Endothelial cells proactively form microvilli-like membrane projections upon intercellular adhesion molecule 1 engagement of leukocyte LFA-1

Specific leukocyte/endothelial interactions are critical for immunity and inflammation, yet the molecular details of this interaction interface remain poorly understood. Thus, we investigated, with confocal microscopy, the distribution dynamics of the central adhesion molecules ICAM-1 and LFA-1 in this context. Monolayers of activated HUVECs stained with fluorescent anti-ICAM-1 Fabs or Chinese hamster ovary-K1 cells expressing ICAM-1-green fluorescent protein were allowed to bind LFA-1-bearing monocytes, neutrophils, or K562 LFA-1 transfectants. ICAM-1 was rapidly relocalized to newly formed microvilli-like membrane projections in response to binding LFA-1 on leukocytes. These ICAM-1-enriched projections encircled the leukocytes extending up their sides and clustered LFA-1 underneath into linear tracks. Projections formed independently of VCAM-1/very late Ag 4 interactions, shear, and proactive contributions from the LFA-1-bearing cells. In the ICAM-1-bearing endothelial cells, projections were enriched in actin but not microtubules, required intracellular calcium, and intact microfilament and microtubule cytoskeletons and were independent of Rho/Rho kinase signaling. Disruption of these projections with cytochalasin D, colchicine, or BAPTA-AM had no affect on firm adhesion. These data show that in response to LFA-1 engagement the endothelium proactively forms an ICAM-1-enriched cup-like structure that surrounds adherent leukocytes but is not important for firm adhesion. This finding leaves open a possible role in leukocyte transendothelial migration, which would be consistent with the geometry and kinetics of formation of the cup-like structure.     

Inside-Out Regulation of ICAM-1 Dynamics in TNF-α-Activated Endothelium

Background

During transendothelial migration, leukocytes use adhesion molecules, such as ICAM-1, to adhere to the endothelium. ICAM-1 is a dynamic molecule that is localized in the apical membrane of the endothelium and clusters upon binding to leukocytes. However, not much is known about the regulation of ICAM-1 clustering and whether membrane dynamics are linked to the ability of ICAM-1 to cluster and bind leukocyte integrins. Therefore, we studied the dynamics of endothelial ICAM-1 under non-clustered and clustered conditions.

Principal Findings

Detailed scanning electron and fluorescent microscopy showed that the apical surface of endothelial cells constitutively forms small filopodia-like protrusions that are positive for ICAM-1 and freely move within the lateral plane of the membrane. Clustering of ICAM-1, using anti-ICAM-1 antibody-coated beads, efficiently and rapidly recruits ICAM-1. Using fluorescence recovery after photo-bleaching (FRAP), we found that clustering increased the immobile fraction of ICAM-1, compared to non-clustered ICAM-1. This shift required the intracellular portion of ICAM-1. Moreover, biochemical assays showed that ICAM-1 clustering recruited beta-actin and filamin. Cytochalasin B, which interferes with actin polymerization, delayed the clustering of ICAM-1. In addition, we could show that cytochalasin B decreased the immobile fraction of clustered ICAM-1-GFP, but had no effect on non-clustered ICAM-1. Also, the motor protein myosin-II is recruited to ICAM-1 adhesion sites and its inhibition increased the immobile fraction of both non-clustered and clustered ICAM-1. Finally, blocking Rac1 activation, the formation of lipid rafts, myosin-II activity or actin polymerization, but not Src, reduced the adhesive function of ICAM-1, tested under physiological flow conditions.

Conclusions

Together, these findings indicate that ICAM-1 clustering is regulated in an inside-out fashion through the actin cytoskeleton. Overall, these data indicate that signaling events within the endothelium are required for efficient ICAM-1-mediated leukocyte adhesion.     

Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes

Ezrin, radixin, and moesin (ERM) regulate cortical morphogenesis and cell adhesion by connecting membrane adhesion receptors to the actin-based cytoskeleton. We have studied the interaction of moesin and ezrin with the vascular cell adhesion molecule (VCAM)-1 during leukocyte adhesion and transendothelial migration (TEM). VCAM-1 interacted directly with moesin and ezrin in vitro, and all of these molecules colocalized at the apical surface of endothelium. Dynamic assessment of this interaction in living cells showed that both VCAM-1 and moesin were involved in lymphoblast adhesion and spreading on the endothelium, whereas only moesin participated in TEM, following the same distribution pattern as ICAM-1. During leukocyte adhesion in static or under flow conditions, VCAM-1, ICAM-1, and activated moesin and ezrin clustered in an endothelial actin-rich docking structure that anchored and partially embraced the leukocyte containing other cytoskeletal components such as alpha-actinin, vinculin, and VASP. Phosphoinositides and the Rho/p160 ROCK pathway, which participate in the activation of ERM proteins, were involved in the generation and maintenance of the anchoring structure. These results provide the first characterization of an endothelial docking structure that plays a key role in the firm adhesion of leukocytes to the endothelium during inflammation.     

Roles of phosphatidylinositol 3-kinase and NF-kappaB in human cytomegalovirus-mediated monocyte diapedesis and adhesion: strategy for viral persistence

Infected peripheral blood monocytes are proposed to play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to tissues, a critical step in the establishment of HCMV persistence and the development of HCMV-associated diseases. We recently provided evidence for a unique strategy involved in viral dissemination: HCMV infection of primary human monocytes promotes their transendothelial migration and differentiation into proinflammatory macrophages permissive for the replication of the original input virus. To decipher the mechanism of hematogenous spread, we focused on the viral dysregulation of early cellular processes involved in transendothelial migration. Here, we present evidence that both phosphatidylinositol 3-kinase [PI(3)K] and NF-kappaB activities were crucial for the HCMV induction of monocyte motility and firm adhesion to endothelial cells. We found that the beta(1) integrins, the beta(2) integrins, intracellular adhesion molecule 1 (ICAM-1), and ICAM-3 were upregulated following HCMV infection and that they played a key role in the firm adhesion of infected monocytes to the endothelium. The viral regulation of adhesion molecule expression is complex, with PI(3)K and NF-kappaB affecting the expression of each adhesion molecule at different stages of the expression cascade. Our data demonstrate key roles for PI(3)K and NF-kappaB signaling in the HCMV-induced cellular changes in monocytes and identify the biological rationale for the activation of these pathways in infected monocytes, which together suggest a mechanism for how HCMV promotes viral spread to and persistence within host organs.     

Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains

During inflammation, leukocytes bind to the adhesion receptors ICAM-1 and VCAM-1 on the endothelial surface before undergoing transendothelial migration, also called diapedesis. ICAM-1 is also involved in transendothelial migration, independently of its role in adhesion, but the molecular basis of this function is poorly understood. Here we demonstrate that, following clustering, apical ICAM-1 translocated to caveolin-rich membrane domains close to the ends of actin stress fibres. In these F-actin-rich areas, ICAM-1 was internalized and transcytosed to the basal plasma membrane through caveolae. Human T-lymphocytes extended pseudopodia into endothelial cells in caveolin- and F-actin-enriched areas, induced local translocation of ICAM-1 and caveolin-1 to the endothelial basal membrane and transmigrated through transcellular passages formed by a ring of F-actin and caveolae. Reduction of caveolin-1 levels using RNA interference (RNAi) specifically decreased lymphocyte transcellular transmigration. We propose that the translocation of ICAM-1 to caveola- and F-actin-rich domains links the sequential steps of lymphocyte adhesion and transendothelial migration and facilitates lymphocyte migration through endothelial cells from capillaries into surrounding tissue.     

