RESUMEN
OBJECTIVE: To explore the in vivo effects of PD-0200347, an alpha(2)delta ligand of voltage gated Ca(2+) channels, on cell signalling in osteoarthritic (OA) chondrocytes from an experimental dog model, and examine the effect of PD-0200347 on the major signalling pathways involved in OA cartilage degradation. METHODS: OA was surgically induced in dogs by sectioning the anterior cruciate ligament. OA dogs were divided into three groups and treated orally with (a) placebo; (b) 15 mg/kg/day PD-0200347, or (c) 90 mg/kg/day PD-0200347. The animals were killed 12 weeks after surgery. Cartilage specimens from femoral condyles and tibial plateaus were processed for immunohistochemistry. Specific antibodies against the phosphorylated form of PKCalpha, Ras, c-Raf, the MAP kinases Erk1/2, p38, JNK, and the transcription factors, CREB and Elk-1, were used. RESULTS: Levels of all the tested signalling mediators were increased in the placebo treated (OA) group compared with the normal group. PD-0200347 treatment significantly reduced the levels of the active forms of PKCalpha, c-Raf, Erk1/2, and Elk-1; however, the levels of the active forms of Ras, p38, JNK, and CREB were not affected by the PD-0200347 treatment. CONCLUSION: The action of PD-0200347 on OA chondrocytes is probably mediated through the inhibition of Erk1/2 activation via a Ras independent mechanism. This effect is associated with reduction of the activation of transcription factors such as Elk-1, which leads to the inhibition of the induction of the major catabolic factors involved in the degradation process of OA cartilage.
Asunto(s)
Condrocitos/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/efectos de los fármacos , Osteoartritis/patología , Proteína Quinasa C-alfa/fisiología , Ácido gamma-Aminobutírico/farmacología , Animales , Proteína de Unión a CREB/metabolismo , Canales de Calcio/efectos de los fármacos , Condrocitos/metabolismo , Modelos Animales de Enfermedad , Perros , Relación Dosis-Respuesta a Droga , Ligandos , MAP Quinasa Quinasa 4/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Osteoartritis/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Elk-1 con Dominio ets/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas ras/metabolismoRESUMEN
In this paper we describe a method for validating therapeutic gene targets in arthritic disease. Ribozymes are catalytic oligonucleotides capable of highly sequence-specific cleavage of RNA. We designed ribozymes that cleave the mRNA encoding stromelysin, a matrix metalloproteinase implicated in cartilage catabolism. Ribozymes were initially screened in cultured fibroblasts to identify sites in the mRNA that were accessible for binding and cleavage. Accessible sites for ribozyme binding were found in various regions of the mRNA, including the 5' untranslated region, the coding region, and the 3' untranslated region. Several ribozymes that mediated sequence-specific and dose-dependent inhibition of stromelysin expression were characterized. Site selection in cell culture was predictive of in vivo bioactivity. An assay for measuring cartilage catabolism in rabbit articular cartilage explants was developed. Ribozymes inhibited IL-1-stimulated stromelysin mRNA expression in articular cartilage explants, yet failed to inhibit proteoglycan degradation. This indicated that up-regulation of stromelysin was not essential for IL-1-induced cartilage catabolism. Broad applications of this approach in therapeutic target validation are discussed.
Asunto(s)
Artritis/enzimología , Artritis/terapia , Marcación de Gen , ARN Catalítico/uso terapéutico , Animales , Artritis/genética , Artritis/metabolismo , Cartílago Articular/enzimología , Cartílago Articular/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Fibroblastos/enzimología , Marcación de Gen/métodos , Humanos , Hidrólisis , Inyecciones Intraarticulares , Masculino , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/fisiología , Inhibidores de la Metaloproteinasa de la Matriz , Técnicas de Cultivo de Órganos , ARN Catalítico/administración & dosificación , ARN Catalítico/metabolismo , Conejos , Reproducibilidad de los Resultados , Especificidad por Sustrato , Membrana Sinovial/enzimología , Membrana Sinovial/metabolismoAsunto(s)
Relaciones Padre-Hijo , Pesar , Esquizofrenia/complicaciones , Suicidio/psicología , Adulto , Anécdotas como Asunto , Humanos , MasculinoRESUMEN
Beta-blockers have proven effective in the treatment of migraine. Dermatologic side effects are extremely rare. We report a patient with migraine who developed an acnelike dermatitis with two different beta-blockers with complete resolution of the acne upon discontinuation of each drug.
