The endothelial diapedesis synapse regulates transcellular migration of human T lymphocytes in a CX3CL1- and SNAP23-dependent manner.

Understanding how cytotoxic T lymphocytes (CTLs) efficiently leave the circulation to target cancer cells or contribute to inflammation is of high medical interest. Here, we demonstrate that human central memory CTLs cross the endothelium in a predominantly paracellular fashion, whereas effector and effector memory CTLs cross the endothelium preferably in a transcellular fashion. We find that effector CTLs show a round morphology upon adhesion and induce a synapse-like interaction with the endothelium where ICAM-1 is distributed at the periphery. Moreover, the interaction of ICAM-1:ß2integrin and endothelial-derived CX3CL1:CX3CR1 enables transcellular migration. Mechanistically, we find that ICAM-1 clustering recruits the SNARE-family protein SNAP23, as well as syntaxin-3 and -4, for the local release of endothelial-derived chemokines like CXCL1/8/10. In line, silencing of endothelial SNAP23 drives CTLs across the endothelium in a paracellular fashion. In conclusion, our data suggest that CTLs trigger local chemokine release from the endothelium through ICAM-1-driven signals driving transcellular migration.

Mechanistic analysis and significance of sphingomyelinase-mediated decreases in transepithelial CFTR currents in nHBEs.

Loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). In the lungs, this manifests as immune cell infiltration and bacterial infections, leading to tissue destruction. Previous work has determined that acute bacterial sphingomyelinase (SMase) decreases CFTR function in bronchial epithelial cells from individuals without CF (nHBEs) and with CF (cfHBEs, homozygous ΔF508-CFTR mutation). This study focuses on exploring the mechanisms underlying this effect. SMase increased the abundance of dihydroceramides, a result mimicked by blockade of ceramidase enzyme using ceranib-1, which also decreased CFTR function. The SMase-mediated inhibitory mechanism did not involve the reduction of cellular CFTR abundance or removal of CFTR from the apical surface, nor did it involve the activation of 5' adenosine monophosphate-activated protein kinase. In order to determine the pathological relevance of these sphingolipid imbalances, we evaluated the sphingolipid profiles of cfHBEs and cfHNEs (nasal) as compared to non-CF controls. Sphingomyelins, ceramides, and dihydroceramides were largely increased in CF cells. Correction of ΔF508-CFTR trafficking with VX445 + VX661 decreased some sphingomyelins and all ceramides, but exacerbated increases in dihydroceramides. Additional treatment with the CFTR potentiator VX770 did not affect these changes, suggesting rescue of misfolded CFTR was sufficient. We furthermore determined that cfHBEs express more acid-SMase protein than nHBEs. Lastly, we determined that airway-like neutrophils, which are increased in the CF lung, secrete acid-SMase. Identifying the mechanism of SMase-mediated inhibition of CFTR will be important, given the imbalance of sphingolipids in CF cells and the secretion of acid-SMase from cell types relevant to CF.

Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation.

The migration of neutrophils from the blood circulation to sites of infection or injury is a key immune response and requires the breaching of endothelial cells (ECs) that line the inner aspect of blood vessels. Unregulated neutrophil transendothelial cell migration (TEM) is pathogenic, but the molecular basis of its physiological termination remains unknown. Here, we demonstrated that ECs of venules in inflamed tissues exhibited a robust autophagic response that was aligned temporally with the peak of neutrophil trafficking and was strictly localized to EC contacts. Genetic ablation of EC autophagy led to excessive neutrophil TEM and uncontrolled leukocyte migration in murine inflammatory models, while pharmacological induction of autophagy suppressed neutrophil infiltration into tissues. Mechanistically, autophagy regulated the remodeling of EC junctions and expression of key EC adhesion molecules, facilitating their intracellular trafficking and degradation. Collectively, we have identified autophagy as a modulator of EC leukocyte trafficking machinery aimed at terminating physiological inflammation.

