ABSTRACT
Despite numerous biomarkers being proposed for rheumatoid arthritis (RA), a gap remains in our understanding of their mechanisms of action. In this study, we discovered a novel role for gelsolin (GSN), an actin-binding protein whose levels are notably reduced in the plasma of RA patients. We elucidated that GSN is a key regulator of NLRP3 inflammasome activation in macrophages, providing a plausible explanation for the decreased secretion of GSN in RA patients. We found that GSN interacts with NLRP3 in LPS-primed macrophages, hence modulating the formation of the NLRP3 inflammasome complex. Reducing GSN expression significantly enhanced NLRP3 inflammasome activation. GSN impeded NLRP3 translocation to the mitochondria; it contributed to the maintenance of intracellular calcium equilibrium and mitochondrial stability. This maintenance is crucial for controlling the inflammatory response associated with RA. Furthermore, the exacerbation of arthritic symptoms in GSN-deficient mice indicates the potential of GSN as both a diagnostic biomarker and a therapeutic target. Moreover, not limited to RA models, GSN has demonstrated a protective function in diverse disease models associated with the NLRP3 inflammasome. Myeloid cell-specific GSN-knockout mice exhibited aggravated inflammatory responses in models of MSU-induced peritonitis, folic acid-induced acute tubular necrosis, and LPS-induced sepsis. These findings suggest novel therapeutic approaches that modulate GSN activity, offering promise for more effective management of RA and a broader spectrum of inflammatory conditions.
ABSTRACT
Leukotriene B4 (LTB4) receptor 1 (BLT1) is a chemotactic G protein-coupled receptor expressed by leukocytes, such as granulocytes, macrophages, and activated T cells. Although there is growing evidence that BLT1 plays crucial roles in immune responses, its role in dendritic cells remains largely unknown. Here, we identified novel DC subsets defined by the expression of BLT1, namely, BLT1hi and BLT1lo DCs. We also found that BLT1hi and BLT1lo DCs differentially migrated toward LTB4 and CCL21, a lymph node-homing chemoattractant, respectively. By generating LTB4-producing enzyme LTA4H knockout mice and CD11c promoter-driven Cre recombinase-expressing BLT1 conditional knockout (BLT1 cKO) mice, we showed that the migration of BLT1hi DCs exacerbated allergic contact dermatitis. Comprehensive transcriptome analysis revealed that BLT1hi DCs preferentially induced Th1 differentiation by upregulating IL-12p35 expression, whereas BLT1lo DCs accelerated T cell proliferation by producing IL-2. Collectively, the data reveal an unexpected role for BLT1 as a novel DC subset marker and provide novel insights into the role of the LTB4-BLT1 axis in the spatiotemporal regulation of distinct DC subsets.
Subject(s)
Dendritic Cells/metabolism , Hypersensitivity/pathology , Inflammation/pathology , Receptors, Leukotriene B4/metabolism , Skin/pathology , Animals , Biomarkers/metabolism , Cell Differentiation/drug effects , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Chemokine CCL21/pharmacology , Dendritic Cells/drug effects , Dermatitis, Atopic/complications , Dermatitis, Atopic/immunology , Dermatitis, Atopic/pathology , Hypersensitivity/complications , Hypersensitivity/immunology , Inflammation/complications , Inflammation/immunology , Interleukin-12/biosynthesis , Leukotriene B4/metabolism , Lymph Nodes/drug effects , Mice, Inbred C57BL , Th1 Cells/drug effects , Th1 Cells/immunology , Transcriptome/geneticsABSTRACT
Receptor activator of nuclear factor-κB ligand (RANKL) is a key mediator of osteoclast differentiation and bone resorption. Osteoblast-lineage cells including osteoblasts and osteocytes express RANKL, which is regulated by several different factors, including hormones, cytokines, and mechanical forces. In vivo and in vitro analyses have demonstrated that various types of mechanosensing proteins on the cell membrane (i.e. mechanosensors) and intracellular mechanosignaling proteins play essential roles in the differentiation and functions of osteoblasts, osteoclasts, and osteocytes via soluble factors, such as sclerostin, Wnt ligands, and RANKL. This section provides an overview of the in vivo and in vitro evidence for the regulation of RANKL expression by mechanosensing and mechanotransduction.
