Study Of Pro-inflammatory Roles Of Activin A

Inflammation is a common disease, and a normal pathological phenomena. When various kinds of pathogenic factors stimulated, the cells, cytokines, extracellular matrix interacted with each other. They could induce the tissues injury or fibroblast cells hyperplasy, then induced tissues and organs fibrosis. The continued development of inflammation can induce organs disorganization and dysfunction, and even failure. It is a serious threaten to humans life and health. Therefore it will be great significance to actively researched for the development of inflammatory mechanisms, and effective treatment to reduce organs disorganization, dysfunction or failure.Activin A, also known as immunosuppressive P, belongs to transforming growth factor-β(TGF-β) superfamily members, and has many biological effects, but the direct role of activin A on inflammatory response cells still has no clear report. In this study, we used activin A to stimulate L929 fibroblast cell line and mouse peritoneal macrophages in vitro and vivo, and to investigate on the direct role of activin A on inflammatory cells.Ⅰ. Materials and Methods1. Animals: 18-22g male C57BL/6 mice.2. Cell: L929 cells from mouse lung fibroblast cell line.3. Detected the secretion of activin A in mouse peritoneal macrophagesPrimary cultured mouse peritoneal macrophages, LPS(1μg/ml) was used to stimulate the cell. The cells cultured medium alone as control. The supernatants of 1,2,4,8-hour cell cultured was collected, then ELISA method was used to test levels of Activin A in supernatants.4. Quantitative Analysis of IL-1β, IL-6Mouse peritoneal macrophages were cultured in medium include Activin A (2～10 ng/ml) 24 h, ELISA detected the IL-1βand IL-6.5. Analysis the pinocytotic activity of macrophage in vitro.Mouse peritoneal macrophages cultured in the 96wells plate.Activin A (2～10 ng/ml) stimulated the cells overnight, then added 0.7% neutral red to culture 1 h, PBS wash cells three times, and measured the luminous absorption value (A490nm). 6. Analysis the phagocytic activity of macrophage in vitro.Mouse peritoneal macrophages cultured in the 96wells plate.Activin A (2～10 ng/ml) stimulated the cells overnight, then added 50μl 2% chicken red blood cell to culture 2 h, PBS rinse, Wright - Giemsa staining, observed by optical microscope.7. Detection the expression of arginase 1 (Arginase 1), MHCⅠ, MHCⅡand CD86 by flow cytometry.Mouse peritoneal macrophages cultured in the 12-wells plate. Activin A (10 ng/ml) stimulated the cells overnight , expression of. cell surface molecules of the macrophages were detected by flow cytometry.8. RT-PCR detection of iNOS mRNA expressionMouse peritoneal macrophages cultured in the 12-wells plate. Activin A (10 ng/ml) stimulated the cells overnight , RT-PCR was used to detect the expression of iNOS mRNA.9. Proliferation of mouse peritoneal macrophagesMouse peritoneal macrophages cultured in the 12-wells plate. Activin A (10 ng/ml) stimulated the cells 24h, MTT method was used to analysis the proliferative ability of primary cultured macrophages, 570 nm absorption was test.10. Analysis the secretion of cytokines in vivo Activin A (10 ng and 20 ng) was injected in abdominal cavity, the peritoneal fluid recovered after 24h, then determined the IL-1βand IL-6.11. Analysis the phagocytic activity of mouse peritoneal macrophage in vivoThe chicken red blood cells was injected in abdominal cavity, then tested the ability of macrophages phagocytose particulate antigen influenced by activin A (10 ng and 20 ng) in vivo.12. Analysis the expression of CD86 in mouse peritoneal macrophagesActivin A (10 ng and 20 ng) was injected in abdominal cavity, the peritoneal macrophages recovered after 24h, flow cytometry was used to test the expression of CD86 in mouse peritoneal macrophages.13. Detection the release of NO and TNFa in L929 cellsThe different doses of activin A were used to stimulate L929 cells, then tested the level of NO and TNFa.14. The expression of FN and TIMP mRNA in L929 cellsActivin A stimulated L929 cells, two-step RT-PCR was used to detect the expression of FN and TIMP mRNA.GAPDH as internal reference.15. Determination of pinocytotic activity of L929 cell stimulated by Activin A Soluble neutral red dye was used to detect the pinocytotic activity of L929 cells stimulated by activin A.16. Determination the proliferation of L929 cellL929 cells cultured in the 96-wells plate, then added activin A (0.8～10 ng/ml) to culture 24h, MTT method was used to analysis the proliferative ability of primary cultured macrophages, 570 nm absorption was test.