Platelet and Erythrocyte Extravasation across Inflamed Corneal Venules Depend on CD18, Neutrophils, and Mast Cell Degranulation.

Platelet extravasation during inflammation is under-appreciated. In wild-type (WT) mice, a central corneal epithelial abrasion initiates neutrophil (PMN) and platelet extravasation from peripheral limbal venules. The same injury in mice expressing low levels of the ß2-integrin, CD18 (CD18hypo mice) shows reduced platelet extravasation with PMN extravasation apparently unaffected. To better define the role of CD18 on platelet extravasation, we focused on two relevant cell types expressing CD18: PMNs and mast cells. Following corneal abrasion in WT mice, we observed not only extravasated PMNs and platelets but also extravasated erythrocytes (RBCs). Ultrastructural observations of engorged limbal venules showed platelets and RBCs passing through endothelial pores. In contrast, injured CD18hypo mice showed significantly less venule engorgement and markedly reduced platelet and RBC extravasation; mast cell degranulation was also reduced compared to WT mice. Corneal abrasion in mast cell-deficient (KitW-sh/W-sh) mice showed less venule engorgement, delayed PMN extravasation, reduced platelet and RBC extravasation and delayed wound healing compared to WT mice. Finally, antibody-induced depletion of circulating PMNs prior to corneal abrasion reduced mast cell degranulation, venule engorgement, and extravasation of PMNs, platelets, and RBCs. In summary, in the injured cornea, platelet and RBC extravasation depends on CD18, PMNs, and mast cell degranulation.

Trypanosoma cruzi Exploits E- and P-Selectins To Migrate Across Endothelial Cells and Extracellular Matrix Proteins.

The Chagas disease parasite Trypanosoma cruzi must extravasate to home in on susceptible cells residing in most tissues. It remains unknown how T. cruzi undertakes this crucial step of its life cycle. We hypothesized that the pathogen exploits the endothelial cell programming leukocytes use to extravasate to sites of inflammation. Transendothelial migration (TEM) starts after inflammatory cytokines induce E-selectin expression and P-selectin translocation on endothelial cells (ECs), enabling recognition by leukocyte ligands that engender rolling cell adhesion. Here, we show that T. cruzi upregulates E- and P-selectins in cardiac ECs to which it binds in a ligand-receptor fashion, whether under static or shear flow conditions. Glycoproteins isolated from T. cruzi (TcEx) specifically recognize P-selectin in a ligand-receptor interaction. As with leukocytes, binding of P-selectin to T. cruzi or TcEx requires sialic acid and tyrosine sulfate, which are pivotal for downstream migration across ECs and extracellular matrix proteins. Additionally, soluble selectins, which bind T. cruzi, block transendothelial migration dose dependently, implying that the pathogen bears selectin-binding ligand(s) that start transmigration. Furthermore, function-blocking antibodies against E- and P-selectins, which act on endothelial cells and not T. cruzi, are exquisite in preventing TEM. Thus, our results show that selectins can function as mediators of T. cruzi transendothelial transmigration, suggesting a pathogenic mechanism that allows homing in of the parasite on targeted tissues. As selectin inhibitors are sought-after therapeutic targets for autoimmune diseases and cancer metastasis, they may similarly represent a novel strategy for Chagas disease therapy.

Regulatory T cells reduce endothelial neutral sphingomyelinase 2 to prevent T-cell migration into tumors.

Endothelial cells are key regulators of transendothelial migration and their secretion of chemokines and expression of adhesion molecules facilitates lymphocyte entry into tissues. Previously, we demonstrated that Tregs can reduce transendothelial migration of T cells into tumors by decreasing endothelial CXCL10 secretion, but the mechanism by which this occurs is still not known. In this study, we aimed to define how Tregs decrease transendothelial migration into tumors. mRNA sequencing of intestinal tumor endothelial cells from Treg depleted mice identified neutral sphingomyelinase 2 (nSMase2) as a gene downregulated in the presence of Tregs. nSMase2 is expressed in human umbilical vein endothelial cells (HUVECs) and was decreased after coculture with Tregs. Furthermore, blocking of nSMase2 activity in vitro decreased VCAM1, CX3CL1, and CXCL10 expression in HUVECs, mirroring the same decrease found in Treg cocultures. In the APCmin/+ mouse model of intestinal cancer, nSMase2 is lower in tumor endothelial cells than in unaffected small intestine and chronic treatment with a nSMase2 inhibitor suppressed the increased migration that is otherwise seen in the absence of Tregs. We conclude that nSMase2 is an important mediator in endothelial cells supporting transendothelial migration, which may be targeted by Tregs to reduce T-cell migration into tumors.

