Department of Ophthalmology, Third Hospital of Hebei Medical University, Shijiazhuang, Hebei 050051, China.
Chin Med J (Engl). 2012 Jun;125(11):2041-7.
Prostaglandin E2 (PGE(2)) is a key modulator of dendritic cells (DCs) function, and cornea-derived transforming growth factor beta 2 (TGF-β(2)) promotes the generation of phenotypically and functionally immature DCs. Therefore, this study was carried out to investigate whether PGE(2) is involved in the suppressive effect on DCs maturation mediated by corneal stroma cells (CSCs) and whether PGE(2) and TGF-β(2) have additive effects in this immunosuppressive mechanism.
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 PGE(2) 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-β(2) 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 (MHCII), 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.
Higher concentration of PGE(2) 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-β(2) 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.
PGE(2) contributes to the suppressive effect on BM-DCs maturation mediated by CSCs in vitro, and PGE(2) and TGF-β(2) have additive effects on the immunosuppression of BM-DCs.
前列腺素 E2（PGE2）是树突状细胞（DC）功能的关键调节剂，角膜衍生的转化生长因子β2（TGF-β2）促进表型和功能不成熟的 DC 的生成。因此，本研究旨在探讨 PGE2 是否参与角膜基质细胞（CSC）介导的 DC 成熟抑制作用，以及 PGE2 和 TGF-β2 在这种免疫抑制机制中是否具有相加作用。
通过各种方案从小鼠中获得骨髓来源的 DC（BM-DC）、脾 T 细胞和 CSC 培养上清液。然后，通过酶联免疫吸附试验分析 CSC 培养上清液中的 PGE2 水平。接着，用脂多糖诱导经 E-前列腺素 2 受体拮抗剂 AH6809 或二甲亚砜预处理的未成熟 BM-DC 成熟，同时加入或不加入 CSC 培养上清液。在平行实验中，向上清液中加入中和 TGF-β2 抗体或正常山羊 IgG。然后，通过流式细胞术分析 DC 成熟的细胞表面标志物，包括 CD80、CD86 和主要组织相容性复合体 II（MHCII）；通过同种异体混合淋巴细胞反应评估刺激 T 淋巴细胞增殖的能力，并通过荧光素异硫氰酸酯-葡聚糖摄取评估内吞作用的功能。
CSC 培养上清液中检测到的 PGE2 浓度高于新鲜培养基。此外，与对照组相比，在成熟阶段用上清液处理后，BM-DC 表达的 CD80、CD86 和 MHCII 较低，刺激 T 细胞增殖的能力较低，内吞作用较高。然而，应用 AH6809 后，BM-DC 部分恢复了 T 细胞刺激能力和 CD86 和 MHCII 的表达，但部分丧失了内吞作用。此外，应用 AH6809 和中和 TGF-β2 抗体后，统计分析结果表明，MHCII 表达和 T 细胞刺激能力的交互作用存在统计学差异。
PGE2 有助于 CSCs 体外介导的 BM-DC 成熟抑制作用，PGE2 和 TGF-β2 对 BM-DC 的免疫抑制作用具有相加作用。
