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Ezrin and moesin are required for efficient T cell adhesion and homing to lymphoid organs.

Chen EJ, Shaffer MH, Williamson EK, Huang Y, Burkhardt JK - PLoS ONE (2013)

Bottom Line: Members of the ezrin, radixin and moesin (ERM) family of actin-binding proteins have been implicated in several aspects of this process, but studies have yielded conflicting results.In vivo, ERM-deficient T cells showed defects in homing to lymphoid organs.Taken together, these results show that ERM proteins are largely dispensable for T cell chemotaxis, but are important for β1 integrin function and homing to lymphoid organs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Pennsylvania, USA.

ABSTRACT
T cell trafficking between the blood and lymphoid organs is a complex, multistep process that requires several highly dynamic and coordinated changes in cyto-architecture. Members of the ezrin, radixin and moesin (ERM) family of actin-binding proteins have been implicated in several aspects of this process, but studies have yielded conflicting results. Using mice with a conditional deletion of ezrin in CD4+ cells and moesin-specific siRNA, we generated T cells lacking ERM proteins, and investigated the effect on specific events required for T cell trafficking. ERM-deficient T cells migrated normally in multiple in vitro and in vivo assays, and could undergo efficient diapedesis in vitro. However, these cells were impaired in their ability to adhere to the β1 integrin ligand fibronectin, and to polarize appropriately in response to fibronectin and VCAM-1 binding. This defect was specific for β1 integrins, as adhesion and polarization in response to ICAM-1 were normal. In vivo, ERM-deficient T cells showed defects in homing to lymphoid organs. Taken together, these results show that ERM proteins are largely dispensable for T cell chemotaxis, but are important for β1 integrin function and homing to lymphoid organs.

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ERM-deficient cells undergo efficient diapedesis in vitro.(A,B) Diapedesis under static conditions. Confluent monolayers of 3B-11 endothelial cells were pre-treated with or without TNFα to upregulate VCAM-1, and wild-type or ERM-deficient T cells were added to the apical surfaces. Cells were imaged in an environmental chamber every 30 seconds for 2 hours. (A) DIC images of cells undergoing diapedesis. Arrowheads indicate leading edges of migrating T cells. White arrows indicate cells migrating along the apical surface of endothelial cells; the same cells are marked with red arrows after passing below the endothelial cell layer. (B) Quantitative analysis of assays carried out as in A. Cells undergoing diapedesis are quantified as a percentage of total moving cells. Data represent mean ± StDev from six experiments. (C) Diapedesis under shear flow conditions. Confluent monolayers of 3B-11 endothelial cells were grown in flow chambers, pre-treated with or without TNFα to upregulate VCAM-1, and wild-type or ERM-deficient T cells were added to the apical surfaces and allowed to interact with endothelial cells under shear stress of 0.5 dyne/cm2. Cells were imaged every 30 seconds for 1 hour, and the percentage of moving cells that underwent diapedesis was determined. Data represent mean ± StDev from three experiments.
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pone-0052368-g006: ERM-deficient cells undergo efficient diapedesis in vitro.(A,B) Diapedesis under static conditions. Confluent monolayers of 3B-11 endothelial cells were pre-treated with or without TNFα to upregulate VCAM-1, and wild-type or ERM-deficient T cells were added to the apical surfaces. Cells were imaged in an environmental chamber every 30 seconds for 2 hours. (A) DIC images of cells undergoing diapedesis. Arrowheads indicate leading edges of migrating T cells. White arrows indicate cells migrating along the apical surface of endothelial cells; the same cells are marked with red arrows after passing below the endothelial cell layer. (B) Quantitative analysis of assays carried out as in A. Cells undergoing diapedesis are quantified as a percentage of total moving cells. Data represent mean ± StDev from six experiments. (C) Diapedesis under shear flow conditions. Confluent monolayers of 3B-11 endothelial cells were grown in flow chambers, pre-treated with or without TNFα to upregulate VCAM-1, and wild-type or ERM-deficient T cells were added to the apical surfaces and allowed to interact with endothelial cells under shear stress of 0.5 dyne/cm2. Cells were imaged every 30 seconds for 1 hour, and the percentage of moving cells that underwent diapedesis was determined. Data represent mean ± StDev from three experiments.

