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Francis W Luscinskas - One of the best experts on this subject based on the ideXlab platform.
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reduced expression of junctional adhesion molecule and platelet endothelial cell adhesion molecule 1 cd31 at human vascular endothelial junctions by cytokines tumor necrosis factor α plus interferon γ does not reduce leukocyte Transmigration under fl
American Journal of Pathology, 2001Co-Authors: Sunil K Shaw, Brandy N Perkins, Yawchyn Lim, Yuan Liu, Asma Nusrat, Frederick J Schnell, Charles A Parkos, Francis W LuscinskasAbstract:The combination of tumor necrosis factor (TNF)-α plus interferon (IFN)-γ has been shown previously to promote redistribution of platelet/endothelial cell adhesion molecule-1 (PECAM-1) (CD31), junctional adhesion molecule (JAM), and VE-cadherin away from lateral junctions of human umbilical vein endothelial cell monolayers. In parallel, neutrophil Transmigration was significantly reduced. Because PECAM-1 and JAM have been implicated in leukocyte Transmigration, the observed redistribution by cytokine activation was presumed to represent the mechanism causing decreased Transmigration under static conditions. The current results confirm that culture of human umbilical vein endothelial cells with TNF-α plus IFN-γ caused a decrease in surface-expressed and junctional-localized JAM and PECAM-1, but did not cause decreased leukocyte Transmigration in an in vitro flow assay. Furthermore, blocking monoclonal antibody to PECAM-1 still significantly reduced monocyte Transmigration, demonstrating that it retains a functional role even though its levels were reduced and redistributed away from junctions, whereas a panel of monoclonal antibodies to JAM failed to reduce leukocyte Transmigration. Given the alterations in junction protein location, permeability function was assessed. IFN-γ alone or TNF-α plus IFN-γ significantly increased permeability, but TNF-α alone did not, suggesting lack of correlation between Transmigration and loss of permeability. In conclusion, cytokine activation induced loss and redistribution of PECAM-1 and JAM away from lateral junctions, but per se does not negatively regulate either neutrophil or monocyte Transmigration under flow.
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real time imaging of vascular endothelial cadherin during leukocyte Transmigration across endothelium
Journal of Immunology, 2001Co-Authors: Sunil K Shaw, Paul S Bamba, Brandy N Perkins, Francis W LuscinskasAbstract:Vascular endothelial-cadherin (VE-cadherin) is a component of the adherens junctions of endothelial cells whose role in endothelial Transmigration of leukocytes has been controversial. Using a VE-cadherin/green fluorescent protein fusion construct (VEcadGFP) that mimics the native molecule, we visualized alterations in endothelial junctional structure in real time during Transmigration of human neutrophils and monocytes in an in vitro flow model. We observed abundant Transmigration occurring exclusively at the cell borders (paracellularly). Surprisingly, Transmigration occurred both through de novo formation of transient gaps in VEcadGFP junctional distribution, and also through preexisting gaps. De novo gaps 4–6 μm in size were formed after a leukocyte arrived at a junction, whereas preexisting gaps were present even before the leukocyte had interacted with the endothelial cells contributing to a junction. Gaps rapidly resealed within 5 min after leukocyte Transmigration. Migrating leukocytes appeared to push aside VEcadGFP in the plane of the junction, and this displaced material subsequently diffused back to refill the junction. To our knowledge, this is the first example where molecular events at the lateral junction have been tracked in real time during Transmigration.
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endothelial dependent mechanisms regulate leukocyte Transmigration a process involving the proteasome and disruption of the vascular endothelial cadherin complex at endothelial cell to cell junctions
Journal of Experimental Medicine, 1997Co-Authors: Jennifer R Allport, Han Ding, Tucker Collins, Mary E Gerritsen, Francis W LuscinskasAbstract:Although several adhesion molecules expressed on leukocytes (β1 and β2 integrins, platelet endothelial cell adhesion molecule 1 [PECAM-1], and CD47) and on endothelium (intercellular adhesion molecule 1, PECAM-1) have been implicated in leukocyte transendothelial migration, less is known about the role of endothelial lateral junctions during this process. We have shown previously (Read, M.A., A.S. Neish, F.W. Luscinskas, V.J. Palambella, T. Maniatis, and T. Collins. 1995. Immunity. 2:493–506) that inhibitors of the proteasome reduce lymphocyte and neutrophil adhesion and Transmigration across TNF-α–activated human umbilical vein endothelial cell (EC) monolayers in an in vitro flow model. The current study examined EC lateral junction proteins, principally the vascular endothelial (VE)–cadherin complex and the effects of proteasome inhibitors (MG132 and lactacystin) on lateral junctions during leukocyte adhesion, to gain a better understanding of the role of EC junctions in leukocyte Transmigration. Both biochemical and indirect immunofluorescence analyses of the adherens junction zone of EC monolayers revealed that neutrophil adhesion, not Transmigration, induced disruption of the VE–cadherin complex and loss of its lateral junction localization. In contrast, PECAM-1, which is located at lateral junctions and is implicated in neutrophil Transmigration, was not altered. These findings identify new and interrelated endothelial-dependent mechanisms for leukocyte Transmigration that involve alterations in lateral junction structure and a proteasome-dependent event(s).
