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1.
Biochem Biophys Res Commun ; 511(2): 318-322, 2019 04 02.
Article in English | MEDLINE | ID: mdl-30803755

ABSTRACT

The Kururu no ß-glu® (KBG) is a commercial hydrothermal-treated Aureobasidium pullulans ß-glucan produced by a unique hydrothermal process that results in high solubility of the ß-glucan. In this study, we examined the biological activities of this reagent. RAW264.7 cells do not express Dictin-1 on the cell surface, but cells still respond to various pathogen molecular patterns. Lipopolysaccharide (LPS) induced nitrogen oxide (NO) synthesis and TNF-α production in RAW264.7 cells, and those were suppressed by KBG in a dose-dependent manner. The major signaling cell surface receptor respond to LPS is the TLR4/MD-2 complex. The UT12 antibody against to the TLR4/MD-2 complex mimics LPS function and induces cell responses. NO generation and TNF-α production were similarly induced in cells by stimulation with the antibody, but those were not suppressed by KBG. Cell responses induced by other TLR ligands, such as CPG (TLR9 ligand) and Pam3CSK4 (TLR1/TLR2 ligand), were also suppressed by KBG. Therefore, the target molecule for KBG is different from TLR receptors and Dictin-1. Although we also examined the suppressive activities of several other ß-glucan products, comparable activities were not detected with other reagents. A unique hydrothermal process may produce the active reagent. Reprocessing KBG increased low molecular weight fractions, and suppressive activities were markedly enhanced. Therefore, low molecular weight fractions obtained by hydrothermal processing of KBG may result in potential reagents that control inflammation induced by various pathogens.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Ascomycota , Macrophages/drug effects , Toll-Like Receptors/immunology , beta-Glucans/pharmacology , Animals , Anti-Inflammatory Agents/chemistry , Ascomycota/chemistry , Inflammation/drug therapy , Inflammation/immunology , Lipopolysaccharides/immunology , Macrophages/immunology , Mice , RAW 264.7 Cells , beta-Glucans/chemistry
3.
Infect Immun ; 84(7): 1986-1993, 2016 07.
Article in English | MEDLINE | ID: mdl-27091927

ABSTRACT

Chronic lower respiratory tract infection with Pseudomonas aeruginosa is difficult to treat due to enhanced antibiotic resistance and decreased efficacy of drug delivery to destroyed lung tissue. To determine the potential for restorative immunomodulation therapies, we evaluated the effect of Toll-like receptor 4 (TLR4) stimulation on the host immune response to Pseudomonas infection in mice. We implanted sterile plastic tubes precoated with P. aeruginosa in the bronchi of mice, administered the TLR4/MD2 agonistic monoclonal antibody UT12 intraperitoneally every week, and subsequently analyzed the numbers of viable bacteria and inflammatory cells and the levels of cytokines. We also performed flow cytometry-based phagocytosis and opsonophagocytic killing assays in vitro using UT12-treated murine peritoneal neutrophils. UT12-treated mice showed significantly enhanced bacterial clearance, increased numbers of Ly6G(+) neutrophils, and increased concentrations of macrophage inflammatory protein 2 (MIP-2) in the lungs (P < 0.05). Depletion of CD4(+) T cells eliminated the ability of the UT12 treatment to improve bacterial clearance and promote neutrophil recruitment and MIP-2 production. Additionally, UT12-pretreated peritoneal neutrophils exhibited increased opsonophagocytic killing activity via activation of the serine protease pathway, specifically neutrophil elastase activity, in a TLR4-dependent manner. These data indicated that UT12 administration significantly augmented the innate immune response against chronic bacterial infection, in part by promoting neutrophil recruitment and bactericidal function.


Subject(s)
Antibodies, Monoclonal/pharmacology , Immunity, Innate/drug effects , Pneumonia, Bacterial/immunology , Pneumonia, Bacterial/microbiology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/immunology , Toll-Like Receptor 4/agonists , Animals , Bacterial Load , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Chronic Disease , Cytokines/metabolism , Disease Models, Animal , Female , Mice , Mice, Knockout , Microbial Viability/immunology , Neutrophils/immunology , Neutrophils/metabolism , Neutrophils/microbiology , Opsonin Proteins/immunology , Phagocytosis/immunology , Pneumonia, Bacterial/drug therapy , Pneumonia, Bacterial/metabolism , Serine Proteases/metabolism
4.
J Immunol ; 190(1): 195-204, 2013 Jan 01.
Article in English | MEDLINE | ID: mdl-23203928

