RESUMEN
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. RAW264.7â¯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 RAW264.7â¯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.
Asunto(s)
Antiinflamatorios/farmacología , Ascomicetos , Macrófagos/efectos de los fármacos , Receptores Toll-Like/inmunología , beta-Glucanos/farmacología , Animales , Antiinflamatorios/química , Ascomicetos/química , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Ratones , Células RAW 264.7 , beta-Glucanos/químicaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Inmunidad Innata/efectos de los fármacos , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/inmunología , Receptor Toll-Like 4/agonistas , Animales , Carga Bacteriana , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Enfermedad Crónica , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Noqueados , Viabilidad Microbiana/inmunología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/microbiología , Proteínas Opsoninas/inmunología , Fagocitosis/inmunología , Neumonía Bacteriana/tratamiento farmacológico , Neumonía Bacteriana/metabolismo , Serina Proteasas/metabolismoRESUMEN
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.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Coinfección/inmunología , Inmunidad Innata , Infecciones por Orthomyxoviridae/inmunología , Infecciones Neumocócicas/inmunología , Neumonía/inmunología , Receptor Toll-Like 4/agonistas , Animales , Peso Corporal , Coinfección/microbiología , Coinfección/patología , Coinfección/virología , Modelos Animales de Enfermedad , Histocitoquímica , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos CBA , Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Infecciones Neumocócicas/patología , Neumonía/microbiología , Neumonía/patología , Neumonía/virología , Streptococcus pneumoniae/inmunología , Análisis de SupervivenciaRESUMEN
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.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Lipopolisacáridos/genética , Mutación Puntual/inmunología , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/biosíntesis , Animales , Subgrupos de Linfocitos B/metabolismo , Línea Celular Tumoral , Membrana Celular/genética , Membrana Celular/inmunología , Células Cultivadas , Células HEK293 , Humanos , Inmunofenotipificación , Lipopolisacáridos/biosíntesis , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Ratas Wistar , Receptor Toll-Like 4/deficienciaRESUMEN
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.
Asunto(s)
Formación de Anticuerpos/inmunología , Antígenos/inmunología , Linfocitos B/inmunología , Receptor Toll-Like 4/agonistas , Adyuvantes Inmunológicos , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Presentación de Antígeno/inmunología , Reacciones Antígeno-Anticuerpo , Reactividad Cruzada/inmunología , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptor Toll-Like 4/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Inmunidad Innata/inmunología , Receptor Toll-Like 4/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Western Blotting , Línea Celular , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Lipopolisacáridos/inmunología , Antígeno 96 de los Linfocitos/efectos de los fármacos , Antígeno 96 de los Linfocitos/inmunología , Antígeno 96 de los Linfocitos/metabolismo , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Receptor Toll-Like 4/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , TransfecciónRESUMEN
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.
Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Células de la Médula Ósea/metabolismo , Médula Ósea/metabolismo , Adipocitos/efectos de los fármacos , Adiponectina/genética , Adiponectina/metabolismo , Tejido Adiposo/citología , Tejido Adiposo/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Proliferación Celular , Células Cultivadas , Dexametasona/farmacología , Ensayo de Inmunoadsorción Enzimática , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Insulina/farmacología , Leptina/genética , Leptina/metabolismo , Técnicas de Cultivo de Órganos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
Asunto(s)
Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/farmacología , Arterias Carótidas/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Glicoproteínas/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Trombosis de las Arterias Carótidas , Línea Celular , Modelos Animales de Enfermedad , Receptor de Proteína C Endotelial , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Compuestos Férricos/metabolismo , Glicoproteínas/inmunología , Humanos , Ratones , Unión Proteica/efectos de los fármacos , Proteína C/metabolismo , Receptores de Superficie Celular , Resonancia por Plasmón de SuperficieRESUMEN
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.
Asunto(s)
Proteínas de Fase Aguda/metabolismo , Proteínas Portadoras/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/inmunología , Antígeno 96 de los Linfocitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Regulación hacia Abajo , Humanos , Inmunidad Innata , Ratones , FN-kappa B/metabolismo , Multimerización de Proteína , Ratas , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
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.
Asunto(s)
Angiopoyetinas/metabolismo , Anticoagulantes/farmacología , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteína C/farmacología , Receptor TIE-2/metabolismo , Antígenos CD/fisiología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Receptor de Proteína C Endotelial , Ensayo de Inmunoadsorción Enzimática , Expresión Génica/efectos de los fármacos , Humanos , Immunoblotting , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , ARN Interferente Pequeño , Receptor PAR-1/antagonistas & inhibidores , Receptor PAR-1/fisiología , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Venas Umbilicales/citología , Proteína de la Zonula Occludens-1RESUMEN
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.