Asunto(s)
Acné Vulgar/inducido químicamente , Antagonistas Adrenérgicos beta/efectos adversos , Trastornos Migrañosos/tratamiento farmacológico , Nadolol/efectos adversos , Propranolol/efectos adversos , Adulto , Femenino , HumanosRESUMEN
OBJECTIVE AND DESIGN: We expressed soluble rat ICAM-1, generated a polyclonal anti-ICAM-1 antibody, and studied ICAM-1 upregulation in lung inflammatory conditions. Bacterial and baculovirus expression systems were employed. MATERIAL: 250 g adult, male Long Evans rats were used. For in vitro studies, rat pulmonary artery endothelial cells (RPAEC), rat alveolar macrophages and aortic rings were stimulated (as described below) and evaluated for ICAM-1 expression. TREATMENT: RPAEC and macrophages were stimulated with lipopolysaccharide (LPS) and recombinant murine tumour necrosis factor alpha (TNFalpha). In vivo immunoglobulin G (IgG) immune complex-induced lung injury was employed. METHODS: Enzyme-linked immunoassay (ELISA) Western and Northern blot analyses and immunohistochemical evaluations were performed. All experiments were done at least in duplicate. Data were analyzed by two-tailed Student's t-test. RESULTS: ICAM-1 expression of RPAEC was time- and dose-dependent, peaking at 6h after LPS-stimulation. LPS and TNFalpha each enhanced ICAM-1 expression on alveolar macrophages (reaching a maximum at 2 h). In IgG immune complex-induced lung injury, ICAM-1 mRNA isolated from whole lung peaked at 4 h, while lung ICAM- I protein peaked at 6 h. CONCLUSIONS: Quantitation of ICAM-1 expression in vitro and in vivo suggests that ICAM-1 plays a central role in two lung inflammatory models. Furthermore, lung ICAM-1 upregulation involves at least two cell types: vascular endothelial cells and alveolar macrophages.
Asunto(s)
Molécula 1 de Adhesión Intercelular/química , Pulmón/química , Animales , Bacterias/metabolismo , Baculoviridae/metabolismo , Northern Blotting , Western Blotting , Línea Celular , Endotelio Vascular/fisiología , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Inmunoglobulina G/inmunología , Inmunohistoquímica , Insectos/citología , Insectos/metabolismo , Molécula 1 de Adhesión Intercelular/biosíntesis , Pulmón/metabolismo , Macrófagos Alveolares/metabolismo , Masculino , Neumonía/metabolismo , Ratas , Ratas Long-Evans , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Intra-articular injection of streptococcal cell wall Ag followed by i.v. challenge ("reactivation") results in a destructive lymphocyte-dependent monoarticular arthritis. To further define the role of immune mechanisms in the model, Abs to Th1 and Th2-related cytokines were evaluated. Treatment of rats with antibodies to IL-4 reduced swelling, while treatment with anti-IL-10 or anti-IFN-gamma either had no effect or slightly enhanced the inflammatory response. These results suggest that Th-2 immune mechanisms may be, at least in part, operative in the model. To more precisely define the role of IL-4, the effects of anti-IL-4 on monocyte chemoattractant protein-1 (MCP-1) expression were evaluated. Initial studies demonstrated that mRNA (as determined by in situ hybridization) and protein (as determined by immunofluorescence) for MCP-1 were detectable in inflamed synovial tissue in a time-dependent manner. Anti-IL-4 treatment significantly reduced the expression of mRNA for MCP-1 24 and 72 h after reactivation. In addition, anti-MCP-1 inhibited swelling and reduced influx of (111)In-labeled T cells. These data suggest that the reactivation model of streptococcal cell wall Ag-induced arthritis is Th-2 dependent, and that an inter-relationship exists between IL-4 and the expression of MCP-1.
Asunto(s)
Artritis/inmunología , Quimiocina CCL2/fisiología , Interferón gamma/fisiología , Interleucina-10/fisiología , Interleucina-4/fisiología , Peptidoglicano/administración & dosificación , Streptococcus/inmunología , Animales , Anticuerpos Bloqueadores/administración & dosificación , Artritis/etiología , Artritis/patología , Movimiento Celular/inmunología , Quimiocina CCL2/análisis , Quimiocina CCL2/genética , Quimiocina CCL2/inmunología , Femenino , Inmunohistoquímica , Hibridación in Situ , Inyecciones Intraarticulares , Inyecciones Intravenosas , Interferón gamma/inmunología , Interleucina-10/inmunología , Interleucina-4/inmunología , Ratas , Ratas Endogámicas Lew , Bazo/citología , Bazo/inmunología , Linfocitos T/patologíaRESUMEN
Intraarticular injection of streptococcal cell wall (SCW) antigen followed by intravenous challenge results in a T cell-mediated monoarticular arthritis ill female Lewis rats. Initial studies showed that this reactivation response to intravenous SCW antigen is dependent on the presence of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) and that the early phase of swelling is neutrophil-dependent. Neutrophil depletion or passive immunization with antibodies to P-selectin or macrophage inflammatory protein-2 reduced the intensity of ankle edema and the influx of neutrophils. After the first few days, however, the arthritic response is mediated primarily by mononuclear cells. Joint tissues showed up-regulation of mRNA for monocyte chemotactic protein-1 (MCP-1), which could be inhibited in part by anti-IL-4; treatment of rats with antibodies to IL-4 or MCP-1 significantly suppressed development of ankle edema and histopathological evidence of inflammation. Antibodies to interferon-gamma or IL-10 had no effect. Treatment with anti-MCP-1 also suppressed influx of (111)In-labeled T cells into the ankle joint. These data suggest that the late, mononuclear-dependent phase of SCW-induced arthritis in female Lewis rats requires cytokines that up-regulate MCP-1, which in turn may facilitate recruitment and extravasation of mononuclear cells into the joint.