ABSTRACT

LPS is recognized by TLR4 and radioprotective 105 kDa in B cells. Susceptibility to LPS in murine B cells is most closely linked to the locus containing the TLR4 gene. However, the molecular mechanism underlying genetic control of LPS sensitivity by this locus has not been fully elucidated. In this study, we revealed that C57BL/6 (B6) B cells respond to mAb-induced, TLR4-specific signals stronger than BALB/c (BALB) B cells, as assessed by proliferation and upregulation of CD69 and CD86. In contrast, BALB B cells were not hyporesponsive to agonistic anti-radioprotective 105 kDa mAb or the TLR9 agonist CpG. Although the level of TLR4 mRNA in BALB B cells was comparable with that in B6 B cells, surface TLR4 expression in BALB B cells was lower than that in B6 B cells. This lower surface expression of BALB TLR4 was also observed when HEK293 and Ba/F3 cells were transfected with a BALB TLR4 expression construct. We identified a V254I mutation as the responsible single nucleotide polymorphism for lower surface expression of BALB TLR4. Furthermore, cotransfection of myeloid differentiation factor-2 increased BALB TLR4 expression, although it was still lower than B6 TLR4 expression. In concordance with reduced expression, Ba/F3 cells transfected with BALB TLR4 and myeloid differentiation factor-2 were hyporesponsive compared with those with B6 TLR4, as assessed by LPS-induced NF-κB activation. In conclusion, we revealed that LPS sensitivity is genetically controlled by the level of surface TLR4 expression on B cells. A V254I mutation accounts for the LPS hyporesponsive phenotype of BALB B cells.


Subject(s)
B-Lymphocyte Subsets/immunology , Lipopolysaccharides/genetics , Point Mutation/immunology , Toll-Like Receptor 4/antagonists & inhibitors , Toll-Like Receptor 4/biosynthesis , Animals , B-Lymphocyte Subsets/metabolism , Cell Line, Tumor , Cell Membrane/genetics , Cell Membrane/immunology , Cells, Cultured , HEK293 Cells , Humans , Immunophenotyping , Lipopolysaccharides/biosynthesis , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Rats , Rats, Wistar , Toll-Like Receptor 4/deficiency
5.
Int Immunol ; 25(2): 117-28, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23075507

ABSTRACT

Stimulation of Toll-like receptor 4 (TLR4) induces not only innate but also adaptive immune responses, and has been suggested to exert adjuvant effects. Additional to such positive effects, pre-stimulation of TLR4 induces endotoxin tolerance where animals are unresponsive to subsequent lethal challenges with lipopolysaccharide (LPS). We examined the effects of pre-stimulation of TLR4 using an agonistic anti-TLR4 mAb (UT12) on antibody production in vivo. Pre-injection of UT12 prior to both primary and secondary immunization completely inhibited antigen-specific antibody responses. Cellular analysis revealed that the inhibition was not due to impairment of T-cell activation. Accordingly, T-helper activities in UT12 pre-injected mice were not impaired. In contrast, B-cell priming was defective in UT12 pre-injected mice. The observation that the expression of activation markers such as CD69 and CD86 on B cells was blocked by UT12 pre-injection supports this. Interestingly, UT12 pre-injection only showed inhibitory effects at the primary and not the secondary immunization. These results provide important information concerning the regulatory mechanisms of antibody production, especially in endotoxin-tolerant states.


Subject(s)
Antibody Formation/immunology , Antigens/immunology , B-Lymphocytes/immunology , Toll-Like Receptor 4/agonists , Adjuvants, Immunologic , Animals , Antibodies, Monoclonal/immunology , Antibody Specificity , Antigen Presentation/immunology , Antigen-Antibody Reactions , Cross-Priming/immunology , Female , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Toll-Like Receptor 4/immunology
6.
Int Immunol ; 24(8): 495-506, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22499954

ABSTRACT

Recognition of LPS by the toll-like receptor 4 (TLR4)/MD-2 complex is a trigger of innate immune defense against bacterial invasion. However, excessive immune activation by this receptor complex causes septic shock and autoimmunity. Manipulation of TLR4 signaling represents a potential therapy that would avoid the detrimental consequences of unnecessary immune responses. In this study, we established two novel mAbs that inhibit LPS-induced human TLR4 activation. HT52 and HT4 mAbs inhibited LPS-induced nuclear factor-κB activation in TLR4/MD-2-expressing Ba/F3-transfected cells and cytokine production and up-regulation of CD86 in the human cell line U373 and PBMCs. These inhibitory activities were stronger than that of HTA125 mAb, which we previously reported. Immunofluorescent and biochemical studies using TLR4 deletion mutants revealed that HT52 and HT4 recognized spatially distinct regions on TLR4 irrespective of MD-2 association. The HT52 and HTA125 epitopes were localized within aa 50-190, while the HT4 epitope was formed only by the full length of TLR4. In addition, we demonstrated that HT52 and HT4 failed to compete with LPS for binding to TLR4/MD-2 but inhibited LPS-induced TLR4 internalization. Inhibitory activities were not due to the interaction with the Fcγ receptor CD32. Our finding that binding of mAbs to at least two distinct regions on TLR4 inhibits LPS-dependent activation provides a novel method for manipulating TLR4 activation and also a rationale for designing drugs targeted to TLR4.


