RESUMEN
BACKGROUND: Chemokines are critical mediators of T-cell homing into inflamed skin. The complex nature of this multicellular response makes it difficult to analyse mechanisms mediating the early responses in vivo. OBJECTIVES: To visualize directly T-cell homing into inflamed skin and its inhibition by blockades using a unique noninvasive confocal microscopy. MATERIALS AND METHODS: A mouse model of allergic contact dermatitis was used. T cells from oxazolone-sensitized and -challenged Balb/c mice were first analysed phenotypically in vitro. CD4 T cells were then labelled with a tracker dye and transferred into Balb/c-SCID mice. The recipient mice were challenged with oxazolone and CD4 T-cell homing into inflamed skin was visualized. RESULTS: T cells with the skin homing receptors CCR4 and CCR10 were increased in the affected skin and draining lymph nodes, and effectively attracted by their specific chemokines CCL17, CCL22 and CCL27 in vitro. Using in vivo imaging, T-cell migration into the inflamed skin was observed at 2 h after application, peaking at 12 h and continuing for 48 h. Simultaneous systemic administration of neutralizing antibodies against CCR4 ligands (CCL17 and CCL22) and CCR10 ligand (CCL27) led to a significant suppression of T-cell migration and skin inflammation. CONCLUSIONS: Our data indicate that these tissue-selective adhesion molecules and chemokine/receptor pathways act in concert to attract specialized T-cell populations to mediate cutaneous inflammation. The in vivo imaging technique can be applicable to other models of cutaneous diseases to help with better understanding of the pathogenesis and monitoring the therapeutic effects.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Movimiento Celular/inmunología , Quimiocinas/inmunología , Dermatitis por Contacto/inmunología , Receptores CCR10/inmunología , Receptores CCR4/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Linfocitos T CD4-Positivos/metabolismo , Inhibición de Migración Celular , Movimiento Celular/fisiología , Quimiocinas/metabolismo , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Modelos Animales , Oxazolona/farmacología , Receptores CCR10/metabolismo , Receptores CCR4/metabolismo , Piel/inmunología , Piel/metabolismo , Estadística como AsuntoRESUMEN
Age-related thymic involution is a multifactorial process related to age-related changes in intrathymic T-cell development and cytokines. In contrast, early thymic involution, because of genetic differences that cause rapid or slow thymic involution at younger age, is less well characterized. Here, we analysed three representative rapid-involuting strains of mice, BXD 8, 18 and 32, compared with three representative slow-involuting strains, BXD 9, 19 and 29, all at 2 months of age. In rapid-involuting strains compared with slow involution strains, thymocyte production, as indicated by CD4+ and CD8+ T-cell receptor recombination excision circle (TREC), were decreased. Rapid-involution strains of mice exhibited a developmental block at the DN1 to DN2 and CD4-CD8- (DN) to CD4+CD8+ (double positive, DP) transition stages. There was also increased susceptibility to H2O2-induced apoptosis, decreased thymic expression of IL-7, decreased expression of an IL-7 downstream anti-apoptosis gene, Bcl-2, and increased expression of a pro-apoptotic gene, Bad. In contrast, IL-7R expression was higher on DN thymocytes of rapid-involution strains. The increased expression of IL-7R was associated with an increased thymocyte proliferation in response to anti-CD3 + IL-7 or anti-CD3 + IL-12 + IL-7. These findings indicate that, even at young age, genetic differences of IL-7/IL-7R regulation pathway in BXD strains of mice can lead to characteristic phenotypic changes that have been previously associated with age-related thymic involution.