Asunto(s)
Antígenos CD/metabolismo , Membrana Celular/metabolismo , Células Endoteliales/metabolismo , Proteína C/metabolismo , Receptores de Superficie Celular/metabolismo , Apoptosis/efectos de los fármacos , Membrana Celular/química , Células Cultivadas , Citoprotección/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/ultraestructura , Receptor de Proteína C Endotelial , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Inflamación/prevención & control , Microscopía Confocal , Microscopía Electrónica , Análisis de Secuencia por Matrices de Oligonucleótidos , Tamaño de la Partícula , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Unión Proteica , Proteína C/farmacología , Receptores de Lisoesfingolípidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sepsis/prevención & control , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
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.
Asunto(s)
Corticoesteroides/farmacología , Antígenos CD/sangre , Lupus Eritematoso Sistémico/sangre , Receptores de Superficie Celular/sangre , Corticoesteroides/uso terapéutico , Adulto , Progresión de la Enfermedad , Receptor de Proteína C Endotelial , Femenino , Humanos , Lupus Eritematoso Sistémico/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Trombomodulina/sangreRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/fisiología , Antígeno 96 de los Linfocitos/inmunología , Receptor Toll-Like 4/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Sitios de Unión de Anticuerpos , Unión Competitiva , Línea Celular , Humanos , Hibridomas , Antígeno 96 de los Linfocitos/biosíntesis , Antígeno 96 de los Linfocitos/deficiencia , Antígeno 96 de los Linfocitos/genética , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Unión Proteica , Receptor Toll-Like 4/biosíntesis , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genéticaRESUMEN
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.
Asunto(s)
Proteínas Recombinantes de Fusión/aislamiento & purificación , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Formación de Anticuerpos , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico/métodos , Escherichia coli/metabolismo , Ratones , Proteínas Recombinantes de Fusión/química , Trombina/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
Asunto(s)
Antígenos CD/metabolismo , Artritis Reumatoide/metabolismo , Endotelio Vascular/metabolismo , Monocitos/metabolismo , Proteína C/metabolismo , Receptores de Superficie Celular/metabolismo , Membrana Sinovial/metabolismo , Resistencia a la Proteína C Activada , Adulto , Análisis de Varianza , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Western Blotting/métodos , Quimiocina CCL2/farmacología , Quimiotaxis de Leucocito , Receptor de Proteína C Endotelial , Endotelio Vascular/inmunología , Femenino , Humanos , Inmunohistoquímica , Lipopolisacáridos/farmacología , Masculino , Persona de Mediana Edad , FN-kappa B/análisis , FN-kappa B/metabolismo , Osteoartritis/metabolismo , Proteína C/análisis , Receptores de Superficie Celular/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Membrana Sinovial/química , Membrana Sinovial/inmunología , Factor de Necrosis Tumoral alfa/análisisRESUMEN
PRIMARY OBJECTIVE: Platelet-derived growth factor-BB (PDGF-BB) has been shown to promote the structural integrity of the vessel wall and to increase wound healing capacity. Aim of the present study was to determine the role of PDGF-BB in the context of outcome of septic patients. Furthermore, the effect of treatment with recombinant human activated protein C (rhAPC) on plasma levels of PDGF-BB in severe sepsis was evaluated as well as the in vitro effect of rhAPC on PDGF-BB-release from human endothelial cells (HUVEC). RESEARCH DESIGN, METHODS AND PROCEDURES: PDGF-BB levels were measured in 46 patients on day 3 of severe sepsis. Twenty-one of these patients received treatment with rhAPC. The in vitro effect of rhAPC on PDGF-BB-messenger RNA synthesis and release of PDGF-BB into supernatants was measured by reverse transcriptase-polymerase chain reaction and ELISA-methods. MAIN OUTCOMES AND RESULTS: Survivors of severe sepsis presented with higher PDGF-BB levels than non-survivors (p < 0.05). Septic patients with PDGF-BB levels below 200 pg/ml were 7.3 times more likely (RR = 7.3, 95% CI: 1.4-44.5; p < 0.05) to die from sepsis than patients with higher PDGF-BB values. RhAPC (1-10 microg/ml) stimulated endothelial PDGF-BB-messenger RNA transcription and PDGF-BB-release in vitro. Plasma levels of PDGF-BB in patients receiving rhAPC were significantly (p < 0.01) higher (median 277.7; 25-75th percentiles: 150.5-414.4 pg/ml) than in patients not treated with rhAPC (median: 125.6; 25-75th percentiles: 55.3-344.7 pg/ml). CONCLUSIONS: The ability of rhAPC to upregulate endothelial PDGF-BB production may represent a new molecular mechanism by which rhAPC controls vessel wall homeostasis and increases tissue healing capacity in severe sepsis. PDGF-BB may serve as useful laboratory marker to predict survival in patients presenting with severe sepsis.