Subject(s)
Antibodies, Monoclonal/immunology , Immunity, Innate/immunology , Toll-Like Receptor 4/immunology , Animals , Antibodies, Monoclonal/pharmacology , Blotting, Western , Cell Line , Enzyme Activation/drug effects , Enzyme Activation/immunology , Flow Cytometry , Fluorescent Antibody Technique , Humans , Immunoprecipitation , Lipopolysaccharides/immunology , Lymphocyte Antigen 96/drug effects , Lymphocyte Antigen 96/immunology , Lymphocyte Antigen 96/metabolism , Mice , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Signal Transduction/immunology , Toll-Like Receptor 4/drug effects , Toll-Like Receptor 4/metabolism , Transfection
7.
Int Immunol ; 22(4): 271-80, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20133493

ABSTRACT

Toll-like receptor (TLR) 4/MD-2 dimerization is thought to be required for the initiation of signaling during innate immune responses. In this study, we examined the molecular mechanisms underlying receptor dimerization in the context of accessory molecules, i.e. CD14 and lipopolysaccharide-binding protein (LBP), to determine whether dimerization is required for the initiation of signaling in response to LPS stimulation. We found that LPS-induced TLR4/MD-2 dimerization occurred only in membrane-associated CD14 (mCD14)-expressing cells. Furthermore, dimerization required LBP, but not soluble CD14 (sCD14), as an essential serum component. LPS-induced signaling as assessed by IkappaB-alpha degradation, however, occurred in mCD14-negative cells in the presence of serum and sCD14. Signaling also occurred in mCD14-positive cells in the absence of serum. Time course studies on mCD14-positive cells have demonstrated that LPS stimulation induces rapid activation of nuclear factor-kappaB and p38 in the presence of LBP (TLR4/MD-2 receptor dimerization) as compared with stimulation without LBP (receptor non-dimerization). This early activation was blocked by inhibitory anti-CD14 mAb. These studies suggest that LPS-induced TLR4/MD-2 receptor dimerization is not essential for signaling but prompts rapid signaling during innate immune responses.


Subject(s)
Acute-Phase Proteins/metabolism , Carrier Proteins/metabolism , Lipopolysaccharide Receptors/metabolism , Lipopolysaccharides/immunology , Lymphocyte Antigen 96/metabolism , Membrane Glycoproteins/metabolism , Toll-Like Receptor 4/metabolism , Animals , Cell Line , Cell Membrane/metabolism , Down-Regulation , Humans , Immunity, Innate , Mice , NF-kappa B/metabolism , Protein Multimerization , Rats , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolism
8.
FASEB J ; 24(3): 873-81, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19858095

ABSTRACT

Activated protein C (APC) is an anticoagulant, approved as a treatment for severe sepsis, that can prevent apoptosis, inflammation, and vascular leakage. The aim of this study was to investigate whether APC protects endothelial barrier function through the angiopoietin (Ang)/Tie2 axis. APC significantly up-regulated gene and protein expression of Tie2 and Ang1 in a dose (0.01-10 microg/ml)- and time (0.5-24 h)-dependent manner in human umbilical vein endothelial cells (HUVECs). Interestingly, it markedly inhibited Ang2 with an IC(50) of approximately 0.1 microg/ml. HUVEC permeability, measured using Evans blue dye transfer, was significantly reduced in the presence of APC, and, in concordance, the tight junction associated protein zona occludens (ZO)-1 was up-regulated and localized peripherally around cells, compared with controls. Smooth muscle cell migration toward APC-stimulated HUVECs was elevated compared with unstimulated cells. Blocking antibodies and small interfering (si) RNA treatment, compared with isotype (IgG) or scrambled siRNA controls, showed that APC requires 3 receptors, the endothelial protein C receptor, protease-activated receptor 1, and Tie2 to perform all these barrier stabilization functions. In summary, this study demonstrates that APC has novel effects on the Ang/Tie2 axis, which enhance endothelial barrier function and are likely to contribute to its therapeutic effect in sepsis and other diseases associated with vascular leakage.-Minhas, N., Xue, M., Fukudome, K., Jackson, C. J. Activated protein C utilizes the angiopoietin/Tie2 axis to promote endothelial barrier function.


Subject(s)
Angiopoietins/metabolism , Anticoagulants/pharmacology , Membrane Proteins/metabolism , Phosphoproteins/metabolism , Protein C/pharmacology , Receptor, TIE-2/metabolism , Antigens, CD/physiology , Cell Movement/drug effects , Cells, Cultured , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Protein C Receptor , Enzyme-Linked Immunosorbent Assay , Gene Expression/drug effects , Humans , Immunoblotting , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , RNA, Small Interfering , Receptor, PAR-1/antagonists & inhibitors , Receptor, PAR-1/physiology , Receptors, Cell Surface/antagonists & inhibitors , Receptors, Cell Surface/physiology , Reverse Transcriptase Polymerase Chain Reaction , Umbilical Veins/cytology , Zonula Occludens-1 Protein
9.
Nat Med ; 9(3): 338-42, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12563316

ABSTRACT

Activated protein C (APC) is a systemic anti-coagulant and anti-inflammatory factor. It reduces organ damage in animal models of sepsis, ischemic injury and stroke and substantially reduces mortality in patients with severe sepsis. It was not known whether APC acts as a direct cell survival factor or whether its neuroprotective effect is secondary to its anti-coagulant and anti-inflammatory effects. We report that APC directly prevents apoptosis in hypoxic human brain endothelium through transcriptionally dependent inhibition of tumor suppressor protein p53, normalization of the pro-apoptotic Bax/Bcl-2 ratio and reduction of caspase-3 signaling. These mechanisms are distinct from those involving upregulation of the genes encoding the anti-apoptotic Bcl-2 homolog A1 and inhibitor of apoptosis protein-1 (IAP-1) by APC in umbilical vein endothelial cells. Cytoprotection of brain endothelium by APC in vitro required endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1), as did its in vivo neuroprotective activity in a stroke model of mice with a severe deficiency of EPCR. This is consistent with work showing the direct effects of APC on cultured cells via EPCR and PAR-1 (ref. 9). Moreover, the in vivo neuroprotective effects of low-dose mouse APC seemed to be independent of its anti-coagulant activity. Thus, APC protects the brain from ischemic injury by acting directly on brain cells.