Asunto(s)
Timo/fisiología , Envejecimiento , Animales , Apoptosis , Complejo CD3/fisiología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Femenino , Interleucina-7/farmacología , Ratones , Ratones Endogámicos , Ratones Transgénicos , Estrés Oxidativo , Fenotipo , Receptores de Antígenos de Linfocitos T/metabolismo , Especificidad de la Especie , Linfocitos T/metabolismo , Timo/citologíaRESUMEN
The elevation of soluble Fas (sFas) in the sera of patients with liver disease suggests a role for sFas in the disease process; whether it is protective or not is controversial. To determine the effects of sFas on Fas-induced liver apoptosis, we manipulated mice to produce sFas by transfecting them in vivo with different amounts of an adenovirus that produces mouse sFas driven by the CMV promoter (AdsFas). Fas-mediated apoptosis was induced by administration of anti-mouse Fas (Jo2; 10 microg/mouse) one week later. The administration of AdsFas (10(3), 10(7), or 10(9) pfu/mouse), which was associated with only minimal side-effects, resulted in a significant reduction in the liver transaminase levels and mortality of the mice on challenge with Jo2, as compared to control mice treated with AdLacZ. However, the protective effect of AdsFas was not complete. The possibility that Jo2-induction of TNF-alpha in the Kupffer cells of the liver contributes to the pathology was therefore tested. Although administration of soluble TNF receptor (sTNFRI) alone did not protect the mice from the lethal effects of Jo2, administration of sTNFRI (200 microg/mouse) after infection with AdsFas (10(9) pfu/mouse) resulted in 100% survival of the mice on challenge with Jo2. To confirm that the production of TNF-alpha by Kupffer cells produce the lethal effects of Jo2 that remained after treatment with AdsFas, these cells were selectively ablated by treatment of the mice with gadolinium chloride prior to challenge with Jo2. This treatment greatly reduced early mortality and hepatocellular damage as well as TNF-alpha production 6 h after injection of Jo2. These results indicate that: (1) AdsFas prevents Jo2-induced apoptosis of hepatocytes; (2) In addition to mediating Fas-mediated apoptosis of hepatocytes, Jo2 can separately induce TNF-alpha production by Kupffer cells resulting in early mortality, and (3) Optimal protection from Jo2-induced mortality can be achieved by protection of liver cells by pretreatment with both AdsFas and sTNFRI.
Asunto(s)
Apoptosis , Terapia Genética , Hepatocitos/metabolismo , Macrófagos del Hígado/metabolismo , Hígado/patología , Receptor fas/genética , Receptor fas/fisiología , Animales , Anticuerpos/inmunología , Femenino , Gadolinio/metabolismo , Hemorragia/etiología , Hemorragia/prevención & control , Hepatitis/etiología , Hepatitis/prevención & control , Inmunohistoquímica , Hígado/enzimología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Solubilidad , Factor de Necrosis Tumoral alfa/biosíntesis , Receptor fas/sangre , Receptor fas/inmunologíaRESUMEN
Lipopolysaccharide and D-galactosamine induced lethality and apoptotic liver injury is dependent upon endogenously produced TNF-alpha. Unlike the response to high dose lipopolysaccharide alone, death in this model is a direct result of hepatocyte apoptosis. In a series of recent studies, we have demonstrated that mortality and hepatic injury following lipopolysaccharide administration in D-galactosamine-sensitized mice is dependent upon secreted 17 kDa TNF-alpha acting primarily through the p55 TNF receptor. Transgenic mice expressing null forms of TNF-alpha, the p55 receptor, or expressing only a cell-associated form of TNF-alpha exhibited no mortality and only modest liver injury when challenged with 8 mg of D-galactosamine and 100 ng of lipopolysaccharide. Although Fas ligand expression is increased in the liver, it appeared to play no significant role in outcome, since mice expressing a mutant form of Fas ligand are still sensitive to LPS- and D-galactosamine-induced lethality. Finally, we have seen significant variation in LPS- and D-galactosamine-mediated lethality among different strains of mice. The non-obese diabetic or NOD mouse is highly resistant to LPS-and D-galactosamine-induced lethality, and this appears to be secondary to a post-receptor defect in p55 TNF receptor signaling. The studies confirm an essential role for TNF-alpha and p55 TNF receptor signaling in the hepatocyte apoptosis and lethality associated with lipopolysaccharide and D-galactosamine administration.
Asunto(s)
Apoptosis/genética , Enfermedad Hepática Inducida por Sustancias y Drogas , Galactosamina/farmacología , Lipopolisacáridos/farmacología , Longevidad/genética , Factor de Necrosis Tumoral alfa/genética , Animales , Caspasa 3 , Caspasas/biosíntesis , Quimioterapia Combinada , Humanos , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Hepatopatías/sangre , Hepatopatías/patología , Longevidad/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones Transgénicos , Necrosis , Proteínas Recombinantes , Especificidad de la Especie , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
OBJECTIVE: To determine whether defective T cell apoptosis is associated with the development of a chronic arthritis subsequent to mycoplasma infection, and to determine whether deletion of T cells can prevent the development of this arthritis. METHODS: B6 wild-type (B6-+/+), B6-lpr/lpr, and B6-gld/gld mice were infected with Mycoplasma pulmonis. The severity of lymphocytic infiltration and joint damage was evaluated, and the degree of recovery of viable mycoplasma from the spleen and joints was determined. Antigen-presenting cells derived from Fas mutant lpr mice (lpr-APC) were transfected ex vivo with an adenovirus (Ad) vector to yield lpr-APC expressing high levels of Fas ligand (lpr-APC-AdFasL), which in turn were transferred intraperitoneally into M pulmonis-infected B6-gld/gld mice. The development of arthritis subsequent to M pulmonis infection and the induction of apoptosis of cells within the synovial tissue and lymph nodes of lpr-APC-AdFasL-treated B6-gld/gld mice were determined. RESULTS: Infection of B6-lpr/lpr and B6-gld/gld mice with M pulmonis resulted in an acute-phase inflammation of the synovium that later developed into a chronic erosive arthritis. Similar infection of B6-+/+ mice resulted only in an acute joint inflammatory response that resolved. Chronic arthritis in B6-gld/gld mice and B6-lpr/lpr was not due to persistent infection, since there were no differences in the rates of clearance of M pulmonis from the joints of B6-gld/gld or B6-lpr/lpr mice compared with B6-+/+ mice. Treatment of infected B6-gld/gld mice with lpr-APC-AdFasL resulted in a significantly decreased incidence of chronic arthritis that was associated with a decrease in lymph node T cells, but not with apoptosis of synovial T cells or fibroblasts. CONCLUSION: Defective Fas/FasL-mediated apoptosis of T cells is an important factor that rendered arthritis-resistant B6 mice susceptible to the development of a chronic erosive arthritis subsequent to mycoplasma infection. In vivo lpr-APC-AdFasL cell-gene therapy is a safe and effective method for inhibiting the development of this arthritis.
