Paracellular and Transcellular Leukocytes Diapedesis Are Divergent but Interconnected Evolutionary Events.

Infiltration of the endothelial layer of the blood-brain barrier by leukocytes plays a critical role in health and disease. When passing through the endothelial layer during the diapedesis process lymphocytes can either follow a paracellular route or a transcellular one. There is a debate whether these two processes constitute one mechanism, or they form two evolutionary distinct migration pathways. We used artificial intelligence, phylogenetic analysis, HH search, ancestor sequence reconstruction to investigate further this intriguing question. We found that the two systems share several ancient components, such as RhoA protein that plays a critical role in controlling actin movement in both mechanisms. However, some of the key components differ between these two transmigration processes. CAV1 genes emerged during Trichoplax adhaerens, and it was only reported in transcellular process. Paracellular process is dependent on PECAM1. PECAM1 emerged from FASL5 during Zebrafish divergence. Lastly, both systems employ late divergent genes such as ICAM1 and VECAM1. Taken together, our results suggest that these two systems constitute two different mechanical sensing mechanisms of immune cell infiltrations of the brain, yet these two systems are connected. We postulate that the mechanical properties of the cellular polarity is the main driving force determining the migration pathway. Our analysis indicates that both systems coevolved with immune cells, evolving to a higher level of complexity in association with the evolution of the immune system.

LncRNA MEG3-210 regulates endometrial stromal cells migration, invasion and apoptosis through p38 MAPK and PKA/SERCA2 signalling via interaction with Galectin-1 in endometriosis.

BACKGROUND: Endometriosis is a benign gynaecological disease with malignant characteristics that severely affects women's quality of life. Long noncoding RNA maternally expressed gene 3 (LncRNA MEG3) is a tumour suppressor that is downregulated in various cancer cells and tissues, and regulates multiple biological processes. Emerging studies have revealed that the interactions between MEG3 and proteins are involved in disease progression. Galectin-1 affects cell motility, signal transduction and vascularization, and is overexpressed in endometriosis. Our study is the first to explore the role of MEG3-210 transcript in endometriosis and to reveal the regulatory mechanism mediated by the interaction between MEG3-210 and Galectin-1. MATERIALS AND METHODS: Endometrial tissues and sera from patients with endometriosis and controls were collected. qRT-PCR was performed to detect the expression of MEG3-210 in the endometrium and endometrial stromal cells (ESCs). The CCK-8 assay, the Transwell assay, flow cytometry and animal models were conducted to evaluate the functions of MEG3-210 in vitro and in vivo. Bioinformatic analysis, Western blot assays, RNA-pull down assays and RNA immunoprecipitation were used to explore the potential mechanism of MEG3-210 in endometriosis. RESULTS: Our results showed that MEG3-210 expression was lower in the eutopic endometrium of women with endometriosis. MEG3-210 downregulation promoted ESCs migration, invasion, anti-apoptosis in vitro and growth of endometriotic lesions in vivo. Furthermore, MEG3-210 downregulation could activate p38 mitogen-activated protein kinase (p38 MAPK) and inhibit cAMP-dependent protein kinase A/sarcoplasmic reticulum Ca2+ ATPase 2 (PKA/SERCA2) signalling, which was mediated by Galectin-1. The protein levels of Galectin-1 in patients with endometriosis were elevated, and Galectin-1 siRNA could reduce the size of lesions. CONCLUSION: MEG3-210 regulates ESCs through p38 MAPK and PKA/SERCA signalling via interaction with Galectin-1. The novel regulatory mechanism may provide new insights into drug therapy and the diagnosis of endometriosis.

Review of functional in vitro models of the blood-cerebrospinal fluid barrier in leukaemia research.

Acute lymphoblastic leukaemia represents the most common paediatric malignancy. Although survival rates approach up to 90% in children, investigation of leukaemic infiltration into the central nervous system (CNS) is essential due to the presence of ongoing fatal complications. Recent in vitro studies mostly employed models of the blood-brain barrier (BBB), as endothelial cells of the microvasculature represent the largest surface between the blood stream and the brain parenchyma. However, crossing the blood-cerebrospinal fluid barrier (BCSFB) within the choroid plexus (CP) has been shown to be a general capability of leukaemic blasts. Hence, in vitro models of the BCSFB to study leukaemic transmigration may be of major importance to understand the development of CNS leukaemia. This review will summarise available in vitro models of the BCSFB employed to study the cellular interactions with leukaemic blasts during cancer cell transmigration into the brain compartment across primary or immortal/immortalised BCSFB cells. It will also provide an outlook on prospective improvements in BCSFB in vitro models by developing barrier-on-a-chip models and brain organoids.