Endothelial Focal Adhesions Are Functional Obstacles for Leukocytes During Basolateral Crawling.

An inflammatory response requires leukocytes to migrate from the circulation across the vascular lining into the tissue to clear the invading pathogen. Whereas a lot of attention is focused on how leukocytes make their way through the endothelial monolayer, it is less clear how leukocytes migrate underneath the endothelium before they enter the tissue. Upon finalization of the diapedesis step, leukocytes reside in the subendothelial space and encounter endothelial focal adhesions. Using TIRF microscopy, we show that neutrophils navigate around these focal adhesions. Neutrophils recognize focal adhesions as physical obstacles and deform to get around them. Increasing the number of focal adhesions by silencing the small GTPase RhoJ slows down basolateral crawling of neutrophils. However, apical crawling and diapedesis itself are not affected by RhoJ depletion. Increasing the number of focal adhesions drastically by expressing the Rac1 GEF Tiam1 make neutrophils to avoid migrating underneath these Tiam1-expressing endothelial cells. Together, our results show that focal adhesions mark the basolateral migration path of neutrophils.

Transmigration of Neutrophils From Patients With Familial Mediterranean Fever Causes Increased Cell Activation.

Familial Mediterranean fever (FMF) is caused by pyrin-encoding MEFV gene mutations and characterized by the self-limiting periods of intense inflammation, which are mainly mediated by a massive influx of polymorphonuclear neutrophils (PMNs) into the inflamed sites. Perturbation of actin polymerization by different pathogens was shown to activate the pyrin inflammasome. Our aim was to test whether cytoskeletal dynamics in the absence of pathogens may cause abnormal activation of PMNs from FMF patients. We also aimed to characterize immunophenotypes of circulating neutrophils and their functional activity. Circulating PMNs displayed heterogeneity in terms of cell size, granularity and immunophenotypes. Particularly, PMNs from the patients in acute flares (FMF-A) exhibited a characteristic of aged/activated cells (small cell size and granularity, up-regulated CXCR4), while PMNs form the patients in remission period (FMF-R) displayed mixed fresh/aged cell characteristics (normal cell size and granularity, up-regulated CD11b, CD49d, CXCR4, and CD62L). The findings may suggest that sterile tissue-infiltrated PMNs undergo reverse migration back to bone marrow and may explain why these PMNs do not cause immune-mediated tissue damage. A multidirectional expression of FcγRs on neutrophils during acute flares was also noteworthy: up-regulation of FcγRI and down-regulation of FcγRII/FcγRIII. We also observed spontaneous and fMPL-induced activation of PMNs from the patients after transmigration through inserts as seen by the increased expression of CD11b and intracellular expression of IL-1ß. Our study suggests heightened sensitivity of mutated pyrin inflammasome towards cytoskeletal modifications in the absence of pathogens.

Proteomic Analysis Revealed the Characteristics of Key Proteins Involved in the Regulation of Inflammatory Response, Leukocyte Transendothelial Migration, Phagocytosis, and Immune Process during Early Lung Blast Injury.