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Factor de Crecimiento Derivado de Plaquetas/metabolismo , Sepsis/sangre , Adulto , Anciano , Antígenos CD , Secuencia de Bases , Becaplermina , Células Cultivadas , Cartilla de ADN/genética , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Receptor de Proteína C Endotelial , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factor de Crecimiento Derivado de Plaquetas/genética , Pronóstico , Proteína C/uso terapéutico , Proteínas Proto-Oncogénicas c-sis , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptor PAR-1/antagonistas & inhibidores , Receptores de Superficie Celular/antagonistas & inhibidores , Proteínas Recombinantes/uso terapéutico , Sepsis/tratamiento farmacológico , Sepsis/genéticaRESUMEN
We have established an agonistic monoclonal antibody, UT12, that induces stimulatory signals comparable to those induced by lipopolysaccharide (LPS) through Toll-like receptor 4 and MD-2. UT12 activated nuclear factor kappaB and induced the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) in peritoneal exudative cells. In addition, mice injected with UT12 rapidly fell into endotoxin shock concomitant with the augmentation of serum TNF-alpha and IL-6 levels, followed by death within 12 h. On the other hand, when the mice were pretreated with a sublethal dose of UT12, the mice survived the subsequent lethal LPS challenges, with significant suppression of serum TNF-alpha and IL-6, indicating that UT12 induced tolerance against LPS. This effect of UT12 was maintained for at least 9 days. In contrast, the tolerance induced by LPS continued for less than 3 days. These results illuminate a novel potential therapeutic strategy for endotoxin shock by the use of monoclonal antibodies against the Toll-like receptor 4/MD-2 complex.
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Anticuerpos Monoclonales/metabolismo , Tolerancia Inmunológica/inmunología , Lipopolisacáridos/inmunología , Antígeno 96 de los Linfocitos/inmunología , Receptor Toll-Like 4/inmunología , Animales , Antígeno 96 de los Linfocitos/agonistas , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Unión Proteica/fisiología , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genéticaRESUMEN
Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria, and is the causative agent of endotoxin shock. LPS induces signal transduction in immune cells when it is recognized by the cell surface complex of toll-like receptor 4 (TLR4) and MD-2. The complex recognizes the lipid A structure in LPS, which is buried in the membrane of the outer envelope. To present the Lipid A structure to the TLR4/MD-2, processing of LPS by LPS-binding protein (LBP) and CD14 is required. In previous studies, we expressed recombinant proteins of human MD-2 and CD14 as fusion proteins with thioredoxin in Escherichia coli, and demonstrated their specific binding abilities to LPS. In this study, we prepared a recombinant fusion protein containing 212 amino terminal residues of human LBP (HLB212) by using the same expression system. The recombinant protein expressed in E. coli was purified as a complex form with host LPS. The binding was not affected by high concentrations of salt, but was prevented by low concentrations of various detergents. Both rough-type LPS lacking the O antigen and smooth-type LPS with the antigen bound to HLBP212. Therefore, oligosaccharide repeats appeared to be unnecessary for the binding. A nonpathogenic penta-acylated LPS also bound to HLBP212, but the binding was weaker than that of the wild type. The hydrophobic interaction between the LBP and acyl chains of lipid A appears to be important for the binding. The recombinant proteins of LPS-binding molecules would be useful for analyzing the defense mechanism against infections.
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Proteínas de Fase Aguda/química , Proteínas de Fase Aguda/aislamiento & purificación , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Escherichia coli , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/aislamiento & purificación , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Escherichia coli/genética , Humanos , Lípido A/química , Lípido A/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Antígeno 96 de los Linfocitos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Antígenos O/química , Antígenos O/metabolismo , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
Fractalkine is a unique endothelial cell-derived chemokine that functions both as a chemoattractant and as an adhesion molecule. Recent findings suggest that fractalkine plays an important role in inflammatory diseases by modulating leucocyte endothelial cell interactions. A modulating effect on the immune system in severe sepsis has been suggested for recombinant human activated protein C (rhAPC). However, a little is known about the effect of rhAPC on the endothelial release of soluble fractalkine. The effect of rhAPC (50 ng/ml to 10 microg/ml) and protein C (in equimolar concentrations) on the synthesis of fraktalkine-mRNA and release of soluble protein in human umbilical vein endothelial cells (HUVEC) was determined by reverse transcription-polymerase chain reaction and by an enzyme-linked immunosorbent assay. rhAPC at supra-pharmacological concentrations (1-10 microg/ml) stimulated fractalkine-messenger RNA-gene transcription and release of soluble fractalkine in a time- and dose-dependent manner, whereas the zymogen protein C was ineffective. As shown by experiments using monoclonal antibodies against the thrombin receptor, protease-activated receptor-1 (PAR-1), PAR-2 and against the endothelial protein C receptor (EPCR), the effect of rhAPC on fractalkine upregulation was mediated by binding to the EPCR-receptor and signalling via PAR-1. These in vitro data demonstrate that induction of fractalkine release is an important response of HUVEC to stimulation with rhAPC and may lead to a better understanding of the molecular pathways involved in the mode of action of rhAPC. Further clinical trials are needed to confirm the in vivo relevance of these data.