Ⅱ. Results1. LPS induced the secretion of activin A in the primary cultured mouse peritoneal macrophageLPS stimulated mouse peritoneal macrophages after 4h, the secretion of Activin A rapidly increased, it suggested that activin A may be participate the regulation of macrophage activity in autocrine/paracrine forms.2. Activin A facilitated the secretion of IL-1βand IL-6 in primary cultured mouse peritoneal macrophageThe direction effect of Activin A can induce IL-1βand IL-6 secretion, and had a dose-dependent manner in mouse peritoneal macrophages. 3. Activin A induced pinocytotic and phagocytic activity of primary cultured mouse peritoneal macrophagesIn mouse peritoneal macrophages, Activin A could not only promote pinocytotic activity of macrophage, but also phagocytose the chicken red blood cells.4. The expression of MHCⅠ, MHCⅡand CD86 influenced by ActivinA in primary cultured mouse peritoneal macrophages The macrophages that Activin A treated, the expression of MHCⅠand MHCⅡdid not change. However, the expression of CD86 was significantly increased.5. Activin A influenced the proliferation of primary cultured mouse peritoneal macrophages in vitroActivin A did not significantly affect the proliferation of mouse macrophages.6. Activin A promoted the activity of peritoneal macrophages in mice in vivoActivin A can induce the secretion of IL-1βand IL-6 in mouse peritoneal macrophages, and facilitate the phagocytic capacity of mouse peritoneal macrophages.7. Activin A promoted the expression of CD86 in mouse peritoneal macrophages in vivo Activin A can increase the expression of CD86 one of the activated hallmarks of mouse peritoneal macrophages.8. Activin A induced activation of typeⅡmacrophagesThe results showed that, LPS can significantly increase the expression of iNOS mRNA, and activin A significantly increase the expression of Arginase 1. The data suggested that activin A may have a selective-induced the activation of M2, through promoting the phagocytic activity of M2 to attend the early response of natural immune response. but did not affect the expression of MHCⅠandⅡ. The results suggest that activin A may not attend the antigen-presenting process of specific immune response.9. Activin A promote the release of NO and TNFa in L929 cells Activin A has a significant promoted role for secretion of NO andTNFαin L929 cells.10. The regulation of activin A about the expression of FN and TIMP mRNA in L929 cellsActivin A significantly increased the expression of FN mRNA, but did not affect the expression of TIMP mRNA. Activin A may be prompted the generation of extracellular matrix, and participate in tissue fibrosis, but may not affect the degradation of extracellular matrix.11 Activin A influenced the pinocytotic activity of L929 cell The experimental results show that L929 cells may intake the soluble substances through pinocytotic way, and activin A can promote the L929 cells pinocytotic capacity.12 Activin A influenced the proliferation of L929 cells Activin A did not affect the proliferation of L929 cells. The results suggested that activin A promote the secretion of inflammatory mediators in L929 cells and the expression of FN mRNA, but didinot influenced the proliferation of L929 cell.Ⅲ. The conclusionsIn conclusion, as a kind of inflammation factor, Activin A could induce the macrophage activation which participated inflammatory response. By means of enhancing the macrophage phagosytosis, promoting the macrophage to take part in the organ innade immune response, Activin A could also play a key role in the early phase of organ defense. Asa multifunction factor, Activin A could promote the L929 to secret the inflammation factors and Cell Extra Medium, which further confirmed as a kind of inflammation factors, Activin A may also play an important part in the development of inflammation and tissue fibration.