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
The P2X(7) receptor (P2X(7)R) is an ATP-gated ion channel expressed by monocytes and macrophages. To directly address the role of this receptor in interleukin (IL)-1 beta post-translational processing, we have generated a P2X(7)R-deficient mouse line. P2X(7)R(-/-) macrophages respond to lipopolysaccharide and produce levels of cyclooxygenase-2 and pro-IL-1 beta comparable with those generated by wild-type cells. In response to ATP, however, pro-IL-1 beta produced by the P2X(7)R(-/-) cells is not externalized or activated by caspase-1. Nigericin, an alternate secretion stimulus, promotes release of 17-kDa IL-1 beta from P2X(7)R(-/-) macrophages. In response to in vivo lipopolysaccharide injection, both wild-type and P2X(7)R(-/-) animals display increases in peritoneal lavage IL-6 levels but no detectable IL-1. Subsequent ATP injection to wild-type animals promotes an increase in IL-1, which in turn leads to additional IL-6 production; similar increases did not occur in ATP-treated, LPS-primed P2X(7)R(-/-) animals. Absence of the P2X(7)R thus leads to an inability of peritoneal macrophages to release IL-1 in response to ATP. As a result of the IL-1 deficiency, in vivo cytokine signaling cascades are impaired in P2X(7)R-deficient animals. Together these results demonstrate that P2X(7)R activation can provide a signal that leads to maturation and release of IL-1 beta and initiation of a cytokine cascade.
Asunto(s)
Eliminación de Gen , Interleucina-1/biosíntesis , Precursores de Proteínas/biosíntesis , Receptores Purinérgicos P2/deficiencia , Receptores Purinérgicos P2/fisiología , Adenosina Trifosfato/farmacología , Animales , Células Cultivadas , Ciclooxigenasa 2 , Inducción Enzimática/efectos de los fármacos , Colorantes Fluorescentes/metabolismo , Marcación de Gen , Inflamación/genética , Interleucina-1/metabolismo , Interleucina-6/biosíntesis , Interleucina-6/metabolismo , Isoenzimas/metabolismo , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Noqueados , Nigericina/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Agonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7RESUMEN
Endothelial function was studied ultrasonographically in a healthy subset of African Americans (blacks) because they have an increased risk of hypertension and vascular disease. Twenty-four healthy black and 28 well-matched white subjects were investigated. Ischemia was induced by inflating a cuff over the forearm to 40 mm Hg higher than systolic pressure for 5 minutes. Brachial artery diameter and blood flow velocity were measured at baseline and at 15, 45, and 60 seconds after deflation by use of an Acuson 128XP10 ultrasonograph with a 7.5 MHz transducer. Mean postischemic dilatation, an index of endothelial function, was 1.76+/-0.56% in blacks and 8.79+/-1.22% in whites (P<0.001). Median postischemic vasodilatation in black men [0% (0% to 2.86%)] was not significantly different to that in black women [0.82% (0% to 3.14%)], whereas white women [11.48% (8.70% to 14.29%)] dilated significantly more than white men [4.20% (2.13% to 5.56%)] (P<0.05). Both groups dilated significantly over baseline diameter to sublingual nitroglycerin administration 18.7+/-2.5% (blacks) and 20.2+/-3.2% (whites; P=NS). Mean hyperemic responses did not differ significantly between the 2 subject groups, nor did they differ between men and women of both ethnic groups. We conclude that endothelium-dependent vasodilatation is significantly impaired in healthy, young blacks compared with whites and that gender differences are not seen in blacks with regard to this phenomenon. An impairment in endothelium-dependent NO generation may be a contributing factor to future hypertension and vascular disease in healthy blacks.
Asunto(s)
Población Negra/genética , Arteria Braquial/fisiología , Adulto , Velocidad del Flujo Sanguíneo/fisiología , Endotelio Vascular/fisiología , Femenino , Humanos , Hipertensión/epidemiología , Hipertensión/genética , Isquemia/fisiopatología , Masculino , Persona de Mediana Edad , Factores Sexuales , Ultrasonografía , Vasodilatación/fisiología , Población Blanca/genéticaRESUMEN
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
A new class of glutathione transferases has been discovered by analysis of the expressed sequence tag data base and sequence alignment. Glutathione S-transferases (GSTs) of the new class, named Omega, exist in several mammalian species and Caenorhabditis elegans. In humans, GSTO 1-1 is expressed in most tissues and exhibits glutathione-dependent thiol transferase and dehydroascorbate reductase activities characteristic of the glutaredoxins. The structure of GSTO 1-1 has been determined at 2.0-A resolution and has a characteristic GST fold (Protein Data Bank entry code ). The Omega class GSTs exhibit an unusual N-terminal extension that abuts the C terminus to form a novel structural unit. Unlike other mammalian GSTs, GSTO 1-1 appears to have an active site cysteine that can form a disulfide bond with glutathione.