Asunto(s)
Apoptosis , Artritis Reactiva/patología , Glicoproteínas de Membrana/genética , Infecciones por Mycoplasma/patología , Enfermedad Aguda , Adenoviridae/genética , Animales , Artritis Reactiva/microbiología , Artritis Reactiva/terapia , Linfocitos B/inmunología , Linfocitos B/microbiología , Enfermedad Crónica , Modelos Animales de Enfermedad , Proteína Ligando Fas , Terapia Genética , Ganglios Linfáticos/citología , Ganglios Linfáticos/microbiología , Masculino , Ratones , Ratones Mutantes , Infecciones por Mycoplasma/complicaciones , Infecciones por Mycoplasma/inmunología , Membrana Sinovial/patología , Linfocitos T/inmunología , Linfocitos T/microbiologíaRESUMEN
Pharmacokinetics and immunogenicity of six different recombinant human soluble p55 tumor necrosis factor (TNF) receptor I (sTNFR-I) constructs were evaluated in juvenile baboons. The constructs included either an sTNFR-I IgG1 immunoadhesin (p55 sTNFR-I Fc) or five different sTNFR-I constructs covalently linked to polyethylene glycol. The constructs were administered intravenously three times, and pharmacokinetics and immunogenicity were examined over 63 days. All of the constructs were immunogenic, with the exception of a 2.6-domain monomeric sTNFR-I. To evaluate whether the nonimmunogenic 2.6-domain monomeric construct could protect baboons against TNF-alpha-induced mortality, baboons were pretreated with 1, 5, or 10 mg/kg body wt and were compared with baboons receiving either placebo or 1 mg/kg body wt of the dimeric 4.0-domain sTNFR-I construct (n = 3 each) before lethal Escherichia coli bacteremia. The monomeric construct protected baboons and neutralized TNF bioactivity, although greater quantities were required compared with the dimeric 4.0-domain sTNFR-I construct. We conclude that E. coli-recombinant-derived human sTNFR-I constructs can be generated with minimal immunogenicity on repeated administration and still protect against the consequences of exaggerated TNF-alpha production.
Asunto(s)
Inmunoglobulina G/metabolismo , Papio/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Secuencia de Aminoácidos , Animales , Recuento de Células Sanguíneas , Clonación Molecular , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Etanercept , Semivida , Hemodinámica/fisiología , Humanos , Inmunoglobulina G/inmunología , Cinética , Datos de Secuencia Molecular , Polietilenglicoles , Receptores del Factor de Necrosis Tumoral/inmunología , Factor de Necrosis Tumoral alfa/farmacocinética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), essential components in the pathogenesis of immunoinflammatory diseases, are strongly induced in monocytes by direct contact with stimulated T lymphocytes. This study demonstrates that adult human serum (HS) but not fetal calf or cord blood serum displays inhibitory activity toward the contact-mediated activation of monocytes by stimulated T cells, decreasing the production of both TNF-alpha and IL-1beta. Fractionation of HS and N-terminal microsequencing as well as electroelution of material subjected to preparative electrophoresis revealed that apolipoprotein A-I (apo A-I), a "negative" acute-phase protein, was the inhibitory factor. Functional assays and flow cytometry analyses show that high-density lipoprotein (HDL)-associated apo A-I inhibits contact-mediated activation of monocytes by binding to stimulated T cells, thus inhibiting TNF-alpha and IL-1beta production at both protein and messenger RNA levels. Furthermore, apo A-I inhibits monocyte inflammatory functions in peripheral blood mononuclear cells activated by either specific antigens or lectins without affecting cell proliferation. These results demonstrate a new anti-inflammatory activity of HDL-associated apo A-I that might have modulating functions in nonseptic conditions. Therefore, because HDL has been shown to bind and neutralize lipopolysaccharide, HDL appears to play an important part in modulating both acute and chronic inflammation. The novel anti-inflammatory function of apo A-I reported here might lead to new therapeutic approaches in inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, and atherosclerosis.
