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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.

Allport JR, Ding H, Collins T, Gerritsen ME, Luscinskas FW - J. Exp. Med. (1997)

Bottom Line: Palambella, T.Collins. 1995.In contrast, PECAM-1, which is located at lateral junctions and is implicated in neutrophil transmigration, was not altered.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Although several adhesion molecules expressed on leukocytes (beta1 and beta2 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-alpha-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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Proteasome inhibitors MG132 and lactacystin prevent neutrophil migration under flow at 1.8 dynes/cm2. Confluent EC monolayers were incubated with TNF-α for 4 h before addition of proteasome inhibitors (MG, 20 μM MG132; LAC, 20 μM lactacystin) or DMSO  carrier (Cont.) for 1 (left) or 2 h (right), and neutrophil adhesion and transmigration were assessed as detailed in Materials and Methods. *P <0.05.
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Figure 1: Proteasome inhibitors MG132 and lactacystin prevent neutrophil migration under flow at 1.8 dynes/cm2. Confluent EC monolayers were incubated with TNF-α for 4 h before addition of proteasome inhibitors (MG, 20 μM MG132; LAC, 20 μM lactacystin) or DMSO carrier (Cont.) for 1 (left) or 2 h (right), and neutrophil adhesion and transmigration were assessed as detailed in Materials and Methods. *P <0.05.

Mentions: To distinguish between the effects of the proteasome inhibitors on expression of endothelial leukocyte adhesion molecules and the process of neutrophil transmigration, MG132, lactacystin, or carrier control were added to 4 h TNF-α–activated EC, and the monolayers further incubated. Under such conditions, both inhibitors have no effect on neutrophil adhesion (Fig. 1) or expression of endothelial adhesion molecules (Table 1 and data not shown), but reduce neutrophil transmigration by >50% (Fig. 1). The effect on transmigration was observed by 60 min, and by 120 min the level of blockade was >70% for MG132 and >60% for lactacystin. A direct effect of the inhibitors on neutrophils is not likely because a 5-min pretreatment of neutrophils with 5 μM MG132 before perfusion did not alter neutrophil adhesion or transmigration (vehicle, 51.5 ± 5.9% transmigrated versus MG132 treated, 51.5 ± 6.3%; n = 3 experiments). We conclude that MG132 and lactacystin, two structurally distinct inhibitors of the proteasome, act on the endothelium to block transmigration, separately from their effect on adhesion molecule expression.


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.

Allport JR, Ding H, Collins T, Gerritsen ME, Luscinskas FW - J. Exp. Med. (1997)

Proteasome inhibitors MG132 and lactacystin prevent neutrophil migration under flow at 1.8 dynes/cm2. Confluent EC monolayers were incubated with TNF-α for 4 h before addition of proteasome inhibitors (MG, 20 μM MG132; LAC, 20 μM lactacystin) or DMSO  carrier (Cont.) for 1 (left) or 2 h (right), and neutrophil adhesion and transmigration were assessed as detailed in Materials and Methods. *P <0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199034&req=5

Figure 1: Proteasome inhibitors MG132 and lactacystin prevent neutrophil migration under flow at 1.8 dynes/cm2. Confluent EC monolayers were incubated with TNF-α for 4 h before addition of proteasome inhibitors (MG, 20 μM MG132; LAC, 20 μM lactacystin) or DMSO carrier (Cont.) for 1 (left) or 2 h (right), and neutrophil adhesion and transmigration were assessed as detailed in Materials and Methods. *P <0.05.
Mentions: To distinguish between the effects of the proteasome inhibitors on expression of endothelial leukocyte adhesion molecules and the process of neutrophil transmigration, MG132, lactacystin, or carrier control were added to 4 h TNF-α–activated EC, and the monolayers further incubated. Under such conditions, both inhibitors have no effect on neutrophil adhesion (Fig. 1) or expression of endothelial adhesion molecules (Table 1 and data not shown), but reduce neutrophil transmigration by >50% (Fig. 1). The effect on transmigration was observed by 60 min, and by 120 min the level of blockade was >70% for MG132 and >60% for lactacystin. A direct effect of the inhibitors on neutrophils is not likely because a 5-min pretreatment of neutrophils with 5 μM MG132 before perfusion did not alter neutrophil adhesion or transmigration (vehicle, 51.5 ± 5.9% transmigrated versus MG132 treated, 51.5 ± 6.3%; n = 3 experiments). We conclude that MG132 and lactacystin, two structurally distinct inhibitors of the proteasome, act on the endothelium to block transmigration, separately from their effect on adhesion molecule expression.

Bottom Line: Palambella, T.Collins. 1995.In contrast, PECAM-1, which is located at lateral junctions and is implicated in neutrophil transmigration, was not altered.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Although several adhesion molecules expressed on leukocytes (beta1 and beta2 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-alpha-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).

Show MeSH
Related in: MedlinePlus