Asunto(s)
Glutatión Transferasa/química , Glutatión Transferasa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/enzimología , Cristalografía por Rayos X , Femenino , Glutatión Transferasa/genética , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Masculino , Mamíferos , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Conformación Proteica , Estructura Secundaria de Proteína , Lugares Marcados de Secuencia , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
The incidence of cardiovascular disease is lower in premenopausal women compared with men; following menopause, the risk of mortality from cardiovascular disease increases in females. Postischemic dilatation of the brachial artery has been used previously as an index of endothelium-mediated vasodilation. Using this index, we examined a group of premenopausal and postmenopausal women, some of whom were on estrogen replacement therapy (ERT). All subjects were normotensive (blood pressure [BP] <140/90 mm Hg) and normoglycemic (blood glucose, <100 mg/dL). Fourteen healthy women (mean age, 27 +/- 0.8 years; mean total cholesterol, 174 +/- 6.7 mg/dL) and fourteen healthy men (mean age, 26 +/- 1.4 years; mean total cholesterol, 181 +/- 7.2 mg/dL) were investigated. Nineteen postmenopausal women were also examined; 11 were on ERT (mean age, 55 +/- 2.1 years; mean total cholesterol, 213 +/- 6.6 mg/dL) and eight were not on ERT (mean age, 60 +/- 3.6 years; mean total cholesterol, 222 +/- 14.4 mg/dL). Ischemia was induced by inflating a cuff over the forearm to a pressure of 40 mm Hg above systolic for 5 minutes. Doppler ultrasonography (Acuson [Mountain View, CA] 128XP/10c ultrasonograph with a 7.5-MHz linear array transducer) was used to measure the brachial artery diameter before inflation and 15 seconds and 45 to 60 seconds following cuff deflation. Flow-mediated dilatation (FMD%) and hyperemia were defined as the percentage increase over basal diameter and basal flow, respectively. Postischemic median dilatation in men was 4.20% (interquartile range, 2.13% to 5.56%) and 11.48% (interquartile range, 8.70% to 14.29%) in age-matched premenopausal women (P < .01). For women on ERT, the postischemic median dilatation was 8.11% (interquartile range, 6.01% to 11.60%), as compared with 2.82% (interquartile range, 1.32% to 3.28%) for women without ERT (P < .01). Premenopausal women showed significantly greater dilatation after ischemia than postmenopausal women without ERT (P < .0001). Hyperemia was similar in all groups. These findings show that postischemic vasodilation of the brachial artery is greater in premenopausal women versus age-matched men; it is decreased in postmenopausal women, and ERT restores it toward normal. The pathophysiology underlying the diminution in postischemic dilatation may be relevant to atherogenesis and coronary artery disease (CAD).
Asunto(s)
Vasos Sanguíneos/anatomía & histología , Terapia de Reemplazo de Estrógeno , Hemodinámica/fisiología , Adulto , Anciano , Vasos Sanguíneos/diagnóstico por imagen , Vasos Sanguíneos/efectos de los fármacos , Femenino , Humanos , Hiperemia/diagnóstico por imagen , Hiperemia/fisiopatología , Masculino , Persona de Mediana Edad , Posmenopausia , Caracteres Sexuales , Ultrasonografía , Vasodilatación/fisiologíaRESUMEN
Lipopolysaccharide-activated human monocytes produce prointerleukin (pro-IL)-1beta but release little of this inflammatory cytokine as the biologically active species. Efficient externalization of mature 17-kDa cytokine requires that the activated monocytes encounter a secondary stimulus such as ATP. To identify cation requirements of the ATP-induced process, lipopolysaccharide-activated monocytes were treated with ATP in media containing different Cl- salts or sucrose. Media devoid of Na+ did not support IL-1beta processing. Titration of NaCl into choline chloride- or sucrose-based media restored 17-kDa IL-1beta production. Na+ replacement, however, was not sufficient to support ATP-induced production of 17-kDa IL-1beta in the presence of >/=37 mM extracellular K+ or Li+. Inhibition by K+ suggests that efflux of this cation is a necessary component of the stimulus-coupled response. The inhibitory effect achieved by Na+ depletion is not due to inactivation of the ATP receptor and is distinct from a caspase-1 inhibitor. Stimulus-coupled IL-1beta posttranslational processing, therefore, requires extracellular Na+ for a step downstream of the initiating stimulus but preceding caspase-1 activation.
