The Research On Interleukin-12 Family Cytokines Expression Regulation By Apoptotic Cells In Macrophage

Objective: Body apoptotic cells removed by phagocytosis(apoptotic cell, apo), avoid potentially poisonous to the cells, immunogenicity, or necrotic elements exposed to the surrounding enviroment trigger an autoimmune response and inflammatory reaction, this is an important defence function of the body. So quickly and effectively remove the apoptosis cells in the body plays a vital role in normal growth and development of the human body and maintain a stable internal environment, apoptotic cells is mainly cleaned by professional phagocytic cells such as macrophages and immature dendritic cells, and also include partly professional cells such as fibroblasts and epithelial cells. The body control inflammation not only by rapid and effective clearance of apoptotic cells, also by the regulation of inflammatory cytokine production. In recent years, there are many experiments to prove the changed expression levels of cytokines associated with many diseases. For instance, interleukin-23(IL-23) highly express in autoimmune diseases such as rheumatoid arthritis(RA), psoriasis and cancer etc. IL-12 play a tumor suppressor role on melanoma, intestinal adenocarcinoma, liver and other tumor. There are a large number of experiments found that phagocytosis of apoptotic cells by macrophages and other antigen-presenting cells will regulate cytokine expression levels,Such as the positive regulation of IL-10 and the negative regulation of IL-12.IL-12 family of IL-12 and IL-23 are not only made up of two subunits(IL-12 p35, IL-12 p40 and IL-23 p19, IL-23 p40), they have a common subunits p40, and they also can be produced by the activated antigen presenting cells.The correlation between disease and IL-12,Il-23 have been reported in many experiments, for which the mechanism has also been a lot of understanding, the present study thus begin to study the control direction and mechanism of IL-12 family cytokines in macrophages after stimulated by apoptotic cells cells, focusing on the cytokine IL-23, and laid the molecular basis for the development of a clear clinical applications and cytokine-related diseases.Methods:1 Jurkat cell were recovered(human peripheral T-cell leukemia), cultured and prepared for apoptotic cells and checked apoptosis by flow cytometry.2 The mouse peritoneal macrophages were seperated and cultured.3 RAW264.7 cells and mouse peritoneal macrophages were Respectively stimulated with medium, apoptotic cells(apo), lipopolysaccharide(LPS) and apo + LPS.The m IL-12p35, m IL-12p40, m IL-23p19 expression were detected by Real-time quantitative PCR.4 The amount of IL-10, prostaglandin 2(PGE2) and transforming growth factor-β(TGF-β) were checked by ELISA.5 The pre-transcriptional Primers were designed for IL-23p19, and IL-23p19 pre-transcriptional expression were checked by Real-time PCR.6 RAW cells were transfected with plasmid containing the IL-23p19 gene promoter, cultured for 48 hours and then lysed cells to detect the amount of the luciferase protein.7 RAW cells were stimulated with apo, apo + LPS, then join the transcription inhibitor DRB + ACD to prevent new RNA producion. In the end detecte the p19 m RNA decay situation in each group at each time point.8 The total and phosphorylated p38 in stimulated macrophages were checked using Western Blotting.9 RAW cells were effected by different concentrations of MAPK p38 signal transduction pathway inhibitor SB203580 then stimulated with LPS. In the end detecte the p19 m RNA by real-time PCR.10 The plasmids were designed which can make RAW cells highly expressed p38,then RAW cells were respectively stimulated by medium,apoptotic cells or LPS. In the end detecte the p19 m RNA by real-time PCR.11 The RAW cells were stimulated by apo+LPS and then divided into two groups, SB203580 joins one group, p19 m RNA level in two groups was measured by Real-time PCR at each time point.Results :1 apoptotic cells were successfully prepared by Jurket cells treated with staurosporine(staurosporine). Provide stimulation conditions for follow-up study.2 The Level of IL-12p35, IL-12p40 and IL-23p19 m RNA which were expressed by peritoneal macrophages with the stimulation of apo + LPS is lower than only with the stimulation of LPS.The Level of IL-12p35, IL-12p40 and IL-23p19 m RNA expressed by RAW cells with the stimulation of apo + LPS is lower than only with the stimulation of LPS. LPS-induced macrophages expressing IL-12p35, IL-12p40 and IL-23p19 are inhibited by apoptosis cells.3 By ELISA assay, apoptotic cells to macrophages secrete IL-10, PGE2 had no effect, but can promote the secretion of TGF-β, and the difference was statistically significant.4 IL-23p19 level was detected after RAW cells stimulated by medium, apo, LPS and apo + LPS with the method of real-time PCR. The results showed that apoptotic cells did not inhibit IL-23p19 transcription. So apoptotic cells m IL-23p19 inhibitory effect is not achieved at the transcriptional level.5 After the plasmid containing the IL-23p19 promoter transfected RAW cells, cultured cells and lysed them to detect luciferase protein. Found that apo + LPS-stimulated RAW cells have the highest fluorescein protein level. Description that apoptotic cells do not inhibit the transcription of IL-23p19 promoter. Further validation that the inhibitory effect that apoptotic cells to m IL-23p19 is not achieved at the transcriptional level.6 Activated RAW cells were added with transcription inhibitor DRB + ACD,and then detected the remaining amount of IL-23p19 m RNA. We found that the L-23p19 m RNA decay rate of LPS-stimulated RAW cells slower than apo + LPS stimulated RAW cells. So apoptotic cells may inhibit the expression of IL-23p19 by promoting IL-23p19 m RNA decay, apoptosis inhibition was achieved at the post-transcriptional level.7 Phosphorylated p38 content in apo + LPS-stimulated RAW cells was significantly less than the LPS-stimulated RAW cells found by Western Blot. These results showed apoptotic cells inhibited p38 phosphorylation, thereby inhibiting the MAPK p38 signal transduction pathway.8 Different concentrations of MAPK p38 signal transduction pathway inhibitor SB203580 were added to RAW cells, and then stimulated with LPS. The results showed that IL-23p19 m RNA levels were inversely proportional to the concentration of SB203580. Description MAPK p38 signal transduction pathway in macrophages expressing IL-23p19 is very important.9 Compared with the RAW cells which were transfected with blank plasmid, the RAW cells which were transfected with p38 plasmid express ed significantly increased IL-23p19 m RNA levels after stimulated by LPS. Further explanation MAPK p38 signal transduction pathway in macrophages expressing IL-23p19 is very important.Conclusions:1 The apoptosis inhibit activated macrophages expressing IL-12p35, IL-12p40, IL-23p19 m RNA.2 The apoptotic cells had no effect on the secretion of IL-10 and PGE2, but promotes the secretion of TGF-β.3 The inhibitory effect of apoptotic cells to m IL-23p19 is not achieved at the transcriptional level, but by accelerating p19 m RNA decay.4 The apoptotic cells inhibit p38 MAPK signal transduction pathway by inhibiting the phosphorylation of p38, and through blocking p38 signal pathway to inhibit IL-23p19 expression.