lipid mediator that acts as a potent chemoattractant for inflammatory leukocytes. Kidney fibrosis is caused by migrating inflammatory cells and kidney-resident cells. Here, we examined the role of the high-affinity LTB4 Receptor BLT1 during development of kidney fibrosis in wild-type (WT) mice and BLT1 knockout (BLT1-/-) mice with unilateral ureteral obstruction (UUO). We found elevated expression of 5-lipoxygenase (5-LOX), which generates LTB4, in the renal tubules of WT and BLT1-/- UUO mice. Accumulation of immunoreactive type I collagen in UUO kidneys of WT mice increased over time; however, the increase was less prominent in BLT1-/- mice. Accumulation of S100A4-positive fibroblasts also increased temporally in WT UUO kidneys, but was again less pronounced in those of BLT1-/- mice. The same was true of mRNA encoding transforming growth factor-β (TGF)-β and fibroblast growth factor (FGF)-2. Finally, accumulation of F4/80-positive macrophages, which secrete TGF-β, also increased temporally in WT UUO and BLT1-/- kidneys, but to a lesser extent in the latter. Following LTB4 stimulation in vitro, macrophages showed increased expression of mRNA encoding TGF-β/FGF-2 and Col1a1, whereas L929 fibroblasts showed increased expression of mRNA encoding α smooth muscle actin (SMA). Bone marrow (BM) transplantation studies revealed that the area positive for type I collagen was significantly smaller in BLT1-/--BM→WT UUO kidneys than in WT-BM→WT kidneys. Thus, LTB4-BLT1 signaling plays a critical role in fibrosis in UUO kidneys by increasing accumulation of macrophages and fibroblasts. Therefore, blocking BLT1 may prevent renal fibrosis.

  • interplay between cxcr2 and blt1 facilitates neutrophil infiltration and resultant keratinocyte activation in a murine model of imiquimod induced psoriasis
    Journal of Immunology, 2014
    Co-Authors: Hayakazu Sumida, Motonao Nakamura, Yoshihiro Kita, Satoshi Ishii, Keisuke Yanagida, Kouji Matsushima, Shinichi Sato, Takao Shimizu
    Abstract:

    Psoriasis is an inflammatory skin disease with accelerated epidermal cell turnover. Neutrophil accumulation in the skin is one of the histological characteristics of psoriasis. However, the precise mechanism and role of neutrophil infiltration remain largely unknown. In this article, we show that orchestrated action of CXCR2 and Leukotriene B4 Receptor BLT1 plays a key role in neutrophil recruitment during the development of imiquimod (IMQ)-induced psoriatic skin lesions in mice. Depletion of neutrophils with anti–Ly-6G Ab ameliorated the disease severity, along with reduced expression of proinflammatory cytokine IL-1β in the skin. Furthermore, CXCR2 and BLT1 coordinately promote neutrophil infiltration into the skin during the early phase of IMQ-induced inflammation. In vitro, CXCR2 ligands augment Leukotriene B4 production by murine neutrophils, which, in turn, amplifies chemokine-mediated neutrophil chemotaxis via BLT1 in autocrine and/or paracrine manners. In agreement with the increased IL-19 expression in IMQ-treated mouse skin, IL-1β markedly upregulated expression of acanthosis-inducing cytokine IL-19 in human keratinocytes. We propose that coordination of chemokines, lipids, and cytokines with multiple positive feedback loops might drive the pathogenesis of psoriasis and, possibly, other inflammatory diseases as well. Interference to this positive feedback or its downstream effectors could be targets of novel anti-inflammatory treatment.

  • A high-affinity monoclonal antibody against the FLAG tag useful for G-protein-coupled Receptor study.
    Analytical biochemistry, 2012
    Co-Authors: Fumiyuki Sasaki, Kazuko Saeki, Toshiaki Okuno, Takao Shimizu, Liu Min, Naoya Onohara, Haruyasu Kato, Takehiko Yokomizo
    Abstract:

    The FLAG sequence (DYKDDDDK) is an artificial sequence widely used to detect, quantify, and purify proteins expressed as FLAG-fusion proteins. Several highly specific monoclonal antibodies for FLAG are commercially available; however, they are not always sensitive enough to detect proteins expressed at low levels and can give rise to unacceptable levels of background signal when used for immunostaining in vitro and in vivo. The current study reports the successful establishment of hybridoma cells that produce an extremely high-affinity antibody to FLAG, namely 2H8 Ab. 2H8 Ab stained FLAG-tagged G-protein-coupled Receptors more strongly than commercially available antibodies in both flow cytometry and immunostaining experiments with no background staining. 2H8 was sensitive enough to detect FLAG-tagged G-protein-coupled Receptors and soluble proteins in crude preparations, which could not be achieved using commercially available antibodies. Only 10 ng of 2H8 Ab was required to immunoprecipitate FLAG-tagged G-protein-coupled Receptors from cell lysates. Of note, 2H8 stained FLAG-tagged BLT2, a low-affinity Leukotriene B4 Receptor, expressed in vivo in the small intestine of mice under control of the villin promoter. Thus, 2H8 Ab is a promising tool for analyzing various FLAG-fusion proteins, particularly G-protein-coupled Receptors, both in vitro and in vivo.

