Hug tightly and say goodbye: role of endothelial ICAM-1 in leukocyte transmigration

Stable adhesion of leukocytes to endothelium is crucial for transendothelial migration (TEM) of leukocytes evoked during inflammatory responses, immune surveillance, and homing and mobilization of hematopoietic progenitor cells. The basis of stable adhesion involves expression of intercellular adhesion molecule-1 (ICAM-1), an inducible endothelial adhesive protein that serves as a counter-receptor for beta(2)-integrins on leukocytes. Interaction of ICAM-1 with beta(2)-integrins enables leukocytes to adhere firmly to the vascular endothelium and subsequently, to migrate across the endothelial barrier. The emerging paradigm is that ICAM-1, in addition to firmly capturing leukocytes, triggers intracellular signaling events that may contribute to active participation of the endothelium in facilitating the TEM of adherent leukocytes. The nature, duration, and intensity of ICAM-1-dependent signaling events may contribute to the determination of the route (paracellular vs. transcellular) of leukocyte passage; these aspects of ICAM-1 signaling may in turn be influenced by density and distribution of ICAM-1 on the endothelial cell surface, the source of endothelial cells it is present on, and the type of leukocytes with which it is engaged. This review summarizes our current understanding of the "ICAM-1 paradigm" of TEM with an emphasis on the signaling events mediating ICAM-1 expression and activated by ICAM-1 engagement in endothelial cells.     

Leukocyte-endothelial-cell interactions in leukocyte transmigration and the inflammatory response

Both the innate and adaptive immune responses are dependent on the migration of leukocytes across endothelial cells. The process of diapedesis, in which the leukocyte crawls between tightly apposed endothelial cells, is a unique and complex process. Several molecules concentrated at the junctions of endothelial cells, originally described as having a role in holding the endothelial monolayer together, have also been shown to have a role in the emigration of leukocytes. Several mechanisms have been proposed for 'loosening' the junctions between endothelial cells to enable leukocyte passage. These leukocyte-endothelial-cell adhesion molecules are probably involved in regulating the signaling as well as the adhesion events of diapedesis. In addition, this Review introduces a new and unified nomenclature for the junctional adhesion molecule (JAM) family.     

Activation of extracellular regulated kinases is required for the increase in airway epithelial permeability during leukocyte transmigration

The goal of this study was to determine whether the extracellular regulated kinases (ERK1/2) are involved in leukocyte transmigration across airway epithelium and the associated changes in epithelial permeability. In vitro, we used formyl-methionyl-leucyl-phenylalanine (fMLP) to induce migration of HL-60 cells (a human leukocyte cell line) across sheets of polarized Calu-3 airway epithelial cells and also to induce migration of human neutrophils across primary cultures of cow tracheal epithelial cells. In both systems, leukocyte migration decreased transepithelial electrical resistance (R(te)), increased epithelial permeability to albumin (P(alb)), and increased ERK1/2 phosphorylation in epithelial cells. Leukocyte migration and the associated changes in R(te), P(alb), and ERK1/2 phosphorylation were inhibited by calphostin C, a blocker of protein kinase C (PKC), and by PD98059 (a blocker of ERK1/2). Leukocyte transmigration in rat tracheas in vivo was induced with fMLP, and was associated with increased P(alb) and phosphorylation of epithelial ERK1/2. Again, migration and the associated changes were inhibited by luminal PD98059 or calphostin C though neither agent affected rat leukocyte migration in Boyden chambers in vitro. We conclude that PKC and ERK1/2 pathways are activated in airway epithelial cells during migration of leukocytes and are important regulators of airway epithelial permeability.     