Asunto(s)
Interleucina-1/metabolismo , Monocitos/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Cationes Monovalentes/farmacología , Senescencia Celular/fisiología , Medios de Cultivo/química , Humanos , Monocitos/efectos de los fármacos , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7 , Sodio/farmacologíaRESUMEN
In response to LPS, peritoneal macrophages produce IL-1, but, for the most part, newly synthesized cytokine molecules remain cell associated. Externalization and proteolytic processing of pro-IL-1 beta can be initiated by extracellular ATP. In this study, kinetics and inhibitor sensitivity of the stimulus-coupled mechanism were investigated with [35S]methionine-labeled macrophages. Optimal ATP concentrations required to promote cytokine post-translational processing suggest the involvement of a P2Z type of receptor. Proteolysis of pro-IL-1 beta initiates within 7.5 min of ATP addition; 17-kDa mature IL-1 beta is observed first intracellularly and subsequently extracellularly. In contrast, ATP-treated cells do not contain 17-kDa IL-1 alpha. Macrophages exposed to ATP continuously or only for a 15-min pulse release IL-1 alpha, IL-1 beta, and lactate dehydrogenase (LDH). Proteolytic maturation of IL-1 beta exceeds that of IL-1 alpha in both formats, but pulsed cells process the externalized cytokines more efficiently. Ethacrynic acid and DIDS (4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid) block ATP-induced proteolysis of pro-IL-1 beta and prevent release of pro-IL-1 alpha/beta and LDH; they do not inhibit ATP-induced K+ (86Rb+) efflux. Ethacrynic acid inhibits release of both forms of IL-1 with a similar concentration dependence; within the arrested cells, procytokines accumulate in a Triton-insoluble fraction. An IL-1 beta-converting enzyme inhibitor blocks proteolysis of IL-1 beta, but it does not prevent release of pro-IL-1 alpha, pro-IL-1 beta, or LDH. These results indicate that ATP stimulates externalization of both IL-1 alpha and IL-1 beta. The ATP-induced cytokine release mechanism is accompanied by cell death and requires activity of an anion transport inhibitor-sensitive component, but this pathway operates independently of cytokine proteolytic processing.
Asunto(s)
Adenosina Trifosfato/fisiología , Interleucina-1/metabolismo , Procesamiento Proteico-Postraduccional , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Adenosina Trifosfato/farmacología , Animales , Caspasa 1 , Cisteína Endopeptidasas/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Ácido Etacrínico/farmacología , Interleucina-1/antagonistas & inhibidores , Transporte Iónico/efectos de los fármacos , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C3H , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Factores de TiempoRESUMEN
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
Interleukin (IL)-1beta produced by monocytes and macrophages is not released via the normal secretory apparatus, and prior to its release, this cytokine must be proteolytically processed to generate a mature biologically active species. Biochemical mechanisms that regulate these posttranslational steps are not well understood. Lipopolysaccharide (LPS) is a poor activator of IL-1 posttranslational processing despite serving as a potent inducer of IL-1 synthesis. For example, freshly isolated human monocytes treated with LPS released <30% of their newly synthesized IL-1beta as the mature 17-kDa cytokine species, and monocytes that were aged overnight in culture prior to LPS treatment released no 17-kDa cytokine. In contrast, addition of extracellular ATP promoted IL-1beta posttranslational processing from both monocyte populations. Previous studies indicated that ATP, acting via surface P2Z-type receptors, promoted major intracellular ionic changes. To explore whether these ionic changes were required for cytokine posttranslational processing, LPS-stimulated human monocytes were maintained in ionically altered media. Hypotonic conditions promoted an efficient and selective release of mature 17-kDa IL-1beta from LPS-activated monocytes in the absence of ATP. In contrast, hypertonic conditions blocked the ATP-induced posttranslational processing reactions. Both hypotonic stress- and ATP-induced processing were blocked when NaI was substituted for NaCl within the medium; substitution with NaSCN or NaNO3 also blocked the ATP response, but these salts were less inhibitory against the hypotonic stimulus. Sodium glucuronate substitution did not inhibit cytokine processing induced by either stimulus. Removal of divalent cations from the medium did not affect the ATP response, but pretreatment of monocytes with the phosphatase inhibitor okadaic acid dose-dependently suppressed ATP-induced IL-1beta posttranslational processing. A volume-induced change to the intracellular ionic environment, therefore, may represent a key element of the mechanism by which IL-1beta posttranslational processing is initiated. The strong dependence of this cytokine release mechanism on chloride anions suggests that selective anion transporters function as important components of this response.