Asunto(s)
Apolipoproteína A-I/farmacología , Comunicación Celular/efectos de los fármacos , Interleucina-1/biosíntesis , Lipoproteínas HDL/fisiología , Monocitos/efectos de los fármacos , Linfocitos T/fisiología , Factor de Necrosis Tumoral alfa/biosíntesis , Reacción de Fase Aguda , Adulto , Animales , Apolipoproteína A-I/aislamiento & purificación , Bovinos , Depresión Química , Diseño de Fármacos , Sangre Fetal , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Recién Nacido , Inflamación , Interleucina-1/genética , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Lipoproteínas HDL/aislamiento & purificación , Ratones , Monocitos/metabolismo , ARN Mensajero/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Tumor necrosis factor receptor (TNFR) p55-knockout (KO) mice are susceptible profoundly to Salmonella infection. One day after peritoneal inoculation, TNFR-KO mice harbor 1,000-fold more bacteria in liver and spleen than wild-type mice despite the formation of well organized granulomas. Macrophages from TNFR-KO mice produce abundant quantities of reactive oxygen and nitrogen species in response to Salmonella but nevertheless exhibit poor bactericidal activity. Treatment with IFN-gamma enhances killing by wild-type macrophages but does not restore the killing defect of TNFR-KO cells. Bactericidal activity of macrophages can be abrogated by a deletion in the gene encoding TNFalpha but not by saturating concentrations of TNF-soluble receptor, suggesting that intracellular TNFalpha can regulate killing of Salmonella by macrophages. Peritoneal macrophages from TNFR-KO mice fail to localize NADPH oxidase-containing vesicles to Salmonella-containing vacuoles. A TNFR-KO mutation substantially restores virulence to an attenuated mutant bacterial strain lacking the type III secretory system encoded by Salmonella pathogenicity island 2 (SPI2), suggesting that TNFalpha and SPI2 have opposing actions on a common pathway of vesicular trafficking. TNFalpha-TNFRp55 signaling plays a critical role in the immediate innate immune response to an intracellular pathogen by optimizing the delivery of toxic reactive oxygen species to the phagosome.
Asunto(s)
Antígenos CD/fisiología , Macrófagos/metabolismo , NADPH Oxidasas/metabolismo , Fagosomas/enzimología , Receptores del Factor de Necrosis Tumoral/fisiología , Salmonella/enzimología , Animales , Antígenos CD/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Fagosomas/ultraestructura , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis TumoralRESUMEN
Tumor necrosis factor-alpha (TNF-alpha) is a mediator of severe inflammatory processes, including rheumatoid arthritis. Suppression of TNF with a soluble type I or type II receptor molecule (TNF-RI or TNF-RII) has the potential to decrease cytokine levels and modulate inflammatory diseases in humans. However, it has recently been reported that treatment of mice with a TNF-RI:Fc immunoadhesin protein augmented Gram positive infections and subsequent mortality. To determine if TNF-alpha blockade with soluble TNF-alpha receptors might alter immune system function, assays were assessed in rodents treated with a dimeric form of the p55 TNF-RI, Tumor Necrosis Factor-binding protein (TNFbp). Administration of TNFbp resulted in suppression of primary and secondary IgG antibody responses and cell-mediated immune function. No treatment-related differences were detected in immune-enhancing assays or non-specific immune function parameters. Bacterial host resistance assays with Listeria monocytogenes, Staphylococcus aureus or Escherichia coli showed an increase in tissue colony counts only with L. monocytogenes challenged animals following TNFbp administration. These results suggest that TNFbp has the capacity to inhibit adaptive immune function in experimental animal models. Studies suggest that while reducing TNF-alpha is important in controlling cytokine-dependent disease states, maintenance of a threshold level may be critical for normal immune function.