Activation of endothelial extracellular signal-regulated kinase is essential for neutrophil transmigration: potential involvement of a soluble neutrophil factor in endothelial activation

During an inflammatory response induced by infection or injury, leukocytes traverse the endothelial barrier into the tissue space. Extravasation of leukocytes is a multistep process involving rolling, tethering, firm adhesion to the endothelium, and finally, transendothelial migration, the least characterized step in the process. The resting endothelium is normally impermeable to leukocytes; thus, during inflammation, intracellular signals that modulate endothelial permeability are activated to facilitate the paracellular passage of leukocytes. Using a static in vitro assay of neutrophil transmigration across human umbilical vein endothelium, a panel of inhibitors of intracellular signaling was screened for their ability to inhibit transmigration. PD98059, a specific inhibitor of extracellular signal-regulated kinase (ERK) 1/2 activation, inhibited both transmigration across TNF-alpha-activated endothelium and transmigration induced by the chemoattractant fMLP in a dose-dependent manner. PD98059 did not inhibit neutrophil chemotaxis in the absence of an endothelial barrier nor neutrophil adhesion to the endothelium, suggesting that its effect was on the endothelium, and furthermore, that endothelial ERK activation may be important for transmigration. We demonstrate in this study that endothelial ERK is indeed activated during neutrophil transmigration and that its activation is dependent on the addition of neutrophils to the endothelium. Further characterization showed that the trigger for endothelial ERK activation is a soluble protein of molecular mass approximately 30 kDa released from neutrophils after activation.     

Intracellular domain of brain endothelial intercellular adhesion molecule-1 is essential for T lymphocyte-mediated signaling and migration

To examine the role of the ICAM-1 C-terminal domain in transendothelial T lymphocyte migration and ICAM-1-mediated signal transduction, mutant human (h)ICAM-1 molecules were expressed in rat brain microvascular endothelial cells. The expression of wild-type hICAM-1 resulted in a significant increase over basal levels in both adhesion and transendothelial migration of T lymphocytes. Endothelial cells (EC) expressing ICAM-1 in which the tyrosine residue at codon 512 was substituted with phenylalanine (hICAM-1(Y512F)) also exhibited increased lymphocyte migration, albeit less than that with wild-type hICAM-1. Conversely, the expression of truncated hICAM-1 proteins, in which either the intracellular domain was deleted (hICAM-1DeltaC) or both the intracellular and transmembrane domains were deleted through construction of a GPI anchor (GPI-hICAM-1), did not result in an increase in lymphocyte adhesion, and their ability to increase transendothelial migration was attenuated. Truncated hICAM-1 proteins were also unable to induce ICAM-1-mediated Rho GTPase activation. EC treated with cell-permeant penetratin-ICAM-1 peptides comprising human or rat ICAM-1 intracellular domain sequences inhibited transendothelial lymphocyte migration, but not adhesion. Peptides containing a phosphotyrosine residue were equipotent in inhibiting lymphocyte migration. These data demonstrate that the intracellular domain of ICAM-1 is essential for transendothelial migration of lymphocytes, and that peptidomimetics of the ICAM-1 intracellular domain can also inhibit this process. Such competitive inhibition of transendothelial lymphocyte migration in the absence of an affect on adhesion further implicates ICAM-1-mediated signaling events in the facilitation of T lymphocyte migration across brain EC. Thus, agents that mimic the ICAM-1 intracellular domain may be attractive targets for novel anti-inflammatory therapeutics.     

The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation

Leukocyte transendothelial migration involves the active participation of the endothelium through the formation of apical membrane protrusions that embrace adherent leukocytes, termed docking structures. Using live-cell imaging, we find that prior to transmigration, endothelial docking structures form around 80% of all neutrophils. Previously we showed that endothelial RhoG and SGEF control leukocyte transmigration. In this study, our data reveal that both full-length Trio and the first DH-PH (TrioD1) domain of Trio, which can activate Rac1 and RhoG, interact with ICAM-1 and are recruited to leukocyte adhesion sites. Moreover, upon clustering of ICAM-1, the Rho-guanine nucleotide exchange factor Trio activates Rac1, prior to activating RhoG, in a filamin-dependent manner. We further show that docking structure formation is initiated by ICAM-1 clustering into ring-like structures, which is followed by apical membrane protrusion. Interestingly, we find that Rac1 is required for ICAM-1 clustering, whereas RhoG controls membrane protrusion formation. Finally, silencing endothelial Trio expression or reducing TrioD1 activity without affecting SGEF impairs both docking structure formation and leukocyte transmigration. We conclude that Trio promotes leukocyte transendothelial migration by inducing endothelial docking structure formation in a filamin-dependent manner through the activation of Rac1 and RhoG.     