Involvement of IbeA in meningitic Escherichia coli K1-induced polymorphonuclear leukocyte transmigration across brain endothelial cells

Transmigration of neutrophil [polymorphonuclear neutrophil (PMN)] across the blood-brain barrier (BBB) is a critical event in the pathogenesis of bacterial meningitis. We have shown that IbeA is able to induce meningitic Escherichia coli invasion of brain microvascular endothelial cells (BMECs), which constitutes the BBB. In this report, we provide evidence that IbeA and its receptor, vimentin, play a key role in E. coli-induced PMN transmigration across BMEC. In vitro and in vivo studies indicated that the ibeA-deletion mutant ZD1 was significantly less active in stimulating PMN transmigration than the parent strain E44. ZD1 was fully complemented by the ibeA gene and its product. E. coli-induced PMN transmigration was markedly inhibited by withaferin A, a dual inhibitor of vimentin and proteasome. These cellular effects were significantly stimulated and blocked by overexpression of vimentin and its head domain deletion mutant in human BMEC, respectively. Our studies further demonstrated that IbeA-induced PMN migration was blocked by bortezomib, a proteasomal inhibitor and correlated with upregulation of endothelial ICAM-1 and CD44 expression through proteasomal regulation of NFκB activity. Taken together, our data suggested that IbeA and vimentin contribute to E. coli K1-stimulated PMN transendothelial migration that is correlated with upregulation of adhesion molecule expression at the BBB.     

Effects of inflammatory cytokines on the permeability of human lung microvascular endothelial cell monolayers and differential eosinophil transmigration

BACKGROUND: Rhinovirus (RV) infections can result in asthma exacerbations in both adults and children. Respiratory epithelium, the primary site of RV replication, responds to the viral infection by generating a variety of cytokines and chemokines capable of promoting airway inflammation and hence might increase asthma severity. Some of these mediators might also affect the permeability of underlying vascular endothelium. OBJECTIVE: We hypothesized that RV infections can promote airway inflammation and thus asthma by enhancing local vascular permeability. METHODS: Confluent human lung microvascular endothelial cell (HMVEC-L) monolayers were used as an in vitro model of vascular endothelium to determine whether cytokines associated with RV-induced infections are capable of modulating endothelial cell permeability as measured by means of transendothelial electrical resistance. Recombinant cytokines and chemokines were added to confluent HMVEC-L monolayers cultured on Transwell filters, and permeability was measured as decreased electrical resistance over time. Eosinophil transendothelial migration was assessed under the same experimental conditions. RESULTS: TNF- alpha, IL-1 beta, and IFN- gamma significantly increased HMVEC-L permeability. In contrast, GM-CSF, G-CSF, IL-8, IL-6, and RANTES had no effect. Although incubation of HMVEC-L monolayers with either TNF-alpha or IL-1beta promoted eosinophil migration, IFN-gamma had no effect, indicating that enhanced permeability alone was not sufficient for eosinophil infiltration. CONCLUSION: Select cytokines, generated in response to RV infection, can increase vascular permeability and might provide a mechanism by which RV infection can lead to edema, cellular infiltration, and inflammation and thus compromised airflow.     

Direct effects of E coli endotoxin on structure and permeability of pulmonary endothelial monolayers and the endothelial layer of intimal explants.

The direct structural, metabolic, and physiologic effects of Escherichia coli endotoxin on bovine pulmonary endothelial monolayers and on the intact endothelial layer of bovine pulmonary artery intimal explants were examined. Endothelial monolayers exposed to E coli endotoxin (0.001, 0.01, 0.1, 1.0, and 10 micrograms/ml) for 24 hours in the absence of bovine fetal calf serum (FCS) showed a dose-dependent response, as demonstrated by number of pyknotic cells and lactate dehydrogenase release that was enhanced by addition of FCS. Prostacyclin production was increased only in the presence of FCS. Endotoxin also caused an increase in permeability. Endothelial cells on nitrocellulose filters placed in chemotaxis chambers with radioactive tracers in the upper well showed a significant 25% increase in rate of equilibration (counts in lower well/counts in upper well) of 3H-water after 2 and 3 hours' incubation with endotoxin (3 hours' endotoxin = 0.89 +/- 0.03 m +/- SE; no endotoxin = 0.69 +/- 0.05) and a 40% increase in equilibration of 125I-albumin at three hours (3 hours' endotoxin = 0.40 +/- 0.03; no endotoxin = 0.27 +/- 0.02). An increase in hydraulic conductance was also seen at 1 hour. Electron microscopy of the endothelial layer of intimal explants showed dilatations in the intercellular junctions and cellular changes representing contraction--increased prominence of cytoplasmic filaments, nuclear crenation, and cytoplasmic protrusions--at 30 and 60 minutes. From 2 hours evidence of cell death was found. Thus, endotoxin causes structural and metabolic changes in pulmonary endothelial cells and an increase in permeability of the endothelial layer. The injury occurs in the absence of FCS but is enhanced by its addition.     

