Trophoblast migration under flow is regulated by endothelial cells

During pregnancy, trophoblasts enter the uterine vasculature and are found in spiral arteries far upstream of uterine capillaries. It is unknown whether trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by intravasation directly into spiral arteries after interstitial migration. We have developed an in vitro system consisting of early gestation macaque monkey trophoblasts cocultured with uterine endothelial cells and have exposed the cells in a parallel plate flow chamber to physiological levels of shear stress. Videomicroscopy followed by quantitative image analysis revealed that the migratory activity (expressed as average displacement and average migration velocity) of trophoblasts cultured on top of endothelial cells remained unchanged between shear stresses of 1-30 dyne/cm(2) whereas activity of trophoblasts alone increased with increasing shear stress. When the direction of migration was assessed at 1 and 7.5 dyne/cm(2), the extent of migration against and with flow was roughly equal for both trophoblasts alone and cocultured trophoblasts. At shear stress levels of 15 and 30 dyne/cm(2), trophoblasts incubated alone showed a significant decrease in migration against flow and corresponding increased migration in the direction of flow. In contrast, trophoblasts cocultured with uterine endothelial cells maintained the same extent of migration against flow at all shear stress levels. Migration against flow was also maintained when trophoblasts were cultured with endothelial cell-conditioned medium or fixed endothelial cells. The results indicate that factors expressed on the surface of uterine endothelial cells and factors released by endothelial regulate trophoblast migration under flow.     

Regulation of trophoblast beta1-integrin expression by contact with endothelial cells

Background  

In human and non-human primates, migratory trophoblasts penetrate the uterine epithelium, invade uterine matrix, and enter the uterine vasculature. Invasive trophoblasts show increased expression of β1 integrin. Since trophoblast migration within the uterine vasculature involves trophoblast attachment to endothelial cells lining the vessel walls, this raises the possibility that cell-cell contact and/or factors released by endothelial cells could regulate trophoblast integrin expression. To test this, we used an in vitro system consisting of early gestation macaque trophoblasts co-cultured on top of uterine microvascular endothelial cells.     

Macaque trophoblast migration is regulated by RANTES

In human and non-human primates, migratory trophoblasts penetrate the uterine epithelium, invade the endometrium, enter the uterine vasculature, and migrate within the arteries. The mechanisms that regulate this directional migration are unknown. We have used early gestation macaque trophoblasts to test the hypothesis that trophoblast migration is regulated by the chemokine, Regulated on Activation T-Cell Expressed and Secreted (RANTES). Immunohistochemical analysis of cryosections of endometrial tissue showed expression of RANTES by stromal cells and vascular cells. Isolated endothelial cells expressed RANTES as determined by immunocytochemistry and RT-PCR analyses. Immunohistochemical analysis of endometrial cryosections showed that the RANTES receptor, CCR5, was expressed by trophoblasts on anchoring villi and by cells within the trophoblastic shell. Cytokeratin-positive/CCR5-positive cells, consistent with trophoblasts, were also found scattered within the stroma and were often clustered around blood vessels. Isolated trophoblast cells expressed CCR5 as determined by immunocytochemistry and RT-PCR analyses. Isolated trophoblasts migrated towards RANTES when cultured in migration chambers and migration was reduced in the presence of anti-CCR5 antibody. When trophoblasts were cultured on dishes coated with recombinant RANTES, expression of beta1 integrin was increased. The RANTES-induced increase in beta1 integrin expression was inhibited by pertussis toxin. These data suggest a role for RANTES and CCR5 in the regulation of trophoblast migration within the endometrium and within the uterine vasculature.     

