RESUMEN
The interleukin-1 (IL-1) receptor antagonist (IL-1ra) is an endogenous antagonist that blocks the effects of the proinflammatory cytokines IL-1alpha and IL-1beta by occupying the type I IL-1 receptor. Here we describe transgenic mice with astrocyte-directed overexpression of the human secreted IL-1ra (hsIL-1ra) under the control of the murine glial fibrillary acidic protein (GFAP) promoter. Two GFAP-hsIL-1ra strains have been generated and characterized further: GILRA2 and GILRA4. These strains show a brain-specific expression of the hsIL-1ra at the mRNA and protein levels. The hsIL-1ra protein was approximated to approximately 50 ng/brain in cytosolic fractions of whole brain homogenates, with no differences between male and female mice or between the two strains. Furthermore, the protein is secreted, inasmuch as the concentration of hsIL-1ra in the cerebrospinal fluid was 13 (GILRA2) to 28 (GILRA4) times higher in the transgenic mice than in the control animals. To characterize the transgenic phenotype, GILRA mice and nontransgenic controls were injected with recombinant human IL-1beta (central injection) or lipopolysaccharide (LPS, peripheral injection). The febrile response elicited by IL-1beta (50 ng/mouse icv) was abolished in hsIL-1ra-overexpressing animals, suggesting that the central IL-1 receptors were occupied by antagonist. The peripheral LPS injection (25 micrograms/kg ip) triggered a fever in overexpressing and control animals. Moreover, no differences were found in LPS-induced (100 and 1,000 micrograms/kg ip; 1 and 6 h after injection) IL-1beta and IL-6 serum levels between GILRA and wild-type mice. On the basis of these results, we suggest that binding of central IL-1 to central IL-1 receptors is not important in LPS-induced fever or LPS-induced IL-1beta and IL-6 plasma levels.
Asunto(s)
Reacción de Fase Aguda/fisiopatología , Encéfalo/metabolismo , Ratones Transgénicos/metabolismo , Sialoglicoproteínas/metabolismo , Animales , Femenino , Fiebre/inducido químicamente , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/sangre , Interleucina-6/sangre , Lipopolisacáridos , Masculino , Ratones , Ratones Endogámicos , Ratones Transgénicos/genética , ARN Mensajero/metabolismo , Sialoglicoproteínas/genéticaRESUMEN
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
The occurrence of the endogenous receptor antagonist for the cytokine interleukin-1 in the rat adrenal gland was analyzed y polymerase chain reaction and by immunohistochemistry using a rabbit polyclonal antiserum. Expression of interleukin-1 receptor antagonist mRNA was demonstrated in both adrenal medulla and cortex, and a marked increase in the transcription was observed after systemic administration of lipopolysaccharides. Interleukin-1 receptor antagonist immunoreactivity was seen in the adrenal medulla, and the immunofluorescence intensity was stronger in the adrenergic, phenylethanolamine N-methyltransferase-positive cells than in the noradrenergic chromaffin cells. The distribution of interleukin-1 receptor antagonist protein is complementary to that of interleukin-1 alpha-like immunoreactivity found in phenylethanolamine N-methyltransferase-negative cells and overlaps with and resembles the distribution of interleukin-1 beta-immunoreactive material. The expression of the interleukin-1 receptor antagonist in the adrenal gland complements previous findings of large constitutive pools of interleukin-1 alpha and interleukin-1 beta in this neuroendocrine organ and also suggests participation of adrenal interleukin-1 receptor antagonist in neuroimmune modulation.
Asunto(s)
Médula Suprarrenal/química , Interleucina-1/análisis , ARN Mensajero/análisis , Sialoglicoproteínas/análisis , Animales , Secuencia de Bases , Técnica del Anticuerpo Fluorescente Indirecta , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/genética , Masculino , Datos de Secuencia Molecular , Conejos , Ratas , Ratas Sprague-Dawley , Receptores de Interleucina-1/agonistas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Sialoglicoproteínas/genéticaRESUMEN
Expression of the cytokine interleukin 1 alpha (IL-1 alpha) was demonstrated in the rat PC12 pheochromocytoma cell line by (i) immunohistochemistry using rabbit polyclonal antisera raised against the recombinant murine IL-1 alpha, (ii) an ELISA, and (iii) a specific cell conversion bioassay based on the use of LBRM33-1A5 cells. IL-1 alpha mRNA was demonstrated in the PC12 cells, by PCR amplification. Constitutive expression of IL-1 alpha in PC12 cells was demonstrated in all experiments, although the cellular levels of IL-1 alpha-like immunoreactivity varied. The expression of IL-1 alpha, as studied at the mRNA level, was inducible by mouse nerve growth factor (7S NGF), and the gene product level was inducible in a dose- and time-dependent fashion by 7S NGF. The maximum induction corresponds to a 600% increase in IL-1 alpha-like immunoreactivity above the expression level found in noninduced cells and occurred after a 3-day incubation of the cells with NGF at 0.75 micrograms/ml of culture medium. The significance of the ability of NGF to induce IL-1 expression lies in the fact that IL-1 itself also acts as a growth factor that promotes glial proliferation and, even more importantly, IL-1 itself induces the expression of NGF at peripheral nerve injury [Lindholm, D., Heumann, R., Meyer, M. & Thoenen, H. (1987) Nature (London) 330, 658-659].