Abstract

Objectives: 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.

Research Design and Methods: CD4 ⁺ T lymphocytes 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.

Results: Diabetic T lymphocytes secreted 2-fold more IFNγ and IL-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)-1α short isoform I.1 (HIF1αI.1) mRNA levels than control. HIF1αI.1 is a negative regulator of lymphocyte activation. S1P significantly increased HIF1α I.1 mRNA levels in both control and diabetic groups. IFNγ production and surface CD69 expression was significantly increased in lymphocytes of HIF1αI.1-deficient mice. S1P did not reduce either CD69 or IFNγ expression in lymphocytes from HIF1αI.1-deficient mice.

Conclusions: S1P acts through the S1P1 receptor and HIF1α I.1 to negatively regulate T lymphocyte activation, providing a potential therapeutic target for prevention of diabetes and its vascular complications.