RESUMEN
The pathophysiology of osteoarthritis (OA) includes the destruction of subchondral bone tissue and inflammation of the synovium. Thus, an effective disease-modifying treatment should act on both of these pathogenetic components. It is known that cSrc kinase is involved in bone and cartilage remodeling, and SYK kinase is associated with the inflammatory component. Thus the aim of this study was to characterize the mechanism of action and efficacy of a small molecule multikinase inhibitor MT-SYK-03 targeting SYK and cSrc kinases among others in different in vitro and in vivo arthritis models. The selectivity of MT-SYK-03 kinase inhibition was assayed on a panel of 341 kinases. The compound was evaluated in a set of in vitro models of OA and in vivo OA and RA models: surgically-induced arthritis (SIA), monosodium iodoacetate-induced arthritis (MIA), collagen-induced arthritis (CIA), adjuvant-induced arthritis (AIA). MT-SYK-03 inhibited cSrc and SYK with IC50 of 14.2 and 23 nM respectively. Only five kinases were inhibited > 90% at 500 nM of MT-SYK-03. In in vitro OA models MT-SYK-03 reduced hypertrophic changes of chondrocytes, bone resorption, and inhibited SYK-mediated inflammatory signaling. MT-SYK-03 showed preferential distribution to joint and bone tissue (in rats) and revealed disease-modifying activity in vivo by halving the depth of cartilage erosion in rat SIA model, and increasing the pain threshold in rat MIA model. Chondroprotective and antiresorptive effects were shown in a monotherapy regime and in combination with methotrexate (MTX) in murine and rat CIA models; an immune-mediated inflammation in rat AIA model was decreased. The obtained preclinical data support inhibition of cSrc and SYK as a viable strategy for disease-modifying treatment of OA. A Phase 2 clinical study of MT-SYK-03 is to be started.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/enzimología , Huesos/efectos de los fármacos , Proteína Tirosina Quinasa CSK/antagonistas & inhibidores , Cartílago/efectos de los fármacos , Osteoartritis/tratamiento farmacológico , Osteoartritis/enzimología , Quinasa Syk/antagonistas & inhibidores , Animales , Artritis Experimental/patología , Resorción Ósea/patología , Condrocitos/patología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Humanos , Inflamación , Concentración 50 Inhibidora , Ácido Yodoacético/farmacología , Receptores de Lipopolisacáridos/biosíntesis , Masculino , Ratones , Monocitos/citología , Sustancias Protectoras/farmacología , Conejos , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Membrana Sinovial/patologíaRESUMEN
Children are more susceptible to Giardia lamblia infection. Cells and hormones contained in human colostrum have an immunoprotective action against giardiasis, but the effects of advanced maternal age on these components are poorly understood. This study analyzed the colostrum of older women to determine melatonin and cortisol levels besides the participation of these hormones on the functional activity of phagocytes against G. lamblia. Colostrum samples were collected from younger (18 to 35 years old) and older (over 36 years old) lactating women. Colostrum samples were subjected to melatonin and cortisol determination, immunophenotyping, quantification of superoxide release, and assessment of phagocytic rate and microbicidal activity of phagocytes treated with hormones and in the presence of G. lamblia. Colostrum from mothers of advanced age contained higher melatonin and cortisol levels and a lower rate of cells expressing CD14+ and CD15+. In the colostru of these older mothers, melatonin increased superoxide release by phagocytes. In both groups, superoxide release by phagocytes treated with cortisol was higher in the presence of G. lamblia. In colostrum from mothers of advanced age, mononuclear (MN) phagocytes treated with melatonin showed higher phagocytosis of G. lamblia and higher microbicidal index. In younger mothers, MN and polymorphonuclear (PMN) colostrum phagocytes exhibited higher rates of G. lamblia elimination when treated with both melatonin and cortisol. In older mothers, cortisol and melatonin regulation for the functional activity of colostrum phagocytes against G. lamblia may represent an additional defense mechanism, relevant for the protection and treatment of parasitic infections in breastfed children.
Asunto(s)
Envejecimiento/inmunología , Calostro/inmunología , Giardia lamblia/inmunología , Giardiasis/inmunología , Hidrocortisona/farmacología , Melatonina/farmacología , Neutrófilos/inmunología , Fagocitosis/inmunología , Adolescente , Adulto , Animales , Niño , Estudios Transversales , Femenino , Giardiasis/parasitología , Giardiasis/prevención & control , Humanos , Hidrocortisona/análisis , Lactancia/fisiología , Antígeno Lewis X/biosíntesis , Receptores de Lipopolisacáridos/biosíntesis , Edad Materna , Melatonina/análisis , Fagocitos/inmunología , Embarazo , Superóxidos/metabolismo , Adulto JovenRESUMEN
OBJECTIVES: To evaluate the anti-inflammatory potential of Pterodon polygalaeflorus hexane extract (HE) and its fractions on macrophage migration in vitro and in vivo. METHODS: Hexane extract from P. polygalaeflorus fruits was fractionated and yielded four fractions. RAW 264.7 cells were treated with samples to evaluate cell viability (MTT assay), cell migration (wound healing and transwell assays), CD14 expression (flow cytometry), iNOS and cytokine mRNA expression (RT-qPCR), NO (Griess reaction) and cytokine (ELISA) production. In vivo migration was evaluated on the thioglycollate-induced peritonitis model. Qualitative analysis was performed by GC-MS. KEY FINDINGS: All fractions inhibited the NO production by LPS-stimulated RAW 264.7 cells. Fr3 and Fr4 presented the lowest IC50 values. The expressions of iNOS and IL-1ß, TNF-α and IL-10 cytokines were inhibited by Fr3 and Fr4, whereas the CD14 expression was only inhibited by Fr3. All the samples inhibited RAW 264.7 migration in the wound healing and transwell assays. Fr3 and Fr4 reduced the migration of Mac-1+ Gr-1- cells to the peritoneum and presented in their compositions: 6α-hydroxy-7ß-acetoxyvouacapan-17ß-oate, methyl 6α,7ß-dihydroxyvouacapan-17ß-oate, methyl 6α-acetoxy-7ß-hydroxyvouacapan-17ß-oate, geranylgeraniol and 14,15-epoxy-geranylgeraniol. CONCLUSIONS: The anti-inflammatory effects of Fr3 and Fr4 involve inhibition of cell migration, iNOS expression and NO production, cytokine expression (mRNA and proteins) and CD14 expression (Fr3).