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cytokine activated human endothelial monolayers support enhanced neutrophil Transmigration via a mechanism involving both endothelial leukocyte adhesion molecule 1 and intercellular adhesion molecule 1
Journal of Immunology, 1991Co-Authors: Francis W Luscinskas, Myron I Cybulsky, Jeanne Marie Kiely, Christopher S Peckins, Vannessa M Davis, Michael A GimbroneAbstract:rIL-1 beta treatment of cultured human endothelial cells (HEC) promotes polymorphonuclear leukocyte (PMN) adhesion and Transmigration. Using in vitro quantitative monolayer adhesion and videomicroscopic Transmigration assays, we have examined the contributions of endothelial-leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and the leukocyte adhesion complex, CD11/CD18, to these processes. Maximal enhancement of PMN adhesion and Transmigration were observed after 4 h of rIL-1 beta treatment, when surface expression of ELAM-1 had peaked and ICAM-1 was modestly increased. Blocking mAb directed to either ELAM-1 or ICAM-1 inhibited greater than 90% of the up-regulated PMN Transmigration. Blocking mAb directed to either CD11a/CD18 (LFA-1, a ICAM-1 counter-receptor), CD11b/CD18 (Mo-1), or CD18 (common beta 2-integrin) also blocked greater than 90% of PMN Transmigration. At later time points (24 or 48 h), ELAM-1 surface expression was markedly decreased, whereas ICAM-1 expression was increased over the 4-h level; PMN adhesion remained elevated (approximately 50 to 60% of 4 h level), but Transmigration returned to levels seen with unactivated HEC. These data indicate that PMN interaction with at least two distinct HEC adhesion molecules is necessary for transendothelial migration and suggests that PMN adhesion and Transmigration, although interrelated, are mechanistically distinct processes.
Sunil K Shaw - One of the best experts on this subject based on the ideXlab platform.
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endothelial cell cortactin coordinates intercellular adhesion molecule 1 clustering and actin cytoskeleton remodeling during polymorphonuclear leukocyte adhesion and Transmigration
Journal of Immunology, 2006Co-Authors: Lin Yang, Sunil K Shaw, Richard M Froio, Jennifer R Kowalski, Patrick W Yacono, Milan Bajmoczi, David E Golan, Sheila M ThomasAbstract:Endothelial cell ICAM-1 interacts with leukocyte β 2 integrins to mediate adhesion and transmit outside-in signals that facilitate leukocyte Transmigration. ICAM-1 redistribution and clustering appear necessary for leukocyte Transmigration, but the mechanisms controlling ICAM-1 redistribution and clustering have not been identified. We recently reported that Src kinase phosphorylation of endothelial cortactin regulates polymorphonuclear cell (PMN) Transmigration. In this study, we tested the hypotheses that the Src family kinase-cortactin pathway mediates association of ICAM-1 with the actin cytoskeleton and that this association is required for ICAM-1 clustering and leukocyte Transmigration. Cross-linking ICAM-1 induced cytoskeletal remodeling and a decrease in ICAM-1 lateral mobility, as assessed by fluorescence recovery after photobleaching. Cytoskeletal remodeling after ICAM-1 cross-linking was reduced by knockdown of cortactin by small interfering RNA, by expression of a cortactin mutant deficient in Src phosphorylation sites (cortactin3F), and by the Src kinase inhibitor PP2. Pretreatment of cytokine-activated human endothelial monolayers with cortactin small interfering RNA significantly decreased both actin and ICAM-1 clustering around adherent PMN and the formation of actin-ICAM-1 clusters required for PMN Transmigration. Our data suggest a model in which tyrosine phosphorylation of cortactin dynamically links ICAM-1 to the actin cytoskeleton, enabling ICAM-1 to form clusters and facilitate leukocyte Transmigration.