Previous studies found that blast injury caused a significant increased expression of interleukin-1, IL-6, and tumor necrosis factor, a significant decrease in the expression of IL-10, an increase in Evans blue leakage, and a significant increase in inflammatory cell infiltration in the lungs. However, the molecular characteristics of lung injury at different time points after blast exposure have not yet been reported. Therefore, in this study, tandem mass spectrometry (TMT) quantitative proteomics and bioinformatics analysis were used for the first time to gain a deeper understanding of the molecular mechanism of lung blast injury at different time points. Forty-eight male C57BL/6 mice were randomly divided into six groups: control, 12 h, 24 h, 48 h, 72 h, and 1 w after low-intensity blast exposure. TMT quantitative proteomics and bioinformatics analysis were performed to analyze protein expression profiling in the lungs from control and blast-exposed mice, and differential protein expression was verified by Western blotting. The results demonstrated that blast exposure induced severe lung injury, leukocyte infiltration, and the production of inflammatory factors in mice. After analyzing the expression changes in global proteins and inflammation-related proteomes after blast exposure, the results showed that a total of 6861 global proteins and 608 differentially expressed proteins were identified, of which 215, 128, 187, 232, and 65 proteins were identified at 12 h, 24 h, 48 h, 72 h, and 1 week after blast exposure, respectively. Moreover, blast exposure-induced 177 differentially expressed proteins were associated with inflammatory responses, which were enriched in the inflammatory response regulation, leukocyte transendothelial migration, phagocytosis, and immune response. Therefore, blast exposure may induce early inflammatory response of lung tissue by regulating the expression of key proteins in the inflammatory process, suggesting that early inflammatory response may be the initiating factor of lung blast injury. These data can provide potential therapeutic candidates or approaches for the development of future treatment of lung blast injury.

Uveal melanoma cells use ameboid and mesenchymal mechanisms of cell motility crossing the endothelium.

Uveal melanomas (UMs) are malignant cancers arising from the pigmented layers of the eye. UM cells spread through the bloodstream, and circulating UM cells are detectable in patients before metastases appear. Extravasation of UM cells is necessary for formation of metastases, and transendothelial migration (TEM) is a key step in extravasation. UM cells execute TEM via a stepwise process involving the actin-based processes of ameboid blebbing and mesenchymal lamellipodial protrusion. UM cancers are driven by oncogenic mutations that activate Gαq/11, and this activates TRIO, a guanine nucleotide exchange factor for RhoA and Rac1. We found that pharmacologic inhibition of Gαq/11 in UM cells reduced TEM. Inhibition of the RhoA pathway blocked amoeboid motility but led to enhanced TEM; in contrast, inhibition of the Rac1 pathway decreased mesenchymal motility and reduced TEM. Inhibition of Arp2/3 complex allowed cells to transmigrate without intercalation, a direct mechanism similar to the one often displayed by immune cells. BAP1-deficient (+/-) UM subclones displayed motility behavior and increased levels of TEM, similar to the effects of RhoA inhibitors. We conclude that RhoA and Rac1 signaling pathways, downstream of oncogenic Gαq/11, combine with pathways regulated by BAP1 to control the motility and transmigration of UM cells.

Reversal of Pathogen-Induced Barrier Defects in Intestinal Epithelial Cells by Contra-pathogenicity Agents.

BACKGROUND: Environmental enteropathy (EE) is associated with stunting, impairment of responses to oral vaccines, and other adverse health consequences in young children throughout the developing world. EE is characterized by chronic low-grade intestinal inflammation and disrupted epithelial barrier integrity, partly resulting from dysregulation of tight junction proteins, observed in other enteropathies such as celiac disease. During EE, this dysregulation of tight junction expression amplifies translocation of pathogenic bacteria across the intestinal mucosa. AIMS: The aim was to determine whether enteropathogen-mediated epithelial barrier failure can be ameliorated using contra-pathogenicity therapies. METHODS: Intestinal epithelial barrier damage was assessed in Caco-2 cells incubated with three important enteropathogens identified in EE patients: Enteropathogenic Escherichia coli (EPEC), Citrobacter rodentium (C. rodentium), and Cryptosporidium parvum (C. parvum). Potential therapeutic molecules were tested to detect effects on transepithelial resistance (TER), bacterial translocation (BT), claudin-4 expression, and regulation of the inflammatory cytokine response. RESULTS: All three enteropathogens compared to uninfected cells, reduced TER (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0007), reduced claudin-4 expression, and permitted BT (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0003) through the monolayer. Zinc, colostrum, epidermal growth factor, trefoil factor 3, resistin-like molecule-ß, hydrocortisone, and the myosin light chain kinase inhibitor ML7 (Hexahydro-1-[(5-iodo-1-naphthalenyl)sulfonyl]-1H-1,4-diazepine hydrochloride); ML7) improved TER (up to 70%) and decreased BT (as much as 96%). Only zinc demonstrated modest antimicrobial activity. CONCLUSION: The enteropathogens impaired intestinal-epithelial barrier integrity with dysregulation of claudin-4 and increased bacterial translocation. Enteropathogen-mediated damage was reduced using contra-pathogenicity agents which mitigated the effects of pathogens without direct antimicrobial activity.

