RESUMEN
One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of amyloid plaques. The main constituent of the amyloid plaques is the amyloid beta-peptide (A beta) shown to activate glial cells in vitro. A growing body of evidence suggests that these cells contribute to neurotoxicity through production of inflammatory cytokines, chemokines, and neurotoxic substances, such as reactive oxygen species (ROS). In this study, mRNA levels of the inflammatory cytokines interleukin (IL)-1alpha and beta, and IL-6 were analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) in rat primary mixed glial cells after treatment with A beta(25-35), a biologically active fragment of A beta peptide with neurotoxic properties. Clear morphological changes of the astrocytes, as well as proliferation and clustering of microglial cells were observed by light and immunofluorescence microscopy after 24 h treatment. Significant increases in IL-1alpha and IL-6 mRNA levels were detected after 24 and 72 h, whereas significantly increased levels of IL-1beta mRNA could only be detected after 4 h treatment. The most pronounced effect was seen on IL-6 mRNA expression, which increased approx two- to threefold after treatment. In addition, increased secretion of IL-6 was detected after 96 h exposure. Recently, association of IL-1alpha and IL-6 gene polymorphism with AD was reported, suggesting that these cytokines may play an important role in the development of the disease. The increased mRNA levels of IL-1alpha and IL-6 in parallel with the morphological changes in the mixed glial-cell cultures support that these cytokines may be involved in A beta-induced gliosis and in the pathogenesis of AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Neuroglía/metabolismo , Fragmentos de Péptidos/metabolismo , Animales , Técnicas de Cultivo de Célula , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Interleucina-1/genética , Interleucina-6/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Regulación hacia ArribaRESUMEN
betaA25-35, a neurotoxic fragment of the Alzheimer beta-amyloid peptide (betaA), acts as a strong inducer of pro-inflammatory cytokines, such as interleukin (IL)-1 and IL-6, in glial cells. Since IL-1 is known to induce expression of both IL-1 and IL-6, we have investigated to what extent the induction of IL-1alpha and IL-6 by betaA25-35, is dependent on the IL-1 receptor type I (IL-1RI), the only known signalling IL-1 receptor. Primary astroglial cell cultures prepared from wild-type and IL-1RI-deficient mice were incubated in the presence of betaA25-35 (100 microM) for 19 h, followed by analysis of mRNA levels of IL-1alpha and IL-6. Cell cultures treated with betaA25-35 showed a significant increase in mRNA levels for IL-1alpha and IL-6 and in addition increased levels of IL-1alpha immunoreactivity. A supersensitive IL-1alpha response was observed in astroglial cell cultures lacking the IL-1 RI as compared to betaA25-35 treated cell cultures from wild-type mice. In contrast the betaA25-35-induced increase of IL-6 was lower in the absence of IL-1RI. In conclusion, these results suggest that a functional IL-1 signal transduction is not necessary for induction of mRNA levels of IL-1alpha and IL-6 in astroglial cell cultures treated with betaA25-35, but that induction of IL-6 involves at least two distinct mechanisms, one of which occurs via activation of the IL-1RI.
