Our overall goal is to elucidate the functional and transcriptomic specialization of endothelial cells (EC) that recruit lymphocytes from the blood, and to define novel mechanisms involved in lymphocyte homing via high endothelial venules (HEV). In addition to differences from capillary (CAP) and other EC, HEV display tissue-specific expression of vascular ?addressins?, adhesion receptors and chemokines that control the organ specificity of lymphocyte homing. To identify genes and transcriptional networks that define and program HEV specialization, we have initiated whole genome expression studies of lymphoid tissue HEV and CAP. Preliminary data mining has identified novel candidate mechanisms for the control of lymphocyte recruitment, including a novel vascular addressin for B cell homing and HEV expression of genes for metabolism of lipid regulators of diapedesis.
Aims i nclude: 1) To comprehensively analyze the segmental (HEV vs CAP) and tissue-specific (skin- draining vs mesenteric lymph node and Peyer?s patch) transcriptional specialization of HEC, and to define the response of HEC to immunization. HEC and CAP from dissociated PLN, MLN and PP will be sorted by FACS based on EC subset markers, and RNA submitted for whole genome expression profiling. The transcriptional response of HEC to polarizing immune responses will be defined by comparison of HEV and CAP from resting vs immunized LN. 2) To elucidate the biosynthetic machinery required for HEV synthesis of a novel carbohydrate vascular addressin for B cell homing. Our gene expression data led to discovery of a ?Gal??2, 6 sialyltransferase-dependent PP HEV ligand for the B cell lectin Siglec2 (CD22). We will use CD22-Fc binding in studies of St6gal1, Cmah and CHST 24 sulfotransferase knockout mice to test the hypothesized roles of HEV-expressed glycan-modifying enzymes in synthesis of the novel addressin. 3) To test the hypothesis that HEV-associated lipid metabolites regulate lymphocyte migration from HEC. Based on transcriptional analyses of enzyme expression by HEC and surrounding cells, we hypothesize that gradients of lipid chemoattractants participate in lymphocyte recruitment via HEV. Lymphocyte diapedesis during short term homing will be quantified, and the effects of deficiency in lymphocyte receptors and/or inhibition of targeted metabolic pathways in recipient lymph nodes will be assessed. Comprehensive analyses of HEV and CAP transcriptomes will open up new areas of investigation in vascular biology and immunology. Elucidation of the mechanisms of vascular control of lymphocyte recruitment may lead to novel targets and approaches for the control of autoimmune and other pathologic inflammation.
Specialized blood vessels called `high endothelial venules' or HEV are the portals through which blood lymphocytes (immune cells) gain access to lymph nodes and gut lymphoid tissues, and to tissue sites of chronic inflammation. To understand HEV better, we will define the genes they express, contrast their gene programs with those of other blood vessel lining cells, and mine the resulting `transcriptomic' data for insights into HEV function. Initial studies have identified new candidate mechanisms for the control of lymphocyte recruitment, including a carbohydrate address code for gut associated HEV, and enzymes that control lymphocyte motility and may allow escape from HEV. Understanding the mechanisms by which HEV control lymphocyte recruitment may lead to novel targets and approaches for the control of autoimmune, cardiovascular and other diseases of inflammation.
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