The Effect Of IL-17A On Bone Marrow-Derived Dendritic Cells By GM-CSF Introduction

Objective:We established a model about mouse bone marrow-derived dendritic cells (BMDC) in vitro which was iduced by GM-CSF and stimulized by LPS. To determine the affect of IL-17A on the BMDC differentiation and maturation, different concentrations of rmIL-17A were added at the stages of differentiation and mature.Methods:Get bone marrow cells from femurs and tibias of6-8weeks Balb/c mice. Disperse marrow cells in RPMI-1640by flushing several times with RPMI-1640. ACK was added to obtain bone marrow precursor cells.2X106cells were cultured containing10%FBS RPMI-1640complete medium supplemented with200U/ml GM-CSF for8days. Every three days, fresh medium was added. The no-adherent cells were harvested and were purified by CD11c+immune magnetic beads. Purified cells following up stimulize with LPS to make them mature. We detected DC costimulatory molecule CD40、CD80、CD86and MHC Ⅱ expression by flow cytometry while the secretion of cytokines IL-12p40、IL-10、IL-6and TNF-α in the supernatant was detected by ELISA.To detect the effect of IL-17A on differentiation and mature of BMDC, at the differentiation GM-CSF induced BMDC, various concentrations of rmIL-17A(10or100ng/ml) were added. After8days, the no-adherent cells were harvested and were purified by CD11c+immune magnetic beads. Purified CD11c+cells following up stimulize by LPS with/without various concentrations of rmIL-17A (50,100or500ng/ml) continue for36hours, flow cytometry was used on costimulatory molecule CD40、CD80、CD86and MHC Ⅱ expressings and ELISA was applied for examing IL-12p40and IL-10.Results:BMDC induced by GM-CSF for6-8days were proliferation and dendritic cells showed typical morphological. LPS can effectively stimulate GM-CSF-induced differentiation of mature BMDC. The expression of costimulatory molecules CD40, CD80, CD86on DC more than the group of No LPS stimulated.The cytokines secretion IL-12p40, IL-10, IL-6and TNF-α CD11c+DC supernatant more than the group of NO LPS stimulated. In order to explore the effect of IL-17A to BMDC progenitors phenotype and differentiation, BMDC precursor cells isolated and rmIL-17A(10ng/ml) or rmIL-17A(100ng/ml) combined GM-CSF was added to culture the cells. The results show that IL-17A can promate the expression of BMDC costimulatory molecules CD40, CD86and MHCII by induction GM-CSF. At the same time, it has a dose-dependent. In order to confirm the effect of IL-17A to fully differentiated BMDC, we added rmIL-17(50,100or500ng/ml) to BMDC induced by GM-CSF for8days.then continue culture36hours. Compared with NO rmIL-17A, BMDC surface costimulatory molecules CD40, CD80, CD86, MHCII and the cytokines secretion in the supernatant IL-12p40and IL-10were not significantly changed of each group. It shows that IL-17A has no function of stimulating fully differentiated BMDC to mature. But IL-17A up-regulates the expression of costimulatory molecules on DC and the secretion of IL-12P40and IL-10in the process of GM-CSF-induced-BMDC with LPS.Conclusions:This experiment uses the method of GM-CSF-inducing and LPS stimulization successfully cultured and amplification DC. IL-17A promotes the development of DC progenitor’s phenotype which is induced by GM-CSF. IL-17A alone has little influence on the differentiation of DC. IL-17A up-regulates the expression of DC costimulatory molecules including CD40, CD80, CD86and MHC Ⅱ which are expressed on mature DC stimulated by LPS. It also up-regulate the level of IL-12p40and IL-10secreted by DC induced by LPS.These findings suggest that IL-17A plays an important role in promoting DC differentiation.