RESUMEN
Lipopolysaccharide (LPS)-activated monocytes and macrophages produce large quantities of pro-interleukin (IL)-1beta but externalize little mature cytokine. Efficient post-translational processing of the procytokine occurs in vitro when these cells encounter a secretion stimulus such as ATP, cytolytic T cells, or hypotonic stress. Each of these stimuli promotes rapid conversion of 31-kDa pro-IL-1beta to its mature 17-kDa species and release of the 17-kDa cytokine. In this study, two novel pharmacological agents, CP-424,174 and CP-412,245, are identified as potent inhibitors of stimulus-coupled IL-1beta post-translational processing. These agents, both diarylsulfonylureas, block formation of mature IL-1beta without increasing the amount of procytokine that is released extracellularly, and they inhibit independently of the secretion stimulus used. Conditioned medium derived from LPS-activated/ATP-treated human monocytes maintained in the absence and presence of CP-424,174 contained comparable quantities of IL-6, tumor necrosis factor-alpha (TNFalpha), and IL-1RA, but 30-fold less IL-1beta was generated in the test agent's presence. As a result of this decrease, monocyte conditioned medium prepared in the presence of CP-424,174 demonstrated a greatly diminished capacity to promote an IL-1-dependent response (induction of serum amyloid A synthesis by Hep3B cells). Oral administration of CP-424,174 to mice resulted in inhibition of IL-1 in the absence of an effect on IL-6 and TNFalpha. These novel agents, therefore, act as selective cytokine release inhibitors and define a new therapeutic approach for controlling IL-1 production in inflammatory diseases.
Asunto(s)
Interleucina-1/biosíntesis , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Adenosina Trifosfato/farmacología , Animales , Citocinas/biosíntesis , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Soluciones Hipotónicas , Técnicas In Vitro , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Metionina/metabolismo , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Pruebas de Precipitina , Compuestos de Sulfonilurea/farmacología , Linfocitos T/efectos de los fármacosRESUMEN
LL-Z1271alpha, a fungal metabolite, dose-dependently inhibited interleukin-1beta (IL-1beta) production in lipopolysaccharide (LPS)-stimulated human whole blood. Oral administration of LL-Z1271alpha to LPS-challenged mice caused significant lowering in the IL-1beta levels in peritoneal cavity. Data presented suggest that LL-Z1271alpha inhibits IL-1beta production by a novel mechanism as the inhibitory activity was not due to effects on caspase-1 (IL-1beta converting enzyme), the ATP-induced release mechanism or a lysosomotrophic effect.
Asunto(s)
Interleucina-1/biosíntesis , Terpenos/farmacología , Adenosina Trifosfato/farmacología , Animales , Inhibidores de Caspasas , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Lipopolisacáridos/farmacología , RatonesRESUMEN
Cultured monocytes and macrophages stimulated with LPS produce large quantities of proIL-1beta, but release little mature cytokine to the medium. The efficiency at which the procytokine is converted to its active 17-kDa species and released extracellularly is enhanced by treating cytokine-producing cells with a secretion stimulus such as ATP or nigericin. To determine whether this need for a secretion stimulus extends to blood, individual donors were bled twice daily for 4 consecutive days, and the collected blood samples were subjected to a two-step IL-1 production assay. LPS-activated blood samples generated cell-free IL-1beta, but levels of the extracellular cytokine were greatly increased by subsequent treatment with ATP or nigericin. Specificity and concentration requirements of the nucleotide triphosphate effect suggests a P2X(7) receptor involvement. Quantities of IL-1beta generated by an individual donor's blood in response to the LPS-only and LPS/ATP stimuli were relatively consistent over the 4-day period. Between donors, consistent differences in cytokine production capacity were observed. Blood samples treated with ATP also demonstrated enhanced IL-18 production, but TNF-alpha levels decreased. Among leukocytes, monocytes appeared to be the most affected cellular targets of the ATP stimulus. These studies indicate that an exogenous stimulus is required by blood for the efficient production of IL-1beta and IL-18, and suggest that circulating blood monocytes constitutively express a P2X(7)-like receptor.
