title: Vascular endothelial growth factor c/vascular endothelial growth factor receptor 3 signaling regulates chemokine gradients and lymphocyte migration from tissues to lymphatics.
abstract: BACKGROUND
  Circulation of leukocytes via blood, tissue and lymph is integral to adaptive immunity. Afferent lymphatics form CCL21 gradients to guide dendritic cells and T cells to lymphatics and then to draining lymph nodes (dLN). Vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 (VEGFR-3) are the major lymphatic growth factor and receptor. We hypothesized these molecules also regulate chemokine gradients and lymphatic migration.
  
  
  METHODS
  CD4 T cells were injected into the foot pad or ear pinnae, and migration to afferent lymphatics and dLN quantified by flow cytometry or whole mount immunohistochemistry. Vascular endothelial growth factor receptor 3 or its signaling or downstream actions were modified with blocking monoclonal antibodies (mAbs) or other reagents.
  
  
  RESULTS
  Anti-VEGFR-3 prevented migration of CD4 T cells into lymphatic lumen and significantly decreased the number that migrated to dLN. Anti-VEGFR-3 abolished CCL21 gradients around lymphatics, although CCL21 production was not inhibited. Heparan sulfate (HS), critical to establish CCL21 gradients, was down-regulated around lymphatics by anti-VEGFR-3 and this was dependent on heparanase-mediated degradation. Moreover, a Phosphoinositide 3-kinase (PI3K)α inhibitor disrupted HS and CCL21 gradients, whereas a PI3K activator prevented the effects of anti-VEGFR-3. During contact hypersensitivity, VEGFR-3, CCL21, and HS expression were all attenuated, and anti-heparanase or PI3K activator reversed these effects.
  
  
  CONCLUSIONS
  Vascular endothelial growth factor C/VEGFR-3 signaling through PI3Kα regulates the activity of heparanase, which modifies HS and CCL21 gradients around lymphatics. The functional and physical linkages of these molecules regulate lymphatic migration from tissues to dLN. These represent new therapeutic targets to influence immunity and inflammation.
authors.affiliation: 1 Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, MD. 2 Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD. 3 Department of Surgery, University of Maryland School of Medicine, Baltimore, MD.
authors.forename: Daiki
authors.initials: D
authors.lastname: Iwami
authors.forename: C Colin
authors.initials: CC
authors.lastname: Brinkman
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Bromberg
mesh_terms: D019264:Adoptive Transfer; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000637:transplantation; D054421:Chemokine CCL21 / Q000378:metabolism*; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D003877:Dermatitis, Contact / Q000276:immunology / Q000378:metabolism / Q000517:prevention & control; D004139:Dinitrofluorobenzene; D004195:Disease Models, Animal; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D005966:Glucuronidase / Q000037:antagonists & inhibitors / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism; D008198:Lymph Nodes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D042601:Lymphatic Vessels / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D012867:Skin / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D042582:Vascular Endothelial Growth Factor C / Q000378:metabolism*; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D000099175:Heparanase
chemical_list: D000911:Antibodies, Monoclonal; D054421:Chemokine CCL21; D004139:Dinitrofluorobenzene; D020536:Enzyme Activators; D005966:Glucuronidase; D006497:Heparan Sulfate; D058539:Phosphatidylinositol 3-Kinase; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D042582:Vascular Endothelial Growth Factor C; D040321:Vascular Endothelial Growth Factor Receptor-3; D000099175:Heparanase; C467483:vascular endothelial growth factor C, mouse