  • the Leukotriene B4 Receptor blt1 is required for the induction of experimental autoimmune encephalomyelitis
    Biochemical and Biophysical Research Communications, 2010
    Co-Authors: Yasuyuki Kihara, Takehiko Yokomizo, Akilco Kunita, Yasuyuki Morishita, Masashi Fukayama, Satoshi Ishii, Takao Shimizu
    Abstract:

    Abstract Leukotriene B4 (LTB4) is a potent chemoattractant and activator of neutrophils, macrophages and T cells. These cells are a key component of inflammation and all express BLT1, a high affinity G-protein-coupled Receptor for LTB4. However, little is known about the neuroimmune functions of BLT1. In this study, we describe a distinct role for BLT1 in the pathology of experimental autoimmune encephalomyelitis (EAE) and TH1/TH17 immune responses. BLT1 mRNA was highly upregulated in the spinal cord of EAE mice, especially during the induction phase. BLT1−/− mice had delayed onset and less severe symptoms of EAE than BLT1+/+ mice. Additionally, inflammatory cells were recruited to the spinal cord of asymptomatic BLT1+/+, but not BLT1−/− mice before the onset of disease. Ex vivo studies showed that both the proliferation and the production of IFN-γ, TNF-α, IL-17 and IL-6 were impaired in BLT1−/− cells, as compared with BLT1+/+ cells. Thus, we suggest that BLT1 exacerbates EAE by regulating the migration of inflammatory cells and TH1/TH17 immune responses. Our findings provide a novel therapeutic option for the treatment of multiple sclerosis and other TH17-mediated diseases.

Kazuko Saeki - One of the best experts on this subject based on the ideXlab platform.

  • Biochemical and immunological characterization of a novel monoclonal antibody against mouse Leukotriene B4 Receptor 1
    2017
    Co-Authors: Fumiyuki Sasaki, Tomoaki Koga, Kazuko Saeki, Toshiaki Okuno, Yasuyuki Ohkawa, Saiko Kazuno, Tsutomu Fujimura, Takehiko Yokomizo
    Abstract:

    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.

  • Leukotriene B4 Receptor type 2 protects against pneumolysin dependent acute lung injury
    Scientific Reports, 2016
    Co-Authors: Tomoaki Koga, Kazuko Saeki, Misako Shigematsu, Ayako Ishimori, Yumiko Ishii, Yoshitaka Taketomi, Mai Ohba, Airi Jowatanabe, Toshiaki Okuno
    Abstract:

    Although pneumococcal infection is a serious problem worldwide and has a high mortality rate, the molecular mechanisms underlying the lethality caused by pneumococcus remain elusive. Here, we show that BLT2, a G protein-coupled Receptor for Leukotriene B4 and 12(S)-hydroxyheptadecatrienoic acid (12-HHT), protects mice from lung injury caused by a pneumococcal toxin, pneumolysin (PLY). Intratracheal injection of PLY caused lethal acute lung injury (ALI) in BLT2-deficient mice, with evident vascular leakage and bronchoconstriction. Large amounts of cysteinyl Leukotrienes (cysLTs), classically known as a slow reactive substance of anaphylaxis, were detected in PLY-treated lungs. PLY-dependent vascular leakage, bronchoconstriction, and death were markedly ameliorated by treatment with a CysLT1 Receptor antagonist. Upon stimulation by PLY, mast cells produced cysLTs that activated CysLT1 expressed in vascular endothelial cells and bronchial smooth muscle cells, leading to lethal vascular leakage and bronchoconstriction. Treatment of mice with aspirin or loxoprofen inhibited the production of 12-HHT and increased the sensitivity toward PLY, which was also ameliorated by the CysLT1 antagonist. Thus, the present study identifies the molecular mechanism underlying PLY-dependent ALI and suggests the possible use of CysLT1 antagonists as a therapeutic tool to protect against ALI caused by pneumococcal infection.