Asunto(s)
Adenosina Trifosfato/agonistas , Interleucina-18/sangre , Interleucina-18/metabolismo , Interleucina-1/sangre , Interleucina-1/metabolismo , Adenosina Trifosfato/sangre , Adyuvantes Inmunológicos/agonistas , Adyuvantes Inmunológicos/sangre , Artritis Reumatoide/sangre , Artritis Reumatoide/inmunología , Ritmo Circadiano/inmunología , Eosinófilos/inmunología , Eosinófilos/metabolismo , Femenino , Humanos , Interleucina-1/biosíntesis , Lipopolisacáridos/sangre , Lipopolisacáridos/farmacología , Activación de Linfocitos , Linfocitos/inmunología , Linfocitos/metabolismo , Masculino , Monocitos/inmunología , Monocitos/metabolismo , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Procesamiento Proteico-Postraduccional/inmunología , Receptores Purinérgicos P2/biosíntesis , Receptores Purinérgicos P2X7RESUMEN
Despite a large production capacity, freshly isolated lipopolysaccharide (LPS)-activated human monocytes release only a small percentage of their newly synthesized interleukin (IL)-1 beta into the medium. Extracellular ATP, acting via surface P2z-type purino-receptors, increases cytokine posttranslational processing. To explore whether this ATP response was affected by culture conditions, monocytes were maintained for different time periods in the absence and presence of various media components including fetal bovine and human sera and recombinant human cytokines. The ability of monocytes to produce radiolabeled pro-IL-1 beta in response to LPS and to posttranslationally process the procytokine after ATP stimulation was affected both by time in culture and by the presence of specific media components. These observations indicate that ATP's ability to promote human monocyte IL-1 beta posttranslational processing is a dynamic process that is subject to regulation by cytokines and/or growth factors. Changes in monocyte/macrophage ATP responsiveness may provide an important regulatory mechanism for the control of IL-1 biological activity in vivo.
Asunto(s)
Interleucina-1/metabolismo , Monocitos/metabolismo , Adenosina Trifosfato/metabolismo , Bioensayo , Células Cultivadas , Senescencia Celular , Medios de Cultivo , Hexoquinasa/metabolismo , Humanos , Interferón gamma/farmacología , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Procesamiento Proteico-Postraduccional , Sialoglicoproteínas/metabolismoRESUMEN
Tenidap [5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H- indole-1-carboxamide], a novel antirheumatic agent, produces a rapid and sustained intracellular acidification when applied to cells in culture. To investigate the mechanism by which this change in ionic homeostasis is achieved, the acidification activities of structural analogs of tenidap were determined, and the movements of [14C]tenidap into and out of cells were explored. The acidification activity of tenidap was enhanced by lowering extracellular pH, suggesting that the free acid species was required for this process. Consistent with this requirement, a non-acidic analog of tenidap did not produce a change in intracellular pH (pHi). In contrast, multihalogenated derivatives of tenidap produced greater changes in pHi than did tenidap, and one analog produced a transient acidification from which the cell recovered; this recovery, however, was blocked by an inhibitor of the Na+/H+ antiporter. Fibroblasts incubated with [14C]tenidap achieved within 5 min a level of cell-associated drug that remained constant during longer incubations. Simultaneous addition of the electrogenic ionophore valinomycin or the P-glycoprotein inhibitor 4-(3,4-dihydro-6,7-dimethoxy-2(1H)-isoquinolinyl)-N-[2-(3,4-dimethoxyphe nyl) ethyl]-6,7-dimethoxy-2-quinazolinamine (CP-100,356) caused a time- and concentration-dependent increase in the level of cell-associated [14C]tenidap; other agents tested did not promote this enhanced cellular accumulation. [14C]Tenidap accumulated by fibroblasts in the presence of CP-100,356 subsequently was released when these cells were placed in a tenidap- and CP-100,356-free medium. Importantly, several agents that are known to inhibit anion transport processes, including alpha-cyano-beta-(1-phenylindol-3-yl) acrylate, 5-nitro-2(3-phenylpropylamino)-benzoic acid, and meclofenamic acid, inhibited efflux of [14C]tenidap. In contrast, ethacrynic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid did not impair the efflux process. Likewise, tenidap analogs that produced a sustained intracellular acidification blocked the efflux of [14C]tenidap, but non-acidifying species did not. These data suggest that movements of tenidap into and/or out of cells is a facilitated process subject to pharmacological intervention. Together, the structural selectivity of the acidification response and the evidence of facilitated transport suggest that the pHi modulating activity of tenidap is dependent on its unique physicochemical properties. Due to the dependence of these physicochemical properties on environmental and cellular conditions, in vivo expression of the acidification activity is likely to occur only within restricted environments that favor this tenidap-induced process.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Antiportadores/efectos de los fármacos , Antiportadores/metabolismo , Indoles/farmacología , Animales , Aniones , Antiinflamatorios no Esteroideos/farmacocinética , Antiportadores/antagonistas & inhibidores , Transporte Biológico Activo/efectos de los fármacos , Células Cultivadas , Fenómenos Químicos , Química Física , Antiportadores de Cloruro-Bicarbonato , Interacciones Farmacológicas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Indoles/farmacocinética , Líquido Intracelular/metabolismo , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Ionóforos/farmacología , Células L , Ratones , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Oxindoles , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno/metabolismo , Relación Estructura-Actividad , Valinomicina/farmacologíaRESUMEN