Asunto(s)
Antiinflamatorios/farmacología , Movimiento Celular/efectos de los fármacos , Citocinas/biosíntesis , Diterpenos/farmacología , Fabaceae/química , Macrófagos/efectos de los fármacos , Extractos Vegetales/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Frutas/química , Receptores de Lipopolisacáridos/biosíntesis , Macrófagos/citología , Ratones , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Extractos Vegetales/químicaRESUMEN
BACKGROUND: High-fat, high-carbohydrate (HFHC) meals are known to induce oxidative and inflammatory stress, an increase in plasma endotoxin concentrations, and an increase in the expression of suppressor of cytokine signaling-3 (SOCS-3). HYPOTHESIS: The intake of a nutritional supplement containing resveratrol and muscadine grape polyphenols reduces HFHC meal-induced oxidative and inflammatory stress and stimulates the activity of the antioxidant transcription factor, NF-E2-related factor-2 (Nrf-2), and its downstream targets. METHODS: Ten normal, healthy subjects were given a 930-kcal HFHC meal either with placebo or with the supplement. Indices of oxidative stress, inflammation, Nrf-2 binding activity, the concentrations of endotoxin (lipopolysaccharide) and lipoprotein binding protein (LBP), and the expression of toll-like receptor 4 (TLR-4), CD14, IL-1ß, TNFα, SOCS-3, Keap-1, NAD(P)H:quinone oxidoreductase-1 (NQO-1), and GST-P1 were measured. RESULTS: The intake of the supplement suppressed the meal-induced elevations of plasma endotoxin and LBP concentrations, the expression of p47(phox), TLR-4, CD14, SOCS-3, IL-1ß, and Keap-1, while enhancing Nrf-2 binding activity and the expression of NQO-1 and GST-P1 genes. CONCLUSION: A supplement containing resveratrol and muscadine polyphenols suppresses the increase in oxidative stress, lipopolysaccharide and LBP concentrations, and expression of TLR-4, CD14, IL-1ß and SOCS-3 in mononuclear cells after an HFHC meal. It also stimulates specific Nrf-2 activity and induces the expression of the related antioxidant genes, NQO-1 and GST-P1. These results demonstrate the acute antioxidant and antiinflammatory effects of resveratrol and polyphenolic compounds in humans in the postprandial state.
Asunto(s)
Antioxidantes/farmacología , Carbohidratos de la Dieta/farmacología , Grasas de la Dieta/efectos adversos , Flavonoides/farmacología , Inflamación/prevención & control , Estrés Oxidativo/efectos de los fármacos , Fenoles/farmacología , Estilbenos/farmacología , Adulto , Western Blotting , Citocinas/biosíntesis , ADN/biosíntesis , ADN/genética , Fallopia japonica/química , Femenino , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Receptores de Lipopolisacáridos/biosíntesis , Lipopolisacáridos/farmacología , Masculino , Factor 2 Relacionado con NF-E2/biosíntesis , Extractos Vegetales/farmacología , Polifenoles , Resveratrol , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/biosíntesis , Receptor Toll-Like 4/biosíntesis , Vitis/químicaRESUMEN
CD14+ peripheral blood monocytes can differentiate into fibroblast-like cells called fibrocytes. Fibrocytes are associated with, and are at least partially responsible for, wound healing and fibrosis in multiple organ systems. In a variety of lesions in vivo, monocytes appear to differentiate into fibrocytes within days. However, in vitro culture conditions can take up to two weeks to generate fibrocytes. In this study, we describe enhanced serum-free conditions that support the rapid differentiation of human and murine fibrocytes. We compared the effect on fibrocyte differentiation of different anti-coagulants used when collecting blood, and for culturing cells, the effects of different commercial media formulations, the addition of a variety of supplements, cell density, conditioned medium, and glass and plastic substrates. We found that both heparin and EDTA were suitable anti-coagulants, but that blood treated with citrate-phosphate dextrose led to a reduced number of fibrocytes. Fibrocyte differentiation was enhanced when the serum-free medium was based on either FibroLife or StemPro formulations. We also found that only positively charged or hydrophilic glass and plastic surfaces provide adequate support for fibrocyte differentiation. Finally, the optimal cell density was 2.5 x 10(5)cells/ml (approximately 800 cells per mm(2)). These results indicate that blood collection, substrates, media, and cell density all influence in vitro fibrocyte differentiation.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Fibroblastos/citología , Monocitos/citología , Animales , Anticoagulantes , Células Cultivadas , Medio de Cultivo Libre de Suero , Ácido Edético , Heparina , Humanos , Receptores de Lipopolisacáridos/biosíntesis , Ratones , Monocitos/metabolismoRESUMEN
Although it has been established that maternal leukocytes traffic from colostrum into the neonatal circulation, the effects of these cells on neonatal immunity are only beginning to be understood. This study examined the effects of maternal colostral leukocytes on development and maturation of neonatal antigen presenting cells. At birth, groups of neonatal calves received whole or cell-free colostrum (CFC) from their respective dams. Peripheral blood samples were obtained over the first 4 weeks of life, and expression of surface markers associated with cellular activation and physiological stress were monitored on monocyte lineage cells. Calves receiving cell-free colostrum at birth expressed elevated levels of CD11a, CD11c, and CD14, compared to calves receiving whole colostrum (C). Calves receiving cell-free colostrum had an elevated number of monocytes in the peripheral blood during the first 2 weeks of life, however, these cells expressed lower levels of expression of CD25 and MHC class I compared to calves receiving whole colostrum. The most significant differences in marker expression occurred within the first 7 days of life.