Novel inhibitors of leukocyte transendothelial migration.

Leukocyte transendothelial migration is one of the most important step in launching an inflammatory immune response and chronic inflammation can lead to devastating diseases. Leukocyte migration inhibitors are considered as promising and potentially effective therapeutic agents to treat inflammatory and auto-immune disorders. In this study, based on previous trioxotetrahydropyrimidin based integrin inhibitors that suboptimally blocked leukocyte adhesion, twelve molecules with a modified scaffold were designed, synthesized, and tested in vitro for their capacity to block the transendothelial migration of immune cells. One of the molecules, namely, methyl 4-((2-(tert-butyl)-6-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene) methyl) phenoxy) methyl) benzoate, (compound 12), completely blocked leukocyte transendothelial migration, without any toxic effects on immune or endothelial cells (IC50 = 2.4 µM). In vivo, compound 12 exhibited significant therapeutic effects in inflammatory bowel disease (IBD)/Crohn's disease, multiple sclerosis, fatty liver disease, and rheumatoid arthritis models. A detailed acute and chronic toxicity profile of the lead compound in vivo did not reveal any toxic effects. Such a type of molecule might therefore provide a unique starting point for designing a novel class of leukocyte transmigration blocking agents with broad therapeutic applications in inflammatory and auto-immune pathologies.

Semaphorin 3F Promotes Transendothelial Migration of Leukocytes in the Inflammatory Response After Survived Cardiac Arrest.

Leukocyte transmigration through the blood vessel wall is a fundamental step of the inflammatory response and requires expression of adhesion molecule PECAM-1. Accumulating evidence implicates that semaphorin (Sema) 3F and its receptor neuropilin (NRP) 2 are central regulators in vascular biology. Herein, we assess the role of Sema3F in leukocyte migration in vitro and in vivo. To determine the impact of Sema3F on leukocyte recruitment in vivo, we used the thioglycollate-induced peritonitis model. After the induction of peritonitis, C57BL/6 mice were intraperitoneally (i.p.) injected daily with recombinant Sema3F or solvent for 3 days. Compared with solvent-treated controls, leukocyte count was increased in the peritoneal lavage of Sema3F-treated mice indicating that Sema3F promotes leukocyte extravasation into the peritoneal cavity. In line with this observation, stimulation of human endothelial cells with Sema3F enhanced the passage of peripheral blood mononuclear cells (PBMCs) through the endothelial monolayer in the transwell migration assays. Conversely, silencing of endothelial Sema3F by siRNA transfection dampened diapedesis of PBMCs through the endothelium in vitro. xMechanistically, Sema3F induced upregulation of adhesion molecule PECAM-1 in endothelial cells and in murine heart tissue shown by immunofluorescence and western blotting. The inhibition of PECAM-1 by blocking antibody HEC7 blunted Sema3F-induced leukocyte migration in transwell assays. SiRNA-based NRP2 knockdown reduced PECAM-1 expression and migration of PBMCs in Sema3F-treated endothelial cells, indicating that PECAM-1 expression and leukocyte migration in response to Sema3F depend on endothelial NRP2. To assess the regulation of Sema3F in human inflammatory disease, we collected serum samples of patients from day 0 to day 7 after survived out-of-hospital cardiac arrest (OHCA, n = 41). First, we demonstrated enhanced migration of PBMCs through endothelial cells exposed to the serum of patients after OHCA in comparison to the serum of patients with stable coronary artery disease or healthy volunteers. Remarkably, serum samples of OHCA patients contained significantly higher Sema3F protein levels compared with CAD patients (CAD, n = 37) and healthy volunteers (n = 11), suggesting a role of Sema3F in the pathophysiology of the inflammatory response after OHCA. Subgroup analysis revealed that elevated serum Sema3F levels after ROSC are associated with decreased survival, myocardial dysfunction, and prolonged vasopressor therapy, clinical findings that determine the outcome of post-resuscitation period after OHCA. The present study provides novel evidence that endothelial Sema3F controls leukocyte recruitment through a NRP2/PECAM-1-dependent mechanism. Sema3F serum concentrations are elevated following successful resuscitation suggesting that Sema3F might be involved in the inflammatory response after survived OHCA. Targeting the Sema3F/NRP2/PECAM-1 pathway could provide a novel approach to abolish overwhelming inflammation after resuscitation.