Subject(s)
Apoptosis , Blood Coagulation Factors/metabolism , Brain Ischemia/physiopathology , Brain/blood supply , Endothelium, Vascular/metabolism , Neuroprotective Agents/metabolism , Protein C/metabolism , Receptors, Cell Surface/metabolism , Tumor Suppressor Protein p53/metabolism , Animals , Antibodies/metabolism , Brain/metabolism , Caspase 3 , Caspases/metabolism , Cells, Cultured , Endothelium, Vascular/cytology , Humans , In Situ Nick-End Labeling , Mice , Oligopeptides , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptor, PAR-1 , Receptors, Thrombin/genetics , Receptors, Thrombin/metabolism , Tumor Suppressor Protein p53/genetics , bcl-2-Associated X Protein
10.
Pathol Int ; 60(4): 259-67, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20403027

ABSTRACT

The precise role of bone marrow adipose tissue (BMAT) in the marrow remains unknown. The purpose of the present study was therefore to describe a novel method for studying BMAT using 3-D collagen gel culture of BMAT fragments, immunohistochemistry, ELISA and real-time reverse transcription-polymerase chain reaction. Mature adipocytes and CD45+ leukocytes were retained for >3 weeks. Bone marrow stromal cells (BMSC) including a small number of lipid-laden preadipocytes and CD44+/CD105+ mesenchymal stem cell (MSC)-like cells, developed from BMAT. Dexamethasone (10 micromol/L), but not insulin (20 mU/mL), significantly increased the number of preadipocytes. Dexamethasone and insulin also promoted leptin production and gene expression in BMAT. Adiponectin production by BMAT was <0.8 ng/mL under all culture conditions. Dexamethasone promoted adiponectin gene expression, while insulin inhibited it. This finding suggests that dexamethasone, but not insulin, may serve as a powerful adipogenic factor for BMAT, in which adiponectin protein secretion is normally very low, and that BMAT may exhibit a different phenotype from that of the visceral and subcutaneous adipose tissues. BMAT-osteoblast interactions were also examined, and it was found that osteoblasts inhibited the development of BMSC and reduced leptin production, while BMAT inhibited the growth and differentiation of osteoblasts. The present novel method proved to be useful for the study of BMAT biology.


Subject(s)
Adipocytes/metabolism , Adipose Tissue/metabolism , Bone Marrow Cells/metabolism , Bone Marrow/metabolism , Adipocytes/drug effects , Adiponectin/genetics , Adiponectin/metabolism , Adipose Tissue/cytology , Adipose Tissue/drug effects , Bone Marrow/drug effects , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Cell Count , Cell Differentiation/drug effects , Cell Proliferation , Cells, Cultured , Dexamethasone/pharmacology , Enzyme-Linked Immunosorbent Assay , Gene Expression/drug effects , Gene Expression/physiology , Humans , Immunohistochemistry , Insulin/pharmacology , Leptin/genetics , Leptin/metabolism , Organ Culture Techniques , Reverse Transcriptase Polymerase Chain Reaction
11.
Haematologica ; 94(3): 387-94, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19211643

ABSTRACT

BACKGROUND: The endothelial protein C receptor plays an important role within the protein C pathway in regulating coagulation and inflammation. Recently, we described that endothelial protein C receptor can be released in vitro in microparticulate form from primary endothelial cells by exogenous activated protein C. Activated protein C bound to this endothelial protein C receptor retains anticoagulant activity and we hypothesize that this microparticulate endothelial protein C receptor-activated protein C complex can also cleave endothelial protease-activated receptor 1 to modulate inflammation and increase cell survival. Our main objective was, therefore, to study the effect that microparticle-associated endothelial protein C receptor-activated protein C has on endothelial function. DESIGN AND METHODS: Mini-arrays were used and probed with cDNA obtained from endothelial cells after treatment with microparticle-associated endothelial protein C receptor-activated protein C and results were confirmed by real time polymerase chain reaction. The functional relevance of changes at gene level were further analyzed by endothelial apoptosis and permeability assays, in the presence and absence of specific blockade of endothelial protein C receptor, protein C and protease-activated receptor 1. RESULTS: Gene profiling of endothelial cells stimulated by 40 nmol/L activated protein C on microparticles showed significant changes in anti-apoptotic and inflammatory pathways. This was accompanied by protease-activated receptor 1-dependent anti-apoptotic and barrier protective effects, the latter of which also involved sphingosine 1-phosphate receptor and vascular endothelial growth factor receptor-2/ kinase insert domain receptor. Protein C blockade reversed these effects showing specificity for activated protein C on microparticles. Furthermore, confocal microscopy and enzyme-linked immunosorbent assay of plasma obtained from septic patients during recombinant activated protein C treatment showed evidence of their presence in vivo. CONCLUSIONS: Activated protein C on microparticle-associated endothelial protein C receptor release can induce protease-activated receptor 1-dependent endothelial effects. The mechanisms underlying barrier protection involve sphingosine 1-phosphate receptor and kinase insert domain receptor.