Asunto(s)
Artritis Experimental/inmunología , Quimiocina CCL2/biosíntesis , Factores Quimiotácticos/inmunología , Citocinas/inmunología , Monocinas/inmunología , Neutrófilos/inmunología , Selectina-P/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos/farmacología , Artritis Experimental/patología , Pared Celular/inmunología , Quimiocina CXCL2 , Edema , Femenino , Inmunización Pasiva , Interferón gamma/inmunología , Interleucina-10/inmunología , Interleucina-4/inmunología , Articulaciones/inmunología , Articulaciones/patología , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas Lew , Streptococcus/inmunología , Transcripción GenéticaRESUMEN
We previously demonstrated a new resonator device structure that achieves Q-factors well above those currently realisable. The new structure consists of a microwave cavity, where the enclosure walls consist of distributed Bragg reflectors (DBRs) in three dimensions, made of low-loss sapphire. Theoretical analysis has demonstrated that the resonator's performance is critically dependent upon accurate alignment of the DBR components, thereby maintaining the desired symmetry of the resonant structure. Breaking of the symmetry causes mixing of the high performance Bragg reflected mode with low-Q hybrid cavity modes. The fabrication tolerances required to achieve the expected resonator performance are met with a precise but simple alignment tool. A pair of these resonators have been built at 9.0 GHz, and have demonstrated unloaded Qs in excess of 700,000 at room temperature. These resonators are incorporated into simple two-port feedback oscillator circuits. Phase noise measurements were performed on the two free-running oscillators.
RESUMEN
Immune arthritis in rat ankle joints was induced by intra-articular injection of streptococcal cell was extract (SCW), followed 21 days later by i.v. injection of SCW. This results in a monoarticular arthritis characterized by an influx of neutrophils and mononuclear cells, a 35-fold increase in urinary excretion of 8-hydroxy-deoxyguanosine (8-OH-dGUA; an index of free radical production), ankle edema, and joint damage/destruction. Neutrophil depletion substantially reduced the intensity of ankle edema. Ab-induced blockade of P-selectin or ICAM-1 also reduced the intensity of ankle edema and the influx of neutrophils. Blockade of TNF-alpha or IL-1 resulted in nearly complete and persistent reduction in ankle edema and profound reductions in the accumulation of neutrophils and mononuclear cells in affected joints. Finally, blocking of macrophage-inflammatory protein-2 reduced ankle edema and neutrophil accumulation during the first 2 days after i.v. challenge with SCW. These data indicate that SCW-induced arthritis is neutrophil dependent and that the recruitment of neutrophils and subsequent joint edema requires ICAM-1, P-selectin, and macrophage-inflammatory protein-2, as well as TNF-alpha and IL-1.
