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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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Chelation of extracellular Ca2+ by EDTA releases  the VE–cadherin complex from  the lateral junctions, but does not  trigger degradation. (A) HUVEC monolayers were washed  and incubated for 2.5 min with 3  mM EDTA in HBSS, and then  fixed for 5 min in ice-cold methanol. VE–cadherin or PECAM-1 was detected by indirect immunofluorescence as detailed in Materials and Methods, and the images  were captured using a cooled charged-coupled device camera. (B) Immunoprecipitation and blotting studies show that incubation with EDTA  does not induce cleavage of VE–cadherin. Confluent EC monolayers  were incubated with TNF-α for 4 h, and then washed twice with DPBS  and incubated in either HBSS (lane 1) or HBSS medium containing 3  mM EDTA for 2.5 (lane 2) or 5 min (lane 3) at 37°C. VE–cadherin was  immunoprecipitated, resolved by SDS-PAGE, and subjected to blotting  as detailed in Fig. 3 legend.
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Figure 5: Chelation of extracellular Ca2+ by EDTA releases the VE–cadherin complex from the lateral junctions, but does not trigger degradation. (A) HUVEC monolayers were washed and incubated for 2.5 min with 3 mM EDTA in HBSS, and then fixed for 5 min in ice-cold methanol. VE–cadherin or PECAM-1 was detected by indirect immunofluorescence as detailed in Materials and Methods, and the images were captured using a cooled charged-coupled device camera. (B) Immunoprecipitation and blotting studies show that incubation with EDTA does not induce cleavage of VE–cadherin. Confluent EC monolayers were incubated with TNF-α for 4 h, and then washed twice with DPBS and incubated in either HBSS (lane 1) or HBSS medium containing 3 mM EDTA for 2.5 (lane 2) or 5 min (lane 3) at 37°C. VE–cadherin was immunoprecipitated, resolved by SDS-PAGE, and subjected to blotting as detailed in Fig. 3 legend.

Mentions: We then performed several experiments to show that neutrophils trigger an endothelial-dependent signal that leads to degradation of specific members of the complex. First, the members of the VE–cadherin complex were clearly disrupted after a 10 min coincubation of TNF-α–activated EC with neutrophil membranes (representing 107 neutrophils, Fig. 4 C, lane 3), which are devoid of granule contents and had only residual elastase activity (0.38% of total elastase activity when compared to intact neutrophils). In addition, disruption of the VE–cadherin complex by incubation of EC monolayers with neutrophil membranes was demonstrated by indirect immunofluorescence studies (Fig. 4 D). Thus, the components that triggered disruption of the VE–cadherin complex are present in neutrophil plasma membranes. This suggests that the degradation is not due to nonspecific degradation via neutrophil granule proteolytic enzymes. Second, transfer of conditioned medium from coincubation of neutrophils with TNF-activated EC to TNF-activated EC did not alter the VE–cadherin complex (Fig. 4 C, lane 4), suggesting degradation is not induced by a soluble factor. Lastly, neutrophil interactions specifically trigger both the disruption and partial degradation of VE– cadherin complex since incubation of EC monolayers with EDTA, which released VE–cadherin from the lateral junctions (Fig. 5 A), did not trigger degradation of VE–cadherin complex (Fig. 5 B). Taken together, these findings suggest that the dissociation and cleavage of VE–cadherin and its associated proteins are carefully controlled endothelial-specific event(s) that are triggered by leukocyte contact.


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)

Chelation of extracellular Ca2+ by EDTA releases  the VE–cadherin complex from  the lateral junctions, but does not  trigger degradation. (A) HUVEC monolayers were washed  and incubated for 2.5 min with 3  mM EDTA in HBSS, and then  fixed for 5 min in ice-cold methanol. VE–cadherin or PECAM-1 was detected by indirect immunofluorescence as detailed in Materials and Methods, and the images  were captured using a cooled charged-coupled device camera. (B) Immunoprecipitation and blotting studies show that incubation with EDTA  does not induce cleavage of VE–cadherin. Confluent EC monolayers  were incubated with TNF-α for 4 h, and then washed twice with DPBS  and incubated in either HBSS (lane 1) or HBSS medium containing 3  mM EDTA for 2.5 (lane 2) or 5 min (lane 3) at 37°C. VE–cadherin was  immunoprecipitated, resolved by SDS-PAGE, and subjected to blotting  as detailed in Fig. 3 legend.
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Related In: Results  -  Collection

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

Figure 5: Chelation of extracellular Ca2+ by EDTA releases the VE–cadherin complex from the lateral junctions, but does not trigger degradation. (A) HUVEC monolayers were washed and incubated for 2.5 min with 3 mM EDTA in HBSS, and then fixed for 5 min in ice-cold methanol. VE–cadherin or PECAM-1 was detected by indirect immunofluorescence as detailed in Materials and Methods, and the images were captured using a cooled charged-coupled device camera. (B) Immunoprecipitation and blotting studies show that incubation with EDTA does not induce cleavage of VE–cadherin. Confluent EC monolayers were incubated with TNF-α for 4 h, and then washed twice with DPBS and incubated in either HBSS (lane 1) or HBSS medium containing 3 mM EDTA for 2.5 (lane 2) or 5 min (lane 3) at 37°C. VE–cadherin was immunoprecipitated, resolved by SDS-PAGE, and subjected to blotting as detailed in Fig. 3 legend.
Mentions: We then performed several experiments to show that neutrophils trigger an endothelial-dependent signal that leads to degradation of specific members of the complex. First, the members of the VE–cadherin complex were clearly disrupted after a 10 min coincubation of TNF-α–activated EC with neutrophil membranes (representing 107 neutrophils, Fig. 4 C, lane 3), which are devoid of granule contents and had only residual elastase activity (0.38% of total elastase activity when compared to intact neutrophils). In addition, disruption of the VE–cadherin complex by incubation of EC monolayers with neutrophil membranes was demonstrated by indirect immunofluorescence studies (Fig. 4 D). Thus, the components that triggered disruption of the VE–cadherin complex are present in neutrophil plasma membranes. This suggests that the degradation is not due to nonspecific degradation via neutrophil granule proteolytic enzymes. Second, transfer of conditioned medium from coincubation of neutrophils with TNF-activated EC to TNF-activated EC did not alter the VE–cadherin complex (Fig. 4 C, lane 4), suggesting degradation is not induced by a soluble factor. Lastly, neutrophil interactions specifically trigger both the disruption and partial degradation of VE– cadherin complex since incubation of EC monolayers with EDTA, which released VE–cadherin from the lateral junctions (Fig. 5 A), did not trigger degradation of VE–cadherin complex (Fig. 5 B). Taken together, these findings suggest that the dissociation and cleavage of VE–cadherin and its associated proteins are carefully controlled endothelial-specific event(s) that are triggered by leukocyte contact.

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