MiR-92a Inhibits the Progress of Osteosarcoma Cells and Increases the Cisplatin Sensitivity by Targeting Notch1.

BACKGROUND: MicroRNAs (miRs) have been implicated in the development and progression of osteosarcoma. Here, we aimed to illustrate the important role of miR-92a on the regulation of OS development which may help to establish a novel strategy for OS diagnosis and treatment. MATERIALS AND METHODS: Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle and apoptosis were assessed by flow cytometry with PI and PI/Annexin-V stain, respectively. The expression of proteins was examined by western blot. qPCR was used to detect the expression of RNA. Cell migration was assayed with transwell assay. RESULTS: MiR-92a inhibited the proliferation and the migration of OS in vitro and reduced the volume of the tumour in vivo. Further, miR-92a enhanced cisplatin sensitivity of OS. MiR-92a directly targeted Notch1. CONCLUSION: Together, our results indicate that miR-92a inhibited cell growth, migration, and enhanced cisplatin sensitivity of OS cell by targeting Notch1.

Is Transcellular Potassium Shifting With Insulin, Albuterol, or Sodium Bicarbonate in Emergency Department Patients With Hyperkalemia Associated With Recurrent Hyperkalemia After Dialysis?

BACKGROUND: Emergency department (ED) treatment of hyperkalemia often involves shifting potassium into the intracellular space. There is uncertainty whether transcellular shifting causes insufficient potassium removal during hemodialysis, resulting in a subsequent need for further medical therapy or multiple sessions of hemodialysis. OBJECTIVE: We sought to determine whether transcellular potassium shifting in ED patients with hyperkalemia who undergo hemodialysis is associated with recurrent hyperkalemia with or without repeat hemodialysis within 24 h. METHODS: This was a retrospective observational study of ED patients with a potassium value > 5.3 mmol/L and ≥1 hemodialysis run. Transcellular shifting medications were defined as albuterol, insulin, and sodium bicarbonate. Primary outcomes were recurrent hyperkalemia with and without repeat hemodialysis within 24 h of the initial dialysis run. Generalized estimating equation models were created for the outcomes using administration of a shifting medication as the primary predictor. RESULTS: Four hundred seventy-nine encounters were identified. In 238 (50%) encounters, a shifting medication was administered. There were 85 outcomes of recurrent hyperkalemia and 36 outcomes of recurrent hyperkalemia with repeat hemodialysis. After adjustment, administration of shifting medications was not associated with recurrent hyperkalemia (adjusted odds ratio 1.26, 95% confidence interval 0.71-2.23) or recurrent hyperkalemia with repeat dialysis (adjusted odds ratio 1.90, 95% confidence interval 0.80-4.48). CONCLUSIONS: Administration of transcellular shifting medications for hyperkalemia in the ED was not associated with either recurrent hyperkalemia after hemodialysis or the need for a second dialysis session within 24 h. Our findings address the uncertainty regarding transcellular potassium shifting before emergent dialysis and support safe ED administration of medications that shift potassium to the intracellular space.

Astrocyte-shed extracellular vesicles regulate the peripheral leukocyte response to inflammatory brain lesions.

Brain injury induces a peripheral acute cytokine response that directs the transmigration of leukocytes into the brain. Because this brain-to-peripheral immune communication affects patient recovery, understanding its regulation is important. Using a mouse model of inflammatory brain injury, we set out to find a soluble mediator for this phenomenon. We found that extracellular vesicles (EVs) shed from astrocytes in response to intracerebral injection of interleukin-1ß (IL-1ß) rapidly entered into peripheral circulation and promoted the transmigration of leukocytes through modulation of the peripheral acute cytokine response. Bioinformatic analysis of the protein and microRNA cargo of EVs identified peroxisome proliferator-activated receptor α (PPARα) as a primary molecular target of astrocyte-shed EVs. We confirmed in mice that astrocytic EVs promoted the transmigration of leukocytes into the brain by inhibiting PPARα, resulting in the increase of nuclear factor κB (NF-κB) activity that triggered the production of cytokines in liver. These findings expand our understanding of the mechanisms regulating communication between the brain and peripheral immune system and identify astrocytic EVs as a molecular regulator of the immunological response to inflammatory brain damage.