Asunto(s)
Artritis/inmunología , Factores Quimiotácticos/fisiología , Molécula 1 de Adhesión Intercelular/fisiología , Monocinas/fisiología , Neutrófilos/fisiología , Selectina-P/fisiología , Peptidoglicano/inmunología , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Artritis/etiología , Quimiocina CXCL2 , Desoxiguanosina/análogos & derivados , Desoxiguanosina/biosíntesis , Desoxiguanosina/orina , Modelos Animales de Enfermedad , Edema/patología , Femenino , Inyecciones Intravenosas , Interleucina-1/fisiología , Neutropenia/inmunología , Peptidoglicano/administración & dosificación , Ratas , Ratas Endogámicas Lew , Factores de Tiempo , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Airway instillation of bacterial lipopolysaccharide (LPS) into rat lungs induces neutrophil accumulation, which is known to be intercellular adhesion molecule-1 (ICAM-1)-dependent. In the present study, ICAM-1 messenger RNA (mRNA) of whole lung was found to increase by 20-fold in this inflammatory model. This increase was reduced by 81% after treatment of animals with anti-tumor necrosis factor-alpha (TNF-alpha) antibody and by 37% after treatment with anti-interleukin-1 (IL-1) antibody. The same interventions reduced whole-lung ICAM-1 protein by 85% and 25%, respectively. The studies were extended to assess the locale in lung of ICAM-I upregulation. Lung vascular ICAM-1 content, which was assessed by vascular fixation of [125I]anti-ICAM-1, rose 4-fold after airway instillation of LPS. This rise was also TNF-alpha-dependent. Under the same experimental conditions, fixation of [125I]anti-ICAM-1 to airway surfaces increased 11-fold in a TNF-alpha-dependent manner. In situ hybridization and immunohistochemical analyses of lung tissue revealed ICAM-1 upregulation in the bronchiolar epithelium and in peribronchiolar smooth muscle. Soluble ICAM-1 could also be detected in bronchoalveolar lavage fluids (BALFs) of animals after intratracheal instillation of LPS. Retrieved alveolar macrophages showed a small, significant, and transient increase in surface expression of ICAM-1. These data indicate, at the very least, a dual compartmentalized (vascular and airway) upregulation of ICAM-1 after airway instillation of LPS. This upregulation requires TNF-alpha and IL-1. The functional significance of upregulated airway ICAM-1 remains to be determined.
Asunto(s)
Molécula 1 de Adhesión Intercelular/genética , Lipopolisacáridos/farmacología , Pulmón/irrigación sanguínea , Pulmón/química , Animales , Anticuerpos/farmacología , Western Blotting , Expresión Génica/efectos de los fármacos , Hibridación in Situ , Molécula 1 de Adhesión Intercelular/análisis , Molécula 1 de Adhesión Intercelular/inmunología , Interleucina-1/inmunología , Pulmón/citología , Macrófagos Alveolares/química , Masculino , Músculo Liso Vascular/química , Músculo Liso Vascular/efectos de los fármacos , Neutrófilos/inmunología , ARN Mensajero/análisis , Ratas , Ratas Endogámicas , Factores de Tiempo , Factor de Necrosis Tumoral alfa/inmunología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Monocyte chemoattractant protein-1 (MCP-1) regulates monocyte accumulation in several macrophage-dependent experimental disease models. In the neonatal brain, activated microglia accumulate rapidly after hypoxic-ischemic injury. These cells produce potentially neurotoxic factors that may contribute to the progression of injury. To determine whether MCP-1 could be one of the molecular signals that influences the microglial response to hypoxic-ischemic injury in the neonatal brain, we examined the impact of acute hypoxic-ischemic injury on MCP-1 mRNA and protein expression. Seven-day-old rats underwent right carotid artery ligation, followed by 3 hours of 8% oxygen exposure, to elicit ipsilateral forebrain hypoxic-ischemic injury. To detect MCP-1 mRNA in situ hybridization assays were performed using 35S-labeled antisense riboprobes generated from rat MCP-1 cDNA. Animals were evaluated 0, 1, 2, 4, 8, 16, 24, 48, and 120 hours after hypoxic exposure (N > or = 3/group). Immunocytochemistry (with a polyclonal rabbit antirat MCP-1 antibody) was used to determine the anatomic and temporal distribution of MCP-1, in samples obtained 10 minutes to 5 days after hypoxic exposure (N > or = 3/group). Monocyte chemoattractant protein-1 mRNA was first detected in periventricular regions of the lesioned hemisphere 1 hour after hypoxia-ischemia; periependymal and intraparenchymal MCP-1 mRNA expression were detected at 4 hours; hybridization signal peaked at 8 to 24 hours; and no MCP-1 mRNA was detected at 48 and 120 hours. In lesioned forebrain, MCP-1 protein expression were consistently detected at 2.5 to 48 hours after hypoxia-ischemia. Many immunoreactive cells appeared to be neurons. These results suggest that in the developing brain, MCP-1 could represent a functionally important molecular signal for the microglial response to hypoxic-ischemic injury.