High Endothelial Venules Accelerate Naive T Cell Recruitment by Tumor Necrosis Factor-Mediated R-Ras Upregulation.

Recruitment of naive T cells to lymph nodes is essential for the development of adaptive immunity. Upon pathogen infection, lymph nodes promptly increase the influx of naive T cells from the circulation in order to screen and prime the T cells. The precise contribution of the lymph node vasculature to the regulation of this process remains unclear. Here we show a role for the Ras GTPase, R-Ras, in the functional adaptation of high endothelial venules to increase naive T cell trafficking to the lymph nodes. R-Ras is transiently up-regulated in the endothelium of high endothelial venules by the inflammatory cytokine tumor necrosis factor (TNF) within 24 hours of pathogen inoculation. TNF induces R-Ras upregulation in endothelial cells via JNK and p38 mitogen-activated protein kinase but not NF-κB. Studies of T cell trafficking found that the loss of function of endothelial R-Ras impairs the rapid acceleration of naive T cell recruitment to the lymph nodes upon inflammation. This defect diminished the ability of naive OT-1 T cells to develop antitumor activity against ovalbumin-expressing melanoma. Proteomic analyses suggest that endothelial R-Ras facilitates TNF-dependent transendothelial migration (diapedesis) of naive T cells by modulating molecular assembly the at T cell-endothelial cell interface. These findings give new mechanistic insights into the functional adaptation of high endothelial venules to accelerate naive T cell recruitment to the lymph nodes.

Once upon a time, inflammation

Inflammation has accompanied humans since their first ancestors appeared on Earth. Aulus Cornelius Celsus (25 BC-50 AD), a Roman encyclopedist, offered a still valid statement about inflammation: "Notae vero inflammationis sunt quatuor: rubor et tumor cum calore and dolore", defining the four cardinal signs of inflammation as redness and swelling with heat and pain. While inflammation has long been considered as a morbid phenomenon, John Hunter (18th century) and Elie Metchnikoff (19th century) understood that it was a natural and beneficial event that aims to address a sterile or an infectious insult. Many other famous scientists and some forgotten ones have identified the different cellular and molecular players, and deciphered the different mechanisms of inflammation. This review pays tribute to some of the giants who made major contributions, from Hippocrates to the late 19th and first half of the 20th century. We particularly address the discoveries related to phagocytes, diapedesis, chemotactism, and fever. We also mention the findings of the various inflammatory mediators and the different approaches designed to treat inflammatory disorders.(AU)

Selective modulation of trans-endothelial migration of lymphocyte subsets in multiple sclerosis patients under fingolimod treatment.

