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Platelet-derived sphingosine 1-phosphate induces migration of Jurkat T cells.

ABSTRACT: BACKGROUND: The migration of T cell to atherosclerotic lesions is proposed to be involved in the pathogenesis of the atherosclerosis. Sphingosine 1-phosphate (S1P), a bioactive lysophospholipid released from activated platelets, exerts a variety of responses such as cell migration and proliferation, and reportedly induces T cell migration. Accordingly, platelet-T cell interactions may exist based on T cell responses triggered by platelet-derived S1P. METHODS: S1P was measured using two-step lipid extraction followed by high-performance liquid chromatography (HPLC) separation while other phospholipids were determined by an enzymatic assay. The expression of S1P and lysophosphatidic acid receptors on Jurkat T cells was examined by RT-PCR and flow cytometry. Jurkat cell migration by S1P and the supernatant of activated platelets (SAP) was evaluated by a modified Boyden's chamber assay. RESULTS: S1P1 receptor was confirmed to be expressed on Jurkat T cell by RT-PCR and flow cytometry. S1P at 10-100 nM induced strong Jurkat cell migration, which was inhibited by the S1P1 (and S1P3) antagonist VPC23019 and the Gi inactivator pertussis toxin (PTX). We found that the supernatant (releasate) of human platelets activated by collagen stimulation, which contains S1P abundantly, induced Jurkat cell migration and that the migration was inhibited by VPC23019 and PTX. In addition, human serum, into which platelet contents (including S1P) are fully released, induced the Jurkat cell migration, which was also inhibited by VPC23019. CONCLUSIONS: Our findings suggest that platelet-derived S1P induces Jurkat T cell migration possibly via S1P1. S1P may be a key molecule involved in the responses triggered by platelet-T cell interactions, including atherosclerosis.

SUBMITTER: Iino J

PROVIDER: S-EPMC4193286 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Platelet-derived sphingosine 1-phosphate induces migration of Jurkat T cells.

Iino Junko J Osada Makoto M Kurano Makoto M Kaneko Makoto M Ohkawa Ryunosuke R Satoh Yumiko Y Okubo Shigeo S Ozaki Yukio Y Tozuka Minoru M Tsuno Nelson H NH Yatomi Yutaka Y

Lipids in health and disease 20140925

<h4>Background</h4>The migration of T cell to atherosclerotic lesions is proposed to be involved in the pathogenesis of the atherosclerosis. Sphingosine 1-phosphate (S1P), a bioactive lysophospholipid released from activated platelets, exerts a variety of responses such as cell migration and proliferation, and reportedly induces T cell migration. Accordingly, platelet-T cell interactions may exist based on T cell responses triggered by platelet-derived S1P.<h4>Methods</h4>S1P was measured using ...[more]

PMID: 25253303

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Project description:Background: The pathophysiology of non-alcoholic steatohepatitis involves hepatocyte lipotoxicity due to excess saturated free fatty acids and concomitant proinflammatory macrophage effector responses. These include the infiltration of macrophages into hepatic cords in response to incompletely understood stimuli. Stressed hepatocytes release an increased number of extracellular vesicles (EVs), which are known to participate in intercellular signaling and coordination of the behavior of immune cell populations via their cargo. We hypothesized that hepatocyte-derived lipotoxic EVs that are enriched in sphingosine 1-phosphate (S1P) are effectors of macrophage infiltration in the hepatic microenvironment. Methods: Lipotoxic EVs were isolated from palmitate treated immortalized mouse hepatocytes and characterized by nanoparticle tracking analysis. Lipotoxic EV sphingolipids were quantified using tandem mass spectrometry. Wildtype and S1P1 receptor knockout bone marrow-derived macrophages were exposed to lipotoxic EV gradients in a microfluidic gradient generator. Macrophage migration toward EV gradients was captured by time-lapse microscopy and analyzed to determine directional migration. Fluorescence-activated cell sorting along with quantitative PCR and immunohistochemistry were utilized to characterize the cell surface expression of S1P1 receptor on intrahepatic leukocytes and hepatic expression of S1P1 receptor, respectively. Results: Palmitate treatment induced the release of EVs. These EVs were enriched in S1P. Palmitate-induced S1P enriched EVs were chemoattractive to macrophages. EV S1P enrichment depended on the activity of sphingosine kinases 1 and 2, such that, pharmacological inhibition of sphingosine kinases 1 and 2 resulted in a significant reduction in EV S1P cargo without affecting the number of EVs released. When exposed to EVs derived from cells treated with palmitate in the presence of a pharmacologic inhibitor of sphingosine kinases 1 and 2, macrophages displayed diminished chemotactic behavior. To determine receptor-ligand specificity, we tested the migration responses of macrophages genetically deleted in the S1P1 receptor toward lipotoxic EVs. S1P1 receptor knockout macrophages displayed a marked reduction in their chemotactic responses toward lipotoxic palmitate-induced EVs. Conclusions:Palmitate-induced lipotoxic EVs are enriched in S1P through sphingosine kinases 1 and 2. S1P-enriched EVs activate persistent and directional macrophage chemotaxis mediated by the S1P1 receptor, a potential signaling axis for macrophage infiltration during hepatic lipotoxicity, and a potential therapeutic target for non-alcoholic steatohepatitis.

Project description:Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD). A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P) stimulated the production of inflammatory mediators such as TNF-? and IL-8 from three-dimensionally cultured human primary keratinocytes (termed "3D keratinocytes"), which form a stratum corneum. PaCDase alone did not affect TNF-? gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-?, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-?, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-?-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-?, mRNA. PaCDase induced NF-?B p65 phosphorylation. The NF-?B inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-?B p65 phosphorylation and reduction in the protein level of the NF-?B inhibitor I?B?. Collectively, these findings suggest that (i) 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii) S1P induces the production of TNF-? via S1P receptors, and (iii) released TNF-? stimulates the production of inflammatory mediators such as IL-8.

Project description: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-alpha, monocyte chemoattractant protein-1, and interleukin-12. Such antiinflammatory actions were also evident in LPS-stimulated 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 kappaB, activator protein-1, and STAT-1 response elements. Using inducible NO synthase promoter-reporter constructs, we found that S1P significantly reduced the nuclear factor kappaB-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.

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Platelet-derived sphingosine 1-phosphate induces migration of Jurkat T cells.

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Platelet-derived sphingosine 1-phosphate induces migration of Jurkat T cells.

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