Asunto(s)
Antígenos CD/farmacología , Inmunidad/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Antígenos CD/química , Dimerización , Infecciones por Escherichia coli/inmunología , Femenino , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunoglobulina G/biosíntesis , Listeriosis/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratas , Ratas Sprague-Dawley , Receptores del Factor de Necrosis Tumoral/química , Receptores Tipo I de Factores de Necrosis Tumoral , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Infecciones Estafilocócicas/inmunologíaRESUMEN
The major limitation of adenovirus is its association with induction of an inflammatory response and relatively short-term production of the gene therapy transgene product. Adeno-associated virus (AAV) is a 4.68-kb single-strand DNA virus that contains ITRs for viral replication and a packaging signal, and also has been engineered to contain therapeutic genes up to 5 kb in length. Transduction of recombinant AAV (rAAV) results in low inflammatory response and long-term expression. We have cloned a low-immunogenic form of human sTNFRI (sTNFRI2.6D) into AAV (rAAVsTNFRI). This vector was analyzed for its ability to transfect and neutralize the effect of TNF-alpha on primary rheumatoid arthritis synovial fibroblast (RASFs). The rAAVsTNFRI was transduced into the cells at 1.8 x 10(1), 1.8 x 10(2), and 1.8 x 10(3) viral particles per cell. There was greater than 90% neutralization of TNF-alpha at 1.8 x 10(3) viral particles/cell. There was a significant decrease in the synovial cell hyperplasia and cartilage and bone destruction in human TNF-alpha transgenic mice treated intraarticularly with rAAVsTNFRI. These results indicate that the low-immunogenic and long-term expressing vector, rAAVsTNFRI, can be used to deliver the soluble TNF-alpha in vitro and in vivo and effectively reduce the severity of arthritis.
Asunto(s)
Antígenos CD/genética , Artritis Reumatoide/patología , Artritis/terapia , Dependovirus/genética , Terapia Genética/métodos , Receptores del Factor de Necrosis Tumoral/genética , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Antígenos CD/metabolismo , Artritis/patología , Artritis Experimental/terapia , Células Cultivadas , Colagenasas/efectos de los fármacos , Colagenasas/metabolismo , Femenino , Fibroblastos/virología , Humanos , Células L , Ratones , Ratones Transgénicos , Músculos/virología , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sinovitis/patología , Sinovitis/terapia , Pruebas de ToxicidadRESUMEN
The dose-dependent increase in mortality in patients with sepsis who are treated with tumor necrosis factor (TNF) p75 soluble receptor Fc conjugate (p75-Fc) remains unexplained. In this study, neutralization of TNF-alpha-induced interleukin (IL)-8 by p75-Fc in whole human blood exhibited a U-shaped inhibition curve, whereas the TNF-soluble p55 receptor, linked to polyethylene glycol (p55-PEG), exhibited a dose-dependent inhibition. Native soluble p75 increased TNF-alpha-induced IL-8, versus a 61% reduction by native p55. Spontaneous IL-8 production was increased by p75-Fc or native p75 but not by p55-PEG or native p55. Unexpectedly, TNF-alpha-stimulated IL-1 receptor antagonist was suppressed by p75-Fc but not by p55-PEG. Studies of binding to TNF trimer revealed that p75-Fc has an affinity 40-fold lower than that of p55-PEG and a faster off rate. Native and p75-Fc pass TNF-alpha to membrane receptors more readily than does native or p55-PEG, which may partly explain the increased mortality in patients with sepsis who are treated with p75-Fc.
Asunto(s)
Antígenos CD/inmunología , Células Sanguíneas/efectos de los fármacos , Interleucina-8/inmunología , Receptores del Factor de Necrosis Tumoral/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Células Sanguíneas/inmunología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-8/análisis , Pruebas de Neutralización , Polietilenglicoles , Receptores Tipo I de Factores de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis Tumoral , Proteínas Recombinantes de Fusión/inmunología , Sialoglicoproteínas/análisis , Sialoglicoproteínas/inmunologíaRESUMEN
BACKGROUND: The purpose of this study was to examine the effects of a steam burn injury on apoptosis in gut-associated lymphoid tissue and to determine whether endogenous glucocorticoid and Fas ligand signaling were involved in this process. METHODS: Histologic analysis, in situ deoxynucleotidyl transferase dUTP nick-end labeling staining and annexin V and 7-amino-actinomycin-D flow cytometry of lymphocyte populations were evaluated in intraepithelial lymphocytes and Peyer's patch. Additional mice were pretreated with a glucocorticoid receptor antagonist (mifepristone) before the steam burn. Similarly, C3H/HeJ-FasL(gld) mice lacking functional Fas ligand were also studied. RESULTS: Apoptosis was significantly increased in intraepithelial lymphocytes and Peyer's patch after the burn injury. Mifepristone pretreatment significantly reduced apoptosis in both T- and B-cell populations in intraepithelial lymphocytes after the burn injury. In contrast, the increased apoptosis seen in B-cells from Peyer's patch was not seen in C3H/HeJFasL(gld) mice, whereas the increased apoptosis in CD8+ T-cells was unaffected. CONCLUSION: Both corticosteroids and FasL contribute to the apoptosis in gut-associated lymphoid tissues early after burn injury.