  • Leukotriene B4 Receptor type 2 blt2 enhances skin barrier function by regulating tight junction proteins
    The FASEB Journal, 2016
    Co-Authors: Yumiko Ishii, Tomoaki Koga, Kazuko Saeki, Fumiyuki Sasaki, Keiko Kitajima, Chikara Meno, Toshiaki Okuno
    Abstract:

    GPCRs are involved in numerous physiologic functions and are important drug targets. Although the epithelial barrier is important for protection from invading pathogens, the correlation between GPC...

  • modulation of Leukotriene B4 Receptor 1 signaling by Receptor for advanced glycation end products rage
    The FASEB Journal, 2016
    Co-Authors: Takako Ichiki, Masakiyo Sakaguchi, Tomoaki Koga, Kazuko Saeki, Toshiaki Okuno, Yasuhiko Yamamoto, Hiroshi Yamamoto, Takehiko Yokomizo
    Abstract:

    Leukotriene B4 (LTB4) Receptor 1 (BLT1), a high-affinity GPCR for LTB4, plays important roles in acute and chronic inflammatory diseases. Although the LTB4-BLT1 axis is known to promote inflammatio...

  • modulation of Leukotriene B4 Receptor 1 signaling by Receptor for advanced glycation end products rage
    The FASEB Journal, 2016
    Co-Authors: Takako Ichiki, Masakiyo Sakaguchi, Tomoaki Koga, Kazuko Saeki, Toshiaki Okuno, Yasuhiko Yamamoto, Hiroshi Yamamoto, Takehiko Yokomizo
    Abstract:

    Leukotriene B4 (LTB4) Receptor 1 (BLT1), a high-affinity GPCR for LTB4, plays important roles in acute and chronic inflammatory diseases. Although the LTB4-BLT1 axis is known to promote inflammation, no studies have defined the binding proteins that modulate LTB4-BLT1 signaling. In this study, the Receptor for advanced glycation end products (RAGE) interacted with BLT1 in human cervical epithelial HeLa cells. RAGE increased LTB4-BLT1-dependent ERK phosphorylation and inhibited LTB4-BLT1-dependent activation of NF-κB and up-regulation of proinflammatory cytokines and chemokines. RAGE-dependent inhibition of NF-κB was blunted by treatment with an MEK inhibitor, suggesting that RAGE suppresses LTB4-BLT1-dependent NF-κB signaling by enhancing the MEK-ERK pathway. Meanwhile, in a chemotaxis assay of mouse bone marrow-derived neutrophils, the velocity of LTB4-dependent neutrophil migration was attenuated by soluble RAGE, which is an inhibitory decoy protein for RAGE signaling, in a dose-dependent manner (0.2-5 μg/ml), or by RAGE deficiency. Furthermore, both LTB4-dependent ERK phosphorylation in neutrophils and LTB4-dependent neutrophil accumulation in a murine peritonitis model were significantly attenuated in RAGE-deficient mice compared with C57BL/6J wild-type mice, indicating that RAGE potentiates LTB4-dependent neutrophil migration by enhancing ERK phosphorylation. Our results demonstrate that RAGE interacts with BLT1 and modulates LTB4-BLT1 signaling through potentiation of the MEK-ERK pathway.-Ichiki, T., Koga, T., Okuno, T., Saeki K., Yamamoto, Y., Yamamoto, H., Sakaguchi, M., Yokomizo, T. Modulation of Leukotriene B4 Receptor 1 signaling by Receptor for advanced glycation end products (RAGE).

Tomoaki Koga - One of the best experts on this subject based on the ideXlab platform.

  • Biochemical and immunological characterization of a novel monoclonal antibody against mouse Leukotriene B4 Receptor 1
    2017
    Co-Authors: Fumiyuki Sasaki, Tomoaki Koga, Kazuko Saeki, Toshiaki Okuno, Yasuyuki Ohkawa, Saiko Kazuno, Tsutomu Fujimura, Takehiko Yokomizo
    Abstract:

    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.

  • Leukotriene B4 Receptor type 2 protects against pneumolysin dependent acute lung injury
    Scientific Reports, 2016
    Co-Authors: Tomoaki Koga, Kazuko Saeki, Misako Shigematsu, Ayako Ishimori, Yumiko Ishii, Yoshitaka Taketomi, Mai Ohba, Airi Jowatanabe, Toshiaki Okuno
    Abstract:

