Effects Of CpG-ODN On TLR-9 Expression And Th1/Th2 Cytokines Secretion From Murine Macrophage Cell Line

BacgroundBronchial asthma(asthma) is a inflammatory lung disease including lymphocyte,eosinophil,mast cell,inflammation matter and many cytokines.Immune response has an important position in asthma pathogenesy. Rccently,someone preferred nosogenic foundation of asthma is the constitutive ratio and function in balance of Th1,Th2 which are the subgroups of T helper cells.The removal of these apoptotic neutrophils and eosinophils through macrophages is one prerequisite of resolution of acute inflammation.Macrophages can also release many cytokines and protein enzyme,participate in asthma inflammation and airway repair and restructure.So it is necessary to study the function of macrophages in asthma and it is a good idea for asthma therapy through adjusting macrophages.CpG-ODN are oligodeoxynucleotides including CpG.It has intensity immunity stimulation like bacteria DNA.It can arose many immune cells,like macrophage and dendritic cell of mammal.DNA.CpG-ODN can induce Th1 cytokines and reduce Th2 cytokines,down-regulate Th2 but induce Th1 response,which can also shift the Th2 responses substantially toward Th1 and modulate the Th1 / Th2 immune response balance.The balance has significant meaning in cure of asthma.The approach of stimulate functionary cell through Toll like receptor-9(TLR-9).After TLR-9 recognises CpG-ODN,it initiate cellactivation through myeloid differentiation factor 88 and nuclear factor,then upregulate major histocompatibility complex(MHC) and costimulatoty molecules expression,secrete cytokine and so on.So it is important to study cytokines secretion in macrophages after CpG-ODN meets TLR-9,and TLR-9 expression,It has signification to know how CpG-ODN adjusts macrophage.Objective(1) The study was designed to investigate the protein and mRNA expression of TLR-9 after CpG-ODN stimulated macrophages.To explore the character in CpG-ODN adjusting macrophages.(2) Investigate secretion on Th1 cytokines like TNF-α,IL-12 and Th2 cytokines like IL-6 before and after CpG-ODN stimulated the murine-macrophages-like Raw264.7 Cells.To know how CpG-ODN adjusts macrophage.Method(1) In this study,we cultivated RAW264.7 cells.They were seeded in 6-well culture plates and grew overnight in DMEM supplemented with 10%fetal calf serum in a Rumidified 5%CO₂ in cubator at 37℃.Then they were treated with DMEM,CpG-ODN,Non-CpG-ODN or CpG-ODN+chloroquine for 24 hours.The expression of TLR-9 mRNA and protein of each group were analyzed by RT-PCR and Western blot respectively.(2) The concentration of TNF-αand IL-6 in the supernatants were e xamined by Radioimmunoasssy(RIA) and the level of IL-12 was analyzed by ELISA.Result(1) After treated for 24 hours,the expression of TLR-9 mRNA in CpG-ODN group and CpG-ODN+chloroquine group were significant higher than that in other groups.(P＜0.05) Using western blotting also prived that the protein expression of TLR-9 in CpG-ODN group and CpG-ODN+chloroquine group were significant higher than that in other groups.(P＜0.05)(2) The level of TNF-αin the supernatant in the CpG-ODN group was higher than that in other groups.(P＜0.05).The levels of IL-6 and IL-12 in the supernatants had no significant difference in these groups (p＞0.05).ConclusionsThe protein and mRNA expression of TLR-9 both incarase after CpG-ODN stimulated macrophages.CpG-ODN can induce Th1 cytokines like TNF-α,but Th2 cytokines like IL-6 and Th1 cytokines like IL-12 not.These results demonstrate that CpG-ODN can up-regulate the expression of TLR-9 and adjust the expression of Th1 /Th2 cytokines secretion from macrophages. It is a good idea for asthma therapy through adjusting macrophages.