Multiple sclerosis (MS) is an autoimmune disorder where auto-aggressive T cells target the central nervous system (CNS), causing demyelination. The trans-endothelial migration of leucocytes across the blood-brain barrier (BBB) is one of the earliest CNS events in MS pathogenesis. We examined the effect of the disease state and treatment with fingolimod on the transmigration of peripheral blood mononuclear cells (PBMCs) in an in vitro BBB model. Patients' leucocyte numbers, subsets and phenotypes were assessed by flow cytometry. As expected, fingolimod treatment induced a significant reduction in T cell and B cell numbers compared to untreated MS patients and healthy controls. Interestingly fingolimod led to a marked reduction of CD4+ and a significant increase in CD8+ cell numbers. In migrated cells, only CD3+ cell numbers were reduced in fingolimod-treated, compared to untreated patients; it had no effect on B cell or monocyte transmigration. T cells were then differentiated into naïve, effector and memory subsets based on their expression of CCR7. This showed that MS patients had increased numbers of effector memory CD4+ cells re-expressing CD45RA (TEMRA) and a decrease in central memory (CM) CD8+ cells. The former was corrected by fingolimod, while the latter was not. CM CD4+ and CD8+ cells migrated across BBB more efficiently in fingolimod-treated patients. We found that while fingolimod reduced the proportions of naïve CD19+ B cells, it significantly increased the proportions of these cells which migrated. When B cells were further stratified based on CD24, CD27 and CD38 expression, the only effect of fingolimod was an enhancement of CD24hiCD27+ B cell migration, compared to untreated MS patients. The migratory capacities of CD8hi Natural Killer (NK), CD8dim NK and NK-T cells were also reduced by fingolimod. While the disease-modifying effects of fingolimod are currently explained by its effect on reducing circulating auto-aggressive lymphocytes, our data suggests that fingolimod may also have a direct though differential effect on the trans-endothelial migration of circulating lymphocyte populations.

HIF-1α is involved in blood-brain barrier dysfunction and paracellular migration of bacteria in pneumococcal meningitis.

Bacterial meningitis is a deadly disease most commonly caused by Streptococcus pneumoniae, leading to severe neurological sequelae including cerebral edema, seizures, stroke, and mortality when untreated. Meningitis is initiated by the transfer of S. pneumoniae from blood to the brain across the blood-cerebrospinal fluid barrier or the blood-brain barrier (BBB). The underlying mechanisms are still poorly understood. Current treatment strategies include adjuvant dexamethasone for inflammation and cerebral edema, followed by antibiotics. The success of dexamethasone is however inconclusive, necessitating new therapies for controlling edema, the primary reason for neurological complications. Since we have previously shown a general activation of hypoxia inducible factor (HIF-1α) in bacterial infections, we hypothesized that HIF-1α, via induction of vascular endothelial growth factor (VEGF) is involved in transmigration of pathogens across the BBB. In human, murine meningitis brain samples, HIF-1α activation was observed by immunohistochemistry. S. pneumoniae infection in brain endothelial cells (EC) resulted in in vitro upregulation of HIF-1α/VEGF (Western blotting/qRT-PCR) associated with increased paracellular permeability (fluorometry, impedance measurements). This was supported by bacterial localization at cell-cell junctions in vitro and in vivo in brain ECs from mouse and humans (confocal, super-resolution, electron microscopy, live-cell imaging). Hematogenously infected mice showed increased permeability, S. pneumoniae deposition in the brain, along with upregulation of genes in the HIF-1α/VEGF pathway (RNA sequencing of brain microvessels). Inhibition of HIF-1α with echinomycin, siRNA in bEnd5 cells or using primary brain ECs from HIF-1α knock-out mice revealed reduced endothelial permeability and transmigration of S. pneumoniae. Therapeutic rescue using the HIF-1α inhibitor echinomycin resulted in increased survival and improvement of BBB function in S. pneumoniae-infected mice. We thus demonstrate paracellular migration of bacteria across BBB and a critical role for HIF-1α/VEGF therein and hence propose targeting this pathway to prevent BBB dysfunction and ensuing brain damage in infections.

Effects of Benzalkonium Chloride and Preservative-Free Composition on the Corneal Epithelium Cells.

