Effect Of Activin A On Inducing Differentiation And Maturation Of Mouse Monocytes/Macrophages

Activin, a multifunctional growth and differentiation factor of the transforming growth factor-β(TGF-β) superfamily, is a dimeric glycoprotein composed of twoβsubunits linked by a disulfide bond. According to the two differentβsubunits connected by disulfide bonds, activin is divided into the following forms:forms including activin A (βA,βA) composed of homodimer ofβA, activin B (βB,βB) composed of homodimer ofβB and activin AB (βA,βB) composed of heterodimer ofβA andβB. Other forms of activin including activin C (βc,βC), activin E(βEβE), activin AC (βA,βC), activin AE (βA,βE) and activin CE (βC,βE are identified recent years. Activin A is studied more than others now because of its broader physiological role. Activin A shows various biological activities in both the gonad and extragonadal tissue with obvious histological specificity. Previous studies have showed that Activin A induces rest mouse macrophages differentiation and secreting NO, but it inhibits macrophages differentiation induced by LPS. It is speculated that its function may be related to the regulation of Activin A on inducing differentiation and maturation of the mouse monocytes/macrophages, but its mechanism has not yet completely clear. In this study, we cultured mouse peritoneal macrophages and peripheral blood mononuclear cells in vitro,in order to explore the effect of activin A on inducing differentiation and maturation of mouse monocytes/ macrophages.1.Activin A induces differentiation of peripheral blood mononuclear cells into dendritic cellsTo investigate the effect of activin A on immature monocyte/macrophage cells, we obtained the mouse peripheral blood mononuclear cells by density gradient centrifugation, then removed the non-adherent lymphocytes by differential attachment method.After the mouse peripheral blood mononuclear cells was incubated with Activin A for one day, three days, five days and seven days, they were stained with Wright's-Giemsa's staining. Cell morphological changes were observed under a light microscope. It was found that Activin A stimulated-cells gradually showed typical DC-like morphology. Their morphology changed from the short spindle to irregular shape with thick and long dendrite which changed more obviously with time going. CD40 and CD 11c, molecular markers of DCs and macrophages, which were detected by flow cytometry, expressed more with treatment of activin A in a dose-dependent. It was also observed that Activin A could obviously promote the IL-12 secretions of mouse peripheral blood mononuclear cells and showed a dose dependently manner.It is well known that mDCs present more and long dendrite while iDCs with a few and small dendrite. Immature DC mainly exists in a variety of solid organs and non-lymphoid epithelial tissues, which membrane surface expresses low CD80, CD86, MHCⅠ,MHCⅡ, CD11c and so on. But it doesn't express CD40. Mature DC are mainly distributed in the lymph nodes and spleen with numerous and long dendrites. In addition to the expression of specific antigen, they also express highly MHCⅠ, MHCⅡand CD80, CD8, CD40 and CD11c other stimulatory molecules and secrete IL-12. The above results showed that Activin A induces differentiation of peripheral blood mononuclear cells into dendritic cells.2. Activin A induces mouse peritoneal macrophages differentiation into M2 macrophagesMouse peritoneal macrophages cultured in vitro were used to further explore the effect of Activin A on mature monocytes/macrophages. Morphologically, mouse peritoneal macrophages stimulated by Activin A presented obviously irregular and polygonal form. Moreover, large macrophages could also been found. However, they don't have typical dendrites as DC. Given the results above, activin A may further induce the activation of mature macrophages, but failed to induce macrophage to differentiate into DC.NO is a very important active factor secreted by monocytes/macrophages in inflammatory response. It can kill bacteria and tumor cells. But excessive NO can also cause tissue inflammatory injury. Both M1 and M2 can secret NO but in different pathway. M1 mainly synthesizes a mass of NO by iNOS; M2 synthesis NO mainly through Arginase 1. Otherwise, M2 macrophages also can produce inhibit factor IL-10, which can reduce the antigen present and down-regulate immune response. In this study, mouse peritoneal macrophages were stimulated by Activin A, and then the levels of IL-10 and NO in cell culture supernatants were detected by ELISA and Griess method. The results showed that Activin A could significantly promote the mouse peritoneal macrophages to secret IL-10 and NO. CD68 is one of the most important cell membrane molecule markers of monocytes/macrophages and highly expresses on activated and mature monocyte/macrophage surface. The expression of CD68 on mouse peritoneal macrophages was further analyzed by flow cytometry. It was found that activin A significantly increased the expression of CD68 on surface of mouse peritoneal macrophages. By the same method, we also analyzed the expression of CD11c on mouse peritoneal macrophages. The results showed that the low expression of CD11c on mouse peritoneal macrophages was not affected by activin A.In order to further determine the classification of mouse peritoneal macrophages after treated by Activin A, the expression of Agrinase 1 on mouse peritoneal macrophages was analyzed by flow cytometry. The results showed that activin A significantly increased the expression of Arginase 1. The data suggested that Activin A may induce the mouse peritoneal macrophages to differentiation into M2 macrophages.In summary, the effect of Activin A on monocytes/macrophages was related to the macrophage activation state. It not only can induce immature monocytes/ macrophages cells to differentiate into dendritic cells to participate acquired immune response, but also can promote the mature mouse peritoneal macrophages to differentiate into the M2 macrophages, which was involved in regulating natural immune responses.