Asunto(s)
Apoptosis/inmunología , Quemaduras/inmunología , Glucocorticoides/metabolismo , Mucosa Intestinal/inmunología , Tejido Linfoide/inmunología , Glicoproteínas de Membrana/metabolismo , Análisis de Varianza , Animales , Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Quemaduras/patología , Modelos Animales de Enfermedad , Proteína Ligando Fas , Femenino , Glucocorticoides/farmacología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Tejido Linfoide/efectos de los fármacos , Tejido Linfoide/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Mifepristona/farmacología , Ganglios Linfáticos Agregados/efectos de los fármacos , Ganglios Linfáticos Agregados/inmunología , Ganglios Linfáticos Agregados/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismoAsunto(s)
Artritis Reumatoide/tratamiento farmacológico , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Ensayos Clínicos Fase II como Asunto , Humanos , Polietilenglicoles/uso terapéutico , Ensayos Clínicos Controlados Aleatorios como Asunto , Receptores del Factor de Necrosis Tumoral/sangre , Receptores Tipo I de Factores de Necrosis Tumoral , Proteínas Recombinantes/sangre , Proteínas Recombinantes/uso terapéutico , Receptores Señuelo del Factor de Necrosis TumoralRESUMEN
Interleukin (IL-1)alpha and IL-1beta are important mediators of inflammation. The binding of IL-1 to interleukin-1 receptor (IL-1R) type 1 is the initial step in IL-1 signal transduction and therefore is a tempting target for anti-inflammatory therapeutics. To advance our understanding of IL-1R1 binding interactions, we have determined the structure of the extracellular domains of IL-1R1 bound to a 21-amino acid IL-1 antagonist peptide at 3.0-A resolution. The antagonist peptide binds to the domain 1/2 junction of the receptor, which is a conserved binding site for IL-1beta and IL-1 receptor antagonist (IL-1ra). This co-crystal structure also reveals that considerable flexibility is present in IL-1R1 because the carboxyl-terminal domain of the receptor is rotated almost 170 degrees relative to the first two domains of the receptor compared with the previously solved IL-1R1.ligand structures. The structure shows an unexpected binding mode for the peptide and may contribute to the design of smaller IL-1R antagonists.
Asunto(s)
Péptidos/química , Péptidos/metabolismo , Receptores de Interleucina-1/metabolismo , Sialoglicoproteínas/química , Sialoglicoproteínas/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Proteína Antagonista del Receptor de Interleucina 1 , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Receptores Tipo I de Interleucina-1 , SpodopteraRESUMEN
Tumor necrosis factor (TNF)-alpha and Fas ligand (FasL) are trimeric proteins that induce apoptosis through similar caspase-dependent pathways. Hepatocytes are particularly sensitive to inflammation-induced programmed cell death, although the contribution of TNF-alpha and/or FasL to this injury response is still unclear. Here, we report that D-galactosamine and lipopolysaccharide-induced liver injury in C57BL/6 mice is associated with increased hepatic expression of both TNF-alpha and FasL mRNA. Pretreatment of mice with a TNF-binding protein improved survival, reduced plasma aspartate aminotransferase concentrations, and attenuated the apoptotic liver injury, as determined histologically and by in situ 3' OH end labeling of fragmented nuclear DNA. In contrast, pretreatment of mice with a murine-soluble Fas fusion protein (Fasfp) had only minimal effect on survival, and apoptotic liver injury was either unaffected or exacerbated depending on the dose of Fasfp employed. Similarly, mice with a spontaneous mutation in FasL (B6Smn.C3H-Fasl(gld) derived from C57BL/6) were equally sensitive to D-galactosamine/lipopolysaccharide-induced shock. We conclude that the shock and apoptotic liver injury after D-galactosamine/lipopolysaccharide treatment are due primarily to TNF-alpha release, whereas increased FasL expression appears to contribute little to the mortality and hepatic injury.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Galactosamina , Lipopolisacáridos , Glicoproteínas de Membrana/fisiología , Receptores del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/fisiología , Animales , Apoptosis , Proteínas Portadoras/farmacología , Fragmentación del ADN , Proteína Ligando Fas , Femenino , Expresión Génica , Hígado/metabolismo , Hígado/patología , Hepatopatías/metabolismo , Hepatopatías/patología , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Mutación , ARN Mensajero/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral , Proteínas Recombinantes de Fusión/farmacología , Receptores Señuelo del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/genética , Receptor fas/genéticaRESUMEN
Lipopolysaccharide and D-galactosamine induced lethality and apoptotic liver injury is dependent on endogenously produced tumor necrosis factor (TNF)-alpha. The present study was undertaken to determine whether membrane-associated or secreted TNF-alpha signaling through the p55 or p75 receptor was responsible for survival and hepatic injury after lipopolysaccharide administration in D-galactosamine-sensitized mice. Transgenic mice expressing null forms of TNF-alpha, the p55 and p75 receptor, and mice expressing only a cell-associated form of TNF-alpha were challenged with 8 mg D-galactosamine and 100 ng lipopolysaccharide. Mortality and apoptotic liver injury were only seen in wild-type and p75 knockout mice. p75 Knockout mice had significantly higher concentrations of plasma TNF-alpha than any other experimental group (P = 0.05) and tended to have the highest mortality and liver injury. In contrast, p55 and TNF-alpha knockout mice and animals expressing only a cell-associated form of TNF-alpha exhibited no mortality or liver injury. We conclude that survival and apoptotic liver injury in response to lipopolysaccharide and D-galactosamine are dependent exclusively on secreted TNF-alpha signaling through the p55 receptor.