Tenidap is a novel anti-inflammatory and anti-arthritic agent that lowers intracellular pH and suppresses anion transport when applied to cells in vitro. Both of these parameters are known to influence pro-inflammatory cell function. To investigate whether tenidap can modulate cellular responses to cytokine stimulation, several in vitro cytokine-driven assays were characterized with respect to their tenidap sensitivity. Human monocytes treated with granulocyte-macrophage colony stimulating factor (GM-CSF) demonstrated an increased production of IL-6 as well as an increased total translational activity. Tenidap dose-dependently inhibited both cytokine-induced responses; the effect on IL-6, however, occurred at lower tenidap concentrations than those required to prevent the increase in total translational activity. In contrast, the known translational inhibitor cycloheximide did not demonstrate selectivity for IL-6; this agent decreased the GM-CSF-induced increase in total translational activity in parallel with its effects on IL-6. GM-CSF-treated monocytes also produced greater amounts of IL-1 beta in response to LPS stimulation than did non-GM-CSF-treated cells, and tenidap again suppressed this cytokine-induced activation. Human Hep3B cells treated with a combination of interleukin (IL)-1 beta and IL-6 demonstrated an acute phase-type of response. These hepatoma cells increased production of the positive acute phase protein serum amyloid A (SAA) while they decreased production of a negative acute phase protein human serum albumin (HSA). Tenidap dose-dependently inhibited the cytokine-induced increase in SAA production without effecting synthesis of HSA or total TCA-precipitable macromolecules. Importantly, the ability of tenidap to alter these various cytokine responses was not shared with piroxicam, a potent cyclooxygenase inhibitor. Finally, human neutrophils treated with either GM-CSF or tumor necrosis factor (TNF)-alpha demonstrated an increased chloride conductance as measured by the loss of radioactive chloride from 36Cl-loaded cells. When tenidap was included within the medium during cytokine stimulation, loss of radioactive chloride was prevented. Thus, tenidap inhibited the cytokine-induced increase in anion transport. Together, these results indicate that tenidap can suppress cellular activation processes induced by a variety of cytokines. This functional antagonism is not dependent on cyclooxygenase inhibition but, rather, appears to link to tenidap's unique ability to alter ionic homeostasis. These in vitro observations, therefore, may help to explain how this novel anti-inflammatory agent acts to lower acute phase proteins and IL-6 levels in man.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Indoles/farmacología , Interleucina-1/biosíntesis , Interleucina-1/farmacología , Interleucina-6/biosíntesis , Interleucina-6/farmacología , Monocitos/inmunología , Carcinoma Hepatocelular , Línea Celular , Células Cultivadas , Cicloheximida/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Lipopolisacáridos/farmacología , Neoplasias Hepáticas , Monocitos/efectos de los fármacos , Oxindoles , Biosíntesis de Proteínas , Células Tumorales CultivadasRESUMEN
The presence of positive acute phase proteins within the circulation of rheumatoid arthritis patients suggests that elevated cytokine production associated with this chronic inflammatory disorder initiates the hepatic acute phase response. Cytokines produced at inflammatory lesions are believed to travel via the circulation to the liver where they induce acute phase protein production by hepatocytes. To test whether serum from rheumatoid arthritis patients contained sufficient levels of cytokines to promote an acute phase response in vitro, a bioassay was developed that employed the human hepatoma cell line Hep3B. These cells produced the acute phase protein serum amyloid A (SAA) in response to a combination of recombinant IL-1 beta and IL-6 or to monocyte conditioned medium. Serum (or plasma) from normal individuals or from rheumatoid arthritis patients did not induce SAA production by Hep3B cells. Moreover, these serum samples did not prevent SAA production induced by monocyte conditioned medium, indicating that they did not contain inhibitors of cytokine activity. Despite the inactivity of serum samples, synovial fluid samples obtained from rheumatoid arthritis patients were active in the hepatocyte bioassay and promoted SAA synthesis. One synovial fluid sample was analysed in detail to identify cytokines responsible for the SAA-inducing activity. Neutralizing antisera against IL-6 and IL-1 beta blocked this activity by > 90% whereas anti-IL1 alpha and anti-TNF-alpha sera were without effect. Absolute cytokine levels within the synovial fluid sample were determined by ELISA; IL-6, IL-beta and TNF-alpha, but not IL-1 alpha, were confirmed to be present. Moreover, the synovial fluid sample contained a large amount of the IL-1 receptor antagonist. These data indicate, therefore, that synovial fluid recovered from an inflamed joint contains all the necessary cytokines in balance with inhibitors to promote SAA production by Hep3B cells. The steady state levels of these factors within the plasma compartment, however, were insufficient to induce the acute phase response by cultured Hep3B cells, suggesting that this system does not mimic the relationship between the circulation and the liver that likely exists in rheumatoid arthritis patients.
