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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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Effect of neutrophil adhesion on the endothelial VE–cadherin complex. (A) Confluent EC monolayers were incubated with media alone or media containing TNF-α for 6 h and washed. Neutrophils  (107) in DPBS-HSA or DPBS-HSA alone were added for 10 min at 37°C  under static conditions. Nonadherent neutrophils were removed by  washing and the monolayers were extracted on ice for 30 min. Proteins  were immunoprecipitated with anti–VE–cadherin mAb as detailed in  Materials and Methods and detected by immunoblotting with specific  mAb. (B) Confluent EC monolayers were treated with media alone or  media containing TNF-α for 4 h, washed twice, and 107 neutrophils  were added. After incubation for 0, 3, or 10 min at 37°C, nonadherent  neutrophils were removed, and monolayers were extracted with lysis  buffer 1 containing 0.5% SDS. Proteins were immunoprecipitated using  specific mAb directed against each individual component and subsequently detected by immunoblotting with a specific mAb. The Mr for  molecular mass standards are shown on the left margin and 0, 3, and 10  refer to the time period of neutrophil–EC coincubation at 37°C. (C) Media alone (lane 1), resting neutrophils (107 cells, lane 2), neutrophil membranes representing 107 cells (lane 3), or conditioned media from coincubations of neutrophils (107 cells) with TNF-α–activated HUVEC (lane 4)  were added to control (lane 1) or TNF-α–activated HUVEC monolayers  (lanes 2–4) and incubated at 37°C for 10 min. HUVEC were washed  twice and extracted with ice-cold lysis buffer 1 (without 0.5% SDS). Proteins were immunoprecipitated with anti–VE–cadherin mAb as detailed  in Materials and Methods, and detected by immunoblotting with specific  mAb. (D) Neutrophil membranes representing 3 × 106 cells were incubated with 4 h TNF-α–activated EC in wells of chamber slides for 10  min at 37°C. The monolayers were washed twice and fixed for 5 min at  4°C with ice-cold methanol. Junctional proteins were detected by indirect immunofluorescence.
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Figure 4: Effect of neutrophil adhesion on the endothelial VE–cadherin complex. (A) Confluent EC monolayers were incubated with media alone or media containing TNF-α for 6 h and washed. Neutrophils (107) in DPBS-HSA or DPBS-HSA alone were added for 10 min at 37°C under static conditions. Nonadherent neutrophils were removed by washing and the monolayers were extracted on ice for 30 min. Proteins were immunoprecipitated with anti–VE–cadherin mAb as detailed in Materials and Methods and detected by immunoblotting with specific mAb. (B) Confluent EC monolayers were treated with media alone or media containing TNF-α for 4 h, washed twice, and 107 neutrophils were added. After incubation for 0, 3, or 10 min at 37°C, nonadherent neutrophils were removed, and monolayers were extracted with lysis buffer 1 containing 0.5% SDS. Proteins were immunoprecipitated using specific mAb directed against each individual component and subsequently detected by immunoblotting with a specific mAb. The Mr for molecular mass standards are shown on the left margin and 0, 3, and 10 refer to the time period of neutrophil–EC coincubation at 37°C. (C) Media alone (lane 1), resting neutrophils (107 cells, lane 2), neutrophil membranes representing 107 cells (lane 3), or conditioned media from coincubations of neutrophils (107 cells) with TNF-α–activated HUVEC (lane 4) were added to control (lane 1) or TNF-α–activated HUVEC monolayers (lanes 2–4) and incubated at 37°C for 10 min. HUVEC were washed twice and extracted with ice-cold lysis buffer 1 (without 0.5% SDS). Proteins were immunoprecipitated with anti–VE–cadherin mAb as detailed in Materials and Methods, and detected by immunoblotting with specific mAb. (D) Neutrophil membranes representing 3 × 106 cells were incubated with 4 h TNF-α–activated EC in wells of chamber slides for 10 min at 37°C. The monolayers were washed twice and fixed for 5 min at 4°C with ice-cold methanol. Junctional proteins were detected by indirect immunofluorescence.

Mentions: To examine whether neutrophil transmigration correlated with any alterations in the VE–cadherin complex, neutrophils were incubated with TNF-α–activated EC monolayers for 10 min, and then the nonadherent neutrophils were removed by washing. Visual inspection of TNF-α–treated EC cultures before lysis showed intact and tightly confluent monolayers with many adherent and transmigrated neutrophils. The ratio of adherent neutrophils to individual EC was 1:2. In contrast, few neutrophils adhered to unactivated EC, and no neutrophils transmigrated. When the levels of VE–cadherin complex were determined in Triton X-100–soluble lysates by immunoprecipitation with anti–VE–cadherin mAb and immunoblotting with specific mAb to each component, dramatic alterations were observed. Neutrophil adhesion and/or migration across TNF-α–activated EC monolayers induced loss of VE–cadherin, β-catenin, and plakoglobin, whereas the level of α-catenin was not decreased (Fig. 4 A, compare lane 3 to 4). Over the course of our experiments, we noted that the native immunoreactive species of β-catenin, plakoglobin, and p120/p100 were always below detectable levels, whereas there was often retention of a small amount of VE–cadherin. Coincubation of control unactivated EC monolayers with unactivated neutrophils consistently had no significant effect on the VE–cadherin complex (compare lane 1 and 2). Since equal numbers of EC were used for each sample, and this ECL detection system is very sensitive, we infer the losses are not due to unequal sample loading.