SPARC is a VCAM-1 counter-ligand that mediates leukocyte transmigration

VCAM-1 is a cell surface molecule, which has been shown to mediate leukocyte adhesion to the endothelium and subsequent transmigration. Although VCAM-1 regulates adhesion through its interaction with VLA-4, VLA-4 does not play a role in VCAM-1-dependent diapedesis, an observation suggesting the presence of a second ligand for VCAM-1. We now report a novel interaction between VCAM-1 and secreted protein acidic and rich in cysteine (SPARC), which induces actin cytoskeletal rearrangement and intercellular gaps, physiological processes known to be important for leukocyte transmigration. The binding of leukocyte-derived SPARC to VCAM-1 was demonstrated to be necessary for leukocyte transmigration through endothelial monolayers (diapedesis) in vitro, and furthermore, SPARC null mice have abnormalities in leukocyte recruitment to the inflamed peritoneum in vivo. These findings provide new insight into the mechanisms of transendothelial leukocyte migration and suggest a potential, targetable interaction for therapeutic intervention.     

The selective and superoxide-independent disruption of intestinal epithelial tight junctions during leukocyte transmigration

Polymorphonuclear leukocyte (PMN) transmigration across cultured intestinal epithelial monolayers has been shown to be associated with a decrease in transepithelial resistance to the passive flow of ions. Using flux techniques, we show that this effect reflects selective, PMN induced alterations in paracellular, as opposed to transcellular, ion permeability. Enhancement of paracellular permeability due to PMN transmigration is not simply due to expansion of the paracellular space resulting from cell death as cytotoxicity does not occur during this process. Thus, permeability alterations accompanying PMN transmigration can be specifically attributed to altered permeability of the rate limiting barrier of the paracellular pathway, the intercellular tight junction. We have also explored the mechanism by which PMN induce transient tight junction dissolution during transmigration. Use of inhibitors of toxic oxygen metabolites or use of PMN from patients with chronic granulomatous disease show that oxygen metabolites are neither required for transmigration or for the permeability abnormality accompanying transmigration. Similarly, use of protease inhibitors suggest that release of proteases by PMN during transmigration is not the basis by which PMN are able to cross tight junctions. Structural studies show that transient intimate PMN-epithelial cell plasma membrane associations and cytoskeletal specializations preceed junctional impalement by PMN. We speculate that such putative adhesion sites serve as the foothold from which PMN may generate the mechanical force necessary to cross tight junctions during transmigration.     

小窝与血管内皮通透性

小窝(caveolae)是调控血管内皮跨细胞通透性的关键分子,通过Cav-1的表达和磷酸化、Src激酶的激活及细胞旁转运调节血管的高通透性。caveolae及Cav-1参与低密度脂蛋白的跨细胞转运,调节血脑屏障,干预肿瘤病理性的血管新生及肺内皮屏障功能,有望为动脉粥样硬化和脑卒中等心脑血管疾病及某些癌症提供新的治疗靶点。     

A new continuous monitoring system for simultaneous measurements of intracellular calcium and either endothelial permeability or leukocyte extravasation

OBJECTIVE AND DESIGN: We have developed a continuous monitoring system that will quantify lymphocyte extravasation or intercellular permeability with intracellular calcium measurements in a single preparation. Materials: Human microvascular endothelial cells, human lymphocytes and histamine. TREATMENT: Endothelial barrier function and intracellular calcium were examined upon application of either human lymphocytes (1 x 10(5) cells/ml) or histamine (1 microM). METHODS: Endothelial cells labeled with FURA-2 were examined for both changes in endothelial barrier function and mobilization of intracellular calcium using fluorescence spectroscopy and ratio imaging, respectively. RESULTS: Increases in endothelial intracellular calcium occur in the first minute and are followed by increases in endothelial intercellular permeability to albumin (5-7 min) or lymphocytes (15-20 min). Approximately 60 min following calcium mobilization, there was a second and larger extravasation. CONCLUSION: The continuous monitoring system expands our understanding of the time course of the permeability and extravasation events to mobilization of endothelial intracellular calcium.     