Evidence of a functional role for interaction between ICAM-1 and nonmuscle alpha-actinins in leukocyte diapedesis

ICAM-1 is involved in both adhesion and extravasation of leukocytes to endothelium during inflammation. It has been shown that the ICAM-1 cytoplasmic domain is important for transendothelial migration of leukocytes but the precise molecular mechanisms involving the intracytoplasmic portion of ICAM-1 is not known. To characterize precisely the molecular scaffolding associated with ICAM-1, we have used the yeast two-hybrid system, and we have identified six different proteins interacting with the ICAM-1 cytoplasmic domain. In this study, we report that the two forms of nonmuscle alpha-actinin (i.e., alpha-actinin 1 and alpha-actinin 4) associate with ICAM-1, and that these interactions are essential for leukocyte extravasation. These interactions were further confirmed by coimmunoprecipitation and immunofluorescence in endothelial cells and in ICAM-1-transfected Chinese hamster ovary cells. The function of these interactions was analyzed by point mutation of charged amino acids located on ICAM-1 cytoplasmic domain. We have identified three charged amino acids (arginine 480, lysine 481, and arginine 486) which are essential in the binding of alpha-actinins to the ICAM-1 cytoplasmic tail. Mutation of these amino acids completely inhibited ICAM-1-mediated diapedesis. Experiments with siRNA inhibiting specifically alpha-actinin 1 or alpha-actinin 4 on endothelial cells indicated that alpha-actinin 4 had a major role in this phenomenon. Thus, our data demonstrate that ICAM-1 directly interacts with cytoplasmic alpha-actinin 1 and 4 and that this interaction is required for leukocyte extravasation.     

Simvastatin modulates TNFalpha-induced adhesion molecules expression in human endothelial cells

Adhesion and transendothelial migration of leukocytes into the vascular wall is a crucial step in atherogenesis. Expression of cell adhesion molecules by endothelial cells plays a leading role in this process. We investigated the effect of simvastatin, an inhibitor of HMG-CoA reductase administered to reduce plasma levels of LDL-cholesterol, on the expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) by human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor alpha (TNFalpha). We found the expression to be significantly inhibited by the drug in a time and concentration-dependent manner and to a greater extent in the case of VCAM-1 as compared with ICAM-1. In TNFalpha-stimulated HUVEC, simvastatin decreased VCAM-1 and ICAM-1 mRNA levels, inhibited TNFalpha-induced activation of nuclear factor kappaB (NF-kappaB) and enhanced expression of peroxisome proliferator-activated receptor alpha (PPARalpha). These effects were associated with reduction of adherence of monocytes and lymphocytes to HUVEC. The present findings suggest that the benefits of statins in vascular disease may include the inhibition of expression of VCAM-1 and ICAM-1 through effects on NF-kappaB.     

Ultrastructural identification and distribution of the adhesion molecules ICAM-1 and LFA-1 in the vascular and extravascular compartments of the human palatine tonsil

Summary Immunohistological analysis of sections prepared from human palatine tonsils revealed marked differences in the distribution of the adhesion molecule, leucocyte function antigen-1 (LFA-1) and its counter receptor, intercellular adhesion molecule-1 (ICAM-1). Light microscopy showed that LFA-1 was restricted to the leucocytes, particularly the lymphocytes. In contrast, staining of ICAM-1 was predominantly confined to the vascular endothelium with the greatest expression seen on the morphologically distinct high endothelial venules in the parafollicular areas; these are the sites that appear to support lymphocyte migration. Electron microscopy revealed that ICAM-1 was present on the luminal and lateral surfaces of the high endothelium and absent from the abluminal surface supported by basal lamina. The ICAM-1 was also absent from those surfaces of the endothelium that were in close contact with intravascular lymphocytes. Other cells stained by the anti-ICM-1 antibody included dendritic cells, plasma cells and epithelial cells in the reticulated crypt epithelium and in the upper strata of the non-keratinised stratified squamous epithelium. The high expression of LFA-1 was most prominent on lymphocytes, low on antigen-presenting cells and activated lymphoid cells, and not detectable on plasma cells, epithelial and endothelial cells. We propose that LFA-1/ICAM-1 binding participates in mediating the transendothelial migration of lymphocytes across the high endothelial venules of palatine tonsil.     