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coordinated redistribution of leukocyte lfa 1 and endothelial cell icam 1 accompany neutrophil Transmigration
Journal of Experimental Medicine, 2004Co-Authors: Sunil K Shaw, Yuan Liu, Asma Nusrat, Michael B Kim, Ravi M Rao, Charles U Hartman, Richard M Froio, Lin Yang, Todd Jones, Charles A ParkosAbstract:The leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) and its endothelial ligand intercellular adhesion molecule (ICAM)-1 play an important role in Transmigration as demonstrated by in vivo and in vitro models of inflammation. Despite the prominent role, little is known concerning the distribution and dynamic behavior of these adhesion molecules during leukocyte Transmigration. Therefore, we examined the spatial and temporal distribution of LFA-1 on neutrophils actively transmigrating tumor necrosis factor-α–activated human umbilical vein endothelial monolayers under shear flow. Upon neutrophil arrest, LFA-1 was evenly distributed. However, once neutrophils initiated Transmigration, LFA-1 rapidly redistributed to form a ringlike cluster at the neutrophil–endothelial junctional interface through which Transmigration occurred. As Transmigration was completed, LFA-1 redistributed to the neutrophil uropod. Endothelial ICAM-1 and JAM-A both colocalized with the ringlike LFA-1 cluster. Further analysis of PMA-stimulated neutrophils, which increase mobility of LFA-1, showed a rapid redistribution of LFA-1 and ICAM-1, but not endothelial JAM-A. Thus, endothelial JAM-A does not appear to contribute to adhesion or Transmigration in this system. This is the first demonstration that neutrophil LFA-1 rapidly redistributes to form a ringlike structure that coclusters with endothelial ICAM-1 as the neutrophil transmigrates.
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reduced expression of junctional adhesion molecule and platelet endothelial cell adhesion molecule 1 cd31 at human vascular endothelial junctions by cytokines tumor necrosis factor α plus interferon γ does not reduce leukocyte Transmigration under fl
American Journal of Pathology, 2001Co-Authors: Sunil K Shaw, Brandy N Perkins, Yawchyn Lim, Yuan Liu, Asma Nusrat, Frederick J Schnell, Charles A Parkos, Francis W LuscinskasAbstract:The combination of tumor necrosis factor (TNF)-α plus interferon (IFN)-γ has been shown previously to promote redistribution of platelet/endothelial cell adhesion molecule-1 (PECAM-1) (CD31), junctional adhesion molecule (JAM), and VE-cadherin away from lateral junctions of human umbilical vein endothelial cell monolayers. In parallel, neutrophil Transmigration was significantly reduced. Because PECAM-1 and JAM have been implicated in leukocyte Transmigration, the observed redistribution by cytokine activation was presumed to represent the mechanism causing decreased Transmigration under static conditions. The current results confirm that culture of human umbilical vein endothelial cells with TNF-α plus IFN-γ caused a decrease in surface-expressed and junctional-localized JAM and PECAM-1, but did not cause decreased leukocyte Transmigration in an in vitro flow assay. Furthermore, blocking monoclonal antibody to PECAM-1 still significantly reduced monocyte Transmigration, demonstrating that it retains a functional role even though its levels were reduced and redistributed away from junctions, whereas a panel of monoclonal antibodies to JAM failed to reduce leukocyte Transmigration. Given the alterations in junction protein location, permeability function was assessed. IFN-γ alone or TNF-α plus IFN-γ significantly increased permeability, but TNF-α alone did not, suggesting lack of correlation between Transmigration and loss of permeability. In conclusion, cytokine activation induced loss and redistribution of PECAM-1 and JAM away from lateral junctions, but per se does not negatively regulate either neutrophil or monocyte Transmigration under flow.