Subject(s)
RANK Ligand/metabolism , Animals , Biomechanical Phenomena , Cellular Microenvironment , Humans , Mechanotransduction, Cellular , Models, Biological , Signal TransductionABSTRACT
Receptor activator of NF-κB (RANK) ligand (RANKL) induces the differentiation of monocyte/macrophage-lineage cells into the bone-resorbing cells called osteoclasts. Because abnormalities in RANKL, its signaling receptor RANK, or decoy receptor osteoprotegerin (OPG) lead to bone diseases such as osteopetrosis, the RANKL/RANK/OPG system is essential for bone resorption. RANKL was first discovered as a T cell-derived activator of dendritic cells (DCs) and has many functions in the immune system, including organogenesis, cellular development. The essentiality of RANKL in the bone and the immune systems lies at the root of the field of "osteoimmunology." Furthermore, this cytokine functions beyond the domains of bone metabolism and the immune system, e.g., mammary gland and hair follicle formation, body temperature regulation, muscle metabolism, and tumor development. In this review, we will summarize the current understanding of the functions of the RANKL/RANK/OPG system in biological processes.
ABSTRACT
Osteocytes function as critical regulators of bone homeostasis by coordinating the functions of osteoblasts and osteoclasts, and are constantly exposed to mechanical force. However, the molecular mechanism underlying the mechanical signal transduction in osteocytes is not well understood. Here, we found that Yoda1, a selective Piezo1 agonist, increased intracellular calcium mobilization and dose-dependently decreased the expression of Sost (encoding Sclerostin) in the osteocytic cell line IDG-SW3. We also demonstrated that mechanical stretch of IDG-SW3 suppressed Sost expression, a result which was abrogated by treatment with the Piezo1 inhibitor GsMTx4, and the deficiency of Piezo1. Furthermore, the suppression of Sost expression was abolished by treatment with an Akt inhibitor. Taken together, these results indicate that the activation of the Piezo1-Akt pathway in osteocytes is required for mechanical stretch-induced downregulation of Sost expression.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Ion Channels/metabolism , Mechanotransduction, Cellular , Osteocytes/cytology , Proto-Oncogene Proteins c-akt/metabolism , Animals , Down-Regulation , Mice , Osteocytes/metabolism , Signal TransductionABSTRACT
Leukotrienes (LTs) are inflammatory mediators derived from arachidonic acid. LTs include the di-hydroxy acid LT (LTB4) and the cysteinyl LTs (CysLTs; LTC4, LTD4 and LTE4), all of which are involved in both acute and chronic inflammation. We and other groups identified a high-affinity LTB4 receptor, BLT1; the LTC4 and LTD4 receptors, CysLT1 and CysLT2; and the LTE4 receptor, GPR99. Pharmacological studies have shown that BLT1 signaling stimulates degranulation, chemotaxis and phagocytosis of neutrophils, whereas CysLT1 and CysLT2 signaling induces airway inflammation by increasing vascular permeability and the contraction of bronchial smooth muscle. Recently, we and other groups suggested that the LTB4-BLT1 axis and the cysteinyl LTs-CysLT1/2 axis are involved in chronic inflammatory diseases including asthma, atopic dermatitis, psoriasis, atherosclerosis, arthritis, obesity, cancer and age-related macular degeneration using animal models for disease and gene knockout mice. This review describes the classical and novel functions of LTs and their receptors in several inflammatory diseases and discusses the potential clinical applications of antagonists for LT receptors and inhibitors of LT biosynthesis.
