Molecular Ophthalmology, Department of Ophthalmology, University of Duisburg-Essen, Essen, Germany.
Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, United States.
Invest Ophthalmol Vis Sci. 2018 Nov 1;59(13):5391-5397. doi: 10.1167/iovs.18-25466.
Graves' orbitopathy (GO) is an autoimmune orbital disorder associated with Graves' disease caused by thyrotropin receptor autoantibodies. Orbital fibroblasts (OFs) and CD40 play a key role in disease pathogenesis. The bioactive lipid sphingosine-1-phosphate (S1P) has been implicated in promoting adipogenesis, fibrosis, and inflammation in OFs. We investigated the role of CD40 signaling in inducing S1P activity in orbital inflammation.
OFs and T cells were derived from GO patients and healthy control (Ctl) persons. S1P abundance in orbital tissues was evaluated by immunofluorescence. OFs were stimulated with CD40 ligand and S1P levels were determined by ELISA. Further, activities of acid sphingomyelinase (ASM), acid ceramidase, and sphingosine kinase were measured by ultraperformance liquid chromatography. Sphingosine and ceramide contents were analyzed by mass spectrometry. Finally, the role for S1P in T-cell attraction was investigated by T-cell migration assays.
GO orbital tissue showed elevated amounts of S1P as compared to control samples. Stimulation of CD40 induced S1P expression in GO-derived OFs, while Ctl-OFs remained unaffected. A significant increase of ASM and sphingosine kinase activities, as well as lipid formation, was observed in GO-derived OFs. Migration assay of T cells in the presence of SphK inhibitor revealed that S1P released by GO-OFs attracted T cells for migration.
The results demonstrated that CD40 ligand stimulates GO fibroblast to produce S1P, which is a driving force for T-cell migration. The results support the use of S1P receptor signaling modulators in GO management.
Graves 眼病（GO）是一种与 Graves 病相关的自身免疫性眼眶疾病，由促甲状腺激素受体自身抗体引起。眼眶成纤维细胞（OFs）和 CD40 在疾病发病机制中起关键作用。生物活性脂质 1-磷酸鞘氨醇（S1P）已被牵涉到促进 OFs 中的脂肪生成、纤维化和炎症。我们研究了 CD40 信号在诱导眼眶炎症中 S1P 活性中的作用。
从 GO 患者和健康对照（Ctl）中分离出 OFs 和 T 细胞。通过免疫荧光评估眼眶组织中 S1P 的丰度。用 CD40 配体刺激 OFs，通过 ELISA 测定 S1P 水平。此外，通过超高效液相色谱法测定酸性鞘氨醇酶（ASM）、酸性神经酰胺酶和鞘氨醇激酶的活性。通过质谱分析鞘氨醇和神经酰胺的含量。最后，通过 T 细胞迁移测定研究了 S1P 在 T 细胞吸引中的作用。
与对照样本相比，GO 眼眶组织显示出 S1P 含量增加。CD40 刺激诱导了 GO 来源的 OFs 中 S1P 的表达，而 Ctl-OFs 则不受影响。在 GO 来源的 OFs 中观察到 ASM 和鞘氨醇激酶活性以及脂质形成的显著增加。在 SphK 抑制剂存在的情况下，T 细胞迁移测定显示 GO-OFs 释放的 S1P 吸引 T 细胞迁移。
结果表明，CD40 配体刺激 GO 成纤维细胞产生 S1P，这是 T 细胞迁移的驱动力。结果支持在 GO 管理中使用 S1P 受体信号调节剂。
