Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse T cell model to characterize transendothelial migration across lymphatics

ABSTRACT: Sphingosine 1-phosphate (S1P) influences T cell migration into and out of secondary lymphoid organs; however, its mechanism of action remains uncertain. Our previous research shows that agonism of the S1P receptor S1P1 inhibits the egress of T lymphocytes from the peripheral tissues into afferent lymphatics. To better define the mechanism of inhibition, we developed an in vitro model to characterize T cell transendothelial migration across lymphatics. Two commercially available endothelial cell lines (MS-1 and SVEC4-10) were characterized by flow cytometry, real time RT-PCR, and Affymetrix Gene Array. These cell lines were grown to confluent monolayers in transwell systems, on either the upper or lower surface of the transwell insert. T cells were isolated from the spleens of (C57BL/6 x C3H/HeJ)F1, S1P1 KO, or S1P1 KO littermate controls, and either treated with the S1P receptor modulator FTY720 or left untreated. Cells were migrated to chemokines (CCL19 or CCL21) for 4 hours, and migration quantified. Flow cytometry, RT-PCR, and array results identified MS-1 as a blood vascular endothelial cell line, expressing high levels of CD31, CD34, and ICAM-1 as well as other endothelial cell markers; while SVEC4-10 closely resemble a lymphatic phenotype, expressing LYVE-1, VEGFR-3, and podoplanin. T cells efficiently migrate across MS-1, whether grown on the upper or lower surface; whereas migration across SVEC4-10 only occurs when cells are grown on the lower surface of the transwell (iSVEC), recapitulating basal (abluminal) to apical (luminal) migration that occurs in vivo. FTY720 inhibits T cell migration across iSVEC, but not across MS-1. Inhibition is due to drug effects only on T cells but not endothelial cells. S1P1 KO T cells treated with FTY720 are not inhibited in their migration across the iSVEC line, showing that S1P1 stimulation is required for migration inhibition. The in vitro model developed here is the first to use endothelial cell lines to analyze the regulation of T cell migration across lymphatic endothelium. The results show there is directional control of T cell migration across lymphatic cells, such that T cells only migrate from a basal to apical direction. Agonism of S1P1 specifically inhibits migration, while absence of the receptor does not. These findings have important implications for the use of S1P1 agonists in transplantation, as inhibition of cell entry into afferent lymphatics and lymph nodes could impede the development of graft rejection. We used microarrays to detail the global gene expression of these two cell lines in order to beter determine their phenotype as blood vascular or lymphatic endothelial cell line for use in our newly-developed in vitro model. Experiment Overall Design: MS-1 or SVEC4-10 endothelial cell lines were grown in culture flasks for 3-5 days in standard culture medium (see protocol below) until reaching confluence. Cells were then gently dissociated from flask and cells were placed in Trizol reagent for RNA isolation.

ORGANISM(S): Mus musculus

SUBMITTER: Levi Ledgerwood

PROVIDER: E-GEOD-9237 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The sphingosine 1-phosphate receptor 1 causes tissue retention by inhibiting the entry of peripheral tissue T lymphocytes into afferent lymphatics.

Ledgerwood Levi G LG Lal Girdhari G Zhang Nan N Garin Alexandre A Esses Steven J SJ Ginhoux Florent F Merad Miriam M Peche Helene H Lira Sergio A SA Ding Yaozhong Y Yang Yu Y He Xingxuan X Schuchman Edward H EH Allende Maria L ML Ochando Jordi C JC Bromberg Jonathan S JS

Nature immunology 20071125 1

Although much is known about the migration of T cells from blood to lymph nodes, less is known about the mechanisms regulating the migration of T cells from tissues into lymph nodes through afferent lymphatics. Here we investigated T cell egress from nonlymphoid tissues into afferent lymph in vivo and developed an experimental model to recapitulate this process in vitro. Agonism of sphingosine 1-phosphate receptor 1 inhibited the entry of tissue T cells into afferent lymphatics in homeostatic an ...[more]

