Podoplanin maintains high endothelial venule integrity by interacting with platelet CLEC-2

Abstract

Circulating lymphocytes continuously enter lymph nodes (LNs) for immune surveillance through specialised blood vessels named high endothelial venules (HEVs)1–5, a process that increases dramatically during immune responses. How HEVs permit lymphocyte transmigration while maintaining vascular integrity is unknown. Here, we report a role for the transmembrane O-glycoprotein podoplanin (PDPN, also known as gp38 and T1α)6–8 in maintaining HEV barrier function. Mice with postnatal deletion of PDPN lost HEV integrity and exhibited spontaneous bleeding in mucosal LNs, and bleeding in the draining peripheral LN after immunisation. Blocking lymphocyte homing rescued bleeding, indicating that PDPN is required to protect the barrier function of HEVs during lymphocyte trafficking. Further analyses demonstrated that PDPN expressed on fibroblastic reticular cells (FRCs)7, which surround HEVs, functions as an activating ligand for platelet C-type lectin-like receptor 2 (CLEC-2)9,10. Mice lacking FRC PDPN or platelet CLEC-2 exhibited significantly reduced levels of VE-cadherin (VE-cad), which is essential for overall vascular integrity11,12, on HEVs. Infusion of wild-type (WT) platelets restored HEV integrity in CLEC-2-deficient mice. Activation of CLEC-2 induced release of sphingosine-1-phosphate (S1P)13,14 from platelets, which promoted expression of VE-cad on HEVs ex vivo. Furthermore, draining peripheral LNs of immunised mice lacking S1P had impaired HEV integrity similar to PDPN- and CLEC-2-deficient mice. These data demonstrate that local S1P release after PDPN-CLEC-2-mediated platelet activation is critical for HEV integrity during immune responses.

CLEC-2 is required for development and maintenance of lymph nodes

“…31 Herzog et al demonstrated that platelets CLEC-2 is necessary to maintain HEV stability postlymphocyte migration. 25 In this manuscript, the author did not report on the impact of the loss of CLEC-2 on the immune response. Here we expand these observations and explore their functional relevance demonstrating that, while loss of CLEC-2 has no direct immunological consequences in the immune responses upon single immunization, LN status after chronic antigenic stimulation is largely impaired when CLEC-2 expression on platelets is missing.…”

“…Clec1b-deficient DCs have been shown to fail to access the Gp38 1 afferent lymphatics, reducing T-cell priming and antigen response in LNs, 24 while a role for CLEC-2 in HEV maintenance was reported during the revision of this manuscript. 25 In our study, we took advantage of the Clec1b PF4-Cre conditional mouse, in which Clec1b expression is selectively deleted on the megakaryocyte/platelet lineage, to dissect the role of CLEC-2 in the development of the LN anlage in the embryo and the maintenance of the LN structure and function in adult life.…”

“…25 To test whether the loss of HEV integrity could affect the long-term B-and T-cell recirculation in the LN, we studied lymphocyte recruitment in Clec1b fl/fl PF4-Cre mice after several rounds of immunization. Mice were immunized in the front paw pad, with 10 mg of alum-precipitated NP-CGG on day 0, day 21, and day 42.…”

Ecto‐5′‐nucleotidase/CD73 enhances endothelial barrier function and sprouting in blood but not lymphatic vasculature

“…Thus, the blood vasculature of all studied organs in CD73-deficient mice has been shown to be leakier to small molecules in multiple models of ischemiareperfusion injury and LPS-induced inflammation when compared to WT mice. Since CD73 is inducible in ischemic and inflammatory stimuli [31,32], and since the steady-state permeability in different vascular beds varies substantially [33], it was relevant to address the role of CD73 in permeability control within the lymphatic system under normal conditions. Our data with the cultured human ECs, and more notably, the in vivo experiments with the CD73-deficient mice clearly showed that CD73 is involved in the control of basal vascular permeability.…”

Mutation of Threonine 34 in Mouse Podoplanin-Fc Reduces CLEC-2 Binding and Toxicity in Vivo While Retaining Anti-lymphangiogenic Activity

“…Also, CLEC-2-positive dendritic cells interact with Pdpn-positive LEC and follicular reticular cells for their migration from the periphery to and through the lymph node where they present antigens to T-cells (18). Very recently, the interaction between CLEC-2-positive plate-lets and Pdpn-positive follicular reticular cells was reported to be involved in maintaining the integrity of high endothelial venules (19).…”