Eosinophil transendothelial migration induced by cytokines. I. Role of endothelial and eosinophil adhesion molecules in IL-1 beta-induced transendothelial migration.

IL-1 beta promotes adhesiveness in human umbilical vein endothelial cells (HuVEC) for eosinophils through expression of adhesion molecules including intercellular adhesion molecules-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1). Using an in vitro endothelial monolayer system, we examined whether IL-1 beta or TNF-alpha can promote eosinophil transendothelial migration. We also evaluated the contributions of ICAM-1, E-selectin, VCAM-1, leukocyte adhesion complex (CD11/18), and very late Ag-4 (CD11b/18) (VLA-4) in this process using blocking mAb, and determined the changes in expression of CD11b and L-selectin on eosinophils that had undergone transmigration. IL-1 beta and TNF-alpha treatment of HuVEC (4 h, 5 ng/ml) induced significant transendothelial migration of eosinophils (a 4.1 +/- 0.4-fold (IL-1 beta) and 2.0 +/- 0.9-fold (TNF-alpha) increase from the spontaneous value of 3.2 +/- 0.3%). Increased CD11b expression and shedding of L-selectin were observed on eosinophils following IL-1 beta-induced eosinophil transendothelial migration. Studies with mAb revealed that blockade of either ICAM-1 or CD11/18 inhibited transmigration, while antibodies against VCAM-1 and VLA-4 had no inhibitory effect. Among antibodies which block beta 2 integrins, anti-CD18 mAb had the best inhibitory effect (88% inhibition). The combined inhibitory effect of anti-CD11a mAb and anti-CD11b mAb was roughly equal to that of anti-CD18, although anti-CD11a (31% inhibition) and anti-CD11b (52% inhibition) were less effective individually. Anti-ICAM-1 by itself inhibited IL-1 beta-induced eosinophil transendothelial migration (24% inhibition) whereas neither anti-E-selectin nor anti-VCAM-1 were effective inhibitors. Interestingly, the combination of anti-E-selectin and anti-VCAM-1 with anti-ICAM-1 inhibited IL-1 beta-induced eosinophil transendothelial migration significantly better (53% inhibition) than anti-ICAM-1 alone. These results suggest that although the initial attachment of eosinophils to IL-1 beta-activated endothelial cells involves VCAM-1, E-selectin, and ICAM-1, the subsequent transendothelial migration process relies heavily on ICAM-1 and CD11/18. Finally, the changes that eosinophils have been observed to undergo during infiltration in vivo, namely increased expression of CD11/18 and shedding of L-selectin, appear to take place as a direct result of the interaction between eosinophils and endothelial cells.     

Lymphatic endothelial murine chloride channel calcium-activated 1 is a ligand for leukocyte LFA-1 and Mac-1

The lymphatic circulation mediates drainage of fluid and cells from the periphery through lymph nodes, facilitating immune detection of lymph-borne foreign Ags. The 10.1.1 mAb recognizes a lymphatic endothelial Ag, in this study purified by Ab-affinity chromatography. SDS-PAGE and mass spectrometry identified murine chloride channel calcium-activated 1 (mCLCA1) as the 10.1.1 Ag, a 90-kDa cell-surface protein expressed in lymphatic endothelium and stromal cells of spleen and thymus. The 10.1.1 Ab-affinity chromatography also purified LFA-1, an integrin that mediates leukocyte adhesion to endothelium. This mCLCA1-LFA-1 interaction has functional consequences, as lymphocyte adhesion to lymphatic endothelium was blocked by 10.1.1 Ab bound to endotheliumor by LFA-1 Ab bound to lymphocytes. Lymphocyte adhesion was increased by cytokine treatment of lymphatic endothelium in association with increased expression of ICAM-1, an endothelial surface protein that is also a ligand for LFA-1. By contrast, mCLCA1 expression and the relative contribution of mCLCA1 to lymphocyte adhesion were unaffected by cytokine activation, demonstrating that mCLCA1 and ICAM-1 interactions with LFA-1 are differentially regulated. mCLCA1 also bound to the LFA-1-related Mac-1 integrin that is preferentially expressed on leukocytes. mCLCA1-mediated adhesion of Mac-1- or LFA-1-expressing leukocytes to lymphatic vessels and lymph node lymphatic sinuses provides a target for investigation of lymphatic involvement in leukocyte adhesion and trafficking during the immune response.     

