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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 do not prevent neutrophil-induced  disruption of the VE–cadherin complex. Neutrophils (107 in 5 ml) were  incubated with EC monolayers that were treated with TNF-α (4 h, 25  ng/ml) followed by 2 h of incubation with MG132 (M), ALLM (A), or  lactacystin (L), or 0.02% DMSO (−). After 10 min at 37°C, EC monolayers were extracted with lysis buffer 1 and processed for detection of the  VE–cadherin complex.
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Figure 7: Proteasome inhibitors do not prevent neutrophil-induced disruption of the VE–cadherin complex. Neutrophils (107 in 5 ml) were incubated with EC monolayers that were treated with TNF-α (4 h, 25 ng/ml) followed by 2 h of incubation with MG132 (M), ALLM (A), or lactacystin (L), or 0.02% DMSO (−). After 10 min at 37°C, EC monolayers were extracted with lysis buffer 1 and processed for detection of the VE–cadherin complex.

Mentions: To investigate whether the degradation of the VE–cadherin complex involved the cellular proteasome system, the effects of proteasome inhibitors were evaluated. As shown in Fig. 7, disruption of the VE–cadherin complex was not prevented by 2 h of incubation with 20 μM lactacystin or MG132, even though such treatments significantly reduced transmigration (Fig. 1). For comparison, the components of the VE–cadherin complex in TNF-α–activated EC remained constant in the absence of neutrophils. In addition, shorter incubations (5 or 30 min) with 50 μM MG132 did not prevent neutrophil-dependent dissociation of the VE– cadherin complex (data not shown). These findings establish that neutrophil adhesion triggers rapid EC-dependent degradation of the VE–cadherin complex through an enzymatic pathway that appears to be independent of the proteasome.


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 do not prevent neutrophil-induced  disruption of the VE–cadherin complex. Neutrophils (107 in 5 ml) were  incubated with EC monolayers that were treated with TNF-α (4 h, 25  ng/ml) followed by 2 h of incubation with MG132 (M), ALLM (A), or  lactacystin (L), or 0.02% DMSO (−). After 10 min at 37°C, EC monolayers were extracted with lysis buffer 1 and processed for detection of the  VE–cadherin complex.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Proteasome inhibitors do not prevent neutrophil-induced disruption of the VE–cadherin complex. Neutrophils (107 in 5 ml) were incubated with EC monolayers that were treated with TNF-α (4 h, 25 ng/ml) followed by 2 h of incubation with MG132 (M), ALLM (A), or lactacystin (L), or 0.02% DMSO (−). After 10 min at 37°C, EC monolayers were extracted with lysis buffer 1 and processed for detection of the VE–cadherin complex.
Mentions: To investigate whether the degradation of the VE–cadherin complex involved the cellular proteasome system, the effects of proteasome inhibitors were evaluated. As shown in Fig. 7, disruption of the VE–cadherin complex was not prevented by 2 h of incubation with 20 μM lactacystin or MG132, even though such treatments significantly reduced transmigration (Fig. 1). For comparison, the components of the VE–cadherin complex in TNF-α–activated EC remained constant in the absence of neutrophils. In addition, shorter incubations (5 or 30 min) with 50 μM MG132 did not prevent neutrophil-dependent dissociation of the VE– cadherin complex (data not shown). These findings establish that neutrophil adhesion triggers rapid EC-dependent degradation of the VE–cadherin complex through an enzymatic pathway that appears to be independent of the proteasome.

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