Asunto(s)
Bovinos/inmunología , Calostro/inmunología , Leucocitos Mononucleares/inmunología , Animales , Animales Recién Nacidos , Presentación de Antígeno , Antígeno CD11a/biosíntesis , Antígeno CD11a/sangre , Antígeno CD11c/biosíntesis , Antígeno CD11c/sangre , Bovinos/sangre , Linaje de la Célula/inmunología , Calostro/citología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Citometría de Flujo/veterinaria , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunoglobulina G/sangre , Inmunofenotipificación/veterinaria , Receptores de Lipopolisacáridos/biosíntesis , Receptores de Lipopolisacáridos/sangre , Óxido Nítrico/biosíntesis , Óxido Nítrico/sangreRESUMEN
OBJECTIVE: To investigate the effects of Qinggan Huoxue Recipe (QGHXR), a compound Chinese herbal medicine, and its decomposed formulas Qinggan Recipe (QGR) and Huoxue Recipe (HXR) on expressions of CD14, Toll like receptor 4 (TLR(4)) and nuclear factor-kappaB (NF-kappaB) in Kupffer cells. METHODS: The isolated primary rat Kupffer cells were treated with lipopolysaccharide (LPS) for a certain period of time, a series of concentrations of drug-containing serums of QGHXR and its decomposed formulas were added, the expressions of NF-kappaB, tumor necrosis factor-alpha (TNF-alpha), CD14, and TLR(4) of the Kupffer cells were detected in different culture conditions by using Western blot, real-time reverse transcription polymerase chain reaction (RT-PCR) and ELISA methods respectively. RESULTS: QGR could down-regulate the expression of membrane receptor CD14, but the expression of NF-kappaB and TNF-alpha were not significantly decreased after QGR treatment. HXR could down-regulate the expression of membrane receptor TLR4 and inhibit the expressions of NF-kappaB and TNF-alpha. QGHXR could down regulate the expressions of membrane receptors CD14 and TLR(4) and inhibit the expressions of NF-kappaB and TNF-alpha. CONCLUSION: QGHXR can protect liver cells by down regulating the expressions of CD14, TLR(4) and NF-kappaB and inhibiting TNF-alpha expression.
Asunto(s)
Medicamentos Herbarios Chinos/farmacología , Macrófagos del Hígado/efectos de los fármacos , Receptores de Lipopolisacáridos/genética , FN-kappa B/genética , Receptor Toll-Like 4/genética , Animales , Western Blotting , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Expresión Génica/efectos de los fármacos , Macrófagos del Hígado/citología , Macrófagos del Hígado/metabolismo , Receptores de Lipopolisacáridos/biosíntesis , Lipopolisacáridos/farmacología , Masculino , FN-kappa B/biosíntesis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Suero , Receptor Toll-Like 4/biosíntesisRESUMEN
Licorice, the roots of Glycyrrhiza inflata, is used by practitioners of alternative medicine to treat individuals with gastric or duodenal ulcers, bronchitis, cough, arthritis, adrenal insufficiency, and allergies. We investigated the anti-inflammatory properties of 4 licorice extracts: extracts of roasted licorice obtained by ethanol (rLE) or water extraction (rLW) and extracts of raw licorice obtained by ethanol (LE) or water extraction (LW). rLE demonstrated strong anti-inflammatory activity through its ability to reduce nitric oxide and prostaglandin E(2) production in the LPS-stimulated mouse macrophage cell, RAW264.7. It also inhibited the production of pro-inflammatory cytokines and CD14 expression on the LPS-stimulated RAW264.7 cells. Further study indicated that LPS-induced degradation and phosphorylation of Ikappa-Balpha, along with DNA-binding of NF-kappaB, was significantly inhibited by rLE exposure in RAW264.7 cells. In the murine model, we found that in vivo exposure to rLE-induced an increase in the survival rate, reduced plasma levels of TNF-alpha and IL-6, and increased IL-10 production in LPS-treated mice. Collectively, these data suggest that the use of rLE may be a useful therapeutic approach to various inflammatory diseases.
