ABSTRACT
Lymphocytes are recruited from blood by high-endothelial venules (HEVs). We performed transcriptomic analyses and identified molecular signatures that distinguish HEVs from capillary endothelium and that define tissue-specific HEV specialization. Capillaries expressed gene programs for vascular development. HEV-expressed genes showed enrichment for genes encoding molecules involved in immunological defense and lymphocyte migration. We identify capillary and HEV markers and candidate mechanisms for regulated recruitment of lymphocytes, including a lymph node HEV-selective transmembrane mucin; transcriptional control of functionally specialized carbohydrate ligands for lymphocyte L-selectin; HEV expression of molecules for transendothelial migration; and metabolic programs for lipid mediators of lymphocyte motility and chemotaxis. We also elucidate a carbohydrate-recognition pathway that targets B cells to intestinal lymphoid tissues, defining CD22 as a lectin-homing receptor for mucosal HEVs.
Subject(s)
Capillaries/metabolism , Endothelium/metabolism , Gene Expression Profiling , Lymphocytes/metabolism , Lymphoid Tissue/blood supply , Venules/metabolism , Animals , Cell Movement/genetics , Endothelial Cells/metabolism , Endothelium/cytology , Female , Flow Cytometry , Gene Ontology , Lymph Nodes/blood supply , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microscopy, Confocal , Oligonucleotide Array Sequence AnalysisABSTRACT
The catecholamine norepinephrine (NE) stimulates T lymphocytes through a beta-adrenergic receptor (ßAR)/adenylyl cyclase (AC)/cyclic AMP (cAMP)/protein kinase A (PKA) pathway, leading to altered cell responsiveness and apoptosis. p38 Mitogen-activated protein kinase (MAPK), a major intracellular signalling mediator for cellular and environmental stressors, is involved in the production of immune modulators and in the regulation of T-cell development, survival and death. In these studies we investigated the relationship among NE signalling, p38 MAPK activity and T-cell death. We showed that NE stimulation of BALB/c mouse thymocytes and S49 thymoma cells selectively increases the dual phosphorylation and activity of p38α MAPK. p38 MAPK activation involves the ßAR, Gs protein, AC, cAMP and PKA, as determined through the use of a ßAR antagonist, activators of AC and cAMP, and S49 clonal mutants deficient in Gs and PKA. Dual phosphorylation of p38 MAPK is also dependent on its own catalytic activity. Inhibition of p38 MAPK activity revealed its involvement in cAMP-mediated activating transcription factor-2 (ATF-2) phosphorylation, Fas ligand messenger RNA (mRNA) up-regulation, and cell death. These results identify a mechanism through which NE stimulation of the ßAR/Gs/PKA pathway activates p38 MAPK, which can be potentiated by autophosphorylation, and leads to changes in T-cell dynamics, in part through the regulation of Fas ligand mRNA expression.
Subject(s)
Norepinephrine/pharmacology , Signal Transduction , T-Lymphocytes/cytology , T-Lymphocytes/immunology , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Apoptosis , Cells, Cultured , Cyclic AMP-Dependent Protein Kinases/metabolism , Enzyme Activation , Fas Ligand Protein/metabolism , GTP-Binding Protein alpha Subunits, Gs/metabolism , Lymphocyte Activation , Male , Mice , Mice, Inbred BALB C , RNA, Messenger/metabolism , Receptors, Adrenergic, beta/metabolism , T-Lymphocytes/enzymology , Thymoma , Thymus Gland/cytology , Tumor Cells, CulturedABSTRACT
Thy-1 is a cell surface protein important in immunologic and neurologic processes, including T cell activation and proliferation, and neuronal outgrowth. In murine thymocytes, Thy-1 is downregulated in response to norepinephrine (NE) through posttranscriptional destabilization of its mRNA mediated by ßAR/AC/cAMP/PKA signaling. In this study we investigated factors involved in NE/cAMP-mediated Thy-1 mRNA destabilization in S49 thymoma cells, and identified a region containing two copies of the AUUUA regulatory element (ARE), a motif commonly associated with mRNA decay, in the Thy-1 mRNA 3' UTR. Insertion of the Thy-1 ARE region into a reporter gene, resulted in cAMP induced destabilization of the reporter gene mRNA. RNA-protein binding studies revealed multiple Thy-1 ARE binding proteins, including AUF1, HuR, and TIAR. RNA silencing of HuR enhanced cAMP-mediated downregulation of Thy-1 mRNA, in contrast, silencing AUF1 had no effect. Immunoblotting revealed multiple proteins phosphorylated by PKA as a result of NE or cAMP signaling. These results reveal that the machinery of NE/cAMP modulation of Thy-1 mRNA decay involves a cAMP responsive ARE in its 3' UTR and multiple site specific ARE binding proteins. These findings add to our knowledge of Thy-1 mRNA regulation and provide insight into the regulation of ARE containing mRNAs, which impacts stress-related immunosuppression.