Trophoblast interactions with endothelial cells are increased by interleukin-1beta and tumour necrosis factor alpha and involve vascular cell adhesion molecule-1 and alpha4beta1

Interactions between fetal extravillous trophoblast cells and maternal uterine cells are of critical importance in successful placentation. In the first trimester, trophoblasts invade the uterine environment and reach the spiral arteries where they interact with vascular cells; however, little is known of the nature of these interactions. We have developed a fluorescent binding assay to investigate the contact between trophoblasts and endothelial cells and to determine its regulation by cytokines and adhesion molecules. Stimulation of an endothelial cell line (SGHEC-7) with interleukin-1beta or tumour necrosis factor-alpha significantly increased adhesion of the first-trimester extravillous trophoblast-derived cell line, SGHPL-4. Using blocking antibodies, vascular cell adhesion molecule-1 (VCAM-1) and integrin alpha4beta1 (VLA-4), but not intercellular adhesion molecule-1 (ICAM-1), were shown to be important in trophoblast binding to activated endothelial cells. SGHPL-4 cells were shown to express HLA-G, alpha4beta1 and ICAM-1 at high levels and LFA-1 and VCAM-1 at lower levels. ICAM-1 and VCAM-1 are expressed on SGHEC-7 cells and their expression was confirmed on primary decidual endothelial cells. In conclusion, we have demonstrated the importance of VCAM-1 and alpha4beta1 in trophoblasts-endothelial interactions. Improved knowledge of the nature of these fetal-maternal interactions will have implications for understanding situations when placentation is compromised.     

Vitronectin receptors are expressed by macaque trophoblast cells and play a role in migration and adhesion to endothelium.

The objective of this work was to develop an in vitro system that would extend the usefulness of the macaque as a model for studying trophoblast invasion and spiral artery modification. We sought to determine whether trophoblast cells isolated from early gestation macaque placentas expressed vitronectin receptors and tested the idea that these receptors play a role in trophoblast migration and adhesion. Cytotrophoblast cells were isolated from 40-100 day macaque placentas, cultured, and characterized by immunofluorescence microscopy and flow cytometry. The cells expressed alphaV, beta3, and beta1 integrins on their surfaces. Immunohistochemical analysis of early gestation placentas and decidua basalis confirmed that intravascular trophoblast cells express alphaVbeta3/beta5. Using migration chambers we found that the trophoblast cells migrated towards vitronectin but not towards bovine serum albumin. This specific migration was blocked by preincubating the trophoblast cells with anti-vitronectin receptor (alphaVbeta3/beta5) antibodies. In other experiments, macaque trophoblast cells adhered to myometrial endothelial cells in a time-dependent manner and adhesion was significantly blocked by antibodies against alphaVbeta3/beta5 integrin. The results suggest that vitronectin receptors expressed by macaque trophoblast cells play a role in the migratory activity of these cells and may also be important in mediating attachment to endothelium.     

Filamin B mediates ICAM-1-driven leukocyte transendothelial migration

During inflammation, the endothelium mediates rolling and firm adhesion of activated leukocytes. Integrin-mediated adhesion to endothelial ligands of the Ig-superfamily induces intracellular signaling in endothelial cells, which promotes leukocyte transendothelial migration. We identified the actin cross-linking molecule filamin B as a novel binding partner for intracellular adhesion molecule-1 (ICAM-1). Immune precipitation as well as laser scanning confocal microscopy confirmed the specific interaction and co-localization of endogenous filamin B with ICAM-1. Importantly, clustering of ICAM-1 promotes the ICAM-1-filamin B interaction. To investigate the functional consequences of filamin B binding to ICAM-1, we used small interfering RNA to reduce filamin B expression in ICAM-1-GFP expressing HeLa cells. We found that filamin B is required for the lateral mobility of ICAM-1 and for ICAM-1-induced transmigration of leukocytes. Reducing filamin B expression in primary human endothelial cells resulted in reduced recruitment of ICAM-1 to endothelial docking structures, reduced firm adhesion of the leukocytes to the endothelium, and inhibition of transendothelial migration. In conclusion, this study identifies filamin B as a molecular linker that mediates ICAM-1-driven transendothelial migration.     

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.     

NF-kappaB activation for constitutive expression of VCAM-1 and ICAM-1 on B lymphocytes and plasma cells.