Asunto(s)
Antiinflamatorios/farmacología , Glycyrrhiza/metabolismo , Lipopolisacáridos/metabolismo , Macrófagos/metabolismo , Animales , Dinoprostona/metabolismo , Femenino , Proteínas I-kappa B/metabolismo , Inflamación , Receptores de Lipopolisacáridos/biosíntesis , Ratones , Ratones Endogámicos ICR , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Extractos VegetalesRESUMEN
Toll-like receptors (TLRs) play a pivotal role in the induction of innate immunity after the transactivation of proinflammatory cytokine genes. However, the responses of TLRs during severe polymicrobial sepsis have not been thoroughly examined. Although dehydroepiandrosterone (DHEA), a steroid hormone, is reported to have an immunomodulatory effect after sepsis, the mechanism responsible for its salutary is not known. To investigate this, male ICR/Jcl mice (5-8 weeks old) were subjected to sepsis by cecal ligation and puncture (CLP) or sham operation. The mice received vehicle or DHEA (40 mg/kg body weight) subcutaneously immediately after the surgery. Plasma IL-10 levels and splenic macrophage TNF-alpha production, as well as the expression levels of CD14, TLR2, and TLR4 mRNAs on splenic macrophages, were assessed 6 h after the surgery. The results indicate that mice with sepsis show a marked increase in the plasma IL-10 levels and a decrease in TNF-alpha production by splenic macrophages. TLR2 and TLR4 mRNA expression levels after CLP were significantly lower compared with those after the sham operation. TNF-alpha production and TLR2 and TLR4 mRNA expression on splenic macrophages are restored with DHEA administration. Furthermore, administration of DHEA after CLP delayed the mortality of animals. These results indicate that the anti-inflammatory phase of sepsis induces a marked down-regulation of TLR expression on splenic macrophages; however, administration of DHEA resulted in the restoration of TLR2 and TLR4 mRNA expression.
Asunto(s)
Deshidroepiandrosterona/fisiología , Macrófagos/citología , Macrófagos/microbiología , Sepsis/microbiología , Bazo/citología , Bazo/microbiología , Receptores Toll-Like/metabolismo , Adyuvantes Inmunológicos/fisiología , Animales , Antiinflamatorios/farmacología , Membrana Celular/metabolismo , ADN Complementario/metabolismo , Deshidroepiandrosterona/química , Deshidroepiandrosterona/farmacología , Regulación hacia Abajo , Citometría de Flujo , Inflamación , Interleucina-10/biosíntesis , Interleucina-10/sangre , Receptores de Lipopolisacáridos/biosíntesis , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , ARN/química , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Receptor Toll-Like 2/biosíntesis , Receptor Toll-Like 4/biosíntesis , Activación Transcripcional , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
A proline-rich polypeptide complex (PRP) with immunoregulatory and procognitive activities shows beneficial effects in Alzheimer's disease (AD). The mechanism of action of PRP in AD is not yet clarified. Here, we present results of the effect of PRP on Vitamin D3-induced phenotypic (CD11b and CD14) and functional (phagocytic) differentiation/maturation of monocytes/macrophages using the premonocytic HL-60 cell line as a model. This cell line can be induced to differentiate into monocyte/macrophage cells by incubation with Vitamin D3. However, when Vitamin D3 was applied together with PRP, a 30-40% inhibition of the expression of the differentiation markers and an over-60% inhibition of phagocytic ability were observed. When PRP was administered to the cells after treatment with Vitamin D3, no attenuation of the differentiation/maturation process of the HL-60 cells was observed. This indicates that PRP affects the early stages of differentiation/maturation of these cells. Our results, therefore, suggest that PRP, which affects the differentiation/maturation processes of cells of monocyte/macrophage lineage, may regulate in this way the inflammatory processes in which these cells participate.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Calostro/química , Complejos Multiproteicos/farmacología , Animales , Antígeno CD11b/biosíntesis , Colecalciferol/farmacología , Femenino , Células HL-60 , Humanos , Receptores de Lipopolisacáridos/biosíntesis , Macrófagos/fisiología , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Embarazo , Ovinos , Vitaminas/farmacologíaRESUMEN
Calreticulin (CRT) is a binding protein for apoptotic N-acetylmuramyl-L-alanyl-D-isoglutamine (L,D-MDP) or peptidoglycan in RK(13) cells. CRT on RK(13) cell surface (srCRT) forms complex(es) with tumor necrosis factor receptor 1 (TNFR1) and TNFR-associated death domain (TRADD) protein of the cell membrane. CRT polyclonal or monoclonal antibody binding to RK(13) srCRT dose-dependently inhibited L,D-MDP-induced apoptosis. In RK(13) cells, L,D-MDP up-regulated the TNFR1.TRADD complex of the plasma membrane and subsequently induced cytosolic TRADD-Fas-associated death domain protein complex. Biotinylated srCRT was capable of calcium-dependent binding of Sepharose-immobilized L,D-MDP or peptidoglycan. However, Toll-like receptors TLR-2 and TLR-4, Nod2, and CD14 of RK(13) cells did not specifically bind Sepharose-immobilized L,D-MDP. High concentrations (5-40 mm) of EGTA dose-dependently inhibited free L,D-MDP binding to purified RK(13) cell CRT and promoted free L,D-MDP dissociation from RK(13) cell CRT.MDP complex. Different concentrations of EGTA (0-40 mm) added to