RESUMO
The purpose of this study was to investigate the in vitro effect of Suppressor Of Cytokine Signaling 1 (SOCS1) overexpression in retinal pigment epithelium (RPE) cells on their activation by pro-inflammatory cytokines IFNγ, TNFα and IL-17. Retinal pigment epithelium cells (ARPE-19) were stably transfected with the control plasmid pIRES2-AcGFP1 or the plasmid pSOCS1-IRES2-AcGFP1. They were stimulated by IFNγ (150ng/ml), TNFα (30ng/ml) or IL-17 (100ng/ml). The levels of SOCS1 mRNA were measured by real-time PCR. Signal Transducer and Activator of Transcription 1 (STAT1) phosphorylation and IκBα expression were analysed by western Blot (WB). IL-8 secretion was analysed by ELISA and expression of MHCII molecules and ICAM-1/CD54 by flow cytometry. Our data show that SOCS1 mRNA overexpression in RPE cells prevents IFNγ-induced SOCS1 mRNA increase and IFNγ-mediated STAT1 phosphorylation. Moreover, SOCS1 overexpression in RPE cells inhibits IFNγ-induced decrease of IL-8 secretion and prevents IFNγ-induced MHC II and ICAM1/CD54 upregulation. However, SOCS1 overexpression does not affect TNFα-induced IκBα degradation nor block TNFα-induced or IL-17-induced IL-8 secretion. On the contrary, IL-17-induced secretion is increased by SOCS1 overexpression. In conclusion, SOCS1 overexpression in RPE cells inhibits some IFNγ-mediated responses that lead to uveitis development. This notion raises the possibility that SOCS1 overexpression could be a novel target for treating non-infectious uveitis. However, some proinflammatory effects of TNFα and IL-17 stimulation on RPE are not blocked by SOCS1 overexpression.
Assuntos
Citocinas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Uveíte/metabolismo , Células Cultivadas , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucina-8/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Fator de Transcrição STAT1 , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Adult macaque Schwann cells were infected using adeno-associated virus type-2-derived vectors expressing the green fluorescent protein reporter gene under the control of the cytomegalovirus, the hybrid cytomegalovirus-betaactin, the myelin basic protein or the tetracycline-inducible promoters. On the basis of green fluorescent protein expression, gene transfer efficiency was compared in resting and dividing conditions following or not following hydroxyurea or etoposide treatment. Hydroxyurea allowed promoter-dependent expression of green fluorescent protein in infected Schwann cells. Etoposide treatment led to a high percentage of green fluorescent protein expressing cells (over 50%) with all promoters tested. When infected cells were grafted into demyelinated nude mice spinal cord, green fluorescent protein expression was only observed with the cytomegalovirus-betaactin and tetracycline-inducible promoters. In addition, adeno-associated virus type-2 infection reduced the grafted cell survival but increased their differentiation.