Cytokine stimulation can activate NF-kappaB that triggers inducible expression of E-selectin, VCAM-1 (Vascular Cell Adhesion Molecule-1) and ICAM-1 (Intercellular Cell Adhesion Molecule-1) in endothelial cells. In the previous study, we have shown that B lymphocytes and plasma cells can express E-selectin by constitutive activation of NF-kappaB. Here we show that human B lymphocytes and ARH-77 plasma cells expressed VCAM-1 and ICAM-1 in a cytokine dispensable mechanism. NF-kappaB antagonists could inhibit their expressions in ARH-77 cells. The activities of NF-kappaB for VCAM-1 and ICAM-1 promoters prior to cytokine stimulation were detected in ARH-77 cells using electrophoretic mobility shift assays. Again, NF-kappaB antagonists could abrogate these promoter activities. Taken together, our results demonstrate that NF-kappaB activation is the underlying molecular mechanism for constitutive expression of E-selectin, VCAM-1, and ICAM-1 on human B lymphocytes and plasma cells.     

Neutrophil transmigration under shear flow conditions in vitro is junctional adhesion molecule-C independent

Endothelial cell junctional adhesion molecule (JAM)-C has been proposed to regulate neutrophil migration. In the current study, we used function-blocking mAbs against human JAM-C to determine its role in human leukocyte adhesion and transendothelial cell migration under flow conditions. JAM-C surface expression in HUVEC was uniformly low, and treatment with inflammatory cytokines TNF-alpha, IL-1beta, or LPS did not increase its surface expression as assessed by FACS analysis. By immunofluorescence microscopy, JAM-C staining showed sparse localization to cell-cell junctions on resting or cytokine-activated HUVEC. Surprisingly, staining of detergent-permeabilized HUVEC revealed a large intracellular pool of JAM-C that showed little colocalization with von Willebrand factor. Adhesion studies in an in vitro flow model showed that functional blocking JAM-C mAb alone had no inhibitory effect on polymorphonuclear leukocyte (PMN) adhesion or transmigration, whereas mAb to ICAM-1 significantly reduced transmigration. Interestingly, JAM-C-blocking mAbs synergized with a combination of PECAM-1, ICAM-1, and CD99-blocking mAbs to inhibit PMN transmigration. Overexpression of JAM-C by infection with a lentivirus JAM-C GFP fusion protein did not increase adhesion or extent of transmigration of PMN or evoke a role for JAM-C in transendothelial migration. These data suggest that JAM-C has a minimal role, if any, in PMN transmigration in this model and that ICAM-1 is the preferred endothelial-expressed ligand for PMN beta(2) integrins during transendothelial migration.     

Tumor cell/endothelial cell tight contact upregulates endothelial adhesion molecule expression mediated by NFκB: Differential role of the shear stress

Cancer metastasis is a multistep process involving cell-cell interactions, but little is known about the adhesive interactions and signaling events during extravasation of tumor cells (TCs). In this study, cell adhesion molecule (CAM) expression was investigated using an in vitro assay, in which TCs were seeded onto an endothelial cell (ECs) monolayer and cocultured during 5 h. Flow cytometry, confocal microscopy as well as western blot analysis indicated that endothelial ICAM-1 (Inter Cellular Adhesion Molecule-1), VCAM-1 (Vascular Adhesion Molecule-1) and E-selectin were up-regulated after TC-EC coculture, whereas no change was observed for CAMs expression in tumor cells. This increased CAMs expression required tight contact between TCs and ECs. Incubation of ECs with the pyrrolidine-dithiocarbamate NFκB inhibitor prior to coculture, fully prevented coculture-induced expression of endothelial CAMs. Using specific blocking antibodies we showed an implication of ICAM-1 and VCAM-1 for TCs extravasation and VCAM-1 for adhesion. Moreover, fluid flow experiments revealed that high shear stress totally abolished coculture-induced as well as TNFα-induced CAMs over-expression. This study suggests that TCs could act as a potent inflammatory stimulus on ECs by inducing CAMs expression via NFκB activation, and that this action can be modulated by shear stress.     