Subject(s)
Arachidonic Acid/pharmacology , Inflammation/drug therapy , Inflammation/metabolism , Receptors, Leukotriene/metabolism , Animals , HumansABSTRACT
Leukotriene B4 receptor 1 (BLT1) triggers the migration of granulocytes and activated T cells; however, its role in B-cell function remains unclear. Here we report that BLT1 is required to induce the production of antigen-specific IgA against oral vaccine through mediating innate immune signals from commensal bacteria. B cells acquire BLT1 expression during their differentiation to IgA+ B cells and plasma cells in Peyer's patches and the small intestinal lamina propria, respectively. BLT1 KO mice exhibited impaired production of antigen-specific fecal IgA to oral vaccine despite normal IgG responses to systemically immunized antigen. Expression of MyD88 was decreased in BLT1 KO gut B cells and consequently led to diminished proliferation of commensal bacteria-dependent plasma cells. These results indicate that BLT1 enhances the proliferation of commensal bacteria-dependent IgA+ plasma cells through the induction of MyD88 and thereby plays a key role in the production of antigen-specific intestinal IgA.
Subject(s)
Epitopes/immunology , Gastrointestinal Microbiome/immunology , Immunity, Innate , Immunoglobulin A, Secretory/immunology , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Receptors, Leukotriene B4/genetics , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Immunization , Intestinal Mucosa/microbiology , Male , Mice , Mice, Knockout , Myeloid Differentiation Factor 88/metabolism , Peyer's Patches/immunology , Peyer's Patches/metabolism , Plasma Cells/immunology , Plasma Cells/metabolism , Receptors, Leukotriene B4/metabolism , Signal Transduction , Vaccines/administration & dosage , Vaccines/immunologyABSTRACT
Bone undergoes a constant reconstruction process of resorption and formation called bone remodeling, so that it can endure mechanical loading. During food ingestion, masticatory muscles generate the required masticatory force. The magnitude of applied masticatory force has long been believed to be closely correlated with the shape of the jawbone. However, both the mechanism underlying this correlation and evidence of causation remain largely to be determined. Here, we established a novel mouse model of increased mastication in which mice were fed with a hard diet (HD) to elicit greater masticatory force. A novel in silico computer simulation indicated that the masticatory load onto the jawbone leads to the typical bone profile seen in the individuals with strong masticatory force, which was confirmed by in vivo micro-computed tomography (micro-CT) analyses. Mechanistically, increased mastication induced Insulin-like growth factor (IGF)-1 and suppressed sclerostin in osteocytes. IGF-1 enhanced osteoblastogenesis of the cells derived from tendon. Together, these findings indicate that the osteocytes balance the cytokine expression upon the mechanical loading of increased mastication, in order to enhance bone formation. This bone formation leads to morphological change in the jawbone, so that the bone adapts to the mechanical environment to which it is exposed.
Subject(s)
Mandible/physiology , Mastication/physiology , Osteocytes/physiology , Osteogenesis/physiology , Animals , Bite Force , Bone Remodeling/physiology , Computer Simulation , Diet/adverse effects , Insulin-Like Growth Factor I/metabolism , Mandible/metabolism , Mice , Mice, Inbred C57BL , Osteocytes/metabolism , Stress, Mechanical , X-Ray Microtomography/methodsABSTRACT
Age-related macular degeneration (AMD), a progressive chronic disease of the central retina, is associated with aging and is a leading cause of blindness worldwide. Here, we demonstrate that leukotriene B4 (LTB4) receptor 1 (BLT1) promotes laser-induced choroidal neovascularization (CNV) in a mouse model for wet-type AMD. CNV was significantly less in BLT1-deficient (BLT1-KO) mice compared with BLT1-WT controls. Expression of several proangiogenic and profibrotic factors was lower in BLT1-KO eyes than in BLT1-WT eyes. LTB4 production in the eyes was substantially increased in the early phase after laser injury. BLT1 was highly expressed in M2 macrophages in vitro and in vivo, and ocular BLT1+ M2 macrophages were increased in the aged eyes after laser injury. Furthermore, M2 macrophages were rapidly attracted by LTB4 and subsequently produced VEGF-A- through BLT1-mediated signaling. Consequently, intravitreal injection of M2 macrophages augmented CNV formation, which was attenuated by BLT1 deficiency. Thus, laser-induced injury to the retina triggered LTB4 production and attracted M2 macrophages via BLT1, leading to development of CNV. A selective BLT1 antagonist (CP105696) and 3 LTB4 inhibitors (zileuton, MK-886, and bestatin) reduced CNV in a dose-dependent manner. CP105696 also inhibited the accumulation of BLT1+ M2 macrophages in the laser-injured eyes of aged mice. Together, these results indicate that the LTB4-BLT1 axis is a potentially novel therapeutic target for CNV of wet-type AMD.
