Macrophage Microvesicles Induces IL-8 Response In THP-1 Cells Through MAPK Pathways Activated

Objective: macrophage Microvesicles(MVs) is a important pathogenesis of COPD、diabetes mellitus and chronic kidney disease and other diseases. The present research has learned that central link in pathogenesis for macrophage MVs is the immune injury caused by excessive inflammatory reaction. But,the specific pathogenesis for macrophage MVs causing excessive inflammatory reaction is unclear. The goal of this experiment is to find whether IL-8 is induced in THP-1 cells when it is treated with macrophage MVs through MAPK pathways activated.Methods:Referring to the method of Yang SR and Nakamura, two cigarette smoke extract is collected by injection syringe and the cigarette smoke extract is poured into 20 ml of serum free medium slowly that can obtained culture medium containing 10% of cigarette smoke extract.THP-1 cells in vitro is treated by culture medium containing 2.5% of cigarette smoke extract and the macrophage MVs is collected after 20 hours.Flow cytometry to detect the macrophage MVs.THP-1 cells in vitro is treated by different concentrations of macrophage MVs respectivelyand IL-8 induced by macrophage MVs is tested by the method of ELISA technique at the different time of 0h、3h、6h、12h、24h.The method of Western blotting is used to test the phosphorylated level of ERK1/2, JNK and p38. Different concentrations(1μM,10μM,30μM) of JNK specific inhibitor SP600125,ERK1/2 specific inhibitor PD98059 and p38 specific inhibitor SB203580 are used to treat with THP-1 cells and then 105/ml of macrophage MVs was added to the medium for another 12 h, the IL-8 was detected by the method of ELISA.Results:(1) THP-1 cells can produces obviously macrophage MVs when it is treated with culture medium containing 2.5% of cigarette smoke extract after 20 hours.(2) macrophage MVs can induces THP-1 cells to produce IL-8, and the production of IL-8 is found to be dose-dependent in a range.At the same concentration of macrophage MVs as the treatment time increasing, the output of IL- 8 increases when the treatment time arrives at 12 h the output of IL- 8 arrives at the peak.(3)The results of Western blotting display that macrophage MVs can activates JNK/MAPK 、p38/MAPK and ERK1/2 /MAPK pathways in THP-1 cells.The level of phosphorylated p38/MAPK 、 phosphorylated ERK1/2 and phosphorylated JNK arrived at the peak when THP-1 cells is treated with macrophage MVs for 30 min.(4) ELISA shows that the specific inhibitors such as SB203580、PD98059 and SP600125 are able to decrease the levels of IL-8. When the concentration of specific inhibitors is 30μM the output of IL-8 decreasing to 31.3%、51.4% and 46.1% severally.Conclusions:(1) THP-1 cells can produce macrophage MVs when it is treated with cigarette smoke extract.(2) THP-1 cells can produce IL-8 when them is treated with macrophage MVs.(3) JNK/MAPK、p38/MAPK and ERK1/2 /MAPK pathways are activated when THP-1 cells is treated with macrophage MVs.The activated JNK/MAPK、p38/MAPK and ERK1/2 /MAPK pathways are related to THP-1 cells products IL-8.