Asunto(s)
Antígenos CD/fisiología , Enfermedad Hepática Inducida por Sustancias y Drogas , Galactosamina , Lipopolisacáridos , Hepatopatías/fisiopatología , Receptores del Factor de Necrosis Tumoral/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Alanina Transaminasa/sangre , Animales , Antígenos CD/genética , Apoptosis , Aspartato Aminotransferasas/sangre , Femenino , Hepatopatías/mortalidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Immune suppression and increased apoptotic loss of circulating lymphocytes have been reported after burn injury. However, little is known about the underlying mechanisms responsible for the increased apoptosis of lymphoid and parenchymal cells in solid organs and the role played by inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha) and Fas ligand (FasL), as well as by glucocorticoids. To evaluate the role of endogenously produced glucocorticoids and FasL, mice subjected to a 20% steam burn were pretreated with a glucocorticoid receptor antagonist (mifepristone) or a neutralizing murine Fas fusion protein. Three and twenty-four hours after burn injury, histological analysis, caspase-3 activity, and in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and phenotyping of lymphocyte populations for apoptosis were evaluated. Burn injury increased the number of apoptotic cells and caspase-3 activity in thymus and spleen, but not in other solid organs. Increased apoptosis was seen in several T and B cell populations from both thymus and spleen. Mifepristone pretreatment significantly reduced the apoptosis and caspase-3 activity after burn injury, whereas blocking FasL activity had only minimal effects. We conclude that corticosteroids, and not FasL, are primarily responsible for the increased caspase-3 activity and apoptosis in thymus and spleen cell populations early after burn injury.
Asunto(s)
Apoptosis/efectos de los fármacos , Quemaduras/enzimología , Caspasas/metabolismo , Dexametasona/farmacología , Glucocorticoides/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Apoptosis/inmunología , Quemaduras/inmunología , Quemaduras/patología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/enzimología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Caspasa 3 , Corticosterona/sangre , Modelos Animales de Enfermedad , Proteína Ligando Fas , Femenino , Citometría de Flujo , Regulación Enzimológica de la Expresión Génica/inmunología , Antagonistas de Hormonas/farmacología , Etiquetado Corte-Fin in Situ , Hígado/inmunología , Hígado/patología , Pulmón/inmunología , Pulmón/patología , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Mifepristona/farmacología , ARN Mensajero/análisis , Organismos Libres de Patógenos Específicos , Bazo/inmunología , Bazo/patología , Timo/inmunología , Timo/patología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
OBJECTIVE: To evaluate the safety, immunogenicity, pharmacokinetics, and efficacy of intravenous administration of tumor necrosis factor binding protein (TNFbp) dimer in patients with rheumatoid arthritis (RA). METHODS: This phase I/II study was a multicenter, randomized, double blind, placebo controlled, ascending dose study evaluating TNFbp dimer administered by i.v. infusion. Thirty-three patients with RA divided into 3 cohorts received TNFbp dimer (30, 100, 300 microg/kg) or placebo during a 5 min infusion at baseline and at 3 and 6 weeks; patients were followed at routine intervals after each infusion through 77 days postinfusion. Pharmacokinetics were analyzed using a log-linear regimen and comparisons were made between half-life after first, 2nd, and 3rd doses. Plasma TNFbp dimer concentrations and serum antibody levels were used in the measurement of pharmacokinetics. RESULTS: Administration of 30 microg/kg of TNFbp dimer was generally well tolerated; the maximum tolerated dose was 100 microg/kg. No serious adverse events were reported. A significant antibody response affected the half-life and clearance of TNFbp dimer at each dose group. Anti-TNFbp antibodies were noncytotoxic and nonagonistic. Clinical evaluations provided evidence of in vivo activity of TNFbp dimer in these patients. CONCLUSION: TNFbp dimer administered to patients with long standing RA resulted in significant antibody production to the study drug. This effect reduced the half-life and clearance of the TNFbp. This TNFbp will not be a viable option for treating patients with RA secondary to immunogenicity.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Adulto , Anciano , Formación de Anticuerpos , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Estudios de Cohortes , Método Doble Ciego , Femenino , Semivida , Humanos , Masculino , Persona de Mediana Edad , Receptores Tipo I de Factores de Necrosis Tumoral , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapéutico , Insuficiencia del Tratamiento , Receptores Señuelo del Factor de Necrosis TumoralRESUMEN