Rolling of Th1 cells via P-selectin glycoprotein ligand-1 stimulates LFA-1-mediated cell binding to ICAM-1

Activated T cells migrate from the blood into nonlymphoid tissues through a multistep process that involves cell rolling, arrest, and transmigration. P-Selectin glycoprotein ligand-1 (PSGL-1) is a major ligand for P-selectin expressed on subsets of activated T cells such as Th1 cells and mediates cell rolling on vascular endothelium. Rolling cells are arrested through a firm adhesion step mediated by integrins. Although chemokines presented on the endothelium trigger integrin activation, a second mechanism has been proposed where signaling via rolling receptors directly activates integrins. In this study, we show that Ab-mediated cross-linking of the PSGL-1 on Th1 cells enhances LFA-1-dependent cell binding to ICAM-1. PSGL-1 cross-linking did not enhance soluble ICAM-1 binding but induced clustering of LFA-1 on the cell surface, suggesting that an increase in LFA-1 avidity may account for the enhanced binding to ICAM-1. Combined stimulation by PSGL-1 cross-linking and the Th1-stimulating chemokine CXCL10 or CCL5 showed a more than additive effect on LFA-1-mediated Th1 cell adhesion as well as on LFA-1 redistribution on the cell surface. Moreover, PSGL-1-mediated rolling on P-selectin enhanced the Th1 cell accumulation on ICAM-1 under flow conditions. PSGL-1 cross-linking induced activation of protein kinase C isoforms, and the increased Th1 cell adhesion observed under flow and also static conditions was strongly inhibited by calphostin C, implicating protein kinase C in the intracellular signaling in PSGL-1-mediated LFA-1 activation. These results support the idea that PSGL-1-mediated rolling interactions induce intracellular signals leading to integrin activation, facilitating Th1 cell arrest and subsequent migration into target tissues.     

Differential utilization of ICAM-1 and VCAM-1 during the adhesion and transendothelial migration of human T lymphocytes.

The comparative roles of the endothelial cell (EC) adhesion receptors VCAM-1 and ICAM-1 during the adhesion and transendothelial migration of T cells were examined. The adhesion of T cells to IL-1-activated EC was markedly, but not completely, inhibited by mAb to VCAM-1 as well as to its counter-receptor, VLA-4, whereas, T cell binding to IL-1-activated EC was not blocked by mAb to ICAM-1 or to its counter-receptor, LFA-1. In contrast, LFA-1/ICAM-1, but not VLA-4/VCAM-1, mediated much, but not all, of the binding of T cells to unstimulated EC. Activation of T cells with phorbol dibutyrate and ionomycin alter the receptor-counter-receptor pairs used for binding to EC. Regardless of the activation status of the EC, the binding of activated T cells was not blocked by mAb to VLA-4 or VCAM-1. Moreover, the binding of activated T cells to EC was blocked to a lesser degree by mAb to LFA-1 than that of resting T cells, and mAb to ICAM-1 blocked binding only modestly. The role of VCAM-1 and ICAM-1 during the transendothelial migration of T cells was also examined. Regardless of the activation status of the T cells or the EC, VCAM-1 was never found to function during transendothelial migration, even when it mediated the binding of resting T cells to IL-1-activated EC. In contrast, ICAM-1 played an important role in transendothelial migration under all of the conditions examined, including situations when T cell-EC binding was not mediated by ICAM-1. Immunoelectron microscopic analysis of transendothelial migration supported the conclusion that ICAM-1 but not VCAM-1 played a central role in this process. Thus, ICAM-1 was prominently and uniformly expressed at all EC membrane sites that were in contact with bound and migrating T cells, whereas VCAM-1 was localized to the luminal surface of IL-1-activated EC, but was often absent from the surface of the EC in contact with T cells undergoing transendothelial migration. These studies confirm that ICAM-1 and VCAM-1 play reciprocal roles in the binding of resting T cells to resting and IL-1-activated EC, respectively, but a less prominent role in the binding of activated T cells. Moreover, ICAM-1 but not VCAM-1 plays a role in transendothelial migration, regardless of the receptor-counter-receptor pairs used for initial binding.     