Asunto(s)
Animales Recién Nacidos/metabolismo , Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Quimiocina CCL2/metabolismo , Hipoxia/metabolismo , Animales , Inmunohistoquímica , Hibridación in Situ , Ratas , Ratas Sprague-DawleyRESUMEN
Activation of the complement cascade and subsequent assembly of the membrane attack complex (MAC) occur in a number of pathophysiological settings. When formed on the surface of endothelial cells in sublytic concentrations, the MAC can induce a number of proinflammatory activities, including the secretion of soluble mediators (eg, interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1) and the up-regulation of cell surface adhesion molecules. Available data indicate that MAC-induced cell activation may occur through several complex signal transduction pathways, but little is known about the intranuclear mechanisms by which complement-derived products promote the up-regulation of inflammatory mediators. Using purified distal complement proteins (C5-9) to assemble functional MAC on early-passage human umbilical vein endothelial cells (HUVECs), we examined mechanisms of MCP-1 and IL-8 induction. Formation of sublytic concentrations of MAC promoted an increase in nuclear factor (NF)-kappa B DNA binding activity within 60 minutes as determined by serial electrophoretic mobility shift assay. Cytosolic to nuclear translocation of NF-kappa B was confirmed by Western immunoblot and immunocytochemical analyses. Formation of the C5b-8 complex also promoted NF-kappa B translocation but to a lesser degree than observed in HUVECs containing complete MAC. No cytosolic to nuclear translocation of the p65 NF-kappa B subunit was observed in unstimulated HUVECs or in cells incubated with the MAC components devoid of C7. Preincubation of HUVECs with pyrrolidine dithiocarbamate prevented MAC-induced increases in IL-8 and MCP-1 mRNA concentrations and protein secretion. A direct cause and effect linkage between MAC assembly and NF-kappa B activation was established through examination of the pharmacological effect of the peptide SN50 on IL-8 and MCP-1 expression. SN50 is a recently engineered 26-amino-acid peptide that contains a lipophilic cell-membrane-permeable motif and a nuclear localization sequence that specifically competes with the nuclear localization sequence of the NF-kappa B p50 subunit. This study provides direct in vitro evidence that the distal complement system (MAC) can promote proinflammatory endothelial cell activation, specifically, increases in IL-8 and MCP-1 mRNA concentrations and protein secretion, and that cytosolic to nuclear translocation of NF-kappa B is necessary for this response.
Asunto(s)
Quimiocina CCL2/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/fisiología , Endotelio Vascular/metabolismo , Interleucina-8/metabolismo , FN-kappa B/metabolismo , Northern Blotting , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , ADN/metabolismo , Endotelio Vascular/efectos de los fármacos , Humanos , Inmunohistoquímica , Lipopolisacáridos/farmacología , Pirrolidinas/farmacología , Tiocarbamatos/farmacología , Factores de Tiempo , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/metabolismoRESUMEN
A new resonator device structure is described that achieves Q-factors well above those currently realizable for conventional room temperature microwave structures. The new structure consists of a microwave cavity, for which the enclosure walls consist of distributed Bragg reflectors (DBR) made of low-loss sapphire. For this configuration, most of the resonant field resides in empty space, with small field strengths in the thin layers of sapphire which comprise the DBR structure. The physical structure takes the form of interpenetrating concentric rings and plates of low-loss sapphire contained in a cylindrical metal enclosure. The theoretical analysis of the DBR resonant structure allows the positions and dimensions of the component rings and plates to be precisely determined for a specified resonant frequency. The resonator Q can be accurately calculated, and plots of the resonant fields clearly show the physical mechanism leading to the observed efficiency of this resonator structure. Experimental results are given for resonators designed at 9.0 and 13.2 GHz. The measured unloaded Q's at room temperature are over 650000 and 450000, respectively.
RESUMEN
Cell surface assembly of the membrane attack complex (MAC) of complement occurs in a variety of pathophysiological settings. Depending upon the density and size distribution of pores formed by the MAC and the functional integrity of membrane regulators of complement activation, the MAC can either cause direct cell lysis or transduce cell activation. We have examined the functional capacity of sublytic concentrations of MAC to induce the secretion of specific alpha- and beta-chemokines from human umbilical vein endothelial cells (HUVECs). Endothelial cell activation by the MAC has particular relevance to complement-dependent inflammatory processes including ischemia-reperfusion injury and acute lung injury. Assembly of sublytic concentrations of the MAC on HUVECs resulted in the sequential secretion of both neutrophil and monocyte chemotactic activities. Analysis of conditioned medium from MAC-bearing HUVECs revealed that the neutrophil chemotactic activity was largely attributable to interleukin (IL)-8, whereas the monocyte chemotactic activity, which was detected later (peak at 8 hours versus 4 hours), was largely attributable to MCP-1. This temporal pattern of MAC-induced secretion of IL-8 and MCP-1 was confirmed using IL-8- and MCP-1-specific enzyme-linked immunosorbent assays. Northern hybridization analysis of HUVECs revealed that MAC deposition was accompanied by an increase in IL-8 and MCP-1 mRNA levels. These data indicate that assembly of sublytic concentrations of the MAC on HUVECs can induce the sequential secretion of both neutrophil and monocyte chemotactic activities and that the former is largely attributable to IL-8 whereas the latter is largely attributable to MCP-1.
