title: Sphingosine-1-phosphate reduces CD4+ T-cell activation in type 1 diabetes through regulation of hypoxia-inducible factor short isoform I.1 and CD69.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Cardiovascular Research Center, University of Virginia, P.O. Box 801394, 415 Lane Rd., MR5, Rm. G123, Charlottesville, VA 22908, USA.
authors.forename: Suseela
authors.initials: S
authors.lastname: Srinivasan
authors.forename: David T
authors.initials: DT
authors.lastname: Bolick
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Lukashev
authors.forename: Courtney
authors.initials: C
authors.lastname: Lappas
authors.forename: Michail
authors.initials: M
authors.lastname: Sitkovsky
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Lynch
authors.forename: Catherine C
authors.initials: CC
authors.lastname: Hedrick
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000187:drug effects / Q000276:immunology*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000187:drug effects / Q000276:immunology*; D016207:Cytokines / Q000276:immunology; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology* / Q000517:prevention & control; D003925:Diabetic Angiopathies / Q000517:prevention & control; D005434:Flow Cytometry; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000172:deficiency / Q000235:genetics*; D037181:Lectins, C-Type; D008213:Lymphocyte Activation / Q000187:drug effects*; D008246:Lysophospholipids / Q000627:therapeutic use*; D051379:Mice; D016688:Mice, Inbred NOD; D018345:Mice, Knockout; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000627:therapeutic use; D013154:Spleen / Q000276:immunology; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D000099192:CD69 Antigens
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D016207:Cytokines; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D037181:Lectins, C-Type; D008246:Lysophospholipids; D034741:RNA, Small Interfering; D013110:Sphingosine; D000099192:CD69 Antigens; C497443:Hif1a protein, mouse; C060506:sphingosine 1-phosphate