| Activin is a member of transforming growth factor-β(TGF-β) superfamily, composed ofβA andβB subunits of inhibin as a homodimer or heterodimer. Activin is termed activin A(βAβA),activin B(βBβB) and activin AB(βAβB) according to various subunits and their biological effects are similar.Activin A is usually used for studying.Activin A plays important roles from ontogenesis to maturation,such as early development of embryo,hematopoiesis,neurogenesis, cell apoptosis and regulation of generation and hormone secretion.As an immunological inhibitor,activin A can suppress activities of activated macrophages.However,we demonstrated that activin A could not only inhibit IL-1βsecretion,but also downregulate CD68 expression on mouse monocyte/ macrophage line RAW264.7 cells,simultaneously. RAW264.7 cells derived from Abelson murine leukemia virus-induced tumor and have potential of differentiation into macrophages or dendritic cells(DCs). CD68 is one of the markers of mature macrophages.It is still unclear that mechanism of CD68 expression inhibition on RAW264.7 cells stimulated by activin A.We speculate that RAW264.7 cells maybe differentiate into DCs by positive effects of activin A or mature into macrophages by suppressive effect of activin A.It has been known that marrow multipotent hemopoietic stem cells have potential to differentiate into DCs.Whether marrow mesenchynal stem cells(BMSCs) can differentiate into DCs,which has not been specifically reported.We recently found that mouse BMSCs could differentiation into monocyte-like cells.In this study,in order to elucidate the effect mechanism in which activin A downregulates CD68 expression on RAW264.7 cells,further explore immunoregulatory function of activin A on monocytes/macrophages differentiation and maturation,we investigate the effect of activin A on mouse monocytes/macrophage line RAW264.7 cells,BMSCs and mouse peritoneal macrophages as follow.1.Activin A induces differentiation of RAW 264.7 cells into DC-like cellsIncubated with activin A for 72 h,RAW264.7 cells was stained with Wright's- Giemsa's staining,and cell morphological changes were observed under a light microscope.We found that activin A stimulated-cells gradually showed typical DC-like morphology that larger,irregular,extensive stretchable growth,whereas cells of control group were round,polygonal or fusiform in shape,few protrusion.CD40,CD11c,CD80,CD86 and MHC-Ⅱmolecules are highly expressed on mature DCs whereas CD14 and CD 68 are mature makers of macrophages.In addition,there are less expressions of CD80,CD86 and MHC-Ⅱand almost no expression of CD40 or CD11c on surface of macrophages, compared with DC.We detected molecular markers of DCs and macrophages by flow cytometry and found that activin A could obviously promote expressions of CD80,CD86,CD11c,CD40 and MHCⅡmolecules and downregulate expressions of CD68 and CD14 cells.NO level represents activation of cells. DCs can also secrete NO in processes of activation and maturation.Therefore,we analyzed NO level by Griess method and found that activin A could obviously promote the NO secretions of RAW264.7 cells.Because IL-12 is a marker of mature DCs,we detected IL-12 level of cell culture supernatants by ELISA and found activin A significantly upregulated IL-12 secretion of RAW264.7 cells. Induction of naive T cell activation is an important function of mature DCs.We analyzed T lymphocyte proliferative response by mixed lymphocyte reaction and the result showed that activin A-stimulated RAW 264.7 cells promoted T cell proliferation.In addition,we examined activin specific signal transduction protein.The results showed that activin A significantly promoted the expressions of activin receptor interacting protein 2b(ARIP2b).Moreover,ARIP2b overexpression obviously upregulated expressions of CD11c and CD80,and increased secretions of NO and IL-12 of RAW264.7 cells.The above results suggest that activin A has effect of induction differentiation and maturation of RAW264.7 cells into DC-like cells and ARIP2b could be a key factor to mediate the signal transduction of activin A-induced differentiation and maturation of monocytes into DCs.2.Activin A induces differentiation of mouse bone marrow-derived mesenchymal stem cells into DC-like cellsAs we found that BMSCs could differentiate into monocytes,we further explored induction effect of activin A on differentiation of BMSCs.Mouse marrow-derived mononuclear cells were isolated by Percoll reagents and cultured in 10%FCS- L-DMEM/F12 medium for long time.CD90+CD71+cells were sorted by flow cytometry with anti-CD90-FITC and anti-CD71-PE.Purified BMSCs were identified by flow cytometry and showed positive for CD90 and CD105,negative for CD34.We choose these CD90-expressed BMSCs as targets to investigate the induction effect of activin A in vitro.We found that expressions of CD11c and CD 86 molecules were obviously upregulated,whereas CD68 was inhibited,and the secretions of NO and IL-12 were significantly increased of BMSCs stimulated by activin A for 48 h.The above results indicated that activin A also could induce differentiation of BMSCs into DC-like cells.3.Effect of activin A on the activation and maturation of mouse peritoneal macrophagesTo further explore the effect of activin A on differentiated mature monocytes/ macrophages,we isolated mouse peritoneal macrophages.IL-1βand NO are important active factors of monocytes/macrophages in inflammatory response. After mouse peritoneal macrophages stimulated by activin A,we respectively detected levels of IL-1βand NO in cell culture supernatants by ELISA and Griess methods.The results showed that activin A could markedly promote NO and IL-1βsecretion of mouse peritoneal macrophages.Phagocytosis is also an important function of macrophages.We detected the phagocytosis of mouse peritoneal macrophages by using chicken red cells.Our results showed that activin A could significantly enhance phagecytosis of mouse peritoneal macrophages,compared with control.We further analyzed molecule expression changes of mouse peritoneal macrophages by flow cytometry,and the result showed that MHCⅡmolecule expression on activin A-treated mouse peritoneal macrophages was not obviously changed,compared with control;Thus,activin A could markedly upregulated the expressions of CD68 and CD14,which are mature makers of macrophages.Therefore,these findings suggested that activin A could promote tissue-derived macrophages further activation,which indicated that activin A could enhance innate immunity response of mature macrophages.The role of activin A might contribute to body response to exogenous antigens of inflammation in early stage.Our results suggested that activin A can induce not only differentiation and maturation of mouse monocyte/macrophage line RAW 264.7 cells into DCs but also differentiation of BMSCs into DCs,indicated that activin A could enhance adaptive immunological response,and further demonstrated the mechanism that activin A downregulated CD68 expression on RAW264.7 cells is that RAW264.7 cells differentiate into DCs.Mature DCs can induce cell mediated immune response through secreting IL-12 to activate Th1 cells;Immature can induce humoral immune response though secreting IL-6 and TGF-βto activate Th2 cells. Therefore,activin A could play roles in resisting virus,tumor and bacterium through promoting Th1 immunologic response.Activin A could also significantly promote activities and maturation of mouse peritoneal macrophages, which suggested that activin A could not only induced differentiation of immature DC,but also enhance monocyte/macrophage innate immunity through promoting tissue-derived macrophages to further mature.Activin A has comprehensive biological functions and plays important regulatory roles in various cell growth,differentiation,development and maturation.This study further revealed that activin A could regulate immunologic response through enhancing adaptive and innate immunity,which provide novel theoretical basis for exploring relative nosogenesis of diseases and seeking therapeutic strategies.
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