Asunto(s)
Péptidos beta-Amiloides/inmunología , Astrocitos/inmunología , Interleucina-1/inmunología , Interleucina-6/inmunología , Fragmentos de Péptidos/inmunología , Receptores de Interleucina-1/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Péptidos beta-Amiloides/farmacología , Animales , Astrocitos/efectos de los fármacos , Células Cultivadas , Interleucina-1/genética , Interleucina-6/genética , Ratones , Ratones Noqueados , Fragmentos de Péptidos/farmacología , Receptores de Interleucina-1/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The endogenous pyrogen interleukin-1 (IL-1) is considered as one of the key molecules in orchestrating the host response of injury and inflammation. IL-1 exerts its effects upon binding to the type I IL-1 receptor (IL-1RI). The IL-1-IL-1RI complex is further thought to associate with the IL-1 receptor accessory protein (IL-1RAcP), which is suggested to be important for most IL-1 signal transduction pathways. With the aim of investigating the importance of the IL-1RAcP in IL-1 signalling, IL-1alpha and IL-1beta induced febrile responses and IL-1beta-mediated activation of NFkappaB in primary astrocyte cultures were examined using IL-1RAcP-deficient (IL-1RAcP KO) and wild type mice, respectively. It was shown that neither recombinant rat IL-1alpha (rrIL-1alpha, 25 microg/kg), recombinant rat IL-1beta (rrIL-1beta, 40 microg/kg) nor recombinant human IL-1beta (rhIL-1beta, 50 microg/kg) injected i.p. could elicit febrile responses in the IL-1RAcP-deficient mice, while the same doses of rrIL-1alpha/beta or rhIL-1beta injected into wild type mice caused normal fever responses. A febrile response could be induced in the IL-1RAcP-deficient mice by i.p. administration of E. coli lipopolysaccharide (LPS, 50 microg/kg) and this response was similar to that obtained in wild type mice. Furthermore, it was shown that rhIL-1beta activated, in a concentration-dependent manner, nuclear translocation of the transcriptional nuclear factor kappa B (NFkappaB) in primary astrocyte cultures prepared from wild type mice, whereas no IL-1beta-induced translocation of NFkappaB could be detected in cultures prepared from IL-1RAcP-deficient mice, as revealed by electrophoretic mobility shift assay (EMSA). The rhIL-1beta-induced NFkappaB complexes were shown to contain p50 but no, or very little, p65 and cRel immunoreactive proteins.
Asunto(s)
Fiebre/fisiopatología , Interleucina-1/farmacología , Interleucina-1/fisiología , FN-kappa B/metabolismo , Proteínas/fisiología , Receptores de Interleucina-1 , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Transporte Biológico/fisiología , Núcleo Celular/metabolismo , Células Cultivadas , Humanos , Proteína Accesoria del Receptor de Interleucina-1 , Ratones , Ratones Mutantes , Ratas , Proteínas Recombinantes/farmacología , Transducción de Señal/fisiologíaRESUMEN
IL-1alpha and IL-1beta are potent inflammatory cytokines that contribute to a number of normal physiologic processes and to the development of a number of inflammatory diseases. Two IL-1R, the type I and type II receptors, have been identified. This work describes the derivation and characterization of mice deficient in expression of the type I IL-1R (IL-1RI). IL-1RI-deficient mice were viable and fertile, but failed to respond to IL-1 in a variety of assays, including IL-1-induced IL-6 and E-selectin expression and IL-1-induced fever. Similar to IL-1beta-deficient mice, IL-1RI-deficient mice had a reduced acute phase response to turpentine. In contrast, IL-1RI-deficient mice had a reduced delayed-type hypersensitivity response and were highly susceptible to infection by Listeria monocytogenes. These data demonstrate that the IL-1RI is essential for all IL-1-mediated signaling events examined, and that both IL-1alpha and IL-1beta are critical to the animals' response to injury and infection. These data also demonstrate that IL-1 function is not required for normal development or homeostasis.