Postarrest stalling rather than crawling favors CD8(+) over CD4(+) T-cell migration across the blood-brain barrier under flow in vitro.

Although CD8(+) T cells have been implied in the pathogenesis of multiple sclerosis (MS), the molecular mechanisms mediating CD8(+) T-cell migration across the blood-brain barrier (BBB) into the central nervous system (CNS) are ill defined. Using in vitro live cell imaging, we directly compared the multistep extravasation of activated CD4(+) and CD8(+) T cells across primary mouse brain microvascular endothelial cells (pMBMECs) as a model for the BBB under physiological flow. Significantly higher numbers of CD8(+) than CD4(+) T cells arrested on pMBMECs under noninflammatory and inflammatory conditions. While CD4(+) T cells polarized and crawled prior to their diapedesis, the majority of CD8(+) T cells stalled and readily crossed the pMBMEC monolayer preferentially via a transcellular route. T-cell arrest and crawling were independent of G-protein-coupled receptor signaling. Rather, absence of endothelial ICAM-1 and ICAM-2 abolished increased arrest of CD8(+) over CD4(+) T cells and abrogated T-cell crawling, leading to the efficient reduction of CD4(+) , but to a lesser degree of CD8(+) , T-cell diapedesis across ICAM-1(null) /ICAM-2(-/-) pMBMECs. Thus, cellular and molecular mechanisms mediating the multistep extravasation of activated CD8(+) T cells across the BBB are distinguishable from those involved for CD4(+) T cells.

Giardia duodenalis induces paracellular bacterial translocation and causes postinfectious visceral hypersensitivity.

Irritable bowel syndrome (IBS) is the most frequent functional gastrointestinal disorder. It is characterized by abdominal hypersensitivity, leading to discomfort and pain, as well as altered bowel habits. While it is common for IBS to develop following the resolution of infectious gastroenteritis [then termed postinfectious IBS (PI-IBS)], the mechanisms remain incompletely understood. Giardia duodenalis is a cosmopolitan water-borne enteropathogen that causes intestinal malabsorption, diarrhea, and postinfectious complications. Cause-and-effect studies using a human enteropathogen to help investigate the mechanisms of PI-IBS are sorely lacking. In an attempt to establish causality between giardiasis and postinfectious visceral hypersensitivity, this study describes a new model of PI-IBS in neonatal rats infected with G. duodenalis At 50 days postinfection with G. duodenalis (assemblage A or B), long after the parasite was cleared, rats developed visceral hypersensitivity to luminal balloon distension in the jejunum and rectum, activation of the nociceptive signaling pathway (increased c-fos expression), histological modifications (villus atrophy and crypt hyperplasia), and proliferation of mucosal intraepithelial lymphocytes and mast cells in the jejunum, but not in the rectum. G. duodenalis infection also disrupted the intestinal barrier, in vivo and in vitro, which in turn promoted the translocation of commensal bacteria. Giardia-induced bacterial paracellular translocation in vitro correlated with degradation of the tight junction proteins occludin and claudin-4. The extensive observations associated with gut hypersensitivity described here demonstrate that, indeed, in this new model of postgiardiasis IBS, alterations to the gut mucosa and c-fos are consistent with those associated with PI-IBS and, hence, offer avenues for new mechanistic research in the field.

[The influence of urothelial carcinoembryonic antigen 1 on invasion and migration of oral carcinoma cell lines].