Asunto(s)
Quimiocina CCL2/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/fisiología , Endotelio Vascular/metabolismo , Interleucina-8/biosíntesis , Membrana Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CCL2/biosíntesis , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , Complejo de Ataque a Membrana del Sistema Complemento/farmacología , Medios de Cultivo Condicionados , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Humanos , Monocitos/fisiología , Pruebas de Neutralización , Neutrófilos/fisiología , ARN Mensajero/análisis , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/metabolismoRESUMEN
Glucan-induced pulmonary granulomatous vasculitis in the rat mimics several human lung diseases (e.g., Wegener's granulomatosis, intravenous talcosis). We sought to clarify the role of intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of glucan-induced granulomatous vasculitis. Immunohistochemical analysis of lung sections from rats with florid vasculitis (48 hours) revealed marked alveolar septal and lesional expression of ICAM-1. An ex vivo binding analysis with isotope-labeled antibodies and lung sections taken at various times up to 48 hours after glucan infusion revealed a progressive increase in whole-lung ICAM-1 expression. In vivo measurements of vascular wall-associated ICAM-1 expression revealed an earlier rise that began less than 6 hours after glucan infusion, peaked at 24 to 48 hours, and then declined to near baseline during the ensuing 24 to 96 hours. To assess whether ICAM-1 expression both within blood vessel walls and within lesions per se is important in granuloma development, we carried out in vivo neutralization experiments with several different routes of administration of antibody to ICAM-1. Monoclonal antibody to rat ICAM-1 was either infused intravenously at time 0 (when glucan was infused), infused intravenously at time 0 and after 24 hours, instilled only intratracheally 24 hours after glucan infusion, or given both intravenously (time = 0 and 24 hours) and intratracheally (time = 24 hours). Infusions of monoclonal antibody to rat ICAM-1 resulted in dose-dependent reductions in mean granuloma number and cross-sectional area. Intrapulmonary instillation of antibody to rat ICAM-1 (via tracheostomy 24 hours after glucan infusion) resulted in a modest reduction in mean granuloma number and cross-sectional area. When antibody to ICAM-1 was both infused and instilled via the trachea, we found an additive reduction in mean granuloma size and number. There was a 12-fold increase in adhesion of ED-1-positive peripheral blood mononuclear cells (monocytes) to granuloma-bearing frozen lung sections prepared 48 hours after glucan infusion. Moreover, 73% of the additional adherent monocytes were bound specifically to granulomas per se. The increase in ex vivo monocyte binding to lung sections prepared at 48 hours was reduced 62% when sections were incubated with monoclonal antibody to ICAM-1. Taken together, these data indicate that ICAM-1 expression in evolving glucan-induced granulomatous vasculitis occurs first within blood vessel walls and then within lesional cells per se. The in vivo blocking studies suggest that ICAM-1 expression in both anatomic sites is important in granuloma development.
Asunto(s)
Granuloma/patología , Molécula 1 de Adhesión Intercelular/biosíntesis , Enfermedades Pulmonares/patología , Pulmón/efectos de los fármacos , Vasculitis/patología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/uso terapéutico , Glucanos , Granuloma/inducido químicamente , Granuloma/tratamiento farmacológico , Inmunohistoquímica , Inyecciones Intravenosas , Molécula 1 de Adhesión Intercelular/inmunología , Intubación Intratraqueal , Pulmón/química , Pulmón/patología , Enfermedades Pulmonares/inducido químicamente , Enfermedades Pulmonares/tratamiento farmacológico , Masculino , Ratas , Vasculitis/inducido químicamenteRESUMEN
OBJECTIVE: To evaluate the effects of the phospholipase A2 (PLA2) inhibitor manoalide on cartilage degradation, stromelysin expression, and inflammatory cell accumulation in rabbits treated intraarticularly with recombinant human interleukin-1 alpha (rHuIL-1 alpha). METHODS: Rabbits were given an intraarticular injection of rHuIL-1 alpha. At various time points over a 24-hour period, the rabbits were euthanized and the articular space was lavaged with sterile PBS. The proteoglycan content of the lavage fluid was measured using a dimethylmethylene blue assay. PLA2 activity and differential cell counts were also measured. The femur was removed and cartilage proteoglycan content determined. In some experiments, levels of synovial stromelysin messenger RNA (mRNA) were assessed. Manoalide or vehicle was administered 30 minutes before the rHuIL-1 alpha injection. RESULTS: The rHuIL-1 alpha-induced arthritic response is characterized by significant