PMID: 18037890

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Project description:Sphingosine 1-phosphate (S1P) influences T cell migration into and out of secondary lymphoid organs; however, its mechanism of action remains uncertain. Our previous research shows that agonism of the S1P receptor S1P1 inhibits the egress of T lymphocytes from the peripheral tissues into afferent lymphatics. To better define the mechanism of inhibition, we developed an in vitro model to characterize T cell transendothelial migration across lymphatics. Two commercially available endothelial cell lines (MS-1 and SVEC4-10) were characterized by flow cytometry, real time RT-PCR, and Affymetrix Gene Array. These cell lines were grown to confluent monolayers in transwell systems, on either the upper or lower surface of the transwell insert. T cells were isolated from the spleens of (C57BL/6 x C3H/HeJ)F1, S1P1 KO, or S1P1 KO littermate controls, and either treated with the S1P receptor modulator FTY720 or left untreated. Cells were migrated to chemokines (CCL19 or CCL21) for 4 hours, and migration quantified. Flow cytometry, RT-PCR, and array results identified MS-1 as a blood vascular endothelial cell line, expressing high levels of CD31, CD34, and ICAM-1 as well as other endothelial cell markers; while SVEC4-10 closely resemble a lymphatic phenotype, expressing LYVE-1, VEGFR-3, and podoplanin. T cells efficiently migrate across MS-1, whether grown on the upper or lower surface; whereas migration across SVEC4-10 only occurs when cells are grown on the lower surface of the transwell (iSVEC), recapitulating basal (abluminal) to apical (luminal) migration that occurs in vivo. FTY720 inhibits T cell migration across iSVEC, but not across MS-1. Inhibition is due to drug effects only on T cells but not endothelial cells. S1P1 KO T cells treated with FTY720 are not inhibited in their migration across the iSVEC line, showing that S1P1 stimulation is required for migration inhibition. The in vitro model developed here is the first to use endothelial cell lines to analyze the regulation of T cell migration across lymphatic endothelium. The results show there is directional control of T cell migration across lymphatic cells, such that T cells only migrate from a basal to apical direction. Agonism of S1P1 specifically inhibits migration, while absence of the receptor does not. These findings have important implications for the use of S1P1 agonists in transplantation, as inhibition of cell entry into afferent lymphatics and lymph nodes could impede the development of graft rejection. We used microarrays to detail the global gene expression of these two cell lines in order to beter determine their phenotype as blood vascular or lymphatic endothelial cell line for use in our newly-developed in vitro model. Keywords: Cell type comparison

Project description:A synthetic analog of sphingosine named FTY720 (Fingolimod), phosphorylated by sphingosine kinase-2, interacts with sphingosine-1-phosphate (S1P) receptors expressed on various cells. FTY720 suppresses the disease activity of multiple sclerosis (MS) chiefly by inhibiting S1P-dependent egress of autoreactive T lymphocytes from secondary lymphoid organs, and possibly by exerting anti-inflammmatory and neuroprotective effects directly on brain cells. However, at present, biological effects of FTY720 on human microglia are largely unknown. We studied FTY720-mediated apoptosis of a human microglia cell line HMO6. The exposure of HMO6 cells to non-phosphorylated FTY720 (FTY720-non-P) induced apoptosis in a dose-dependent manner with IC50 of 10.6±2.0 microM, accompanied by the cleavage of caspase-7 and caspase-3 but not of caspase-9. The apoptosis was inhibited by Z-DQMD-FMK, a caspase-3 inhibitor, but not by Pertussis toxin, a Gi protein inhibitor, suramin, a S1P3/S1P5 inhibitor, or W123, a S1P1 competitive antagonist, although HMO6 expressed S1P1, S1P2, and S1P3. Furthermore, both phosphorylated FTY720 (FTY720-P) and SEW2871, a S1P1 selective agonist did not induce apoptosis of HMO6. Genome-wide gene expression profiling and molecular network analysis indicated activation of transcriptional regulation by sterol regulatory element-binding protein (SREBP) in FTY720-non-P-treated HMO6 cells. Western blot verified activation of SREBP2 in these cells, and apoptosis was enhanced by pretreatment with simvastatin, an activator of SREBP2, and by overexpression of the N-terminal fragment of SREBP2. These observations suggest that FTY720-non-P-induced apoptosis of HMO6 human microglia is independent of S1P receptor binding, and positively regulated by the SREBP2-dependent proapoptotic signaling pathway. The HMO6 cell line was established by immortalizing cultured microglia isolated from human embryonic telencephalon tissues with a retroviral vector PASK1.2 encoding v-myc oncogene (Nagai et al. Neurobiol. Dis. 8: 1057-1068, 2001). HMO6 cells express the markers of the microglia/macrophage lineage cells, including CD11b, CD68, CD86, HLA-ABC, HLA-DR, and ricinus communis agglutinin lectin-1 (RCA), serving as a model of human microglia both in vitro and in vivo. The cells were exposed for 2 hours to 10 microM FTY720-non-P dissolved in dimethyl sulfoxide (DMSO) or ethanol or exposed to the vehicle.

Dataset Information

Transcription profiling of mouse T cell model to characterize transendothelial migration across lymphatics

Publications

The sphingosine 1-phosphate receptor 1 causes tissue retention by inhibiting the entry of peripheral tissue T lymphocytes into afferent lymphatics.

Similar Datasets

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