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real time imaging of vascular endothelial cadherin during leukocyte Transmigration across endothelium
Journal of Immunology, 2001Co-Authors: Sunil K Shaw, Paul S Bamba, Brandy N Perkins, Francis W LuscinskasAbstract:Vascular endothelial-cadherin (VE-cadherin) is a component of the adherens junctions of endothelial cells whose role in endothelial Transmigration of leukocytes has been controversial. Using a VE-cadherin/green fluorescent protein fusion construct (VEcadGFP) that mimics the native molecule, we visualized alterations in endothelial junctional structure in real time during Transmigration of human neutrophils and monocytes in an in vitro flow model. We observed abundant Transmigration occurring exclusively at the cell borders (paracellularly). Surprisingly, Transmigration occurred both through de novo formation of transient gaps in VEcadGFP junctional distribution, and also through preexisting gaps. De novo gaps 4–6 μm in size were formed after a leukocyte arrived at a junction, whereas preexisting gaps were present even before the leukocyte had interacted with the endothelial cells contributing to a junction. Gaps rapidly resealed within 5 min after leukocyte Transmigration. Migrating leukocytes appeared to push aside VEcadGFP in the plane of the junction, and this displaced material subsequently diffused back to refill the junction. To our knowledge, this is the first example where molecular events at the lateral junction have been tracked in real time during Transmigration.
Kimberly M. Stroka - One of the best experts on this subject based on the ideXlab platform.
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human neutrophil cytoskeletal dynamics and contractility actively contribute to trans endothelial migration
PLOS ONE, 2013Co-Authors: Kimberly M. Stroka, Heather N Hayenga, Helim ArandaespinozaAbstract:Transmigration through the endothelium is a key step in the immune response. In our recent work, the mechanical properties of the subendothelial matrix and biophysical state of the endothelium have been identified as key modulators of leukocyte trans-endothelial migration. Here, we demonstrated that neutrophil contractile forces and cytoskeletal dynamics also play an active biophysical role during Transmigration through endothelial cell-cell junctions. Using our previously-established model for leukocyte Transmigration, we first discovered that >93% of human neutrophils preferentially exploit the paracellular mode of Transmigration in our in vitro model, and that is independent of subendothelial matrix stiffness. We demonstrated that inhibition of actin polymerization or depolymerization completely blocks Transmigration, thus establishing a critical role for neutrophil actin dynamics in Transmigration. Next, inhibition of neutrophil myosin II-mediated contractile forces renders 44% of neutrophils incapable of retracting their trailing edge under the endothelium for several minutes after the majority of the neutrophil transmigrates. Meanwhile, inhibition of neutrophil contractile forces or stabilization of microtubules doubles the time to complete Transmigration for the first neutrophils to cross the endothelium. Notably, the time to complete Transmigration is significantly reduced for subsequent neutrophils that cross through the same path as a previous neutrophil and is less dependent on neutrophil contractile forces and microtubule dynamics. These results suggest that the first neutrophil induces a gap in endothelial cell-cell adhesions, which "opens the door" in the endothelium and facilitates Transmigration of subsequent neutrophils through the same hole. Collectively, this work demonstrates that neutrophils play an active biophysical role during the Transmigration step of the immune response.
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endothelial cell substrate stiffness influences neutrophil Transmigration via myosin light chain kinase dependent cell contraction
Blood, 2011Co-Authors: Kimberly M. Stroka, Helim ArandaespinozaAbstract:A vast amount of work has been dedicated to the effects of shear flow and cytokines on leukocyte Transmigration. However, no studies have explored the effects of substrate stiffness on Transmigration. Here, we investigated important aspects of endothelial cell contraction-mediated neutrophil Transmigration using an in vitro model of the vascular endothelium. We modeled blood vessels of varying mechanical properties using fibronectin-coated polyacrylamide gels of varying physiologic stiffness, plated with human umbilical vein endothelial cell (HUVEC) monolayers, which were activated with tumor necrosis factor-α. Interestingly, neutrophil Transmigration increased with increasing substrate stiffness below the endothelium. HUVEC intercellular adhesion molecule-1 expression, stiffness, cytoskeletal arrangement, morphology, and cell-substrate adhesion could not account for the dependence of Transmigration on HUVEC substrate stiffness. We also explored the role of cell contraction and observed that large holes formed in endothelium on stiff substrates several minutes after neutrophil Transmigration reached a maximum. Further, suppression of contraction through inhibition of myosin light chain kinase normalized the effects of substrate stiffness by reducing Transmigration and eliminating hole formation in HUVECs on stiff substrates. These results provide strong evidence that neutrophil Transmigration is regulated by myosin light chain kinase-mediated endothelial cell contraction and that this event depends on subendothelial cell matrix stiffness.