accumulation of inflammatory cells, loss of proteoglycan from the condylar cartilage, and induction of mRNA for stromelysin. PLA2 activity was also elevated in synovial fluids from rHuIL-1 alpha-injected joints. Pretreatment with manoalide (0.3 mg/joint) significantly inhibited PLA2 activity in the synovial fluid, prevented the loss of proteoglycan from the condylar cartilage, and reduced proteoglycan levels in lavage fluids. However, manoalide either had no effect on, or stimulated, cell accumulation. To assess the relationship between the induction of PLA2 and stromelysin, levels of stromelysin mRNA were measured in synovial tissue from manoalide- and vehicle-treated joints. Stromelysin message levels were significantly suppressed in a dose-dependent manner. CONCLUSION: These studies demonstrate that manoalide is a potent inhibitor of inflammation and cartilage catabolism, and suggest that PLA2 is involved in the pathophysiology of rHuIL-1 alpha-induced arthritis in rabbits.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Cartílago/efectos de los fármacos , Metaloendopeptidasas/genética , Inhibidores de Fosfodiesterasa/farmacología , Fosfolipasas A/antagonistas & inhibidores , Líquido Sinovial/citología , Terpenos/farmacología , Animales , Artritis/inducido químicamente , Cartílago/metabolismo , Recuento de Células/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Humanos , Inyecciones Intraarticulares , Interleucina-1 , Masculino , Metaloproteinasa 3 de la Matriz , Proteínas de Neoplasias/genética , Fosfolipasas A/metabolismo , Fosfolipasas A2 , ARN Mensajero/metabolismo , Conejos , Proteínas Recombinantes , Líquido Sinovial/enzimologíaRESUMEN
The known accumulation of macrophages in corpora lutea (CL) at the time of luteal regression prompted us to investigate whether the chemoattractant protein monocyte chemoattractant protein-1 (MCP-1) is expressed in the rat CL. On the day of confirmed mating (Day 0 of pregnancy), regressing CL from the previous (nonfertile) estrous cycle contained immunodetectable MCP-1 and numerous monocytes/macrophages, whereas the newly formed CL of pregnancy, within the same ovary, contained little MCP-1 and few monocytes/macrophages. MCP-1 diminished in the regressing CL on Days 3 and 9 of pregnancy, although numerous monocytes/macrophages remained. The CL of pregnancy on Days 3 and 9 of pregnancy contained minimal MCP-1 and relatively few monocytes/macrophages. By Days 17 and 21 of pregnancy, however, prior to parturition and prior to an accumulation of monocytes/macrophages, expression of MCP-1 increased in the CL of pregnancy. Northern blots revealed a resurgence of luteal MCP-1 mRNA on Day 21 of pregnancy: 3805 +/- 1077 on Day 21 vs. 1059 +/- 177 on Day 9 (p < 0.05; expressed as densitometric units relative to beta-actin). In conclusion, the expression of MCP-1 in the rat CL in association with, or preceding, the appearance of monocytes/macrophages at the time of luteal regression is consistent with the known role of MCP-1 as a potent chemoattractant for monocytes/macrophages. This suggests that MCP-1 might have a prominent role in the immunological process of luteal regression.
Asunto(s)
Quimiocina CCL2/genética , Cuerpo Lúteo/metabolismo , Expresión Génica , Animales , Secuencia de Bases , Northern Blotting , Quimiocina CCL2/análisis , Cuerpo Lúteo/química , Cuerpo Lúteo/citología , Femenino , Inmunohistoquímica , Luteólisis , Macrófagos , Datos de Secuencia Molecular , Monocitos , Embarazo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Catalytic RNA molecules, or ribozymes, have generated significant interest as potential therapeutic agents for controlling gene expression. Although ribozymes have been shown to work in vitro and in cellular assays, there are no reports that demonstrate the efficacy of synthetic, stabilized ribozymes delivered in vivo. We are currently utilizing the rabbit model of interleukin 1-induced arthritis to assess the localization, stability, and efficacy of exogenous antistromelysin hammerhead ribozymes. The matrix metalloproteinase stromelysin is believed to be a key mediator in arthritic diseases. It seems likely therefore that inhibiting stromelysin would be a valid therapeutic approach for arthritis. We found that following intraarticular administration ribozymes were taken up by cells in the synovial lining, were stable in the synovium, and reduced synovial interleukin 1 alpha-induced stromelysin mRNA. This effect was demonstrated with ribozymes containing various chemical modifications that impart nuclease resistance and that recognize several distinct sites on the message. Catalytically inactive ribozymes were ineffective, thus suggesting a cleavage-mediated mechanism of action. These results suggest that ribozymes may be useful in the treatment of arthritic diseases characterized by dysregulation of metalloproteinase expression.