Dulbecco's modified essential medium with 1.8 mm calcium or phosphate-buffered saline with 0.18 mm calcium have different effects on medium free calcium concentrations but have identical inhibiting effects on L,D-MDP-induced apoptosis. More inhibition of the L,D-MDP-induced apoptotic DNA ladders and caspase-3 activity in RK(13) cells was obtained with EGTA pretreatment (83%) than just EGTA + L,D-MDP (47%). The knocking down of srCRT by antisense oligonucleotide CRTAS121 (250 nmol/ml) and stealth small interfering RNA CRT_siR479 (150 pm/ml) for 2 days (44 and 66%, respectively), resulted in the inhibition of L,D-MDP-induced caspase-3 activity (47 and 65%, respectively). The results suggest that (a) the binding of L,D-MDP to srCRT is calcium-dependent, i.e. on srCRT-bound calcium, and (b) it is srCRT, not TLR-2, TLR-4, Nod2 or CD14, that mediates L,D-MDP-induced RK(13) cell apoptosis through activating the TNFR1. TRADD-Fas-associated death domain protein apoptotic pathway.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/farmacología , Apoptosis , Calreticulina/fisiología , Adyuvantes Inmunológicos/farmacología , Animales , Anticuerpos Monoclonales/química , Bioensayo , Biotinilación , Western Blotting , Calcio/metabolismo , Calreticulina/metabolismo , Caspasa 3 , Caspasas/metabolismo , Línea Celular , Membrana Celular/metabolismo , Separación Celular , Citosol/metabolismo , ADN/química , Ácido Egtácico/química , Ácido Egtácico/farmacología , Retículo Endoplásmico/metabolismo , Citometría de Flujo , Humanos , Inmunoglobulina G/química , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/química , Receptores de Lipopolisacáridos/biosíntesis , Glicoproteínas de Membrana/química , Ratones , Proteína Adaptadora de Señalización NOD2 , Oligonucleótidos Antisentido/química , Peptidoglicano/química , Unión Proteica , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Conejos , Ratas , Receptores de Superficie Celular/química , Sefarosa/química , Sefarosa/farmacología , Espectrometría de Fluorescencia , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Regulación hacia ArribaRESUMEN
Magnolol (MG) and honokiol (HK), two lignans showing anti-inflammatory and anti-oxidant properties and abundantly available in the medicinal plants Magnolia officinalis and M. obovata, were found to enhance HL-60 cell differentiation initiated by low doses of 1,25-dihydroxyvitamin D3 (VD3) and all-trans-retinoic acid (ATRA). Cells expressing membrane differentiation markers CD11b and CD14 were increased from 4% in non-treated control to 8-16% after being treated with 10-30 microM MG or HK. When added to 1 nM VD3, MG or HK increased markers expressing cells from approximately 30% to 50-80%. When either MG or HK was added to 20 nM ATRA, only CD11b, but not CD14, expressing cells were increased from 9% to 24-70%. Under the same conditions, adding MG or HK to VD3 or ATRA treatment further enlarged the G0/G1 cell population and increased the expression of p27(Kip1), a cyclin-dependent kinase inhibitor. Pharmacological studies using PD098059 (a MEK inhibitor), SB203580 (a p38 MAPK inhibitor) and SP600125 (a JNK inhibitor) suggested that the MEK pathway was important for VD3 and ATRA-induced differentiation and also its enhancement by MG or HK, the p38 MAPK pathway had a inhibitory effect and the JNK pathway had little influence. It is evident that MG and HK are potential differentiation enhancing agents which may allow the use of low doses of VD3 and ATRA in the treatment for acute promyelocytic leukemia.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Antineoplásicos Fitogénicos/farmacología , Compuestos de Bifenilo/farmacología , Calcitriol/farmacología , Agonistas de los Canales de Calcio/farmacología , Diferenciación Celular/efectos de los fármacos , Lignanos/farmacología , Tretinoina/farmacología , Antígeno CD11b/biosíntesis , Fase G1/efectos de los fármacos , Células HL-60 , Humanos , Receptores de Lipopolisacáridos/biosíntesis , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fase de Descanso del Ciclo Celular/efectos de los fármacosRESUMEN
Betulinic acid (BA) is a pentacyclic triterpene found in a number of medicinal plants and has been shown to cause apoptosis in a number of cell lines. We report here that BA may also have an effect on HL-60 cell differentiation. BA was cytotoxic to HL-60 cells with an IC50 of 5.7 microM after a 72-h treatment. Flow cytometry analysis showed that after exposure to 1-12 microM of BA for 72 h, approximately 10% of viable cells were in the sub-G1, presumably apoptotic, phase. At the same time differentiation was induced in approximately 10% (at 1 microM BA) to a maximum of 20% (at 6 microM BA) of cells as judged by the NBT-reduction test, and the expression of membrane markers CD11b and CD14. On the other hand, at 1 and 5 nM, 1alpha,25-dihydroxyvitamin D3 (DHD3) induced differentiation in approximately 10 and 70% of cells, respectively. At 1 nM DHD3, the addition of 1 microM BA increased differentiated cells from 10 to 43% and with 3 microM BA the increase was to 80%. BA also enhanced the effects of DHD3 in the expansion of the G1 cell population with a concomitant decrease of S phase cells. The effects of DHD3 and BA on CD11b and CD14 expression were inhibited by PD98059, a MEK inhibitor. Our results suggest that BA may enhance the effect of DHD3 in inducing mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase-mediated HL-60 cell differentiation.