Purpose: Benzalkonium Chloride (BAK) is reported to have the potential to damage the cornea. We developed a composition with broad-spectrum antimicrobial activity without preservatives by combining trometamol, boric acid, and ethylenediaminetetraacetic acid (TBE). This study aimed at evaluating the corneal damage caused by TBE and comparing it with that caused by BAK. Methods: SV40-immortalized human corneal epithelial cell line (HCE-T) was treated with BAK or TBE, and the cell viability was measured. The exposure time that caused 50% cell death (CDT50) was calculated. Transepithelial electrical resistance (TEER) was measured before and after treatment with BAK or TBE. Occludin was detected with immunostaining and Western blotting after treatment with BAK or TBE. The effect of BAK or TBE on membrane-associated mucins was evaluated with rose bengal (RB) staining. Results: In the BAK group, cell viability decreased in a dose-dependent manner. The viability of the TBE group was significantly greater than that of the BAK group. The CDT50 of the TBE group is greater than that of the BAK groups. In the BAK groups, the recovery of TEER was delayed in a dose-dependent manner, whereas in the TBE group, the recovery occurred earlier. Localization of occludin was disrupted, and the amount of occludin was significantly reduced among the cells exposed to BAK. The area stained with RB in the BAK groups increased, whereas that in the TBE group did not increase. Conclusion: These results suggest that the application of TBE would be useful for developing preservative-free ophthalmic preparations that offer both sufficient safety and antimicrobial activity.

Platelets docking to VWF prevent leaks during leukocyte extravasation by stimulating Tie-2.

Neutrophil extravasation requires opening of the endothelial barrier but does not necessarily cause plasma leakage. Leaks are prevented by contractile actin filaments surrounding the diapedesis pore, keeping this opening tightly closed around the transmigrating neutrophils. We have identified the receptor system that is responsible for this. We show that silencing, or gene inactivation, of endothelial Tie-2 results in leak formation in postcapillary venules of the inflamed cremaster muscle at sites of neutrophil extravasation, as visualized by fluorescent microspheres. Leakage was dependent on neutrophil extravasation, because it was absent upon neutrophil depletion. We identified the Cdc42 GTPase exchange factor FGD5 as a downstream target of Tie-2 that is essential for leakage prevention during neutrophil extravasation. Looking for the Tie-2 agonist and its source, we found that platelet-derived angiopoietin-1 (Angpt1) was required to prevent neutrophil-induced leaks. Intriguingly, blocking von Willebrand factor (VWF) resulted in vascular leaks during transmigration, indicating that platelets interacting with endothelial VWF activate Tie-2 by secreting Angpt1, thereby preventing diapedesis-induced leakiness.

Cleaved endocan acts as a biologic competitor of endocan in the control of ICAM-1-dependent leukocyte diapedesis.

Dysregulated leukocyte diapedesis is a major contributor to acute severe inflammatory states like sepsis and acute respiratory distress syndrome, which are common conditions in critically ill subjects. Endocan is a circulating proteoglycan that binds to the leukocyte integrin LFA-1 and blocks its interaction with its endothelial ligand ICAM-1, subsequently leading to the inhibition of leukocyte recruitment. Recent data have highlighted the hypothetic role of p14, endocan's major catabolite found in the bloodstream of septic patients, as a potential antagonist of endocan, thus participating in the regulation of acute inflammation. We hereby characterize the role of p14 as a biologic competitor of endocan, through assessment of its molecular interactions with LFA-1, endocan, and ICAM-1, as well as its effects on human leukocyte trafficking. Using immunodetection assay, we report that p14 can bind to LFA-1, thus inhibiting the interaction between LFA-1 and endocan, which in turn leads to the restoration of the ICAM-1/LFA-1 interaction. In primary human T cells trafficking assays, we underline the absence of effect of p14 on ICAM-1-dependent adhesion and migration, as well as on transendothelial migration. However, in those models, p14 reverses the antimigratory effect of endocan. To conclude, our study supports the hypothesis of an antagonistic role of p14 versus endocan in its effect on the LFA-1/ICAM-1-dependent human leukocyte recruitment.