OBJECTIVE: To determine the potential for additive or synergistic effects of combination therapy with the recombinant anticytokine agents interleukin-1 receptor antagonist (IL-1Ra) and PEGylated soluble tumor necrosis factor receptor type I (PEG sTNFRI) in established type H collagen-induced arthritis (CIA) and developing adjuvant-induced arthritis (AIA) in rats. METHODS: Rats with established CIA or developing AIA were treated with various doses of IL-1Ra in a slow-release hyaluronic acid vehicle or with PEG sTNFRI, either alone or in combination with the IL-1Ra. The effects of treatment were monitored by sequential caliper measurements of the ankle joints or hind paw volumes, final paw weights, and histologic evaluation with particular emphasis on bone and cartilage lesions. RESULTS: Combination therapy with IL-1Ra and PEG sTNFRI in rats with CIA resulted in an additive effect on clinical and histologic parameters when moderately to highly efficacious doses of each protein were administered. Greater-than-additive effects were seen when an inactive dose of IL-1Ra was given in combination with moderately to minimally active doses of PEG sTNFRI. Plasma levels associated with the latter effect (for both proteins) were similar to those seen in rheumatoid arthritis (RA) patients in clinical trials with these agents. Combination therapy in the AIA model generally resulted in additive effects, but some parameters showed a greater-than-additive benefit. CONCLUSION: The results provide preclinical support for the hypothesis that IL-1Ra administered in combination with PEG sTNFRI might provide substantially more clinical benefit to RA patients than either agent alone at blood levels that are currently achievable in patients.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Citocinas/antagonistas & inhibidores , Inmunoglobulina G/uso terapéutico , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Sialoglicoproteínas/uso terapéutico , Animales , Artritis Experimental/tratamiento farmacológico , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Etanercept , Femenino , Proteína Antagonista del Receptor de Interleucina 1 , Masculino , Ratas , Ratas Endogámicas Lew , Sialoglicoproteínas/sangreRESUMEN
The proinflammatory cytokine, tumour necrosis factor alpha (TNFalpha) has been shown to play a pivotal part in mediating acute and chronic inflammation. The activities of TNFalpha are modulated by the proteolytic shedding of the soluble extracellular domains of the two TNF receptors, p55 sTNF-RI and p75 sTNF-RII. Amgen Inc has cloned and expressed a recombinant form of a natural inhibitor of TNFalpha, referred to as recombinant human soluble TNF receptor type I (r-Hu-sTNF-RI, sTNF-RI). sTNF-RI is an E coli recombinant, monomeric form of the soluble TNF-type I receptor. A high molecular weight polyethylene glycol (PEG) molecule is attached at the N-terminus position to form the molecule intended for clinical evaluations (PEG sTNF-RI). Preclinical studies to date demonstrate that PEG sTNF-RI is efficacious in rodent models of chronic inflammatory disease including rheumatoid arthritis and Crohn's disease at doses as low as 0.3 mg/kg given every other day. This dose results in plasma concentrations of 0.3 to 0.5 microg/ml. Higher doses with correspondingly higher plasma concentrations yield higher efficacy. It has also demonstrated efficacy in E coli lipopolysaccharide, and Staphylococcus enterotoxin B mediated models of acute inflammation in rodents and primates. Pharmacokinetic studies in mice, rats, cynomolgus monkeys, baboons, and chimpanzees have been conducted with PEG sTNF-RI. Absorption from a subcutaneous dose was slow, with the time to reach maximal plasma concentrations of 24-48 hours in rats, and in monkeys, and 3-29 hours in chimpanzees. The initial volume of distribution of PEG sTNF-RI was essentially equivalent to that of plasma (40 ml/kg). This suggests the protein does not appear to extensively distribute from the systemic circulation with a volume of distribution at steady state (Vss) less than 200 ml/kg in all species studied. These results are consistent with previous experience with PEGylated proteins in which PEGylation decreases both the rate of absorption and the plasma clearance of human recombinant proteins in animals and humans. The use of a PEG molecule will probably provide a more advantageous dosing schedule (that is, less frequent dosing) for the patient compared with a non-PEG sTNF-RI.