摘要
Astrocytes are the most numerous cells of the mammalian brain and have a wide range of functions within the central nervous system (CNS). Astrocytes have the capacity to secrete and respond to a variety of cytokines including interleukin 1 (IL 1), interleukin 6 (IL 6) and tumor necrosis factor α (TNFα), which play key roles in the body's response to infection, tissue injury and inflammation. Interleukin 1 receptor antagonist (IL 1ra) is a naturally occurring endogenous inhibitors that can block the actions of IL 1. Objective To study the effects of IL 1ra on TNFα and IL 6 secretions which were produced by astrocytes in response to IL 1β. Methods Primary astrocytes culture was established from neonatal Wistar rat cerebra by modified McCarthy and de Vellis techniques. Culture medium was Dulbecco's modified essential medium (DMEM), high glucose formula supplemented with glucose to a final concentration of 6 g/L. After 11 days the pure astrocytes were obtained by mechanical dislodging and trypsinization. The purity was more than 99% positive for glial fibrillary acidic protein (GFAP), an intracellular antigen unique to astrocytes. The astrocyteswere plated at 5×10 5 cells / well into 6 well plates, and switched to 2 ml of DMEM containing 2% FBS each well. Then the cells were treated in triplicate with control medium alone, rrIFN γ (100 U/ml), hrIL 1β (10-1000 U/ml), hrIL 1ra (100-1000 U/ml), and combinations of the above according to different requirements. Supernatants were collected after 18 h and cytokine levels were measured by bioassay method (cell line B9 for IL 6 and cell line L929 for TNFα). The astrocytes were used up to the age of 3 weeks. Results IL 1β was a strong inducer of cytokine productions by astrocytes in vitro. IL 1β could stimulate the astrocytes to secrete TNFα; IL 1β alone, as well as IFN γ could not significantly induce astrocytes to produce IL 6 , but an augment effect was observed when astrocytes were pretreated with IFN γ for 8 h before the addition of IL 1β; IL 1ra could reduce the TNFα and IL 6 production of astrocytes induced by IL 1β (P< 0.05 ). A little inhibition was observed at equimolar ratios, whereas if at a 10 fold molar excess of IL 1ra to IL 1, 48% of IL 6 secretion and 77% of TNFα secretion induced by IL 1 were inhibited. Conclusion There is a quite complicated cytokine network in the CNS and that IL 1ra has an ability to block the effect of IL 1 on other inflammatory cytokines synthesis by astrocytes, such as TNFα and IL 6. Besides the direct affect, IL 1ra can also affect the production of other cytokines through the network relation. It may help to explain the mechanism of the potential therapeutic function of IL 1ra on traumatic and inflammatory diseases.
Abstract Astrocytes are the most numerous cells of the mammalian brain and have a wide range of functions within the central nervous system (CNS). Astrocytes have the capacity to secrete and respond to a variety of cytokines including interleukin 1 (IL 1), interleukin 6 (IL 6) and tumor necrosis factor α (TNFα), which play key roles in the body's response to infection, tissue injury and inflammation. Interleukin 1 receptor antagonist (IL 1ra) is a naturally occurring endogenous inhibitors that can block the actions of IL 1. Objective To study the effects of IL 1ra on TNFα and IL 6 secretions which were produced by astrocytes in response to IL 1β. Methods Primary astrocytes culture was established from neonatal Wistar rat cerebra by modified McCarthy and de Vellis techniques. Culture medium was Dulbecco's modified essential medium (DMEM), high glucose formula supplemented with glucose to a final concentration of 6 g/L. After 11 days the pure astrocytes were obtained by mechanical dislodging and trypsinization. The purity was more than 99% positive for glial fibrillary acidic protein (GFAP), an intracellular antigen unique to astrocytes. The astrocyteswere plated at 5×10 5 cells / well into 6 well plates, and switched to 2 ml of DMEM containing 2% FBS each well. Then the cells were treated in triplicate with control medium alone, rrIFN γ (100 U/ml), hrIL 1β (10-1000 U/ml), hrIL 1ra (100-1000 U/ml), and combinations of the above according to different requirements. Supernatants were collected after 18 h and cytokine levels were measured by bioassay method (cell line B9 for IL 6 and cell line L929 for TNFα). The astrocytes were used up to the age of 3 weeks. Results IL 1β was a strong inducer of cytokine productions by astrocytes in vitro. IL 1β could stimulate the astrocytes to secrete TNFα; IL 1β alone, as well as IFN γ could not significantly induce astrocytes to produce IL 6 , but an augment effect was observed when astrocytes were pretreated with IFN γ for 8 h before the addition of IL 1β; IL 1ra could reduce the TNFα and IL 6 production of astrocytes induced by IL 1β (P< 0.05 ). A little inhibition was observed at equimolar ratios, whereas if at a 10 fold molar excess of IL 1ra to IL 1, 48% of IL 6 secretion and 77% of TNFα secretion induced by IL 1 were inhibited. Conclusion There is a quite complicated cytokine network in the CNS and that IL 1ra has an ability to block the effect of IL 1 on other inflammatory cytokines synthesis by astrocytes, such as TNFα and IL 6. Besides the direct affect, IL 1ra can also affect the production of other cytokines through the network relation. It may help to explain the mechanism of the potential therapeutic function of IL 1ra on traumatic and inflammatory diseases.
