RESUMEN
INTRODUCTION: Neuromedin U (NMU) is a neuropeptide with pro-inflammatory activity. The primary goal of this study was to determine if NMU promotes autoantibody-induced arthritis. Additional studies addressed the cellular source of NMU and sought to define the NMU receptor responsible for its pro-inflammatory effects. METHODS: Serum containing arthritogenic autoantibodies from K/BxN mice was used to induce arthritis in mice genetically lacking NMU. Parallel experiments examined whether NMU deficiency impacted the early mast-cell-dependent vascular leak response induced by these autoantibodies. Bone-marrow chimeric mice were generated to determine whether pro-inflammatory NMU is derived from hematopoietic cells or stromal cells. Mice lacking the known NMU receptors singly and in combination were used to determine susceptibility to serum-transferred arthritis and in vitro cellular responses to NMU. RESULTS: NMU-deficient mice developed less severe arthritis than control mice. Vascular leak was not affected by NMU deficiency. NMU expression by bone-marrow-derived cells mediated the pro-arthritogenic effect. Deficiency of all of the known NMU receptors, however, had no impact on arthritis severity and did not affect the ability of NMU to stimulate intracellular calcium flux. CONCLUSIONS: NMU-deficient mice are protected from developing autoantibody-induced inflammatory arthritis. NMU derived from hematopoietic cells, not neurons, promotes the development of autoantibody-induced inflammatory arthritis. This effect is mediated by a receptor other than the currently known NMU receptors.
Asunto(s)
Artritis/inmunología , Autoanticuerpos/inmunología , Neuropéptidos/inmunología , Receptores de Neurotransmisores/inmunología , Animales , Artritis/genética , Artritis/metabolismo , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Calcio/inmunología , Calcio/metabolismo , Femenino , Masculino , Mastocitos/inmunología , Mastocitos/metabolismo , Mastocitos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones Transgénicos , Neuropéptidos/deficiencia , Neuropéptidos/genética , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Receptores de Neurotensina/deficiencia , Receptores de Neurotensina/genética , Receptores de Neurotensina/inmunología , Receptores de Neurotransmisores/deficiencia , Receptores de Neurotransmisores/genética , Bazo/inmunología , Bazo/metabolismo , Bazo/patologíaRESUMEN
The distribution of neurotensin receptor 1 immunoreactivity in the rat brain was studied using an antibody against the amino-terminal of the receptor expressed as a fusion protein with glutathione-S transferase. Affinity purified antibodies detected the fusion protein and the complete neurotensin receptor sequence expressed in Escherichia coli. The immunostaining was abolished by preabsorption with the amino-terminal fusion protein. Immunoreactive neurotensin receptor 1 immunoreactivity was detected on cell bodies and their processes in a number of CNS regions. In agreement with previous binding studies neurotensin receptor 1 immunoreactivity was particularly localised in cell bodies in the basal forebrain, nucleus basalis and substantia nigra. At the electron microscope level immunoreactivity was found both in axonal bouton and dendrites and spines in the basal forebrain indicating that neurotensin may act both pre- and post-synaptically. There were several regions such as the substantia gelatinosa, ventral caudate-putamen and the lateral reticular nucleus where the neurotensin receptor 1 positive cells had not previously been reported, indicating that distribution of this receptor is widespread.
Asunto(s)
Anticuerpos Monoclonales , Sistema Nervioso Central/química , Receptores de Neurotensina/análisis , Animales , Especificidad de Anticuerpos , Western Blotting , Sistema Nervioso Central/ultraestructura , Femenino , Hipotálamo/química , Hipotálamo/ultraestructura , Inmunohistoquímica , Masculino , Mesencéfalo/química , Mesencéfalo/ultraestructura , Prosencéfalo/química , Prosencéfalo/ultraestructura , Ratas , Ratas Wistar , Receptores de Neurotensina/química , Receptores de Neurotensina/inmunologíaRESUMEN
Neurotensin (NT) has been shown to be involved in neuroendocrine regulation, and the presence of both the peptide and its receptors has been demonstrated in the hypothalamus. In the present study, we show that hypothalamic neurons in primary cultures express the neurotensin receptor (NTR) and we examined a possible regulation of this receptor by glucocorticoids and activators of adenylate cyclase. In the hypothalamic cultures, 125I-NT bound to a single class of binding sites, presenting a selectivity similar to that observed for the high-affinity NTR previously described in the adult rat brain. Radioautographic studies demonstrated that these 125I-NT binding sites were present on 3% of the neurons. A 48-h treatment with forskolin (fsk) decreased 125I-NT binding by 30%. No effect of dexamethasone (dex) alone was found on that parameter. However, a combined treatment with both agents led to a 40% decrease in 125I-NT binding, corresponding to a reduced number of binding sites, and to a 68% decrease in the amount of NTR mRNA. In parallel, the dex plus forsk treatment increased NT release in the incubation medium. Moreover, the decreases in 125I-NT binding and NTR mRNA induced by this treatment were abolished in the presence of an anti-NT antibody or SR 48692, a non-peptidic antagonist of NTR, suggesting that the down-regulation of NTR observed after dex plus fsk treatment was mediated by the release of endogenous NT. Agonist-induced down-regulation of the NTR in this system was confirmed by the application of an exogenous NT analogue, JMV 449. The present findings indicate that, in hypothalamic cultures, dex and fsk indirectly down-regulate NTR expression via the release of endogenous NT.