Asunto(s)
Calcitriol/farmacología , Células HL-60/citología , Triterpenos/farmacología , Apoptosis/efectos de los fármacos , Antígeno CD11b/biosíntesis , Antígeno CD11b/genética , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Activación Enzimática/fisiología , Flavonoides/farmacología , Fase G1/efectos de los fármacos , Fase G1/fisiología , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Humanos , Receptores de Lipopolisacáridos/biosíntesis , Receptores de Lipopolisacáridos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , Triterpenos Pentacíclicos , Triterpenos/antagonistas & inhibidores , Triterpenos/química , Ácido BetulínicoRESUMEN
In the present study, we investigated the in vitro effect of saucernetin-7, which is a dineolignan isolated from Saururus chinensis, on the proliferation, cell cycle-regulation and differentiation of HL-60 human promyelocytic leukemia cells. Saucernetin-7 potently inhibited the proliferation of HL-60 cells in both a dose- and time-dependent manner with an IC50, approximately 5 microM. DNA flow-cytometry indicated that saucernetin-7 markedly induced a G1 phase arrest of HL-60 cells. Among the G1 phase cell cycle-related proteins, the levels of cyclin-dependent kinase (CDK)6 and cyclin D1 were reduced by saucernetin-7, whereas the steady-state levels of CDK2, CDK4, cyclin D2, cyclin D3 and cyclin E were unaffected. The protein and mRNA levels of a CDK inhibitor p21CIP1/WAF1, but not p27KIP1, were markedly increased by saucernetin-7 and p21CIP1/WAF1 induction is likely to occur at the transcriptional level because actinomycin D blocked this induction. In addition, saucernetin-7 markedly enhanced the binding of p21CIP1/WAF1 with CDK2 and CDK6, resulting in the reduced activity of both kinases and the hypophosphorylation of Rb protein. We furthermore suggest that saucernetin-7 is a potent inducer of the differentiation of HL-60 cells, based on observations such as a reduction of the nitroblue tetrazolium level, an increase in the esterase activities and phagocytic activity, morphology changes, and the expression of CD14 and CD66b surface antigens. In conclusion, the onset of saucernetin-7-induced the G0/G1 arrest of HL-60 cells prior to the differentiation is linked to a sharp up-regulation of the p21CIP1/WAF1 level and a decrease in the CDK2 and CDK6 activities. This is the first report demonstrating that saucernetin-7 potently inhibits the proliferation of human promyelocytic HL-60 cells via the G1 phase cell cycle arrest and differentiation induction.
Asunto(s)
Furanos/metabolismo , Lignanos , Extractos Vegetales/metabolismo , Extractos Vegetales/uso terapéutico , Saururaceae/metabolismo , Animales , Antígenos CD , Antígenos de Neoplasias/biosíntesis , Western Blotting , Quinasas CDC2-CDC28/metabolismo , Moléculas de Adhesión Celular/biosíntesis , Ciclo Celular , Diferenciación Celular , División Celular , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Fase G1 , Proteínas Ligadas a GPI , Glutatión Transferasa/metabolismo , Células HL-60 , Humanos , Concentración 50 Inhibidora , Receptores de Lipopolisacáridos/biosíntesis , Ratones , Modelos Químicos , Fagocitosis , Fosforilación , Pruebas de Precipitina , Fase de Descanso del Ciclo Celular , Proteína de Retinoblastoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sales de Tetrazolio/farmacología , Tiazoles/farmacología , Factores de Tiempo , Transcripción GenéticaRESUMEN
Platycodon grandiflorum, a traditional oriental herbal medicine, is known to have immunostimulatory and antitumor effects. PG, a polysaccharide isolated from P. grandiflorum, has been reported to activate macrophages and B cells. Here, we investigated the membrane receptor and intracellular signaling responsible for the activation of macrophages by PG. PG induced the production of nitric oxide (NO) and the mRNA expression of iNOS in RAW 264.7 cells. To investigate the membrane receptor involved in the activation of NO production, we examined the effect of PG on the production of NO in mouse peritoneal macrophages isolated from wild type C3H/HeN and functional Toll-like receptor 4 (TLR4)-deficient C3H/HeJ mice. PG induced NO production by macrophages isolated from C3H/HeN mice, but had no effect on NO production by macrophages isolated from C3H/HeJ mice. Moreover, monoclonal antibodies directed to TLR4 blocked PG-mediated induction of NO production. In addition, LBP and sCD14 was also found to be involved in the activation of NO production by PG. To further investigate, we examined the effect of PG on the activation of DNA binding of NF-kappa B, which is a downstream transcriptional regulator of TLR4. PG caused degradation of I kappa B and activation of DNA binding of NF-kappa B. In addition, TPCK, a specific NF-kappa B inhibitor, abolished PG-mediated induction of DNA binding of NF-kappa B, production of NO and mRNA expression of iNOS, demonstrating the involvement of NF-kappa B in PG-mediated macrophage activation. Taken together, these results suggest that PG-mediated induction of NO production and iNOS mRNA expression in macrophages is mediated, at least in part, by TLR4/NF-kappa B signaling pathway.
