Circulation of leukocytes via blood, tissue and lymph is integral to adaptive immunity. Afferent lymphatics form CCL21 gradients to guide DC and T cells to lymphatics and then to draining lymph nodes (dLN). VEGF-C and VEGFR-3 are the major lymphatic growth factor and receptor. We hypothesized these molecules also regulate chemokine gradients and lymphatic migration.

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. VEGFR-3 or its signaling or downstream actions were modified with blocking mAbs or other reagents.

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 PI3Kα inhibitor disrupted HS and CCL21 gradients,while 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.

VEGF-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.