Asunto(s)
Interleucina-1/metabolismo , Monocitos/metabolismo , Procesamiento Proteico-Postraduccional , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/farmacología , Bumetanida/farmacología , Calcio/metabolismo , Furosemida/farmacología , Humanos , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Ácido Ocadaico/farmacología , Presión Osmótica , Quinina/farmacología , Transducción de SeñalRESUMEN
LPS-activated murine peritoneal macrophages produce IL-1 beta but externalize little mature cytokine in the absence of a secondary stimulus, and CTLs previously were reported to serve this capacity. The release of 17-kDa IL-1 beta from LPS-activated BALB/c macrophages occurred rapidly after the addition of C57/B1-derived allogeneic CTLs; within 30 min of coculture, mature IL-1 beta was observed in the medium, and maximum release was achieved within 4 h. CTL-induced post-translational processing was efficient, and >80% of newly synthesized pro-IL-1 beta was released into the medium as the 17-kDa species. Externalization of IL-1 beta required active recognition of the macrophage target by the CTL preparation; C57/B1 CTLs promoted the release of mature IL-1 beta from allogeneic BALB/c macrophages, but not from syngeneic C57/B1 macrophages. In contrast, extracellular ATP promoted mature IL-1 beta release from both macrophage populations. CTL-induced cytokine post-translational processing was blocked by anion transport inhibitors, including 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, UK5099, and the anti-inflammatory agent tenidap. An analogue of tenidap, CP-100,829, was more effective as an inhibitor of both IL-1 beta post-translational processing and anion transport. In contrast, the close structural analogue CP-236,492 inhibited neither process. Tenidap's activity was reversible and was not mimicked by cyclooxygenase inhibitors or by cycloheximide. Therefore, tenidap disrupted CTL-induced IL-1 beta post-translational processing by a mechanism dependent on anion transport inhibition. Multiple stimuli are likely to operate in vivo to promote IL-1 beta post-translational processing, and anion transport inhibitors such as tenidap that suppress cytokine processing independently of the initiating stimulus thus represent attractive candidates as therapeutic regulators of IL-1 production.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Indoles/farmacología , Interleucina-1/antagonistas & inhibidores , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/metabolismo , Animales , Aniones/antagonistas & inhibidores , Proteínas Portadoras/inmunología , Técnicas de Cocultivo , Citotoxicidad Inmunológica/efectos de los fármacos , Indoles/química , Transporte Iónico/efectos de los fármacos , Transporte Iónico/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Oxindoles , Procesamiento Proteico-Postraduccional/inmunología , Factores de TiempoRESUMEN
Cytokine-suppressing anti-inflammatory drugs (CSAIDs) are reported to inhibit production of proinflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) by affecting a stress-induced kinase. To gain a better understanding of the selectivity and cellular dynamics of this type of inhibitor, we studied in vitro the prototype member of this class of agents, SKF86002. Lipopolysaccharide (LPS)-activated human monocytes treated with SKF86002 produced less proIL-1 beta but normal amounts of the noncytokine lysozyme. Two-dimensional gel analysis indicated that only eight polypeptides produced by monocytes were decreased by SKF86002. Inhibition of IL-1 beta production was achieved by affecting two separate steps in this cytokine's biogenesis. First, SKF86002 lowered proIL-1 beta synthesis. By pulse-chase analysis, this effect was localized to a posttranscriptional site of action; maximal inhibition was observed when SKF86002 was added at the time of cytokine translation. Exposure of monocytes to SKF86002 for > 2 hr led to a loss of IL-1 beta inhibitory activity, suggesting that these cells adapted to this agent. Moreover, LPS-activated monocytes that were pretreated with granulocyte-macrophage colony-stimulating factor were less sensitive to the proIL-1 beta inhibitory effect of SKF86002, and production of proIL-1 beta by cytokine-stimulated human fibroblasts was impaired only modestly by the CSAID. A second effect of SKF86002 was to inhibit release of IL-1 beta into the medium in response to high concentrations of LPS; this effect is observed only with freshly isolated human monocytes as other IL-1 beta-producing cells do not release significant cytokine in response to LPS. The ability of SKF86002 to inhibit this posttranslational mechanism was mimicked by lysosomotrophic agents such as chloroquine, quinacrine, and methylamine. In contrast, chloroquine, and quinacrine were not effective inhibitors of monocyte proIL-1 beta translation. Thus, SKF86002 inhibits IL-1 beta production by affecting at least two distinct steps in the biosynthesis of this cytokine. Manifestation of these two effects, however, is dependent on the length of time for which cells are exposed to this agent and the nature of the cytokine-producing cellular system.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Imidazoles/farmacología , Interleucina-1/biosíntesis , Tiazoles/farmacología , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Interleucina-1/antagonistas & inhibidores , Interleucina-1/metabolismo , Cinética , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Biosíntesis de ProteínasRESUMEN