Subject(s)
Leukotriene B4/metabolism , Macrophages/metabolism , Macular Degeneration/metabolism , Neovascularization, Pathologic/metabolism , Receptors, Leukotriene B4/metabolism , Animals , Benzopyrans/pharmacology , Carboxylic Acids/pharmacology , Choroidal Neovascularization , Disease Models, Animal , Eye/radiation effects , Eye Injuries , Hydroxyurea/analogs & derivatives , Hydroxyurea/pharmacology , Indoles/pharmacology , Lasers/adverse effects , Leucine/analogs & derivatives , Leucine/pharmacology , Leukotriene B4/antagonists & inhibitors , Leukotriene B4/genetics , Macrophages/drug effects , Macular Degeneration/pathology , Male , Mice , Mice, Knockout , Neovascularization, Pathologic/drug therapy , Receptors, Leukotriene B4/genetics , Signal TransductionABSTRACT
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.
Subject(s)
Antibodies, Monoclonal, Murine-Derived/immunology , Leukotriene B4/immunology , Receptors, Leukotriene B4/antagonists & inhibitors , Animals , Antibodies, Monoclonal, Murine-Derived/chemistry , Antibodies, Monoclonal, Murine-Derived/pharmacology , CHO Cells , Calcium Signaling/drug effects , Cell Differentiation/immunology , Cricetinae , Cricetulus , Granulocytes/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Monocytes/immunology , Protein Structure, Secondary , Receptors, Leukotriene B4/chemistry , Receptors, Leukotriene B4/immunology , Th1 Cells/immunologyABSTRACT
DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues.
Subject(s)
Antibodies, Monoclonal/immunology , Immunoprecipitation/methods , Oligopeptides/immunology , Protein Engineering/methods , Animals , Antibodies, Monoclonal/genetics , Humans , MiceABSTRACT
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 GPCRs and epithelial barrier function remains unknown. Leukotriene B4 (LTB4) receptor type 2 (BLT2), mainly expressed in epithelial cells, is a GPCR for 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) and LTB4. In our study, BLT2 localized at the lateral membrane in BLT2-overexpressing Madin-Darby canine kidney (MDCK) II cells and in the small intestine of BLT2-transgenic mice. BLT2-deficient mice exhibited higher transepidermal water loss and were more sensitive to epicutaneous sensitization. MDCK-BLT2 cells recovered transepithelial electrical resistance (TER) after a calcium switch faster than did MDCK-Mock cells, and 12-HHT stimulation accelerated TER recovery only in MDCK-BLT2 cells. Quantitative PCR and immunoblot analyses revealed that the 12-HHT/BLT2 axis up-regulated claudin-4 (CLDN4) expression in MDCK-BLT2 cells and human primary keratinocytes, and CLDN4 knockdown abolished 12-HHT-dependent TER recovery. Acceleration of TER recovery and induction of CLDN4 expression by 12-HHT stimulation were abolished by inhibition of Gαi protein or p38 MAPK. These results show that 12-HHT/BLT2 enhances epithelial barrier function by increasing CLDN4 expression via the Gαi protein-p38 MAPK pathway.