OBJECTIVE: To investigate the effects of long chain non-coding RNA urothelial carcinoembryonic antigen 1(UCA1) on invasion, migration and proliferation abilities in oral squamous cell carcinoma cell lines SCC15 and CAL27. METHODS: Small interfering RNA of UCA1(UCA1-siRNA) was transfected into SCC15 and CAL27 cell lines by Lipofectamine(TM) 3000 to silence UCA1 , and transfected negtive control si-RNA served as a control. The effect of UCA1-siRNA was detected by quantitative real time-polymerase chain reaction(qRT-PCR) to confirm the successful inhibition of UCA1 by siRNA. The matrix metalloproteinase 9(MMP-9) protein level was detected by Western blotting analysis. The effect of siRNA on cell proliferation and invasion was assessed by transwell migration assay and wound healing assay. Cell counting kit-8(CCK-8) assay was carried out to estimate the proliferation of two cell lines with different expression levels of UCA1. RESULTS: Expressions of UCA1 of SCC15 and CAL27 were successfully suppressed after transfected with siRNA which verified by qRT-PCR, and the efficiency of downregulation of SCC15 and CAL27 was 86.45%(P<0.001)and 78.24%(P<0.001), respectively. The migration, invasion and proliferation of SCC15 and CAL27 cell lines after transfected with siRNA were obviously restrained. The number of migration and invasion of CAL27 cells were 719.20±92.36 versus 208.00±25.58 (P=0.000 7) and 363.40 ± 45.96 versus 164.80 ± 24.68(P= 0.005 2), respectively, the number of migration and invasion of SCC15 cells were 437.20±54.75 vs 145.80±23.31(P=0.001 1) and 249.80±38.41 vs 63.80±11.11 (P=0.001 6), respectively (UCA1-si compare to negtive control), the relative proliferation rates of SCC15 and CAL27 were SCC15: R24 h=0.870, R48 h=0.863, R72 h=0.64, R96 h=0.732; CAL27: R24 h=0.913, R48 h=0.829, R72 h=0.756, R96 h= 0.705(P<0.05), and MMP-9 expression level was decreased by UCA1-siRNA compared with negative control. CONCLUSIONS: UCA1 could enhance the ability of invasion and migration of SCC15 and CAL27 cell lines via regulating MMP-9 protein expression, which suggests that UCA1 might play a pivotal role in oral squamous cell carcinoma invasion and progression.

Temporal studies into attachment, VE-cadherin perturbation, and paracellular migration of human umbilical mesenchymal stem cells across umbilical vein endothelial monolayers.

Mesenchymal stem cells from Wharton's jelly of human umbilical cords (WJ-MSC) are a valuable alternate source of stem cells. Their role in situ and whether they can interact and cross intact endothelial monolayers requires elucidation. The aim of this study was to investigate the dynamic interactions between WJ-MSC and human umbilical vein endothelial cells (HUVEC), including attachment, transit times, extravasation pathway, and the effects of WJ-MSC on junctional vascular endothelial (VE)-cadherin. HUVEC were grown to near confluence in endothelial media and to full confluence in mixed media before the addition of PKH26-labelled WJ-MSC. Time lapse fluorescence microscopy showed stem cells undergoing membrane blebbing followed by amoeboid movement on HUVEC monolayers before rounding up and changing shape toward the spindle-shaped morphology during/after transmigration to subendothelial positions. Cells demonstrated a time lag of 60 min before paracellular extravasation, confirmed by confocal microscopy. Forty-six percent of attached cells crossed in the first 2 h. By 16 h, a majority of cells had transmigrated with >96% of cells crossing by 22 h. There were concomitant changes in endothelial junctional VE-cadherin with statistically significant increases in discontinuous staining at 2 h, return to control values at 16 h, even as from 22 h onward HUVEC displayed increased percentage of junctions with continuous staining and upregulation of protein. Our data suggests that WJ-MSC crosses the endothelial barrier through the paracellular pathway and can influence junctional organization of HUVEC with discreet perturbation of VE-cadherin preceding transmigration followed by upregulation once the adluminal side is reached. The latter may reflect a perivascular support function of WJ-MSC in the umbilical cord.

Invadopodia are required for cancer cell extravasation and are a therapeutic target for metastasis.

Tumor cell extravasation is a key step during cancer metastasis, yet the precise mechanisms that regulate this dynamic process are unclear. We utilized a high-resolution time-lapse intravital imaging approach to visualize the dynamics of cancer cell extravasation in vivo. During intravascular migration, cancer cells form protrusive structures identified as invadopodia by their enrichment of MT1-MMP, cortactin, Tks4, and importantly Tks5, which localizes exclusively to invadopodia. Cancer cells extend invadopodia through the endothelium into the extravascular stroma prior to their extravasation at endothelial junctions. Genetic or pharmacological inhibition of invadopodia initiation (cortactin), maturation (Tks5), or function (Tks4) resulted in an abrogation of cancer cell extravasation and metastatic colony formation in an experimental mouse lung metastasis model. This provides direct evidence of a functional role for invadopodia during cancer cell extravasation and distant metastasis and reveals an opportunity for therapeutic intervention in this clinically important process.