Interplay between rolling and firm adhesion elucidated with a cell-free system engineered with two distinct receptor-ligand pairs

The firm arrest of leukocytes to the endothelium during inflammation is known to be mediated by endothelial intercellular adhesion molecules (ICAMs) binding to activated integrins displayed on leukocyte surface. Selectin-ligand interactions, which mediate rolling, are believed to be important for facilitating firm adhesion, either by activating integrins or by facilitating the transition to firm adhesion by making it easier for integrins to bind. Although leukocytes employ two distinct adhesion molecules that mediate different states of adhesion, the fundamental biophysical mechanisms by which two pairs of adhesion molecules facilitate cell adhesion is not well understood. In this work, we attempt to understand the interaction between two molecular systems using a cell-free system in which polystyrene microspheres functionalized with the selectin ligand, sialyl Lewis(X) (sLe(X)), and an antibody against ICAM-1, aICAM-1, are perfused over P-selectin/ICAM-1 coated surfaces in a parallel plate flow chamber. Separately, sLe(X)/P-selectin interactions support rolling and aICAM-1/ICAM-1 interactions mediate firm adhesion. Our results show that sLe(X)/aICAM-1 microspheres will firmly adhere to P-selectin/ICAM-1 coated surfaces, and that the extent of firm adhesion of microspheres is dependent on wall shear stress within the flow chamber, sLe(X)/aICAM-1 microsphere site density, and P-selectin/ICAM-1 surface density ratio. We show that P-selectin's interaction with sLe(X) mechanistically facilitates firm adhesion mediated by antibody binding to ICAM-1: the extent of firm adhesion for the same concentration of aICAM-1/ICAM-1 interaction is greater when sLe(X)/P-selectin interactions are present. aICAM-1/ICAM-1 interactions also stabilize rolling by increasing pause times and decreasing average rolling velocities. Although aICAM-1 is a surrogate for beta(2)-integrin, the kinetics of association between aICAM-1 and ICAM-1 is within a factor of 1.5 of activated integrin binding ICAM-1, suggesting the findings from this model system may be insightful to the mechanism of leukocyte firm adhesion. In particular, these experimental results show how two molecule systems can interact to produce an effect not achievable by either system alone, a fundamental mechanism that may pervade leukocyte adhesion biology.     

Vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells

Vascular adhesion protein-1 (VAP-1) is an amine oxidase and adhesion receptor that is expressed by endothelium in the human liver. The hepatic sinusoids are perfused by blood at low flow rates, and sinusoidal endothelium lacks selectin expression and has low levels of CD31, suggesting that VAP-1 may play a specific role in lymphocyte recruitment to the liver. In support of this we now report the constitutive expression of VAP-1 on human hepatic sinusoidal endothelial cells (HSEC) in vitro and demonstrate that VAP-1 supports adhesion and transmigration of lymphocytes across these cells under physiological shear stress. These are the first studies to report the function of VAP-1 on primary human endothelial cells. Under static conditions lymphocyte adhesion to unstimulated HSEC was dependent on VAP-1 and ICAM-2, whereas adhesion to TNF-alpha-stimulated HSEC was dependent on ICAM-1, VCAM-1, and VAP-1. Under conditions of flow, blocking VAP-1 reduced lymphocyte adhesion to TNF-alpha-treated HSEC by 50% and significantly reduced the proportion of adherent lymphocytes that transmigrated across cytokine or LPS-activated endothelium. In addition, inhibition of the amine oxidase activity of VAP-1 reduced both adhesion and transmigration of lymphocytes to a level similar to that seen with VAP-1 Ab. Thus, VAP-1 can support transendothelial migration as well as adhesion, and both functions are dependent on its enzymatic activity. In the absence of selectins and CD31, VAP-1 may play a specific role in lymphocyte recruitment via hepatic sinusoidal endothelium. Moreover, since VAP-1 is induced on nonhepatic endothelium in response to inflammation, its ability to support lymphocyte transendothelial migration may be an important systemic function of VAP-1.     

