Lymphatic vessels are key routes for the recirculation of fluid and cells that enter tissues from blood vessels. This function of lymphatics is important for maintenance of normal tissue homeostasis and in inflammatory diseases and other conditions with extensive fluid and cell efflux (1
). Lymphatics are also routes for spreading cancer cells (1
) and for antigen-presenting cells trafficking from tissues to lymph nodes in immune surveillance (4
). Imbalances in efflux and recirculation of fluid or cells can result in lymphedema or disturbed immune responses.
Fluid entry into lymphatics is driven largely by hydrostatic and colloidal osmotic pressure gradients (6
). A prevailing view is that much of the endothelium of initial lymphatics has incomplete or no intercellular junctions (8
). The loosely apposed but overlapping borders of endothelial cells are thought to function as “primary valves” that provide unidirectional fluid flux into lymphatics (11
). When lymphatic endothelial cells are pulled apart by anchoring filaments tensioned by interstitial forces, lymph flows along its pressure gradient into lymphatics (8
The properties of leukocyte entry into lymphatics differ from those for fluid, as leukocyte influx is a selective process. Dendritic cells, macrophages, and lymphocytes enter lymphatics, but neutrophils and erythrocytes generally do not. Leukocytes are attracted by chemokines from lymphatic endothelial cells and interact with complementary adhesion molecules that govern adhesion and migration (4
). Yet, the specific routes cells use to cross the lymphatic endothelium are at an early stage of understanding.
Evidence that lymphatic endothelial cells make junctional proteins comes from gene profiling data, which document the expression of platelet/endothelial cell adhesion molecule–1 (PECAM-1; also known as CD31), junctional adhesion molecule–A (JAM-A), and occludin in cultured cells (14
). Multiple adhesion molecules have also been reported at intercellular junctions in specialized “retothelial” cells of lymph node sinuses (19
Among the unresolved questions about the entry of fluid and cells into lymphatics are the following: (a) how can the integrity of initial lymphatics be maintained if junctions are not present between endothelial cells; (b) if junctions are present, how does fluid enter without repetitive disruption of the junctions; (c) do leukocytes and fluid enter at the same sites; and (d) what is the relation of the distinctive oak leaf shape of endothelial cells of initial lymphatics (9
) to sites of fluid and cell entry?
Based on this background, we sought to learn whether the properties of fluid and cell entry into initial lymphatics could be explained by the specialization of junctions between endothelial cells instead of the absence of junctions. We compared the distribution and composition of junctional proteins in initial lymphatics to those of conventional intercellular junctions in collecting lymphatics and blood vessels. Of particular interest were vascular endothelial cadherin (VE-cadherin) of adherens junctions, tight junction proteins, and the endothelial adhesion molecule PECAM-1. After learning that initial lymphatics had unusual, discontinuous endothelial junctions, we tested junctional integrity and plasticity after inhibition of VE-cadherin, deletion of PECAM-1, or increased fluid and cell flux in inflammation.
The studies exploited the attributes of the mouse tracheal mucosa, where both lymphatics and blood vessels are abundant, easily visualized, and readily compared under baseline conditions or after inflammatory stimuli in wild-type or genetically altered mice (21
). High resolution confocal microscopic imaging of three-dimensional whole mounts revealed discontinuous, button-like junctions (buttons) in the endothelium of initial lymphatics that contained proteins typical of both adherens junctions and tight junctions but were structurally unlike the zipper-like junctions (zippers) elsewhere. These findings suggest that regions between buttons in initial lymphatics are openings where fluid can enter without repetitive formation and dissolution of intercellular junctions.