    Although pneumococcal infection is a serious problem worldwide and has a high mortality rate, the molecular mechanisms underlying the lethality caused by pneumococcus remain elusive. Here, we show that BLT2, a G protein-coupled Receptor for Leukotriene B4 and 12(S)-hydroxyheptadecatrienoic acid (12-HHT), protects mice from lung injury caused by a pneumococcal toxin, pneumolysin (PLY). Intratracheal injection of PLY caused lethal acute lung injury (ALI) in BLT2-deficient mice, with evident vascular leakage and bronchoconstriction. Large amounts of cysteinyl Leukotrienes (cysLTs), classically known as a slow reactive substance of anaphylaxis, were detected in PLY-treated lungs. PLY-dependent vascular leakage, bronchoconstriction, and death were markedly ameliorated by treatment with a CysLT1 Receptor antagonist. Upon stimulation by PLY, mast cells produced cysLTs that activated CysLT1 expressed in vascular endothelial cells and bronchial smooth muscle cells, leading to lethal vascular leakage and bronchoconstriction. Treatment of mice with aspirin or loxoprofen inhibited the production of 12-HHT and increased the sensitivity toward PLY, which was also ameliorated by the CysLT1 antagonist. Thus, the present study identifies the molecular mechanism underlying PLY-dependent ALI and suggests the possible use of CysLT1 antagonists as a therapeutic tool to protect against ALI caused by pneumococcal infection.

  • Leukotriene B4 Receptor type 2 blt2 enhances skin barrier function by regulating tight junction proteins
    The FASEB Journal, 2016
    Co-Authors: Yumiko Ishii, Tomoaki Koga, Kazuko Saeki, Fumiyuki Sasaki, Keiko Kitajima, Chikara Meno, Toshiaki Okuno
    Abstract:

    GPCRs are involved in numerous physiologic functions and are important drug targets. Although the epithelial barrier is important for protection from invading pathogens, the correlation between GPC...

  • modulation of Leukotriene B4 Receptor 1 signaling by Receptor for advanced glycation end products rage
    The FASEB Journal, 2016
    Co-Authors: Takako Ichiki, Masakiyo Sakaguchi, Tomoaki Koga, Kazuko Saeki, Toshiaki Okuno, Yasuhiko Yamamoto, Hiroshi Yamamoto, Takehiko Yokomizo
    Abstract:

    Leukotriene B4 (LTB4) Receptor 1 (BLT1), a high-affinity GPCR for LTB4, plays important roles in acute and chronic inflammatory diseases. Although the LTB4-BLT1 axis is known to promote inflammatio...

  • modulation of Leukotriene B4 Receptor 1 signaling by Receptor for advanced glycation end products rage
    The FASEB Journal, 2016
    Co-Authors: Takako Ichiki, Masakiyo Sakaguchi, Tomoaki Koga, Kazuko Saeki, Toshiaki Okuno, Yasuhiko Yamamoto, Hiroshi Yamamoto, Takehiko Yokomizo
    Abstract:

    Leukotriene B4 (LTB4) Receptor 1 (BLT1), a high-affinity GPCR for LTB4, plays important roles in acute and chronic inflammatory diseases. Although the LTB4-BLT1 axis is known to promote inflammation, no studies have defined the binding proteins that modulate LTB4-BLT1 signaling. In this study, the Receptor for advanced glycation end products (RAGE) interacted with BLT1 in human cervical epithelial HeLa cells. RAGE increased LTB4-BLT1-dependent ERK phosphorylation and inhibited LTB4-BLT1-dependent activation of NF-κB and up-regulation of proinflammatory cytokines and chemokines. RAGE-dependent inhibition of NF-κB was blunted by treatment with an MEK inhibitor, suggesting that RAGE suppresses LTB4-BLT1-dependent NF-κB signaling by enhancing the MEK-ERK pathway. Meanwhile, in a chemotaxis assay of mouse bone marrow-derived neutrophils, the velocity of LTB4-dependent neutrophil migration was attenuated by soluble RAGE, which is an inhibitory decoy protein for RAGE signaling, in a dose-dependent manner (0.2-5 μg/ml), or by RAGE deficiency. Furthermore, both LTB4-dependent ERK phosphorylation in neutrophils and LTB4-dependent neutrophil accumulation in a murine peritonitis model were significantly attenuated in RAGE-deficient mice compared with C57BL/6J wild-type mice, indicating that RAGE potentiates LTB4-dependent neutrophil migration by enhancing ERK phosphorylation. Our results demonstrate that RAGE interacts with BLT1 and modulates LTB4-BLT1 signaling through potentiation of the MEK-ERK pathway.-Ichiki, T., Koga, T., Okuno, T., Saeki K., Yamamoto, Y., Yamamoto, H., Sakaguchi, M., Yokomizo, T. Modulation of Leukotriene B4 Receptor 1 signaling by Receptor for advanced glycation end products (RAGE).