Subject(s)
Antigens, CD/metabolism , Cell Membrane/metabolism , Endothelial Cells/metabolism , Protein C/metabolism , Receptors, Cell Surface/metabolism , Apoptosis/drug effects , Cell Membrane/chemistry , Cells, Cultured , Cytoprotection/drug effects , Endothelial Cells/drug effects , Endothelial Cells/ultrastructure , Endothelial Protein C Receptor , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Expression Profiling , Humans , Inflammation/prevention & control , Microscopy, Confocal , Microscopy, Electron , Oligonucleotide Array Sequence Analysis , Particle Size , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Binding , Protein C/pharmacology , Receptors, Lysosphingolipid/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sepsis/prevention & control , Vascular Endothelial Growth Factor Receptor-2/metabolism
12.
Ann Rheum Dis ; 66(12): 1574-80, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17491095

ABSTRACT

OBJECTIVES: (1) To investigate whether inflammatory synovial tissues from patients with rheumatoid arthritis (RA) express endothelial protein C receptor (EPCR) and (2) to determine the major cell type(s) that EPCR is associated with and whether EPCR functions to mediate the effects of activated protein C (APC) on these cells. METHODS: EPCR, CD68 and PC/APC in synovial tissues were detected by immunostaining and in situ PCR. Monocytes were isolated from peripheral blood of patients with RA and treated with APC, lipopolysaccharide (LPS), and/or EPCR blocking antibody RCR252. Cells and supernatants were collected for RT-PCR, western blotting, enzyme-linked immuosorbent assay and chemotaxis assay. RESULTS: EPCR was expressed by both OA and RA synovial tissues but was markedly increased in RA synovium. EPCR was colocalised with PC/APC mostly on CD68 positive cells in synovium. In RA monocytes, APC upregulated EPCR expression and reduced monocyte chemoattractant protein-1-induced chemotaxis of monocytes by approximately 50%. APC also completely suppressed LPS-stimulated NF-kappaB activation and attenuated TNF-alpha protein by more than 40% in RA monocytes. The inhibitory effects of APC were reversed by RCR252, indicating that EPCR is required. CONCLUSIONS: Our results demonstrate for the first time that EPCR is expressed by synovial tissues, particularly in RA, where it co-localises with PC/APC on monocytes/macrophages. In addition, APC inhibits the migration and activation of RA monocytes via EPCR. These inhibitory effects on RA monocytes suggest that PC pathway may have a beneficial therapeutic effect in RA.


Subject(s)
Antigens, CD/metabolism , Arthritis, Rheumatoid/metabolism , Endothelium, Vascular/metabolism , Monocytes/metabolism , Protein C/metabolism , Receptors, Cell Surface/metabolism , Synovial Membrane/metabolism , Activated Protein C Resistance , Adult , Analysis of Variance , Antigens, CD/analysis , Antigens, Differentiation, Myelomonocytic/analysis , Blotting, Western/methods , Chemokine CCL2/pharmacology , Chemotaxis, Leukocyte , Endothelial Protein C Receptor , Endothelium, Vascular/immunology , Female , Humans , Immunohistochemistry , Lipopolysaccharides/pharmacology , Male , Middle Aged , NF-kappa B/analysis , NF-kappa B/metabolism , Osteoarthritis/metabolism , Protein C/analysis , Receptors, Cell Surface/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Synovial Membrane/chemistry , Synovial Membrane/immunology , Tumor Necrosis Factor-alpha/analysis
13.
Circ Res ; 95(1): 34-41, 2004 Jul 09.
Article in English | MEDLINE | ID: mdl-15166095

ABSTRACT

Activated protein C (APC), a natural anticoagulant, has recently been demonstrated to activate the mitogen-activated protein kinase (MAPK) pathway in endothelial cells in vitro. Because the MAPK pathway is implicated in endothelial cell proliferation, it is possible that APC induces endothelial cell proliferation, thereby causing angiogenesis. We examined this possibility in the present study. APC activated the MAPK pathway, increased DNA synthesis, and induced proliferation in cultured human umbilical vein endothelial cells dependent on its serine protease activity. Antibody against the endothelial protein C receptor (EPCR) inhibited these events. Early activation of the MAPK pathway was inhibited by an antibody against protease-activated receptor-1, whereas neither late and complete activation of the MAPK pathway nor endothelial cell proliferation were inhibited by this antibody. APC activated endothelial nitric oxide synthase (eNOS) via phosphatidylinositol 3-kinase-dependent phosphorylation, followed by activation of protein kinase G, suggesting that APC bound to EPCR might activate the endothelial MAPK pathway by a mechanism similar to that of VEGF. APC induced morphogenetic changes resembling tube-like structures of endothelial cells, whereas DIP-APC did not. When applied topically to the mouse cornea, APC clearly induced angiogenesis in wild-type mice, but not in eNOS knockout mice. These in vitro events induced by APC might at least partly explain the angiogenic activity in vivo. This angiogenic activity of APC might contribute to maintain proper microcirculation in addition to its antithrombotic activity.