Asunto(s)
Artritis Reumatoide/inmunología , Interleucina-1/farmacología , Interleucina-6/farmacología , Monocitos/fisiología , Proteína Amiloide A Sérica/biosíntesis , Líquido Sinovial/inmunología , Artritis Reumatoide/sangre , Artritis Reumatoide/fisiopatología , Carcinoma Hepatocelular , Línea Celular , Medios de Cultivo Condicionados , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-1/análisis , Interleucina-1/fisiología , Interleucina-6/análisis , Interleucina-6/fisiología , Neoplasias Hepáticas , Valores de Referencia , Líquido Sinovial/fisiología , Células Tumorales CultivadasRESUMEN
Tenidap is a novel anti-inflammatory and antiarthritic agent that in clinical studies of rheumatoid arthritis patients, displays symptomatic efficacy superior to nonsteroidal anti-inflammatory drugs (NSAIDs) and equivalent to combinations of NSAIDs and second line agents. Clinical and preclinical biochemical studies have demonstrated that tenidap combines cytokine modulation with suppression of prostaglandin biosynthesis. To better understand tenidap's mechanism of action, in vitro studies of intracellular pH (pHi) were conducted. In cells loaded with the pH-sensitive fluorescence dye 2',7'-bis-(2-carboxyethyl)-5-(and -6) carboxyfluorescein, tenidap, but not NSAIDs, caused a rapid and sustained acidification of the cytoplasmic compartment. Tenidap did not act as a proton ionophore, as it did not dissipate the low pH within lysosomes. Mammalian cells regulate pHi through the concerted action of a number of specific transport proteins, including sodium-proton antiporters and chloride-bicarbonate exchangers. Tenidap did not alter pHi via inhibition of the sodium-proton antiporter, but inhibited activity of chloride-bicarbonate exchangers, as did UK5099, a known anion-transport inhibitor that also lowers pHi. This similar activity suggests that the pHi change is coupled to anion transport inhibition. As a result of the pHi change, tenidap affected pH-dependent cellular activities. Tenidap inhibited mannose 6-phosphate receptor-mediated endocytosis, inhibited protein synthesis, and stimulated accumulation of the amino acid leucine. Effects on these cellular processes rapidly reversed when tenidap was removed from the culture medium. Tenidap's in vitro activities were highly dependent on the medium composition; protein content, pH, and bicarbonate concentration all were important factors that influenced activity. These results indicate that tenidap is a potent anion-transport inhibitor and modulator of pHi. Within the appropriate cell or tissue microenvironment, these activities may contribute to tenidap's novel therapeutic profile.
Asunto(s)
Aniones/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Citoplasma/efectos de los fármacos , Indoles/farmacología , Animales , Bicarbonatos/metabolismo , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Cloruros/metabolismo , Endocitosis/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Células L , Lisosomas/fisiología , Ratones , Oxindoles , Biosíntesis de Proteínas , Receptor IGF Tipo 2/fisiología , Intercambiadores de Sodio-Hidrógeno/efectos de los fármacosRESUMEN
Guanidinothiazolecarboxamides (GTCs) are a novel class of antitumor agents found to be systemically active against experimental pulmonary metastases of 3LL Lewis lung carcinoma. A series of substituted benzothiazole GTCs were found to produce enhancement of survival in this model by using 8 days of intraperitoneal dosing initiated 2 days after intravenous tumor challenge. Quantitative structure-activity relationships have been discovered in the GTC series with survival enhancement correlated to substituent parameters. Optimal correlations were found between the probit transform of the drug-induced increased lifespan (ILS) and field and pi parameters. Among the most effective analogues in this series was N-(5-fluorobenzothiazol-2-yl)-2-guanidinothiazole-4-carboxam ide.