Migration of human hematopoietic progenitor cells across bone marrow endothelium is regulated by vascular endothelial cadherin.

The success of stem cell transplantation depends on the ability of i.v. infused stem cells to engraft the bone marrow, a process referred to as homing. Efficient homing requires migration of CD34(+) cells across the bone marrow endothelium, most likely through the intercellular junctions. In this study, we show that loss of vascular endothelial (VE)-cadherin-mediated endothelial cell-cell adhesion increases the permeability of monolayers of human bone marrow endothelial cells (HBMECs) and stimulates the transendothelial migration of CD34(+) cells in response to stromal cell-derived factor-1alpha. Stromal cell-derived factor-1alpha-induced migration was dependent on VCAM-1 and ICAM-1, even in the absence of VE-cadherin function. Cross-linking of ICAM-1 to mimic the leukocyte-endothelium interaction induced actin stress fiber formation but did not induce loss of endothelial integrity, whereas cross-linking of VCAM-1 increased the HBMEC permeability and induced gaps in the monolayer. In addition, VCAM-1-mediated gap formation in HBMEC was accompanied by and dependent on the production of reactive oxygen species. These data suggest that modulation of VE-cadherin function directly affects the efficiency of transendothelial migration of CD34(+) cells and that activation of ICAM-1 and, in particular, VCAM-1 plays an important role in this process through reorganization of the endothelial actin cytoskeleton and by modulating the integrity of the bone marrow endothelium through the production of reactive oxygen species.     

Rho-GTPase signaling in leukocyte extravasation

Leukocyte transendothelial migration (TEM) is one of the crucial steps during inflammation. A better understanding of the key molecules that regulate leukocyte extravasation aids to the development of novel therapeutics for treatment of inflammation-based diseases, such as atherosclerosis and rheumatoid arthritis. The adhesion molecules ICAM-1 and VCAM-1 are known as central mediators of TEM. Clustering of these molecules by their leukocytic integrins initiates the activation of several signaling pathways within the endothelium, including a rise in intracellular Ca²⁺, activation of several kinase cascades, and the activation of Rho-GTPases. Activation of Rho-GTPases has been shown to control adhesion molecule clustering and the formation of apical membrane protrusions that embrace adherent leukocytes during TEM. Here, we discuss the potential regulatory mechanisms of leukocyte extravasation from an endothelial point of view, with specific focus on the role of the Rho-GTPases.     

Rho-GTPase signaling in leukocyte extravasation: An endothelial point of view

Leukocyte transendothelial migration (TEM) is one of the crucial steps during inflammation. A better understanding of the key molecules that regulate leukocyte extravasation aids to the development of novel therapeutics for treatment of inflammation-based diseases, such as atherosclerosis and rheumatoid arthritis. The adhesion molecules ICAM-1 and VCAM-1 are known as central mediators of TEM. Clustering of these molecules by their leukocytic integrins initiates the activation of several signaling pathways within the endothelium, including a rise in intracellular Ca²⁺, activation of several kinase cascades, and the activation of Rho-GTPases. Activation of Rho-GTPases has been shown to control adhesion molecule clustering and the formation of apical membrane protrusions that embrace adherent leukocytes during TEM. Here, we discuss the potential regulatory mechanisms of leukocyte extravasation from an endothelial point of view, with specific focus on the role of the Rho-GTPases.