Toluene diisocyanate enhances human bronchial epithelial cells' permeability partly through the vascular endothelial growth factor pathway

Background Toluene diisocyanate (TDI) is a recognized chemical asthmogen; yet, the mechanisms of its toxicity have not been elucidated.
Objective To investigate the influence of TDI on the permeability of human bronchial epithelial cell (HBE; HBE135-E6E7) monolayers in vitro , and the expression of vascular endothelial growth factor (VEGF) in these cells.
Methods TDI–human serum albumin (HSA) conjugates were prepared by a modification of Son's method. Fluorescein isothiocyanate-labelled dextran and transmission electron microscopy were used to evaluate the effects of TDI–HSA on HBE135-E6E7 permeability. RT-PCR and ELISA were used to evaluate VEGF gene expression and protein release from HBE135-E6E7 cells stimulated by TDI–HSA. A VEGF-neutralizing antibody was used in monolayer permeability experiments to determine the role of the VEGF pathway in this process.
Results TDI–HSA significantly increased the permeability coefficients of HBE135-E6E7 monolayers ( P <0.01). TDI–HSA treatment significantly increased the expression of VEGF165 and VEGF189 genes ( P <0.01). ELISA showed that TDI significantly induces VEGF release from HBE135-E6E7 cells. Cells treated with TDI–HSA and VEGF-neutralizing antibody had significantly lower permeability coefficients than cells treated with TDI–HSA only ( P <0.01), but still significantly higher than control cells ( P <0.01). Cells treated with TDI–HSA had fewer tight junctions (TJs) than control and HSA-treated cells, and addition of the anti-VEGF antibody did not restore the original number of TJs.
Conclusion TDI increases the permeability of HBE cell monolayers, partly through a VEGF-mediated pathway. This suggests the importance of VEGF in TDI-induced pulmonary diseases, but shows that other pathways may be involved in the pathogenic process.     

Rabbit cationic protein enhances leukocyte adhesiveness.

Cationic protein purified from rabbit peritoneal polymorphonuclear leukocytes (PMN) was demonstrated to incite autoaggregation of the rabbit PMN and promote adhesiveness of human PMN to endothelial cells. PMN aggregation induced by supernatants derived from secretory PMN was blocked by a specific anticationic protein antibody. These studies reveal that a positively charged protein derived from the PMN can alter surface properties of the PMN itself and imply a role for this protein in PMN immobilization at inflammatory sites.     

Inhibition of endothelial interleukin-8 production and neutrophil transmigration by Staphylococcus aureus beta-hemolysin

Neutrophils play a crucial role in the host response to infection with Staphylococcus aureus, which is a major human pathogen capable of causing life-threatening disease. Interleukin-8 (IL-8) is a potent chemoattractant and activator of neutrophils. We previously reported that S. aureus secretes a factor that suppresses IL-8 production by human endothelial cells. Here we isolated an inhibitor of IL-8 production from the supernatant and identified it as staphylococcal beta-hemolysin. Beta-hemolysin reduced IL-8 production without cytotoxicity to endothelial cells. Pretreatment with beta-hemolysin decreased the expression of both IL-8 mRNA and protein induced by tumor necrosis factor alpha (TNF-alpha). Migration of neutrophils across TNF-alpha-activated endothelium was also inhibited by beta-hemolysin. In contrast, beta-hemolysin had no effect on intercellular adhesive molecule 1 expression in activated endothelial cells. These results showed that beta-hemolysin produced by S. aureus interferes with inflammatory signaling in endothelial cells and may help S. aureus evade the host immune response.     