Asunto(s)
Inflamación/fisiopatología , Interleucina-1/farmacología , Receptores de Interleucina-1/deficiencia , Reacción de Fase Aguda/fisiopatología , Animales , Células Cultivadas , Susceptibilidad a Enfermedades , Selectina E/biosíntesis , Selectina E/genética , Femenino , Fiebre/inducido químicamente , Fibroblastos/efectos de los fármacos , Marcación de Gen , Hipersensibilidad Tardía/fisiopatología , Interleucina-1/toxicidad , Interleucina-6/biosíntesis , Interleucina-6/genética , Listeriosis/inmunología , Masculino , Ratones , Ratones Noqueados , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/fisiología , Receptores Tipo I de Interleucina-1 , Transducción de Señal , Trementina/toxicidadRESUMEN
Interleukin 1 beta (IL-1 beta) is a highly inducible proinflammatory cytokine. It is processed to its mature, secreted 17-kDa form by a cysteine endoprotease; the interleukin 1 beta converting enzyme (ICE). Regulation of IL-1 beta levels can be achieved both at transcriptional and translational level and in particular at the posttranslational, ICE catalysed, level. Thus, we examined ICE activity in rats under conditions of systemic stimulation by intraperitoneal (i.p.) injections of lipopolysaccharide (LPS) from E. coli, which are known to dramatically alter IL-1 beta mRNA and protein levels. ICE mRNA levels and endoprotease activity have also been found to be differentially regulated in the rat adrenal gland and rat brain after i.p. injections of LPS. An induction in ICE mRNA levels could be seen in the adrenal gland, the pituitary and in the hypothalamus after LPS treatment as measured by reverse transcription-polymerase chain reaction (RT-PCR), whereas the ICE endoprotease activity was increased in the pituitary and decreased in the hippocampus and in the adrenal gland. The discrepancy between increased mRNA level for ICE and decreased enzyme activity in the adrenals might be explained by the induction of ICE isoforms, some of which might be inhibitory for the enzyme activity and induced by LPS, yielding as a net effect a suppression of ICE activity.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Isoenzimas/metabolismo , Lipopolisacáridos/farmacología , Glándulas Suprarrenales/enzimología , Animales , Caspasa 1 , Cisteína Endopeptidasas/genética , Inducción Enzimática/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Hipotálamo/efectos de los fármacos , Hipotálamo/enzimología , Interleucina-1/metabolismo , Isoenzimas/genética , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Especificidad de Órganos , Células PC12/efectos de los fármacos , Células PC12/enzimología , Hipófisis/efectos de los fármacos , Hipófisis/enzimología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , RatasRESUMEN
Pro interleukin-1 beta converting enzyme (ICE) activity in the pituitary was found to be significantly increased 4 h after intraperitoneal injection of E. coli lipopolysaccharides, when distribution and inducibility of the enzyme was studied in the adult rat brain and the adrenal gland, using an artificial fluorescence peptide substrate. The same lipopolysaccharide treatment induced ICE mRNA levels in the pituitary, adrenal gland and hypothalamus as studied by reverse transcript-polymerase chain reaction.
Asunto(s)
Glándulas Suprarrenales/efectos de los fármacos , Cisteína Endopeptidasas/genética , Hipocampo/efectos de los fármacos , Hipotálamo/efectos de los fármacos , Lipopolisacáridos/farmacología , ARN Mensajero/biosíntesis , Glándulas Suprarrenales/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caspasa 1 , Inducción Enzimática , Hipocampo/enzimología , Hipotálamo/enzimología , Interleucina-1/metabolismo , Masculino , Datos de Secuencia Molecular , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Ratas , Ratas Sprague-Dawley , Valores de ReferenciaRESUMEN
A new method for the synthesis of dehydroalanine (delta Ala)-containing peptides has been developed by combining solid phase peptide synthesis (tert-butyloxycarbonyl/HF-chemistry) with solution synthesis. A sequence from cyanobacterial hepatotoxin microcystin, Ac-D-gamma-Glu-[N-Me-delta Ala]-D-Ala-Leu amide was chosen as a model peptide. The precursor for the synthesis of the dehydroalanine-containing peptide, Ac-D-gamma-Glu-[N,S-diMeCys]-D-Ala-Leu, was synthesized on a solid phase followed by sulfonium salt formation on the resin. The resulting S,S-dimethylated peptide was cleaved from the resin with liquid HF. The HPLC-purified S,S-dimethylated cysteine-containing precursor peptide was subjected to beta-elimination in solution catalysed by DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) in methanol. The final product, Ac-D-gamma-Glu-[N-Me-delta Ala]-D-Ala-Leu amide, was purified by HPLC, and analysed by mass spectrometry and 1H NMR spectroscopy. The stability of the model peptide under acidic, neutral and basic conditions has been studied.