Asunto(s)
Inulina/aislamiento & purificación , Activación de Macrófagos/efectos de los fármacos , Glicoproteínas de Membrana/efectos de los fármacos , Raíces de Plantas/química , Platycodon/química , Polisacáridos/aislamiento & purificación , Receptores de Superficie Celular/efectos de los fármacos , Proteínas de Fase Aguda , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Proteínas Portadoras , Comunicación Celular/inmunología , Línea Celular , ADN/antagonistas & inhibidores , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Inulina/farmacología , Receptores de Lipopolisacáridos/biosíntesis , Receptores de Lipopolisacáridos/sangre , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Activación de Macrófagos/inmunología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Medicina Tradicional de Asia Oriental , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C3H , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , FN-kappa B/inmunología , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Extractos Vegetales/química , Polimixina B/farmacología , Polisacáridos/farmacología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Receptor Toll-Like 4 , Receptores Toll-Like , Clorometilcetona de Tosilfenilalanila/farmacologíaRESUMEN
Endothelial cells play a critical role in monocyte differentiation. Platelets also affect terminal maturation of monocytes in vitro. P-selectin is an important adhesion molecule expressed on both endothelial cells and activated platelets. We investigated its effects on human peripheral blood monocyte differentiation under the influence of different cytokines. Generation of dendritic-like cells (DLCs) from peripheral blood monocytes was promoted by immobilized P-selectin in the presence of M-CSF and IL-4 as judged by dendritic cell (DC) morphology; increased expression of CD1a, a DC marker; low phagocytic activity; and high alloreactivity to naive T cells. In contrast to typical DCs, DLCs expressed CD14 and FcgammaRIII (CD16). These features link the possible identity of DLCs to that of an uncommon CD14(+)CD16(+)CD64(-) monocyte subset found to be expanded in a variety of pathological conditions. Functionally, DLCs generated by P-selectin in combination with M-CSF plus IL-4 primed naive allogeneic CD4(+) T cells to produce significantly less IFN-gamma than cells generated by BSA in the presence of M-CSF and IL-4. P-selectin effects on enhancing CD14(+)CD16(+) DLC generation were completely abrogated by pretreatment of cells with the protein kinase C delta inhibitor rottlerin, but not by classical protein kinase C inhibitor Gö6976. Immobilized P-selectin also inhibited macrophage differentiation in response to M-CSF alone as demonstrated by morphology, phenotype, and phagocytosis analysis. The effects of P-selectin on macrophage differentiation were neutralized by pretreatment of monocytes with Ab against P-selectin glycoprotein ligand 1. These results suggest a novel role for P-selectin in regulating monocyte fate determination.
Asunto(s)
Células Dendríticas/citología , Inhibidores de Crecimiento/fisiología , Receptores de Lipopolisacáridos/biosíntesis , Macrófagos/citología , Monocitos/citología , Selectina-P/fisiología , Receptores de IgG/biosíntesis , Regulación hacia Arriba/inmunología , Adyuvantes Inmunológicos/fisiología , Diferenciación Celular/inmunología , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Humanos , Isoenzimas/fisiología , Macrófagos/inmunología , Monocitos/enzimología , Monocitos/inmunología , Proteína Quinasa C/fisiología , Proteína Quinasa C-delta , Proteínas Recombinantes/farmacologíaRESUMEN
In human blood two monocyte populations can be distinguished, i.e., the CD14(++)CD16(-)DR(+) classical monocytes and the CD14(+)CD16(+)DR(++) proinflammatory monocytes that account for only 10% of all monocytes. We have studied TNF production in these two types of cells using three-color immunofluorescence and flow cytometry on whole peripheral blood samples stimulated with either LPS or with the bacterial lipopeptide S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys(4)-OH,trihydrochloride (Pam3Cys). After stimulation with LPS the median fluorescence intensity for TNF protein was 3-fold higher in the proinflammatory monocytes when compared with the classical monocytes. After stimulation with Pam3Cys they almost exclusively responded showing 10-fold-higher levels of median fluorescence intensity for TNF protein. The median fluorescence intensity for Toll-like receptor 2 cell surface protein was found 2-fold higher on CD14(+)CD16(+)DR(++) monocytes, which may explain, in part, the higher Pam3Cys-induced TNF production by these cells. When analyzing secretion of TNF protein into the supernatant in PBMCs after depletion of CD16(+) monocytes we found a reduction of LPS-induced TNF by 28% but Pam3Cys-induced TNF was reduced by 64%. This indicates that the minor population of CD14(+)CD16(+) monocytes are major producers of TNF in human blood.