Subject(s)
MAP Kinase Signaling System/physiology , Receptors, Leukotriene B4/metabolism , Skin/metabolism , Tight Junctions/metabolism , Animals , Claudin-4/biosynthesis , Claudin-4/genetics , Dogs , Fatty Acids, Unsaturated/pharmacology , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Humans , MAP Kinase Signaling System/drug effects , Madin Darby Canine Kidney Cells , Mice , Mice, Knockout , Receptors, Leukotriene B4/genetics , Skin/cytology , Tight Junctions/genetics , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Age is a major risk factor in age-related macular degeneration (AMD), but the underlying cause is unknown. We find increased Rho-associated kinase (ROCK) signaling and M2 characteristics in eyes of aged mice, revealing immune changes in aging. ROCK isoforms determine macrophage polarization into M1 and M2 subtypes. M2-like macrophages accumulated in AMD, but not in normal eyes, suggesting that these macrophages may be linked to macular degeneration. M2 macrophages injected into the mouse eye exacerbated choroidal neovascular lesions, while M1 macrophages ameliorated them, supporting a causal role for macrophage subtypes in AMD. Selective ROCK2 inhibition with a small molecule decreased M2-like macrophages and choroidal neovascularization. ROCK2 inhibition upregulated M1 markers without affecting macrophage recruitment, underlining the plasticity of these macrophages. These results reveal age-induced innate immune imbalance as underlying AMD pathogenesis. Targeting macrophage plasticity opens up new possibilities for more effective AMD treatment.
Subject(s)
Macrophages/metabolism , rho-Associated Kinases/metabolism , Aging , Animals , Bone Marrow Cells/cytology , Cell Differentiation/drug effects , Cell Polarity , Cells, Cultured , Choroid/blood supply , Choroidal Neovascularization , Cytokines/pharmacology , Humans , Macrophages/cytology , Macular Degeneration/metabolism , Macular Degeneration/pathology , Male , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Signal Transduction , rho-Associated Kinases/antagonists & inhibitorsABSTRACT
Leukotriene B4 (LTB4) has been implicated in the pathogenesis of allergic diseases. BLT2, a low-affinity LTB4 receptor, is activated by LTB4 and 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT). Although the high-affinity LTB4 receptor BLT1 has been shown to exert proinflammatory roles, the role of BLT2 in allergic inflammation has not been clarified. To study the function of BLT2 in development of asthma, we used mice model of ovalbumin (OVA)-induced allergic airway disease. The 12-HHT levels were elevated in bronchoalveolar lavage (BAL) fluids of OVA-sensitized/challenged wild-type mice. BLT2-deficient mice exhibited enhanced eosinophilia in BAL fluids after OVA exposure. Interleukin (IL)-13 levels in BAL fluids and IL-13-producing CD4(+) T cells in the lungs were elevated in BLT2-deficient mice compared to wild-type mice, whereas the levels of IL-4, IL-5, and interferon (IFN)-γ in BAL fluids and serum OVA-specific IgE were comparable. Transfection of BLT2-specific small interfering RNA enhanced IL-13 production in CD4(+) T cells in vitro. Expression of BLT2 mRNA in CD4(+) T cells was significantly reduced in patients with asthma compared to healthy control subjects. These findings indicate that BLT2 has a protective role in allergic airway inflammation and that diminished BLT2 expression in CD4(+) T cells may contribute to the pathophysiology of asthma.