IL-1 beta is an important inflammatory mediator produced by monocytes and macrophages after LPS stimulation. In the absence of a secondary stimulus, however, little IL-1 beta is released into the medium. Previously, ATP was shown to promote the release and proteolytic maturation of IL-1 beta from LPS-stimulated murine peritoneal macrophages. Tenidap, a new anti-inflammatory and antiarthritic agent, inhibited the release and maturation of IL-1 beta induced in vitro by ATP treatment of murine peritoneal macrophages. Tenidap's inhibitory activity was mimicked by other agents that blocked anion transport, such as UK5099 and DIDS. In contrast, cyclooxygenase-inhibiting nonsteroidal anti-inflammatory drugs, such as piroxicam and naproxen, did not impair ATP-induced post-translational processing. Human monocytes responded to LPS to produce IL-1 beta, but externalized little of their newly synthesized cytokine. ATP at concentrations > or = 2 mM promoted IL-1 beta release from these cells. The degree to which the released cytokine was proteolytically processed to its biologically active 17-kDa species, however, depended on the pH of the medium; a greater processing efficiency was observed at slightly acidic (pH 6.9) values. Tenidap and other anion transport inhibitors effectively prevented the ATP response of cultured human monocytes. Likewise, LPS-stimulated human alveolar macrophages responded to ATP by releasing 17-kDa IL-1 beta, and tenidap inhibited this response. The ATP-induced release and maturation of IL-1 beta from human monocytes and macrophages, therefore, was suppressed by anion transport inhibitors, suggesting that anion conductance is a necessary component of the ATP-promoted externalization mechanism. In view of IL-1's importance as an inflammatory mediator, tenidap may demonstrate novel anti-inflammatory activities by virtue of its inhibition of the post-translational release and maturation of this cytokine.
Asunto(s)
Aniones/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Indoles/farmacología , Interleucina-1/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Peritoneales/metabolismo , Monocitos/metabolismo , Adenosina Trifosfato/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/farmacología , Citoplasma/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Monocitos/efectos de los fármacos , Oxindoles , Procesamiento Proteico-Postraduccional/efectos de los fármacosRESUMEN
Lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages produce large quantities of interleukin (IL)-1 beta but in the absence of a secondary stimulus little of this cytokine is proteolytically processed to its mature biologically active state and externalized. The potassium-proton ionophore nigericin and ATP are known to promote the maturation and release of IL-1 beta from LPS-stimulated cells. We investigated the mechanisms by which these agents act in an attempt to understand requirements of the post-translational processing. Like nigericin, the ionophores A204 and lasalocid induced the release and maturation of IL-1 beta. The electrogenic potassium ionophore valinomycin, however, did not stimulate these post-translational events. Addition of nigericin or lasalocid to LPS-stimulated cells produced a rapid intracellular acidification; A204, however, did not alter pH, indicating that an acidification was not necessary for activation of IL-1 beta maturation. Macrophages treated with ATP became rounded and swollen, and after 30 min of treatment their appearance was comparable with cells treated with nigericin. Post-translational maturation and release of IL-1 beta began immediately after ATP addition. The majority of the 17-kDa mature IL-1 beta produced within the first 30 min of treatment was recovered extracellularly; in contrast, during this same time period the 35-kDa IL-1 beta precursor and the cytoplasmic marker enzyme lactate dehydrogenase and the lysosomal enzyme beta-N-acetylglucosaminidase remained cell-associated. ATP, therefore, promoted both the proteolytic maturation of IL-1 beta and the release of the biologically active species in the absence of cell lysis. Longer incubations with ATP caused cytolysis as judged by the release of the cytoplasmic enzymes. ADP was less active than ATP at initiating the post-translational maturation and release of IL-1 beta and AMP, GTP, and UTP were totally inactive, ATP, nigericin, A204, and lasalocid promoted a rapid and complete loss of the potassium analog 86Rb+ from cells that were preloaded with this cation; valinomycin-treated cells released only a portion of the radiolabeled cation. Agents that promoted the maturation and release of IL-1 beta from LPS-stimulated macrophages, therefore, shared an ability to mobilize intracellular potassium. Macrophages treated with ATP or nigericin in medium that contained KCl rather than NaCl failed to proteolytically activate and to release IL-1 beta. These data suggest that ATP and nigericin induce a net decrease in intracellular levels of K+ which is necessary for activation of the post-translational maturation of IL-1 beta.