Development of intimal lesions after leukocyte migration into the vascular wall.

A model was developed to study the role of leukocytes in the development of vascular lesions. Implantation of an endotoxin-soaked cotton thread in the adventitia on the ventral side of the rat femoral artery resulted in leukocyte migration into the vessel wall exclusively in the ventral half of the vessel. Leukocyte migration occurred from both the luminal and adventitial side and consisted of neutrophils and mononuclear cells. Smooth muscle cell rich intimal lesions localized to the ventral half of the vessel were first observed 1 week after implantation. Lesions remained localized to the ventral half of the vessel wall through the 6th week. When leukocyte migration into the vessel wall was inhibited by treatment with dexamethasone, lesion development did not occur. These results suggest that leukocytes can stimulate smooth muscle cell migration into the intima and result in intimal lesion formation.     

Signaling mechanisms regulating endothelial permeability

The microvascular endothelial cell monolayer localized at the critical interface between the blood and vessel wall has the vital functions of regulating tissue fluid balance and supplying the essential nutrients needed for the survival of the organism. The endothelial cell is an exquisite "sensor" that responds to diverse signals generated in the blood, subendothelium, and interacting cells. The endothelial cell is able to dynamically regulate its paracellular and transcellular pathways for transport of plasma proteins, solutes, and liquid. The semipermeable characteristic of the endothelium (which distinguishes it from the epithelium) is crucial for establishing the transendothelial protein gradient (the colloid osmotic gradient) required for tissue fluid homeostasis. Interendothelial junctions comprise a complex array of proteins in series with the extracellular matrix constituents and serve to limit the transport of albumin and other plasma proteins by the paracellular pathway. This pathway is highly regulated by the activation of specific extrinsic and intrinsic signaling pathways. Recent evidence has also highlighted the importance of the heretofore enigmatic transcellular pathway in mediating albumin transport via transcytosis. Caveolae, the vesicular carriers filled with receptor-bound and unbound free solutes, have been shown to shuttle between the vascular and extravascular spaces depositing their contents outside the cell. This review summarizes and analyzes the recent data from genetic, physiological, cellular, and morphological studies that have addressed the signaling mechanisms involved in the regulation of both the paracellular and transcellular transport pathways.     

Effect of streptavidin-biotin on endothelial vasoregulation and leukocyte adhesion

The current study examines whether the adhesion promoting arginine-glycine-aspartate-streptavidin mutant (RGD-SA) also affects two important endothelial cell (EC) functions in vitro: vasoregulation and leukocyte adhesion. EC adherent to surfaces via fibronectin (Fn) or Fn plus RGD-SA were subjected to laminar shear flow and media samples were collected over a period of 4h to measure the concentration of nitric oxide (NO), prostacyclin (PGI(2)), and endothelin-1 (ET-1). Western blot analysis was used to quantify the levels of endothelial-derived nitric oxide synthase (eNOS) and cyclooxygenase II (COX II). In a separate set of experiments, fluorescent polymorphonuclear leukocyte (PMN) adhesion to EC was quantified for EC with and without exposure to flow preconditioning. When cell adhesion was supplemented with the SA-biotin system, flow-induced production of NO and PGI(2) increased significantly relative to cells adherent on Fn alone. Previous exposure of EC to shear flow also significantly decreased PMN attachment to SA-biotin supplemented EC, but only after 2h of exposure to shear flow. The observed decrease in PMN-EC adhesion was negated by NG-nitro-L-arginine methyl ester (L-NAME), an antagonist of NO synthesis, but not by indomethacin, an inhibitor to PGI(2) synthesis, indicating the induced effect of PMN-EC interaction is primarily NO-dependent. Results from this study suggest that the use of SA-biotin to supplement EC adhesion encourages vasodilation and PMN adhesion in vitro under physiological shear-stress conditions. We postulate that the presence of SA-biotin more efficiently transmits the shear-stress signal and amplifies the downstream events including the NO and PGI(2) release and leukocyte-EC inhibition. These results may have ramifications for reducing thrombus-induced vascular graft failure.