Subject(s)
Asthma/immunology , Eosinophilia/immunology , Lung/immunology , Receptors, Leukotriene B4/immunology , Adult , Aged , Animals , Asthma/genetics , Asthma/metabolism , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CHO Cells , Calcium/immunology , Calcium/metabolism , Cricetinae , Cricetulus , Cytokines/immunology , Cytokines/metabolism , Eosinophilia/genetics , Eosinophilia/metabolism , Fatty Acids, Unsaturated/immunology , Fatty Acids, Unsaturated/metabolism , Humans , Leukotriene B4/immunology , Leukotriene B4/metabolism , Lung/metabolism , Lung/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , RNA Interference , Receptors, Leukotriene B4/deficiency , Receptors, Leukotriene B4/genetics , Reverse Transcriptase Polymerase Chain Reaction , Young AdultABSTRACT
BLT1 is a high-affinity receptor for leukotriene B4 (LTB4) that is a potent lipid chemoattractant for myeloid leukocytes. The role of LTB4/BLT1 axis in tumor immunology, including cytokine-based tumor vaccine, however, remains unknown. We here demonstrated that BLT1-deficient mice rejected subcutaneous tumor challenge of GM-CSF gene-transduced WEHI3B (WGM) leukemia cells (KO/WGM) and elicited robust antitumor responses against second tumor challenge with WEHI3B cells. During GM-CSF-induced tumor regression, the defective LTB4/BLT1 signaling significantly reduced tumor-infiltrating myeloid-derived suppressor cells, increased the maturation status of dendritic cells in tumor tissues, enhanced their CD4(+) T-cell stimulation capacity and migration rate of dendritic cells that had phagocytosed tumor-associated antigens into tumor-draining lymph nodes, suggesting a positive impact on GM-CSF-sensitized innate immunity. Furthermore, KO/WGM mice displayed activated adaptive immunity by attenuating regulatory CD4(+) T subsets and increasing numbers of Th17 and memory CD44(hi)CD4(+) T subsets, both of which elicited superior antitumor effects as evidenced by adoptive cell transfer. In vivo depletion assays also revealed that CD4(+) T cells were the main effectors of the persistent antitumor immunity. Our data collectively underscore a negative role of LTB4/BLT1 signaling in effective generation and maintenance of GM-CSF-induced antitumor memory CD4(+) T cells.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Leukemia, Experimental/immunology , Receptors, Leukotriene B4/immunology , Signal Transduction/immunology , Adaptive Immunity , Adoptive Transfer , Animals , CD4-Positive T-Lymphocytes/pathology , Cell Differentiation , Cell Line, Tumor , Cell Movement , Dendritic Cells/immunology , Dendritic Cells/pathology , Female , Gene Expression Regulation, Neoplastic/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Immunity, Innate , Immunologic Memory , Leukemia, Experimental/genetics , Leukemia, Experimental/pathology , Leukotriene B4/immunology , Leukotriene B4/metabolism , Lymphocyte Activation , Male , Mice , Mice, Knockout , Receptors, Leukotriene B4/deficiency , Receptors, Leukotriene B4/genetics , Signal Transduction/genetics , Transduction, GeneticABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/immunology , Blotting, Western , Microscopy, Confocal , Peptides/chemistry , Receptors, G-Protein-Coupled/immunology , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Cricetulus , Hybridomas/cytology , Hybridomas/metabolism , Immunoprecipitation , Mice , NIH 3T3 Cells , Oligopeptides , Receptors, G-Protein-Coupled/chemistryABSTRACT
The present study investigated the growth of human fibrosarcoma (HT-1080) and fibroblast (SF-TY) cells in combination with water-soluble (WS) and high molecular component (HMC) fractions prepared from Reishi (R), Rokkaku-Reishi (2R) and Apple Rokkaku-Reishi (A2R). Each WS fraction exhibited dose-and time-dependent inhibition of the growth of the HT-1080 and SF-TY cells. The extracts exhibited marked antiproliferative activity against the HT-1080 cells. The HMC fractions inhibited cell growth dose-and time-dependently in the HT-1080 cells only, and not in the SF-TY cells, suggesting that HMC fractions selectively inhibit HT-1080 cells. Among the HMC fractions, A2R is a strong candidate for anti-tumor targeting since its fraction exhibited better inhibition than the R and 2R fractions. Furthermore, the volume of the A2R fraction was approximately five times greater than that of the others, and included four proteins (molecular mass 9, 13, 22 and 40 kDa) detected by SDS-PAGE. Three of these (13, 22 and 40 kDa) were confirmed to be glycosylated with the Periodic Acid-Schiff Stain kit. These results suggest that A2R may possess anti-tumor activity and, in particular, that the protein components of A2R may act to selectively inhibit the growth of HT-1080 cells.