Structure and biomechanics of the endothelial transcellular circumferential invasion array in tumor invasion.

Cancer cells breach the endothelium not only through cell-cell junctions but also via individual endothelial cells (ECs), or transcellular invasion. The underlying EC forms a circular structure around the transcellular invasion pore that is dependent on myosin light chain kinase (MLCK) and myosin II regulatory light chain (RLC) phosphorylation. Here we offer mechanistic insights into transcellular invasive array formation amid persistent tensile force from activated EC myosin. Fluorescence recovery after photobleaching (FRAP) experiments, sarcomeric distance measurements using super-resolution microscopy and electron microscopy provide details about the nature of the myosin II invasion array. To probe the relationship between biomechanical forces and the tension required to maintain the curvature of contractile filaments, we targeted individual actin-myosin fibers at the invasion site for photoablation. We showed that adjacent filaments rapidly replace the ablat11ed structures. We propose that the transcellular circumferential invasion array (TCIA) provides the necessary constraint within the EC to blunt the radial compression from the invading cancer cell.

Similarities and differences in the regulation of leukocyte extravasation and vascular permeability.

Leukocyte extravasation is regulated and mediated by a multitude of adhesion and signaling molecules. Many of them enable the capturing and docking of leukocytes to the vessel wall. Others allow leukocytes to crawl on the apical surface of endothelial cells to appropriate sites of exit. While these steps are well understood and the adhesion molecules mediating these interactions are largely identified, a still growing number of adhesion receptors mediate the diapedesis process, the actual migration of leukocytes through the endothelial cell layer, and the underlying basement membrane. In most cases, it is not known which molecular processes they actually mediate, whether they enable the migration of leukocytes through the endothelial cell layer or whether they are involved in the destabilization of endothelial junctions. In addition, leukocytes are able to circumvent junctions and transcytose directly through the body of endothelial cells. While this latter route indeed exists, recent work has highlighted in vivo the junctional pathway as the prevalent way of leukocyte exit in various inflamed tissues. Recent work elucidating molecular mechanisms that regulate endothelial junctions and thereby leukocyte extravasation and vascular permeability will be discussed.

Intestinal barrier dysfunction triggered by invasive bacteria.

The ability to control uptake across the mucosa and to protect the gut from harmful substances present in the lumen is defined as intestinal barrier function. Two routes are usually distinguished for transepithelial transport. The paracellular route allows the passage of ions and small molecules and is mainly regulated by tight junctions (TJ). The transcellular route concerns large molecules or small particles (including bacteria) and is mediated by cell endocytosis and intracellular vesicular traffic. Enteropathogenic bacteria increase the transcellular permeability, especially in the follicle-associated epithelium. They also modulate TJ opening via the redistribution of TJ proteins and the activation of the myosin light chain kinase (MLCK). This review focuses on the molecular mechanisms involved in the bacteria-induced barrier defect and briefly discusses their consequences in human diseases.

ß2 integrin-mediated crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed blood-brain barrier.

In acute neuroinflammatory states such as meningitis, neutrophils cross the blood-brain barrier (BBB) and contribute to pathological alterations of cerebral function. The mechanisms that govern neutrophil migration across the BBB are ill defined. Using live-cell imaging, we show that LPS-stimulated BBB endothelium supports neutrophil arrest, crawling, and diapedesis under physiological flow in vitro. Investigating the interactions of neutrophils from wild-type, CD11a(-/-), CD11b(-/-), and CD18(null) mice with wild-type, junctional adhesion molecule-A(-/-), ICAM-1(null), ICAM-2(-/-), or ICAM-1(null)/ICAM-2(-/-) primary mouse brain microvascular endothelial cells, we demonstrate that neutrophil arrest, polarization, and crawling required G-protein-coupled receptor-dependent activation of ß2 integrins and binding to endothelial ICAM-1. LFA-1 was the prevailing ligand for endothelial ICAM-1 in mediating neutrophil shear resistant arrest, whereas Mac-1 was dominant over LFA-1 in mediating neutrophil polarization on the BBB in vitro. Neutrophil crawling was mediated by endothelial ICAM-1 and ICAM-2 and neutrophil LFA-1 and Mac-1. In the absence of crawling, few neutrophils maintained adhesive interactions with the BBB endothelium by remaining either stationary on endothelial junctions or displaying transient adhesive interactions characterized by a fast displacement on the endothelium along the direction of flow. Diapedesis of stationary neutrophils was unchanged by the lack of endothelial ICAM-1 and ICAM-2 and occurred exclusively via the paracellular pathway. Crawling neutrophils, although preferentially crossing the BBB through the endothelial junctions, could additionally breach the BBB via the transcellular route. Thus, ß2 integrin-mediated neutrophil crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed BBB.