ICAM-2 and lipid rafts disappear from the basal plasma membrane of uterine epithelial cells during early pregnancy in rats

Adhesion molecules are redistributed in rat uterine epithelial cells (UECs) during early pregnancy for endometrial receptivity and implantation. Intercellular adhesion molecule-2 (ICAM-2) is located as an oligomer on the basal plasma membrane of non-receptive UECs on day 1 of pregnancy and colocalizes with the lipid raft marker flotillin-2. At the time of implantation in rats and in ovariectomized rats primed with progesterone, ICAM-2 disappears from the basal plasma membrane and lipid rafts redistribute to the apical membrane. The loss of ICAM-2 might render UECs less adherent to the underlying basal lamina and more prone to apoptosis. Flotillin-2 in the apical plasma membrane at the time of implantation might provide an anchoring point for several adhesion molecules that are known to localize to this region at this time. We suggest that flotillin-2 is involved with adhesion between UECs and the implanting blastocyst, whereas ICAM-2 is associated with the ability for UECs to be removed at the time of implantation.     

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.     

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.     

Membrane-type 1-matrix metalloproteinase regulates intracellular adhesion molecule-1 (ICAM-1)-mediated monocyte transmigration

We examined the mechanism regulating intercellular cell adhesion molecule-1 (ICAM-1)-dependent monocyte transendothelial migration. Monocyte migration through endothelial cells expressing ICAM-1 alone was comparable to that of tumor necrosis factor-alpha-treated cells. Transmigration was reduced in ICAM-1 lacking the cytoplasmic tail and in tyrosine to alanine substitutions at Tyr-485 and Tyr-474. Tissue inhibitors of matrix metalloproteinases (TIMPs) -2 and -3 blocked transmigration, whereas TIMP-1 was ineffective. This profile suggested a role for membrane-type matrix metalloproteinases (MT-MMPs) in transmigration. Inhibitory antibodies and small interference RNA directed against MT1-MMP blocked transmigration, whereas overexpression of MT1-MMP in endothelial cells or monocytes promoted transmigration. MT1-MMP mediated the ectodomain cleavage of ICAM-1 that was blocked by TIMP-2 and -3. Overexpression of MT1-MMP rescued function in ICAM-1Y485A, and to a lesser extent in the cytoplasmic tail-deleted ICAM-1. In a binding assay, wild-type ICAM-1 bound to purified MT1-MMP while ICAM-1 mutants bound poorly. MT1-MMP co-localized with ICAM-1 at distinct structures in endothelial cells. MT1-MMP localization with cells expressing ICAM-1 mutations was reduced and diffused. These results indicate that the cytoplasmic tail of ICAM-1 regulates leukocyte transmigration through MT1-MMP interaction.     

Expression and polarization of intercellular adhesion molecule-1 on human intestinal epithelia: consequences for CD11b/CD18-mediated interactions with neutrophils.

BACKGROUND: Epithelial dysfunction and patient symptoms in inflammatory intestinal diseases such as ulcerative colitis and Crohn's disease correlate with migration of neutrophils (PMN) across the intestinal epithelium. In vitro modeling of PMN transepithelial migration has revealed distinct differences from transendothelial migration. By using polarized monolayers of human intestinal epithelia (T84), PMN transepithelial migration has been shown to be dependent on the leukocyte integrin CD11b/CD18 (Mac-1), but not on CD11a/CD18 (LFA-1). Since intercellular adhesion molecule-I (ICAM-1) is an important endothelial counterreceptor for these integrins, its expression in intestinal epithelia and role in PMN-intestinal epithelial interactions was investigated. MATERIALS AND METHODS: A panel of antibodies against different domains of ICAM-1, polarized monolayers of human intestinal epithelia (T84), and natural human colonic epithelia were used to examine the polarity of epithelial ICAM-1 surface expression and the functional role of ICAM-1 in neutrophil-intestinal epithelial adhesive interactions. RESULTS: While no surface expression of ICAM-1 was detected on unstimulated T84 cells, interferon-gamma (IFN gamma) elicited a marked expression of ICAM-1 that selectively polarized to the apical epithelial membrane. Similarly, apically restricted surface expression of ICAM-1 was detected in natural human colonic epithelium only in association with active inflammation. With or without IFN gamma pre-exposure, physiologically directed (basolateral-to-apical) transepithelial migration of PMN was unaffected by blocking monoclonal antibodies (mAbs) to ICAM-1. In contrast, PMN migration across IFN gamma-stimulated monolayers in the reverse (apical-to-basolateral) direction was inhibited by anti-ICAM-1 antibodies. Adhesion studies revealed that T84 cells adhered selectively to purified CD11b/CD18 and such adherence, with or without IFN gamma pre-exposure, was unaffected by ICAM-1 mAb. Similarly, freshly isolated epithelial cells from inflamed human intestine bound to CD11b/CD18 in an ICAM-1-independent fashion. CONCLUSIONS: These data indicate that ICAM-1 is strictly polarized in intestinal epithelia and does not represent a counterreceptor for neutrophil CD11b/CD18 during physiologically directed transmigration, but may facilitate apical membrane-PMN interactions after the arrival of PMN in the intestinal lumen.     

