Background Prostaglandin E2 （PGE2） is a key modulator of dendritic cells （DCs） function, and cornea-derived transforming growth factor beta 2 （TGF-β2） promotes the generation of phenotypically and functionally ...Background Prostaglandin E2 （PGE2） is a key modulator of dendritic cells （DCs） function, and cornea-derived transforming growth factor beta 2 （TGF-β2） promotes the generation of phenotypically and functionally immature DCs. Therefore, this study was carried out to investigate whether PGE2 is involved in the suppressive effect on DCs maturation mediated by corneal stroma cells （CSCs） and whether PGE2 and TGF-β2 have additive effects in this immunosuppressive mechanism. Methods Bone marrow-derived DCs （BM-DCs）, splenic T cells and CSCs culture supernatant were obtained from mice via various protocols. After that, the level of PGE2 in CSCs culture supernatant was analyzed by enzyme-linked immunosorbent assay. Then, immature BM-DCs pretreated by E-prostanoid 2 receptor antagonist AH6809 or dimethyl sulfoxide were induced to mature in the presence of lipopolysaccharide, with or without CSCs culture supernatant. In parallel experiments, neutralizing TGF-β2 antibody or normal goat IgG was added into the supernatant. Next, the cellular surface markers for DCs maturation, including CD80, CD86, and major histocompatibility complex class Ⅱ （MHC Ⅱ）, were analyzed by flow cytometry; the capability of stimulating the proliferation of T lymphocytes was evaluated by allogeneic mixed lymphocyte reactions and the function of endocytosis was assessed by fluorescein isothiocyanate-dextran uptake. Results Higher concentration of PGE2 was detected in CSCs culture supernatant than in the fresh medium. In addition, compared with control group, after treated with the supernatant in the mature stage, BM-DCs displayed lower expression of CD80, CD86 and MHC Ⅱ, lower T cell stimulatory capacity and higher endocytosis function. However, after the application of AH6809, BM-DCs partially regained T cell stimulatory capacity and expression of CD86 and MHC Ⅱ, but partially lost endocytic activity. Moreover, after the application of AH6809 and neutralizing TGF-β2 antibody, the result of statistical analysis indicated that there was a statistical difference of interaction in the expression of MHC Ⅱ and T cell stimulatory capacity. Conclusions PGE2 contributes to the suppressive effect on BM-DCs maturation mediated by CSCs in vitro, and PGE2 and TGF-β2 have additive effects on the immunosuppression of BM-DCs.展开更多
文摘Background Prostaglandin E2 （PGE2） is a key modulator of dendritic cells （DCs） function, and cornea-derived transforming growth factor beta 2 （TGF-β2） promotes the generation of phenotypically and functionally immature DCs. Therefore, this study was carried out to investigate whether PGE2 is involved in the suppressive effect on DCs maturation mediated by corneal stroma cells （CSCs） and whether PGE2 and TGF-β2 have additive effects in this immunosuppressive mechanism. Methods Bone marrow-derived DCs （BM-DCs）, splenic T cells and CSCs culture supernatant were obtained from mice via various protocols. After that, the level of PGE2 in CSCs culture supernatant was analyzed by enzyme-linked immunosorbent assay. Then, immature BM-DCs pretreated by E-prostanoid 2 receptor antagonist AH6809 or dimethyl sulfoxide were induced to mature in the presence of lipopolysaccharide, with or without CSCs culture supernatant. In parallel experiments, neutralizing TGF-β2 antibody or normal goat IgG was added into the supernatant. Next, the cellular surface markers for DCs maturation, including CD80, CD86, and major histocompatibility complex class Ⅱ （MHC Ⅱ）, were analyzed by flow cytometry; the capability of stimulating the proliferation of T lymphocytes was evaluated by allogeneic mixed lymphocyte reactions and the function of endocytosis was assessed by fluorescein isothiocyanate-dextran uptake. Results Higher concentration of PGE2 was detected in CSCs culture supernatant than in the fresh medium. In addition, compared with control group, after treated with the supernatant in the mature stage, BM-DCs displayed lower expression of CD80, CD86 and MHC Ⅱ, lower T cell stimulatory capacity and higher endocytosis function. However, after the application of AH6809, BM-DCs partially regained T cell stimulatory capacity and expression of CD86 and MHC Ⅱ, but partially lost endocytic activity. Moreover, after the application of AH6809 and neutralizing TGF-β2 antibody, the result of statistical analysis indicated that there was a statistical difference of interaction in the expression of MHC Ⅱ and T cell stimulatory capacity. Conclusions PGE2 contributes to the suppressive effect on BM-DCs maturation mediated by CSCs in vitro, and PGE2 and TGF-β2 have additive effects on the immunosuppression of BM-DCs.