Asunto(s)
Artritis/inducido químicamente , Articulación de la Rodilla/efectos de los fármacos , Metaloendopeptidasas/biosíntesis , ARN Catalítico/farmacología , Membrana Sinovial/efectos de los fármacos , Animales , Artritis/fisiopatología , Secuencia de Bases , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica , Hibridación in Situ , Inyecciones Intraarticulares , Interleucina-1/farmacología , Masculino , Metaloproteinasa 3 de la Matriz , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , ARN Catalítico/metabolismo , ARN Mensajero/biosíntesis , Conejos , Ribonucleasas/metabolismoRESUMEN
Intravenous infusion of particulate yeast cell wall glucan into rats results in the synchronous development of angiocentric pulmonary granulomas that are composed almost entirely of monocytes and macrophages. Previous studies indicate that locally produced monocyte chemoattractant protein-1 (MCP-1) is required for full granuloma development. Because tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 (IL-1) can induce MCP-1 production in a variety of cell types, we sought to determine their potential regulatory roles in this model. A single infusion of anti-TNF-alpha antibody at the time of glucan infusion (time 0) markedly reduced MCP-1 mRNA levels at 1 and 6 hours but not at later time points; there was no effect on granuloma size or number measured at 48 hours. When multiple infusions of anti-TNF-alpha antibody were administered over a 23-hour period (0 to 23 hours), MCP-1 mRNA was reduced through 24 hours, there was a significant reduction in peak bronchoalveolar lavage fluid MCP-1 activity at 48 hours, and there were marked reductions in granuloma size and number at 48 hours. Similar results were observed in animals that received infusions of anti-IL-1 beta. Infusion of anti-IL-1 beta at time 0 resulted in moderate reductions in MCP-1 mRNA at 1 and 6 hours and had no effect on granuloma size or number measured at 48 hours. When multiple infusions of anti-IL-1 beta were administered over a 23-hour period (0 to 23 hours), MCP-1 mRNA was reduced through 24 hours, there was a moderate reduction in peak bronchoalveolar lavage fluid MCP-1 activity at 48 hours, and there were marked reductions in granuloma size and number at 48 hours. A single infusion of anti-TNF-alpha and anti-IL-1 beta together at time 0 resulted in marked reductions in whole lung MCP-1 and mRNA at 1 and 6 hours, but not at 24 hours. Multiple combined infusions of anti-TNF-alpha and anti-IL-1 beta over a 23-hour period resulted in additive reductions in MCP-1 mRNA through 24 hours, bronchoalveolar lavage fluid MCP-1 activity at 48 hours, and granuloma size and number at 48 hours. These data suggest that locally produced TNF-alpha and IL-1 beta play regulatory roles in glucan-induced pulmonary granulomatous vasculitis through the modulation of local MCP-1 production.
Asunto(s)
Anticuerpos/farmacología , Factores Quimiotácticos/metabolismo , Granuloma/metabolismo , Interleucina-1/farmacología , Enfermedades Pulmonares/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Líquido del Lavado Bronquioalveolar , Quimiocina CCL2 , Glucanos , Granuloma/inducido químicamente , Granuloma/patología , Interleucina-1/metabolismo , Pulmón/metabolismo , Enfermedades Pulmonares/inducido químicamente , Enfermedades Pulmonares/patología , Masculino , ARN Mensajero/metabolismo , Ratas , Organismos Libres de Patógenos Específicos , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Chemotactic cytokines coordinate the recruitment of leukocytes into the lung during pulmonary inflammation. In a previous study, we determined that rat pulmonary alveolar macrophages (PAMs) facilitate monocyte recruitment and activation in the lung during acute inflammatory lung injury, in part, through the inducible expression of monocyte chemoattractant protein-1 (MCP-1). MCP-1 is an 11 to 15 kD basic peptide that specifically mediates monocyte chemotaxis and activation. Inflammatory mediators that regulate the expression and secretion of MCP-1 by rat PAMs have not been identified. We determined that stimulation of resident rat PAMs with bacterial lipopolysaccharide (LPS), murine tumor necrosis factor-alpha, or human interleukin-1 beta resulted in the inducible expression of MCP-1 mRNA and the secretion of biologically active MCP-1. In contrast, phorbol myristate acetate, a nonphysiologic leukocyte activator, was significantly less effective in stimulating either enhanced MCP-1 mRNA expression or secretion of MCP-1. These results indicate that the expression of MCP-1 mRNA and the secretion of MCP-1 by rat PAMs are regulated by bacterial products (LPS) and inflammatory cytokines. Further, these results suggest PAMs are regulated by bacterial products (LPS) and inflammatory cytokines. Further, these results suggest that resident PAMs, through elaboration of MCP-1, may play a pivotal role in regulating recruitment and activation of monocytes in the lung during acute inflammatory lung injury.