FURIN Expression in Vascular Endothelial Cells Is Modulated by a Coronary Artery Disease-Associated Genetic Variant and Influences Monocyte Transendothelial Migration.

Background Genome-wide association studies have shown an association between the single-nucleotide polymorphism rs17514846 on chromosome 15q26.1 and coronary artery disease susceptibility. The underlying biological mechanism is, however, not fully understood. rs17514846 is located in the FES Upstream Region (FURIN) gene, which is expressed in vascular endothelial cells (ECs). We investigated whether rs17514846 has an influence on FURIN expression in ECs and whether FURIN affects EC behavior. Methods and Results Quantitative reverse transcription-polymerase chain reaction analysis showed that cultured vascular ECs from individuals carrying the coronary artery disease risk allele of rs17514846 had higher FURIN expression than cells from noncarriers. In support, luciferase reporter analyses in ECs indicated that the risk allele had higher transcriptional activity than the nonrisk allele. Electrophoretic mobility shift assays using EC nuclear protein extracts detected a DNA-protein complex with allele-specific differential binding of a nuclear protein. Knockdown of FURIN in ECs reduced endothelin-1 secretion, nuclear factor-κB activity, vascular cell adhesion molecule-1, and MCP1 (monocyte chemotactic protein-1) expression and monocyte-endothelial adhesion and transmigration. A population-based study showed an association of the rs17514846 risk allele with higher circulating MCP1 levels and greater carotid intima-media thickness. Conclusions The coronary artery disease risk variant at the 15q26.1 locus modulates FURIN expression in vascular ECs. FURIN levels in ECs affect monocyte-endothelial adhesion and migration.

Actin-Binding Protein Cortactin Promotes Pathogenesis of Experimental Autoimmune Encephalomyelitis by Supporting Leukocyte Infiltration into the Central Nervous System.

Leukocyte entry into the central nervous system (CNS) is essential for immune surveillance but is also the basis for the development of pathologic inflammatory conditions within the CNS, such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). The actin-binding protein, cortactin, in endothelial cells is an important player in regulating the interaction of immune cells with the vascular endothelium. Cortactin has been shown to control the integrity of the endothelial barrier and to support neutrophil transendothelial migration in vitro and in vivo in the skin. Here we use cortactin gene-inactivated male and female mice to study the role of this protein in EAE. Inducing EAE by immunization with a myelin oligodendrocyte glycoprotein peptide (MOG35-55) revealed an ameliorated disease course in cortactin gene-deficient female mice compared with WT mice. However, proliferation capacity and expression of IL-17A and IFNγ by cortactin-deficient and WT splenocytes did not differ, suggesting that the lack of cortactin does not affect induction of the immune response. Rather, cortactin deficiency caused decreased vascular permeability and reduced leukocyte infiltration into the brains and spinal cords of EAE mice. Accordingly, cortactin gene-deficient mice had smaller numbers of proinflammatory cuffs, less extensive demyelination, and reduced expression levels of proinflammatory cytokines within the neural tissue compared with WT littermates. Thus, cortactin contributes to the development of neural inflammation by supporting leukocyte transmigration through the blood-brain barrier and, therefore, represents a potential candidate for targeting CNS autoimmunity.SIGNIFICANCE STATEMENT Multiple sclerosis is an autoimmune neuroinflammatory disorder, based on the entry of inflammatory leukocytes into the CNS where these cells cause demyelination and neurodegeneration. Here, we use a mouse model for multiple sclerosis, experimental autoimmune encephalomyelitis, and show that gene inactivation of cortactin, an actin binding protein that modulates actin dynamics and branching, protects against neuroinflammation in experimental autoimmune encephalomyelitis. Leukocyte infiltration into the CNS was inhibited in cortactin-deficient mice, and lack of cortactin in cultured primary brain endothelial cells inhibited leukocyte transmigration. Expression levels of proinflammatory cytokines in the CNS and induction of vascular permeability were reduced. We conclude that cortactin represents a novel potential target for the treatment of multiple sclerosis.