Subject(s)
Endothelium, Vascular/enzymology , Extracellular Signal-Regulated MAP Kinases/metabolism , MAP Kinase Signaling System , Neovascularization, Physiologic , Protein C/pharmacology , Animals , Antibodies/pharmacology , Blood Coagulation Factors/immunology , Cell Proliferation , Cells, Cultured , Cyclic GMP/metabolism , DNA/biosynthesis , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Humans , MAP Kinase Signaling System/drug effects , Mice , Nitric Oxide/biosynthesis , Protein C/physiology , Protein Kinase Inhibitors/pharmacology , Receptor, PAR-1/immunology , Receptors, Cell Surface/immunology
14.
J Invest Dermatol ; 125(6): 1279-85, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16354200

ABSTRACT

Activated protein C (APC) is a natural anticoagulant and inhibitor of inflammation that can stimulate keratinocyte wound repair in vitro and promote wound healing in vivo. The signaling mechanisms, however, are unknown and a keratinocyte receptor for APC has not been identified. Here, we show that cultured human keratinocytes from neonatal foreskins express the endothelial protein C receptor (EPCR). EPCR was also strongly expressed by lower epidermal layers of neonatal foreskin as determined by immunohistochemistry. In cultured keratinocytes, EPCR expression was upregulated by the addition of APC and inhibited by tumor necrosis factor-alpha. Addition of APC stimulated cell proliferation, production of matrix metalloproteinase-2, activation of ERK and p38 kinase signaling pathways, and expression of protease-activated receptor (PAR)-1. A monoclonal antibody, RCR252, which blocks APC binding to EPCR, or a blocking antibody to PAR-1, abolished APC's effects on keratinocytes. In summary, this study demonstrates that EPCR, a major receptor of protein C pathway, is expressed by human keratinocytes, and facilitates APC's function on keratinocytes via activation of PAR-1 pathway. Our findings highlight a possible new role for the protein C pathway in skin physiology and help elucidate the mechanisms of action by which APC promotes wound healing.


Subject(s)
Antigens/metabolism , Blood Coagulation Factors/metabolism , Glycoproteins/metabolism , Keratinocytes/physiology , Protein C/pharmacology , Receptor, PAR-1/metabolism , Receptors, Cell Surface/metabolism , Wound Healing/physiology , Antigens/genetics , Antigens, CD , Blood Coagulation Factors/genetics , Blotting, Western , Cell Division , Cell Survival/drug effects , Cells, Cultured , Endothelial Protein C Receptor , Glycoproteins/genetics , Humans , Infant, Newborn , Keratinocytes/cytology , Keratinocytes/drug effects , RNA, Messenger/genetics , Receptor, PAR-1/drug effects , Receptor, PAR-1/genetics , Receptors, Cell Surface/genetics , Tumor Necrosis Factor-alpha/pharmacology , Wound Healing/drug effects
15.
Thromb Haemost ; 93(4): 743-50, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15841323

ABSTRACT

Prostacyclin (PGI(2)) has beneficial cytoprotective properties, is a potent inhibitor of platelet aggregation and has been reported to improve microcirculatory blood flow during sepsis. The formation of PGI(2) in response to proinflammatory cytokines is catalysed by the inducible cyclooxygenase (COX) isoform COX-2. Recombinant human activated protein C (rhAPC, drotrecogin alfa (activated)) was shown to have multiple biological activities in vitro and to promote resolution of organ dysfunction in septic patients. Whether rhAPC exerts its beneficial effects by modulating prostanoid generation is unknown up to now. It was therefore the aim of the study to examine the in vitro effect of rhAPC on COX-2-mRNA-expression and PGI(2) release from human umbilical vein endothelial cells (HUVEC). We found that rhAPC, at supra-therapeutical concentrations (500 ng/ml-20 microg/ml), upregulated the amount of COX-2-mRNA in HUVEC at t=3-9 h and caused a time- and dose-dependent release of 6-keto PGF(1 alpha), the stable hydrolysis product of prostacyclin. RhAPC further increased the stimulating effect of tumor necrosis factor-alpha (TNF-alpha) and thrombin on COX-2-mRNA-levels. Transcript levels of cyclooxygenase-1 (COX-1) and prostaglandin 12 synthase, however, were unaffected by the stimulation with rhAPC or thrombin. The upregulatory effect on COX2-mRNA levels was specific for rhAPC since the zymogen protein C in equimolar concentrations had no effect on COX-2-mRNA-levels or 6-keto PGF(1 alpha)-release. Western Blot analysis revealed an increase of COX-2-protein content in HUVEC after treatment with rhAPC. As shown by experiments using monoclonal antibodies against the thrombin receptor PAR-1 (mAb=ATAP2) and against the endothelial protein C receptor (EPCR; mAb=RCR-252), the effect of rhAPC on COX-2-mRNA upregulation was mediated by binding to the EPCR-receptor and signaling via PAR-1. These results demonstrate that induction of COX-2-expression is an important response of HUVEC to stimulation with rhAPC and may represent a new molecular mechanism, by which rhAPC promotes upregulation of prostanoid production in human endothelium.