Asunto(s)
Adenosina Trifosfato/farmacología , Interleucina-1/metabolismo , Nigericina/farmacología , Potasio/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Transporte Biológico/efectos de los fármacos , Citoplasma/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Lasalocido/farmacología , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C3H , Monensina/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Radioisótopos de Rubidio/metabolismoRESUMEN
Mouse peritoneal macrophages stimulated with LPS produce large amounts of pro-IL-1 beta. When these cells were pulse-labeled with [35S]methionine, however, little labeled cytokine appeared in the medium after a chase, and that which was externalized was not processed to its mature biologically active form. In an effort to promote proteolytic maturation of IL-1 beta, macrophages were treated with agents that were expected to compromise their viability. The calcium ionophore A23187 and the detergent saponin caused complete release of nonprocessed 35-kDa pro-IL-1 beta and liberation into the extracellular medium of the cytoplasmic marker enzyme LDH and the lysosomal enzyme beta-N-acetylglucosaminidase. Hypotonic lysis resulted in the release of a 20-kDa IL-1 beta species that was distinct from the 17-kDa mature species. Importantly, incubation of the murine macrophages with the potassium/proton ionophore nigericin led to a quantitative conversion of pro-IL-1 beta to a 17-kDa species. The N-terminus of this nigericin-derived product possessed the amino acid sequence expected for mature biologically active IL-1 beta. Monensin, an ionophore similar to nigericin, did not induce release or proteolysis of IL-1 beta. Complete release of mature IL-1 beta required concentrations of nigericin in excess of 2 microM and a minimum of 10 min of treatment. Mature 17-kDa IL-1 beta was observed within the nigericin-treated cells before their lysis. Nigericin's effect was not limited to mouse peritoneal macrophages, inasmuch as the ionophore also induced release and proteolytic maturation of IL-1 beta produced by LPS-stimulated human peripheral blood monocytes. Treatment of macrophages with LPS and nigericin, therefore, results in a unique series of intracellular events that promote formation of mature 17-kDa IL-1 beta.
Asunto(s)
Interleucina-1/metabolismo , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Nigericina/farmacología , Animales , Exocitosis/efectos de los fármacos , Humanos , Técnicas In Vitro , Interleucina-1/química , Lipopolisacáridos/administración & dosificación , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C3H , Peso Molecular , Monocitos/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacosRESUMEN
The ability of tumor-promoting phorbol diesters to inhibit both insulin receptor tyrosine kinase activity and its intracellular signaling correlates with the phosphorylation of the insulin receptor beta subunit on serine and threonine residues. In the present studies, mouse 3T3 fibroblasts transfected with a human insulin receptor cDNA and expressing greater than one million of these receptors per cell were labeled with [32P]phosphate and treated with or without 100 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). Phosphorylated insulin receptors were immunoprecipitated and digested with trypsin. Alternatively, insulin receptors affinity purified from human term placenta were phosphorylated by protein kinase C prior to trypsin digestion of the 32P-labeled beta subunit. Analysis of the tryptic phosphopeptides from both the in vivo and in vitro labeled receptors by reversed-phase HPLC and two-dimensional thin-layer separation revealed that PMA and protein kinase C enhanced the phosphorylation of a peptide with identical chromatographic properties. Partial hydrolysis and radiosequence analysis of the phosphopeptide derived from insulin receptor phosphorylated by protein kinase C indicated that the phosphorylation of this tryptic peptide occurred specifically on a threonine, three amino acids from the amino terminus of the tryptic fragment. Comparison of these data with the known, deduced receptor sequence suggested that the receptor-derived tryptic phosphopeptide might be Ile-Leu-Thr(P)-Leu-Pro-Arg. Comigration of a phosphorylated synthetic peptide containing this sequence with the receptor-derived phosphopeptide confirmed the identity of the tryptic fragment. The phosphorylation site corresponds to threonine 1336 in the human insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)