Leukocytes Breach Endothelial Barriers by Insertion of Nuclear Lobes and Disassembly of Endothelial Actin Filaments.

The endothelial cytoskeleton is a barrier for leukocyte transendothelial migration (TEM). Mononuclear and polymorphonuclear leukocytes generate gaps of similar micron-scale size when squeezing through inflamed endothelial barriers in vitro and in vivo. To elucidate how leukocytes squeeze through these barriers, we co-tracked the endothelial actin filaments and leukocyte nuclei in real time. Nuclear squeezing involved either preexistent or de novo-generated lobes inserted into the leukocyte lamellipodia. Leukocyte nuclei reversibly bent the endothelial actin stress fibers. Surprisingly, formation of both paracellular gaps and transcellular pores by squeezing leukocytes did not require Rho kinase or myosin II-mediated endothelial contractility. Electron-microscopic analysis suggested that nuclear squeezing displaced without condensing the endothelial actin filaments. Blocking endothelial actin turnover abolished leukocyte nuclear squeezing, whereas increasing actin filament density did not. We propose that leukocyte nuclei must disassemble the thin endothelial actin filaments interlaced between endothelial stress fibers in order to complete TEM.

M-sec regulates polarized secretion of inflammatory endothelial chemokines and facilitates CCL2-mediated lymphocyte transendothelial migration.

Activation of endothelial cells by IL-1ß triggers the expression of multiple inflammatory cytokines and leukocyte-attracting chemokines. The machineries involved in the secretion of these inducible proteins are poorly understood. With the use of genome-wide transcriptional analysis of inflamed human dermal microvascular endothelial cells, we identified several IL-1ß-induced candidate regulators of these machineries and chose to focus our study on TNF-α-induced protein 2 (myeloid-secretory). The silencing of myeloid-secretory did not affect the ability of inflamed endothelial cells to support the adhesion and crawling of effector T lymphocytes. However, the ability of these lymphocytes to complete transendothelial migration across myeloid-secretory-silenced human dermal microvascular endothelial cells was inhibited significantly. These observed effects on lymphocyte transendothelial migration were recovered completely when exogenous promigratory chemokine CXCL12 was overlaid on the endothelial barrier. A polarized secretion assay suggested that the silencing of endothelial myeloid-secretory impairs T effector transendothelial migration by reducing the preferential secretion of endothelial-produced CCL2, a key transendothelial migration-promoting chemokine for these lymphocytes, into the basolateral endothelial compartment. Myeloid-secretory silencing also impaired the preferential secretion of other endothelial-produced inflammatory chemokines, as well as cytokines, such as IL-6 and GM-CSF, into the basolateral endothelial compartment. This is the first evidence of a novel inflammation-inducible machinery that regulates polarized secretion of endothelial CCL2 and other inflammatory chemokines and cytokines into basolateral endothelial compartments and facilitates the ability of endothelial CCL2 to promote T cell transendothelial migration.

Transendothelial migration of effector T cells across inflamed endothelial barriers does not require heparan sulfate proteoglycans.

Leukocyte diapedesis is a chemotactic multistep process that requires optimal chemoattractant presentation by the endothelial barrier. Recent studies have described a critical role for heparan sulfate glycosaminoglycans (HSGAGs) in the presentation and functions of chemokines essential for lymphocyte interactions with the lymph node vasculature. We wished to test whether HS expression by a prototypic endothelial cell type, i.e. human umbilical vein endothelial cells (HUVECs), is critical for their ability to support neutrophil and lymphocyte adhesion and transendothelial migration (TEM) under shear flow. We found that HUVECs deposit HS GAGs mainly at their basolateral compartments in both their resting and inflamed states. We next inactivated the key enzyme involved in HS biosynthesis, exostosin-1 (Ext1). Silencing Ext1 resulted in a complete loss of HS biosynthesis; nonetheless, TNF-α and IL-1ß stimulation of key adhesion molecules and inflammatory chemokines necessary for neutrophil or lymphocyte adhesion and TEM remained intact. Ext1 silencing reduced neutrophil arrest and markedly impaired TEM, consistent with a role of basolateral HS GAGs in directing neutrophil crossing of inflamed endothelial barriers. Strikingly, however, the TEM of effector T cells across identically Ext1-silenced HUVECs remained normal. Importantly, the biosynthesis of the main promigratory chemokines for effector T cells and neutrophils, respectively, CCL2 and CXCL1, and their vesicle distributions were also Ext1 independent. These results suggest that transmigrating neutrophils must respond to chemokines transiently presented by apical and basolateral endothelial HS GAGs. In contrast, effector T cells can integrate chemotactic TEM signals directly from intra-endothelial chemokine stores rather than from externally deposited chemokines.