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)

Effect of neutrophil adhesion on the endothelial VE–cadherin complex. (A) Confluent EC monolayers were incubated with media alone or media containing TNF-α for 6 h and washed. Neutrophils  (107) in DPBS-HSA or DPBS-HSA alone were added for 10 min at 37°C  under static conditions. Nonadherent neutrophils were removed by  washing and the monolayers were extracted on ice for 30 min. Proteins  were immunoprecipitated with anti–VE–cadherin mAb as detailed in  Materials and Methods and detected by immunoblotting with specific  mAb. (B) Confluent EC monolayers were treated with media alone or  media containing TNF-α for 4 h, washed twice, and 107 neutrophils  were added. After incubation for 0, 3, or 10 min at 37°C, nonadherent  neutrophils were removed, and monolayers were extracted with lysis  buffer 1 containing 0.5% SDS. Proteins were immunoprecipitated using  specific mAb directed against each individual component and subsequently detected by immunoblotting with a specific mAb. The Mr for  molecular mass standards are shown on the left margin and 0, 3, and 10  refer to the time period of neutrophil–EC coincubation at 37°C. (C) Media alone (lane 1), resting neutrophils (107 cells, lane 2), neutrophil membranes representing 107 cells (lane 3), or conditioned media from coincubations of neutrophils (107 cells) with TNF-α–activated HUVEC (lane 4)  were added to control (lane 1) or TNF-α–activated HUVEC monolayers  (lanes 2–4) and incubated at 37°C for 10 min. HUVEC were washed  twice and extracted with ice-cold lysis buffer 1 (without 0.5% SDS). Proteins were immunoprecipitated with anti–VE–cadherin mAb as detailed  in Materials and Methods, and detected by immunoblotting with specific  mAb. (D) Neutrophil membranes representing 3 × 106 cells were incubated with 4 h TNF-α–activated EC in wells of chamber slides for 10  min at 37°C. The monolayers were washed twice and fixed for 5 min at  4°C with ice-cold methanol. Junctional proteins were detected by indirect immunofluorescence.
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Related In: Results  -  Collection

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Figure 4: Effect of neutrophil adhesion on the endothelial VE–cadherin complex. (A) Confluent EC monolayers were incubated with media alone or media containing TNF-α for 6 h and washed. Neutrophils (107) in DPBS-HSA or DPBS-HSA alone were added for 10 min at 37°C under static conditions. Nonadherent neutrophils were removed by washing and the monolayers were extracted on ice for 30 min. Proteins were immunoprecipitated with anti–VE–cadherin mAb as detailed in Materials and Methods and detected by immunoblotting with specific mAb. (B) Confluent EC monolayers were treated with media alone or media containing TNF-α for 4 h, washed twice, and 107 neutrophils were added. After incubation for 0, 3, or 10 min at 37°C, nonadherent neutrophils were removed, and monolayers were extracted with lysis buffer 1 containing 0.5% SDS. Proteins were immunoprecipitated using specific mAb directed against each individual component and subsequently detected by immunoblotting with a specific mAb. The Mr for molecular mass standards are shown on the left margin and 0, 3, and 10 refer to the time period of neutrophil–EC coincubation at 37°C. (C) Media alone (lane 1), resting neutrophils (107 cells, lane 2), neutrophil membranes representing 107 cells (lane 3), or conditioned media from coincubations of neutrophils (107 cells) with TNF-α–activated HUVEC (lane 4) were added to control (lane 1) or TNF-α–activated HUVEC monolayers (lanes 2–4) and incubated at 37°C for 10 min. HUVEC were washed twice and extracted with ice-cold lysis buffer 1 (without 0.5% SDS). Proteins were immunoprecipitated with anti–VE–cadherin mAb as detailed in Materials and Methods, and detected by immunoblotting with specific mAb. (D) Neutrophil membranes representing 3 × 106 cells were incubated with 4 h TNF-α–activated EC in wells of chamber slides for 10 min at 37°C. The monolayers were washed twice and fixed for 5 min at 4°C with ice-cold methanol. Junctional proteins were detected by indirect immunofluorescence.
Mentions: To examine whether neutrophil transmigration correlated with any alterations in the VE–cadherin complex, neutrophils were incubated with TNF-α–activated EC monolayers for 10 min, and then the nonadherent neutrophils were removed by washing. Visual inspection of TNF-α–treated EC cultures before lysis showed intact and tightly confluent monolayers with many adherent and transmigrated neutrophils. The ratio of adherent neutrophils to individual EC was 1:2. In contrast, few neutrophils adhered to unactivated EC, and no neutrophils transmigrated. When the levels of VE–cadherin complex were determined in Triton X-100–soluble lysates by immunoprecipitation with anti–VE–cadherin mAb and immunoblotting with specific mAb to each component, dramatic alterations were observed. Neutrophil adhesion and/or migration across TNF-α–activated EC monolayers induced loss of VE–cadherin, β-catenin, and plakoglobin, whereas the level of α-catenin was not decreased (Fig. 4 A, compare lane 3 to 4). Over the course of our experiments, we noted that the native immunoreactive species of β-catenin, plakoglobin, and p120/p100 were always below detectable levels, whereas there was often retention of a small amount of VE–cadherin. Coincubation of control unactivated EC monolayers with unactivated neutrophils consistently had no significant effect on the VE–cadherin complex (compare lane 1 and 2). Since equal numbers of EC were used for each sample, and this ECL detection system is very sensitive, we infer the losses are not due to unequal sample loading.

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