Asunto(s)
Cisteína/análogos & derivados , Proteínas de Drosophila , Antígenos HLA-DR/biosíntesis , Receptores de Lipopolisacáridos/biosíntesis , Monocitos/inmunología , Monocitos/metabolismo , Receptores de IgG/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Adyuvantes Inmunológicos/farmacología , Células Cultivadas , Cisteína/farmacología , Técnica del Anticuerpo Fluorescente , Antígenos HLA-DR/sangre , Humanos , Separación Inmunomagnética , Inmunofenotipificación , Inflamación/inmunología , Inflamación/patología , Receptores de Lipopolisacáridos/sangre , Lipopolisacáridos/farmacología , Lipoproteínas/farmacología , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Monocitos/clasificación , ARN Mensajero/biosíntesis , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Receptores de IgG/sangre , Receptores Inmunológicos/metabolismo , Receptor Toll-Like 2 , Receptores Toll-Like , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In this study, we describe human FDF03, a novel member of the Ig superfamily expressed as a monomeric 44-kDa transmembrane glycoprotein and containing a single extracellular V-set Ig-like domain. Two potential secreted isoforms were also identified. The gene encoding FDF03 mapped to chromosome 7q22. FDF03 was mostly detected in hemopoietic tissues and was expressed by monocytes, macrophages, and granulocytes, but not by lymphocytes (B, T, and NK cells), indicating an expression restricted to cells of the myelomonocytic lineage. FDF03 was also strongly expressed by monocyte-derived dendritic cells (DC) and preferentially by CD14+/CD1a- DC derived from CD34+ progenitors. Moreover, flow cytometric analysis showed FDF03 expression by CD11c+ blood and tonsil DC, but not by CD11c- DC precursors. The FDF03 cytoplasmic tail contained two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. When overexpressed in pervanadate-treated U937 cells, FDF03 was tyrosine-phosphorylated and recruited Src homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP)-2 and to a lesser extent SHP-1. Like engagement of the ITIM-bearing receptor LAIR-1/p40, cross-linking of FDF03 inhibited calcium mobilization in response to CD32/FcgammaRII aggregation in transfected U937 cells, thus demonstrating that FDF03 can function as an inhibitory receptor. However, in contrast to LAIR-1/p40, cross-linking of FDF03 did not inhibit GM-CSF-induced monocyte differentiation into DC. Thus, FDF03 is a novel ITIM-bearing receptor selectively expressed by cells of myeloid origin, including DC, that may regulate functions other than that of the broadly distributed LAIR-1/p40 molecule.
Asunto(s)
Células Dendríticas/metabolismo , Granulocitos/metabolismo , Inmunoglobulinas/química , Glicoproteínas de Membrana/biosíntesis , Monocitos/metabolismo , Receptores Inmunológicos/biosíntesis , Homología de Secuencia de Aminoácido , Adulto , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Señalización del Calcio/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Cromosomas Humanos Par 7 , Clonación Molecular , ADN Complementario/aislamiento & purificación , Células Dendríticas/inmunología , Granulocitos/inmunología , Humanos , Inmunoglobulinas/genética , Integrina alfaXbeta2/biosíntesis , Péptidos y Proteínas de Señalización Intracelular , Receptores de Lipopolisacáridos/biosíntesis , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Monocitos/citología , Monocitos/inmunología , Familia de Multigenes/inmunología , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/metabolismo , ARN Mensajero/biosíntesis , Receptores de IgG/antagonistas & inhibidores , Receptores de IgG/fisiología , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Proteínas Tirosina Fosfatasas con Dominio SH2 , Solubilidad , Células U937 , Dominios Homologos src/inmunologíaRESUMEN
Extracts of the vine-like plant Tripterygium wilfordii (TW) have been widely used in China as an immunosuppressant and anti-inflammatory drug for the treatments of rheumatoid arthritis, lupus erythematosus and other inflammatory disorders. In this study the molecular mechanisms of action of three TW extracts (ethanol, aqueous, polysaccharide) on the expression of inflammatory cytokines and adhesion molecules were investigated by RT-PCR and immunofluorescence binding techniques. The lipopolysaccharide (LPS)-mediated stimulatory effects of tumor necrosis factor-alpha (TNF-alpha) cytokine production and cell adhesion molecule (CD11c, CD18, CD14, CD54) expression in human monocytic THP-1 cells were modulated by treatments of the TW extracts or tacrolimus (FK506). The TW polysaccharide moiety exhibited more profound immunosuppressive properties than the aqueous and ethanol extracts. Biochemical characterization of the polysaccharide moiety revealed a major molecular weight of 22 kDa (viz. PSP22). The PSP22 was found to be a potential immunosuppressant that manifests the necessary immunomodulating properties.
Asunto(s)
Medicamentos Herbarios Chinos , Molécula 1 de Adhesión Intercelular/biosíntesis , Monocitos/metabolismo , Polisacáridos/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis , Antígenos CD18/biosíntesis , Línea Celular , Humanos , Inmunosupresores/farmacología , Integrina alfaXbeta2/biosíntesis , Receptores de Lipopolisacáridos/biosíntesis , Monocitos/efectos de los fármacos , Extractos Vegetales/farmacología , TripterygiumRESUMEN
Silica and related substances such as silicate have been proven to possess "adjuvant effects". We have previously reported a finding of polyclonal human T cell activation induced by silicate as a superantigen in vitro. In this study, we observed activation-induced cell death in human lymphocytes after stimulation with chrysotile, a kind of silicate. Apoptotic cells were detected flow cytometrically using the TUNEL assay, and the maximum appearance of TUNEL positive cells occurred on day 4 of incubation. Simultaneously the manifestation of small-sized cells in the specimens increased implying apoptosis. Fas expression on lymphocytes increased to day 3 of incubation with chrysotile, and then spontaneously decreased on day 4 when remarkable apoptosis could be detected. Based on these results it is conceivable that activation-induced cell death occurred through Fas-Fas ligand interaction in lymphocytes after stimulation with silicate in a concentration with which no acute cytotoxicity has been detected. Whether and how the repeated apoptosis in definite clones of lymphocytes causes the induction of sFas synthesis need clarification.