Subject(s)
Blood Coagulation Factors/metabolism , Endothelium, Vascular/metabolism , Prostaglandin-Endoperoxide Synthases/genetics , Protein C/pharmacology , Receptor, PAR-1/metabolism , Receptors, Cell Surface/metabolism , Up-Regulation/drug effects , Cells, Cultured , Cyclooxygenase 2 , Endothelium, Vascular/cytology , Epoprostenol/metabolism , Humans , Membrane Proteins , Prostaglandin-Endoperoxide Synthases/drug effects , Prostaglandins/metabolism , Protein Binding , RNA, Messenger/drug effects , Recombinant Proteins , Thrombin/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Up-Regulation/genetics
16.
Clin Vaccine Immunol ; 20(7): 977-85, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23637040

ABSTRACT

Coinfection with bacteria is a major cause of mortality during influenza epidemics. Recently, Toll-like receptor (TLR) agonists were shown to have immunomodulatory functions. In the present study, we investigated the effectiveness and mechanisms of the new TLR4 agonistic monoclonal antibody UT12 against secondary pneumococcal pneumonia induced by coinfection with influenza virus in a mouse model. Mice were intranasally inoculated with Streptococcus pneumoniae 2 days after influenza virus inoculation. UT12 was intraperitoneally administered 2 h before each inoculation. Survival rates were significantly increased and body weight loss was significantly decreased by UT12 administration. Additionally, the production of inflammatory mediators was significantly suppressed by the administration of UT12. In a histopathological study, pneumonia in UT12-treated mice was very mild compared to that in control mice. UT12 increased antimicrobial defense through the acceleration of macrophage recruitment into the lower respiratory tract induced by c-Jun N-terminal kinase (JNK) and nuclear factor kappaB (NF-κB) pathway-dependent monocyte chemoattractant protein 1 (MCP-1) production. Collectively, these findings indicate that UT12 promoted pulmonary innate immunity and may reduce the severity of severe pneumonia induced by coinfection with influenza virus and S. pneumoniae. This immunomodulatory effect of UT12 improves the prognosis of secondary pneumococcal pneumonia and makes UT12 an attractive candidate for treating severe infectious diseases.


Subject(s)
Antibodies, Bacterial/immunology , Coinfection/immunology , Immunity, Innate , Orthomyxoviridae Infections/immunology , Pneumococcal Infections/immunology , Pneumonia/immunology , Toll-Like Receptor 4/agonists , Animals , Body Weight , Coinfection/microbiology , Coinfection/pathology , Coinfection/virology , Disease Models, Animal , Histocytochemistry , Humans , Male , Mice , Mice, Inbred C3H , Mice, Inbred CBA , Orthomyxoviridae/immunology , Orthomyxoviridae Infections/pathology , Pneumococcal Infections/pathology , Pneumonia/microbiology , Pneumonia/pathology , Pneumonia/virology , Streptococcus pneumoniae/immunology , Survival Analysis
17.
Thromb Haemost ; 103(6): 1239-44, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20352165

ABSTRACT

The endothelial protein C receptor (EPCR) plays an anticoagulant role by improving protein C activation. Although low levels of activated protein C (APC) constitute a thrombosis risk factor, the relationship between modulating EPCR function and thrombosis has not been addressed so far. Monoclonal antibodies (mAb) against murine EPCR were raised, and their ability to block protein C/APC binding was tested. The ferric chloride carotid artery injury model in mice was chosen to test the effect of anti-EPCR mAb on thrombus formation. The time to total occlusion of the vessel was analysed in three groups, given an isotype control mAb (IC), a blocking (RCR-16) or a non-blocking (RCR-20) anti-EPCR mAb. RCR-16 prevented the interaction between protein C/APC and EPCR as demonstrated by surface plasmon resonance and flow cytometry, and inhibited the activation of protein C on the endothelium. IC and RCR-20 were unable to induce such effects. In vivo , RCR-16 shortened the time to total vessel occlusion with respect to IC [13.4 +/- 1.0 (mean +/- SD) and 17.8 +/- 3.2 minutes, respectively, p<0.001]. Occlusive thrombi lasting for more than one hour were observed in all RCR-16-treated animals, but only in 43% of IC-treated ones. Results with RCR-20 were indistinguishable from those observed with IC. For the first time, a direct relationship between blocking EPCR and thrombosis is demonstrated. Blocking anti-EPCR autoantibodies can predispose to thrombosis episodes and may constitute a new therapeutic target.