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Leukocyte Transmigration is Mediated by Endothelial Cell Contractile Forces and Substrate Stiffness
Biophysical Journal, 2011Co-Authors: Kimberly M. Stroka, Helim Aranda-espinozaAbstract:Leukocyte Transmigration through the vascular endothelium is a crucial step in the normal immune response. However, in the cardiovascular disease (CVD) of atherosclerosis, an excess of leukocytes adhere to and transmigrate through the endothelium, and progression of this disease is associated with arterial stiffening and variance in mechanical force transduction. In this study we investigated the mechanics of leukocyte Transmigration in an in vitro model of the vascular endothelium. We modeled healthy versus diseased blood vessels through manipulation of substrate stiffness using polyacrylamide gels, coated with extracellular matrix protein and plated with human umbilical vein endothelial cell (HUVEC) monolayers. The HUVEC monolayers were activated with tumor necrosis factor-alpha to mimic inflammatory conditions. We observed that leukocyte Transmigration through HUVEC monolayers increases with stiffness below the endothelium, and we hypothesized that substrate stiffness changes the biophysical properties of the endothelium to produce this effect. Using an array of biophysical techniques, we first evaluated the adhesion protein expression, stiffness, morphology, cytoskeletal arrangement, and cell-substrate adhesion of HUVEC monolayers as a function of substrate stiffness; however, none of these properties could account for the Transmigration behavior. We also explored the role of endothelial cell-cell adhesion and myosin light chain kinase (MLCK)-dependent endothelial cell contraction. We observed that (1) decreasing cell-cell adhesion increases Transmigration on soft substrates and (2) inhibition of MLCK and endothelial cell contraction normalizes the effects of substrate stiffness by reducing leukocyte Transmigration on stiff substrates without affecting Transmigration on soft substrates. These results provide strong evidence that neutrophil Transmigration is regulated by MLCK-mediated generation of gaps at cell borders through endothelial cell contractile forces which depend on substrate stiffness.
Helim Arandaespinoza - One of the best experts on this subject based on the ideXlab platform.
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human neutrophil cytoskeletal dynamics and contractility actively contribute to trans endothelial migration
PLOS ONE, 2013Co-Authors: Kimberly M. Stroka, Heather N Hayenga, Helim ArandaespinozaAbstract:Transmigration through the endothelium is a key step in the immune response. In our recent work, the mechanical properties of the subendothelial matrix and biophysical state of the endothelium have been identified as key modulators of leukocyte trans-endothelial migration. Here, we demonstrated that neutrophil contractile forces and cytoskeletal dynamics also play an active biophysical role during Transmigration through endothelial cell-cell junctions. Using our previously-established model for leukocyte Transmigration, we first discovered that >93% of human neutrophils preferentially exploit the paracellular mode of Transmigration in our in vitro model, and that is independent of subendothelial matrix stiffness. We demonstrated that inhibition of actin polymerization or depolymerization completely blocks Transmigration, thus establishing a critical role for neutrophil actin dynamics in Transmigration. Next, inhibition of neutrophil myosin II-mediated contractile forces renders 44% of neutrophils incapable of retracting their trailing edge under the endothelium for several minutes after the majority of the neutrophil transmigrates. Meanwhile, inhibition of neutrophil contractile forces or stabilization of microtubules doubles the time to complete Transmigration for the first neutrophils to cross the endothelium. Notably, the time to complete Transmigration is significantly reduced for subsequent neutrophils that cross through the same path as a previous neutrophil and is less dependent on neutrophil contractile forces and microtubule dynamics. These results suggest that the first neutrophil induces a gap in endothelial cell-cell adhesions, which "opens the door" in the endothelium and facilitates Transmigration of subsequent neutrophils through the same hole. Collectively, this work demonstrates that neutrophils play an active biophysical role during the Transmigration step of the immune response.
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endothelial cell substrate stiffness influences neutrophil Transmigration via myosin light chain kinase dependent cell contraction
Blood, 2011Co-Authors: Kimberly M. Stroka, Helim ArandaespinozaAbstract:A vast amount of work has been dedicated to the effects of shear flow and cytokines on leukocyte Transmigration. However, no studies have explored the effects of substrate stiffness on Transmigration. Here, we investigated important aspects of endothelial cell contraction-mediated neutrophil Transmigration using an in vitro model of the vascular endothelium. We modeled blood vessels of varying mechanical properties using fibronectin-coated polyacrylamide gels of varying physiologic stiffness, plated with human umbilical vein endothelial cell (HUVEC) monolayers, which were activated with tumor necrosis factor-α. Interestingly, neutrophil Transmigration increased with increasing substrate stiffness below the endothelium. HUVEC intercellular adhesion molecule-1 expression, stiffness, cytoskeletal arrangement, morphology, and cell-substrate adhesion could not account for the dependence of Transmigration on HUVEC substrate stiffness. We also explored the role of cell contraction and observed that large holes formed in endothelium on stiff substrates several minutes after neutrophil Transmigration reached a maximum. Further, suppression of contraction through inhibition of myosin light chain kinase normalized the effects of substrate stiffness by reducing Transmigration and eliminating hole formation in HUVECs on stiff substrates. These results provide strong evidence that neutrophil Transmigration is regulated by myosin light chain kinase-mediated endothelial cell contraction and that this event depends on subendothelial cell matrix stiffness.
