Abstract-Activated macrophages acquire a proinflammatory (classic) or antiinflammatory (alternative) phenotype that influences atherosclerosis. The present study investigated whether sphingosine-1-phosphate (S1P), with its known antiinflammatory effects, could regulate the inflammatory phenotype of lipopolysaccharide (LPS)-stimulated mouse macrophages. Activation of macrophages by LPS significantly increases proinflammatory cytokine secretion. Pretreatment of macrophages with 500 nmol/L S1P markedly reduced LPS-mediated secretion of tumor necrosis factor-␣, monocyte chemoattractant protein-1, and interleukin-12. Such antiinflammatory actions were also evident in LPSstimulated macrophages treated with the S1P1 receptor-specific agonist SEW2871. Pharmacological antagonism of the S1P1 receptor on macrophages using the S1P1-specific antagonist VPC44116 also blocked proinflammatory cytokine secretion in response to LPS. Studies using bone marrow-derived macrophages from S1P2-deficient mice revealed that the S1P2 receptor did not play a pivotal role in this process. Thus, activation of the S1P1 receptor in mouse macrophages limits the expression of proinflammatory cytokines. Furthermore, we demonstrated that S1P increased arginase I activity and inhibited LPS-induced inducible NO synthase activity in LPS-treated macrophages, again through S1P1 receptor activation on macrophages. Analysis of a 1.7-kb region of the murine inducible NO synthase promoter revealed the presence of putative nuclear factor B, activator protein-1, and STAT-1 response elements. Using inducible NO synthase promoter-reporter constructs, we found that S1P significantly reduced the nuclear factor B-mediated induction of inducible NO synthase. These findings demonstrate an important role for S1P in the regulation of macrophage phenotypic switching. Therefore, we conclude that S1P promotes the production of an alternative antiinflammatory macrophage phenotype through activation of the macrophage S1P1 receptor. (Circ Res. 2008;102:950-958.)Key Words: macrophage Ⅲ sphingosine-1-phosphate Ⅲ arginase I Ⅲ iNOS Ⅲ NFB Ⅲ inflammation A therosclerosis is a chronic inflammatory disease. 1 Monocytes/macrophages play key roles in the initiation and progression of atherosclerosis, and can alter their phenotype in response to changes in the local cytokine environment. 2 Macrophages can be distinctly activated to either a classically activated, or M1, phenotype by proinflammatory molecules such as interferon-␥ and lipopolysaccharide (LPS) or to an "alternatively activated," or M2, phenotype by Th-2 antiinflammatory cytokines such as interleukin (IL)-4. 3 Macrophages can also display a M2b activated phenotype when stimulated by immune complexes and LPS. 4 The M1 and M2 phenotypes are specifically distinguished by the cytokines produced and by the way in which arginine is processed, whereas the M2b phenotype is mainly distinguished by high IL-10 and low IL-12 levels. In classically activated macrophages, Th-1 cytokines trigger the induction of inducible NO synt...

Background-Mice

Aims/hypothesis. Hyperglycaemia is a primary cause of vascular complications in diabetes. A hallmark of these vascular complications is endothelial cell dysfunction, which is partly due to the reduced production of nitric oxide. The aim of this study was to investigate the regulation of endothelial nitric oxide synthase (eNOS) activity by acute and chronic elevated glucose. Methods. Human aortic endothelial cells were cultured in 5.5 mmol/l (NG) or 25 mmol/l glucose (HG) for 4 h, 1 day, 3 days or 7 days. Mouse aortic endothelial cells were freshly isolated from C57BL/6J control and diabetic db/db mice. The expression and activity of eNOS were measured using quantitative PCR and nitrite measurements respectively. The binding of activator protein-1 (AP-1) to DNA in nuclear extracts was determined using electrophoretic mobility-shift assays. Results. Acute exposure (4 h) of human aortic endothelial cells to 25 mmol/l glucose moderately increased eNOS activity and eNOS mRNA and protein expression. In contrast, chronic exposure to elevated glucose (25 mmol/l for 7 days) reduced total nitrite levels (46% reduction), levels of eNOS mRNA (46% reduction) and eNOS protein (65% reduction). In addition, AP-1 DNA binding activity was increased in chronic HGcultured human aortic endothelial cells, and this effect was reduced by the specific inhibition of reactive oxygen species production through the mitochondrial electron transport chain. Mutation of AP-1 sites in the human eNOS promoter reversed the effects of HG. Compared with C57BL/6J control mice, eNOS mRNA levels in diabetic db/db mouse aortic endothelial cells were reduced by 60%. This decrease was reversed by the overexpression of manganese superoxide dismutase using an adenoviral construct. Conclusions/interpretation. In diabetes, the expression and activity of eNOS is regulated through glucosemediated mitochondrial production of reactive oxygen species and activation of the oxidative stress transcription factor AP-1. Abbreviations: AP-1, activator protein-1 · EC, endothelial cell · eNOS, endothelial nitric oxide synthase · FBS, fetal bovine serum · HG, high glucose · MnSOD, manganese superoxide dismutase · MOI, multiplicity of infection · NF-κB, nuclear factor-κB · NG, normal glucose · ROS, reactive oxygen species · UCP-1, uncoupling protein-1