Subject(s)
Antibodies, Blocking/pharmacology , Antibodies, Monoclonal/pharmacology , Carotid Arteries/drug effects , Endothelium, Vascular/drug effects , Glycoproteins/antagonists & inhibitors , Animals , Antibodies, Monoclonal/isolation & purification , Carotid Arteries/metabolism , Carotid Arteries/pathology , Carotid Artery Thrombosis , Cell Line , Disease Models, Animal , Endothelial Protein C Receptor , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Ferric Compounds/metabolism , Glycoproteins/immunology , Humans , Mice , Protein Binding/drug effects , Protein C/metabolism , Receptors, Cell Surface , Surface Plasmon Resonance
18.
Mod Rheumatol ; 19(2): 173-9, 2009.
Article in English | MEDLINE | ID: mdl-19067109

ABSTRACT

To assess the effects of disease activity of systemic lupus erythematosus (SLE) and high-dose corticosteroids on endothelial injuries, the significance of soluble endothelial cell protein C receptor (sEPCR) and soluble thrombomodulin (sTM) was analyzed. Serum levels of sEPCR and sTM were measured by enzyme-linked immunosorbent assay (ELISA) cross-sectionally in 97 SLE patients, 49 patients with other rheumatic diseases and 22 normal subjects. The changes in these levels upon corticosteroid treatment were also analyzed in 41 patients. The levels of sEPCR and sTM were both higher in SLE and other rheumatic disease patients than in normal subjects. When low-dose corticosteroids were used, both the level of sEPCR and the ratio of positive tests for sEPCR were significantly higher in active SLE patients than in inactive patients [median 2.30 ng/ml (range 0.00-147.10 ng/ml) vs 0.00 ng/ml (0.00-58.90 ng/ml) and 53.5 vs 13.0%, respectively] (P < 0.005). Moreover, the ratio of positive tests for sEPCR was higher after corticosteroid treatment in 9 of 19 (47.3%) SLE patients compared to other rheumatic diseases (3/22; 13.6%). Although the mean level of sTM was significantly higher in active SLE patients than in inactive patients, the ratio of positive tests for sTM was not affected by disease activity or corticosteroids. In conclusion, the positive test for sEPCR is a more sensitive biomarker than that for sTM in reflecting endothelial injuries caused by active disease and often by corticosteroids in SLE.


Subject(s)
Adrenal Cortex Hormones/pharmacology , Antigens, CD/blood , Lupus Erythematosus, Systemic/blood , Receptors, Cell Surface/blood , Adrenal Cortex Hormones/therapeutic use , Adult , Disease Progression , Endothelial Protein C Receptor , Female , Humans , Lupus Erythematosus, Systemic/drug therapy , Male , Middle Aged , Thrombomodulin/blood
19.
Protein Expr Purif ; 56(1): 138-44, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17703948

ABSTRACT

A recombinant protein of murine tumor necrosis factor (TNF)-alpha was expressed in Escherichia coli (E. coli) by using a pET Trx Fusion System. The fusion protein was effectively solubilized and purified by Ni-affinity chromatography. A high concentration of thrombin quickly and specifically cleaved the introduced site between the tags and the target fragment. We found that thrombin tightly bound to an ion-exchange resin, CM-Sepharose, under conditions avoiding adsorption of most proteins. By passing through the column, thrombin was quickly removed from the reaction mixtures. These methods appear to be widely potentially useful to remove the tags from recombinant fusion proteins. Prepared recombinant TNF demonstrated cytotoxic effects to L929 cells at very low concentrations with an EC50 value of 0.19+/-0.02 pM. In addition, immunization of a rabbit with the protein induced a neutralizing antibody. The methods used in this study appear to be useful to prepare significant amount of soluble functional recombinant proteins in E. coli.


Subject(s)
Recombinant Fusion Proteins/isolation & purification , Tumor Necrosis Factor-alpha/biosynthesis , Animals , Antibody Formation , Chromatography, Affinity , Chromatography, Ion Exchange/methods , Escherichia coli/metabolism , Mice , Recombinant Fusion Proteins/chemistry , Thrombin/metabolism , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolism
20.
Hybridoma (Larchmt) ; 26(6): 393-9, 2007 Dec.
Article in English | MEDLINE | ID: mdl-18158784

ABSTRACT

Ligands for toll-like receptors (TLR) are known to induce a variety of immune responses. Selective induction of desirable responses would be important for the treatment of individual diseases with various pathogenesis. For this purpose, we established six MAbs against the TLR4/MD-2 complex (UT MAbs) from TLR4(-/-) mice or MD-2(-/-) mice. Three MAbs (UT12, 18, and 22) induced NF-kappaB activation and production of pro-inflammatory cytokines, but the other three (UT15, 41, and 49) did not induce such cell responses. Unlike lipopolysaccharide (LPS), agonistic UT MAbs did not require serum components for the functions. UT41 and UT49 recognized TLR4 in the absence of MD-2. On the other hand, the other four MAbs reacted to the TLR4/MD-2 complex, but not to solo TLR4. Agonistic UT MAbs shared the epitopes, but non-agonistic UT15 reacted to distinct epitope on the complex. UT MAbs appear to be useful analyzing the molecular mechanism of TLR signaling and will contribute to the development of novel immunotherapies.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/physiology , Lymphocyte Antigen 96/immunology , Toll-Like Receptor 4/immunology , Animals , Antibodies, Monoclonal/biosynthesis , Binding Sites, Antibody , Binding, Competitive , Cell Line , Humans , Hybridomas , Lymphocyte Antigen 96/biosynthesis , Lymphocyte Antigen 96/deficiency , Lymphocyte Antigen 96/genetics , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , Mice, SCID , Protein Binding , Toll-Like Receptor 4/biosynthesis , Toll-Like Receptor 4/deficiency , Toll-Like Receptor 4/genetics
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