Srinivasan Suseela, Bolick David T, Lukashev Dmitriy, Lappas Courtney, Sitkovsky Michail, Lynch Kevin R, Hedrick Catherine C
Cardiovascular Research Center, University of Virginia, P.O. Box 801394, 415 Lane Rd., MR5, Rm. G123, Charlottesville, VA 22908, USA.
Diabetes. 2008 Feb;57(2):484-93. doi: 10.2337/db07-0855. Epub 2007 Nov 14.
Non-obese diabetic (NOD) mice develop spontaneous type 1 diabetes. We have shown that sphingosine-1-phosphate (S1P) reduces activation of NOD diabetic endothelium via the S1P1 receptor. In the current study, we tested the hypothesis that S1P could inhibit CD4(+) T-cell activation, further reducing inflammatory events associated with diabetes.
CD4(+) T-cells were isolated from diabetic and nondiabetic NOD mouse splenocytes and treated in the absence or presence of S1P or the S1P1 receptor-specific agonist, SEW2871. Lymphocyte activation was examined using flow cytometry, cytokine bead assays, and a lymphocyte:endothelial adhesion assay.
Diabetic T-cells secreted twofold more gamma-interferon (IFN-gamma) and interleukin-17 than nondiabetic lymphocytes. Pretreatment with either S1P or SEW2871 significantly reduced cytokine secretion by approximately 50%. Flow cytometry analysis showed increased expression of CD69, a marker of lymphocyte activation, on diabetic T-cells. Both S1P and SEW2871 prevented upregulation of CD69 on CD4(+) cells. Quantitative RT-PCR showed that lymphocytes from diabetic NOD mice had 2.5-fold lower hypoxia-inducible factor (HIF)-1alpha short isoform I.1 (HIF1alphaI.1) mRNA levels than control. HIF1alphaI.1 is a negative regulator of lymphocyte activation. S1P significantly increased HIF1alpha I.1 mRNA levels in both control and diabetic groups. IFN-gamma production and surface CD69 expression was significantly increased in lymphocytes of HIF1alphaI.1-deficient mice. S1P did not reduce either CD69 or IFN-gamma expression in lymphocytes from HIF1alphaI.1-deficient mice.
S1P acts through the S1P1 receptor and HIF1alpha I.1 to negatively regulate T-cell activation, providing a potential therapeutic target for prevention of diabetes and its vascular complications.
非肥胖型糖尿病（NOD）小鼠会自发发展为1型糖尿病。我们已经表明，1-磷酸鞘氨醇（S1P）通过S1P1受体减少NOD糖尿病内皮细胞的活化。在当前研究中，我们检验了S1P可抑制CD4(+) T细胞活化，进而减少与糖尿病相关的炎症事件这一假说。
从糖尿病和非糖尿病NOD小鼠脾细胞中分离出CD4(+) T细胞，并在不存在或存在S1P或S1P1受体特异性激动剂SEW2871的情况下进行处理。使用流式细胞术、细胞因子珠检测法和淋巴细胞-内皮细胞黏附检测法检测淋巴细胞活化情况。
糖尿病T细胞分泌的γ干扰素（IFN-γ）和白细胞介素-17比非糖尿病淋巴细胞多两倍。用S1P或SEW2871预处理可使细胞因子分泌显著减少约50%。流式细胞术分析显示，糖尿病T细胞上淋巴细胞活化标志物CD69的表达增加。S1P和SEW2871均能阻止CD4(+)细胞上CD69的上调。定量逆转录聚合酶链反应显示，糖尿病NOD小鼠的淋巴细胞中缺氧诱导因子（HIF）-1α短异构体I.1（HIF1αI.1）的mRNA水平比对照组低2.5倍。HIF1αI.1是淋巴细胞活化的负调节因子。S1P在对照组和糖尿病组中均显著提高了HIF1αI.1的mRNA水平。在HIF1αI.1缺陷小鼠的淋巴细胞中，IFN-γ的产生和表面CD69的表达显著增加。S1P并未降低HIF1αI.1缺陷小鼠淋巴细胞中CD69或IFN-γ的表达。
S1P通过S1P1受体和HIF1αI.1发挥作用，对T细胞活化进行负调节，为预防糖尿病及其血管并发症提供了一个潜在的治疗靶点。