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.     

Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecule-1 (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells

Intercellular adhesion molecule-1 (ICAM-1) on the surface of cultured umbilical vein and saphenous vein endothelial cells was upregulated between 2.5- and 40-fold by rIL-1, rTNF, LPS and rIFN gamma corresponding to up to 5 X 10(6) sites/cell. Endothelial cell ICAM-1 was a single band of 90 kD in SDS-PAGE. Purified endothelial cell ICAM-1 reconstituted into liposomes and bound to plastic was an excellent substrate for both JY B lymphoblastoid cell and T lymphoblast adhesion. Adhesion to endothelial cell ICAM-1 in planar membranes was blocked completely by monoclonal antibodies to lymphocyte function associated antigen-1 (LFA-1) or ICAM-1. Adhesion to artificial membranes was most sensitive to ICAM-1 density within the physiological range found on resting and stimulated endothelial cells. Adhesion of JY B lymphoblastoid cells, normal and genetically LFA-1 deficient T lymphoblasts and resting peripheral blood lymphocytes to endothelial cell monolayers was also assayed. In summary, LFA-1 dependent (60-90% of total adhesion) and LFA-1-independent basal adhesion was observed and the use of both adhesion pathways by different interacting cell pairs was increased by monokine or lipopolysaccharide stimulation of endothelial cells. The LFA-1-dependent adhesion could be further subdivided into an LFA-1/ICAM-1-dependent component which was increased by cytokines and a basal LFA-1-dependent, ICAM-1-independent component which did not appear to be affected by cytokines. We conclude that ICAM-1 is a regulated ligand for lymphocyte-endothelial cell adhesion, but at least two other major adhesion pathways exist.     

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.     

Effect of shear stress on migration and integrin expression in macaque trophoblast cells

During fetal development, trophoblast cells enter endometrial capillaries, migrate within the uterine vasculature, and eventually reside within spiral arteries of the uterus. This invasive activity is accompanied by upregulation of trophoblast beta1 integrin expression. Fluid mechanical shear stress regulates migration and expression of adhesion molecules in vascular endothelial cells, but nothing is known about the effects of shear stress on trophoblast cells. We tested the hypothesis that shear stress regulates the motility and beta1 integrin expression of trophoblast cells. Early gestation macaque trophoblast cells were cultured in 1 x 1-mm square cross-section capillary tubes within which the flow field was determined using three-dimensional computational fluid dynamic simulations. Trophoblast cells in the capillary tubes were exposed to a steady shear stress of 7.5, 15, or 30 dyn/cm2 for up to 24 h. In the absence of flow, trophoblast cells were highly dynamic with constant nondirectional positional shifts but with no net cell migration. Exposure of the cells to shear stress within 24-72 h of cell plating significantly increased the level of this activity and led to net cell migration in the direction of flow. Shear stress also increased the expression and altered the topography of beta1 integrin. These results suggest that shear stress regulates trophoblast motility and beta1 integrin expression in vitro.     

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.