The Spiegelmer NOX-A12, a novel CXCL12 inhibitor, interferes with chronic lymphocytic leukemia cell motility and causes chemosensitization.

The CXC chemokine ligand (CXCL12, or stromal cell-derived factor-1 as previously known) plays a critical role for homing and retention of chronic lymphocytic leukemia (CLL) cells in tissues such as the bone marrow (BM). In tissues, stromal cells constitutively secrete and present CXCL12 via cell-surface-bound glycosaminoglycans (GAGs), thereby attracting CLL cells and protecting them from cytotoxic drugs, a mechanism that may account for residual disease after conventional CLL therapy. NOX-A12, an RNA oligonucleotide in L-configuration (Spiegelmer) that binds and neutralizes CXCL12, was developed for interference with CXCL12 in the tumor microenvironment and for cell mobilization. Here, we examined effects of NOX-A12 on CLL cell migration and drug sensitivity. We found that NOX-A12 effectively inhibited CXCL12-induced chemotaxis of CLL cells. In contrast, NOX-A12 increased CLL migration underneath a confluent layer of BM stromal cells (BMSCs) due to interference with the CXCL12 gradient established by BMSCs. In particular, NOX-A12 competes with GAGs such as heparin for CXCL12 binding, leading to the release of CXCL12 from stromal cell-surface-bound GAGs, and thereby to neutralization of the chemokine. Furthermore, NOX-A12 sensitizes CLL cells toward bendamustine and fludarabine in BMSC cocultures. These data demonstrate that NOX-A12 effectively interferes with CLL cell migration and BMSC-mediated drug resistance, and establishes a rationale for clinical development of NOX-A12 in combination with conventional agents in CLL.

Regulatory role of membrane-bound form interleukin-15 on human uterine microvascular endothelial cells in circulating CD16(-) natural killer cell extravasation into human endometrium.

Interleukin (IL)-15 plays a major role in accumulation of unique CD16(-) natural killer (NK) cells in the human endometrium, partly via selective extravasation of peripheral blood (PB) counterparts from local microvascular circulation. While IL-15 exhibits a chemotactic activity for PB CD16(-) NK cells, IL-15 attenuates their binding capacity to dermatan sulfate, the major CD62L ligand expressed on human uterine microvascular endothelial cells (HUtMVECs). These findings suggest that premature action of IL-15 interferes with CD62L-dependent tethering/rolling of PB CD16(-) NK cells on HUtMVECs, which is an early critical process of leukocyte extravasation. In this study, we investigated the mechanisms underlying the IL-15 regulation in the initial CD62L-dependent contact between PB CD16(-) NK cells and HUtMVECs. Unlike other candidate molecules, recombinant IL-15 downregulated CD62L expression on freshly isolated PB CD16(-) NK cells. IL-12 and IL-10, the two known upregulators of CD62L on CD16(-) NK cells, were not detectable in HUtMVECs and endometrial perivascular stromal cells. Binding to immobilized dermatan sulfate increased surface IL-15 receptor-alpha chain expression on CD16(-) NK cells. Under ovarian steroid stimulation, IL-15 was detectable on the surface, but not in the supernatant, of cultured HUtMVECs. Ovarian steroid-induced IL-15 expression on HUtMVECs was not attenuated by chondroitinase ABC (which degrades chondroitin sulfate-A and -C and dermatan sulfate) or sodium acetate buffer (which dissociates cytokines from their cognate receptors). These results suggest that HUtMVECs secrete a less soluble form of IL-15 into local microcirculation. Instead, HUtMVECs bear a membrane-bound form IL-15 under the influence of ovarian steroids, which may be favorable for preventing downregulation of CD62L on PB CD16(-) NK cells and facilitating their initial contact with HUtMVECs.