Charles A Parkos - One of the best experts on this subject based on the ideXlab platform.
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coordinated redistribution of leukocyte lfa 1 and endothelial cell icam 1 accompany neutrophil Transmigration
Journal of Experimental Medicine, 2004Co-Authors: Sunil K Shaw, Yuan Liu, Asma Nusrat, Michael B Kim, Ravi M Rao, Charles U Hartman, Richard M Froio, Lin Yang, Todd Jones, Charles A ParkosAbstract:The leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) and its endothelial ligand intercellular adhesion molecule (ICAM)-1 play an important role in Transmigration as demonstrated by in vivo and in vitro models of inflammation. Despite the prominent role, little is known concerning the distribution and dynamic behavior of these adhesion molecules during leukocyte Transmigration. Therefore, we examined the spatial and temporal distribution of LFA-1 on neutrophils actively transmigrating tumor necrosis factor-α–activated human umbilical vein endothelial monolayers under shear flow. Upon neutrophil arrest, LFA-1 was evenly distributed. However, once neutrophils initiated Transmigration, LFA-1 rapidly redistributed to form a ringlike cluster at the neutrophil–endothelial junctional interface through which Transmigration occurred. As Transmigration was completed, LFA-1 redistributed to the neutrophil uropod. Endothelial ICAM-1 and JAM-A both colocalized with the ringlike LFA-1 cluster. Further analysis of PMA-stimulated neutrophils, which increase mobility of LFA-1, showed a rapid redistribution of LFA-1 and ICAM-1, but not endothelial JAM-A. Thus, endothelial JAM-A does not appear to contribute to adhesion or Transmigration in this system. This is the first demonstration that neutrophil LFA-1 rapidly redistributes to form a ringlike structure that coclusters with endothelial ICAM-1 as the neutrophil transmigrates.
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reduced expression of junctional adhesion molecule and platelet endothelial cell adhesion molecule 1 cd31 at human vascular endothelial junctions by cytokines tumor necrosis factor α plus interferon γ does not reduce leukocyte Transmigration under fl
American Journal of Pathology, 2001Co-Authors: Sunil K Shaw, Brandy N Perkins, Yawchyn Lim, Yuan Liu, Asma Nusrat, Frederick J Schnell, Charles A Parkos, Francis W LuscinskasAbstract:The combination of tumor necrosis factor (TNF)-α plus interferon (IFN)-γ has been shown previously to promote redistribution of platelet/endothelial cell adhesion molecule-1 (PECAM-1) (CD31), junctional adhesion molecule (JAM), and VE-cadherin away from lateral junctions of human umbilical vein endothelial cell monolayers. In parallel, neutrophil Transmigration was significantly reduced. Because PECAM-1 and JAM have been implicated in leukocyte Transmigration, the observed redistribution by cytokine activation was presumed to represent the mechanism causing decreased Transmigration under static conditions. The current results confirm that culture of human umbilical vein endothelial cells with TNF-α plus IFN-γ caused a decrease in surface-expressed and junctional-localized JAM and PECAM-1, but did not cause decreased leukocyte Transmigration in an in vitro flow assay. Furthermore, blocking monoclonal antibody to PECAM-1 still significantly reduced monocyte Transmigration, demonstrating that it retains a functional role even though its levels were reduced and redistributed away from junctions, whereas a panel of monoclonal antibodies to JAM failed to reduce leukocyte Transmigration. Given the alterations in junction protein location, permeability function was assessed. IFN-γ alone or TNF-α plus IFN-γ significantly increased permeability, but TNF-α alone did not, suggesting lack of correlation between Transmigration and loss of permeability. In conclusion, cytokine activation induced loss and redistribution of PECAM-1 and JAM away from lateral junctions, but per se does not negatively regulate either neutrophil or monocyte Transmigration under flow.