Mentions: In order to enter lymph nodes, T cells must first undergo integrin-mediated adhesion to endothelial cells, and then extravasate by passing through the constricted spaces between endothelial cells, a process termed diapedesis. We therefore assessed the ability of ERM-deficient T cells to undergo diapedesis (Figure 6A). Since ERM-deficient T cells were impaired in their ability to respond to β1 integrin ligands, we selected an endothelial cell line that expresses the β1 integrin ligand VCAM-1 in response to inflammatory stimuli, but lacks β2 integrin ligands ([45] and data not shown). Confluent monolayers of murine 3B-11 endothelial cells were either left untreated or treated with TNF-α to induce upregulation of VCAM-1 (confirmed by flow cytometry, data not shown). Control or ERM-deficient T cells were then placed on top of each monolayer, and diapesesis was imaged using DIC microscopy. As shown in Figure 6A and Video S2, T cells migrating on top of the endothelial monolayer are highly refractile, while cells that have passed beneath the monolayer are readily identifiable based on loss of refractility. Quantitative analysis showed that diapedesis of both control and ERM-deficient T cells was infrequent on untreated monolayers (Figure 6B). In contrast, approximately 50% of T cells applied to TNF-α treated monolayers passed through during the 2 hour imaging period. No difference in rate or frequency of diapedesis was observed between wild-type and ERM-deficient T cells.


Ezrin and moesin are required for efficient T cell adhesion and homing to lymphoid organs.

Chen EJ, Shaffer MH, Williamson EK, Huang Y, Burkhardt JK - PLoS ONE (2013)

ERM-deficient cells undergo efficient diapedesis in vitro.(A,B) Diapedesis under static conditions. Confluent monolayers of 3B-11 endothelial cells were pre-treated with or without TNFα to upregulate VCAM-1, and wild-type or ERM-deficient T cells were added to the apical surfaces. Cells were imaged in an environmental chamber every 30 seconds for 2 hours. (A) DIC images of cells undergoing diapedesis. Arrowheads indicate leading edges of migrating T cells. White arrows indicate cells migrating along the apical surface of endothelial cells; the same cells are marked with red arrows after passing below the endothelial cell layer. (B) Quantitative analysis of assays carried out as in A. Cells undergoing diapedesis are quantified as a percentage of total moving cells. Data represent mean ± StDev from six experiments. (C) Diapedesis under shear flow conditions. Confluent monolayers of 3B-11 endothelial cells were grown in flow chambers, pre-treated with or without TNFα to upregulate VCAM-1, and wild-type or ERM-deficient T cells were added to the apical surfaces and allowed to interact with endothelial cells under shear stress of 0.5 dyne/cm2. Cells were imaged every 30 seconds for 1 hour, and the percentage of moving cells that underwent diapedesis was determined. Data represent mean ± StDev from three experiments.
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Related In: Results  -  Collection

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pone-0052368-g006: ERM-deficient cells undergo efficient diapedesis in vitro.(A,B) Diapedesis under static conditions. Confluent monolayers of 3B-11 endothelial cells were pre-treated with or without TNFα to upregulate VCAM-1, and wild-type or ERM-deficient T cells were added to the apical surfaces. Cells were imaged in an environmental chamber every 30 seconds for 2 hours. (A) DIC images of cells undergoing diapedesis. Arrowheads indicate leading edges of migrating T cells. White arrows indicate cells migrating along the apical surface of endothelial cells; the same cells are marked with red arrows after passing below the endothelial cell layer. (B) Quantitative analysis of assays carried out as in A. Cells undergoing diapedesis are quantified as a percentage of total moving cells. Data represent mean ± StDev from six experiments. (C) Diapedesis under shear flow conditions. Confluent monolayers of 3B-11 endothelial cells were grown in flow chambers, pre-treated with or without TNFα to upregulate VCAM-1, and wild-type or ERM-deficient T cells were added to the apical surfaces and allowed to interact with endothelial cells under shear stress of 0.5 dyne/cm2. Cells were imaged every 30 seconds for 1 hour, and the percentage of moving cells that underwent diapedesis was determined. Data represent mean ± StDev from three experiments.
Mentions: In order to enter lymph nodes, T cells must first undergo integrin-mediated adhesion to endothelial cells, and then extravasate by passing through the constricted spaces between endothelial cells, a process termed diapedesis. We therefore assessed the ability of ERM-deficient T cells to undergo diapedesis (Figure 6A). Since ERM-deficient T cells were impaired in their ability to respond to β1 integrin ligands, we selected an endothelial cell line that expresses the β1 integrin ligand VCAM-1 in response to inflammatory stimuli, but lacks β2 integrin ligands ([45] and data not shown). Confluent monolayers of murine 3B-11 endothelial cells were either left untreated or treated with TNF-α to induce upregulation of VCAM-1 (confirmed by flow cytometry, data not shown). Control or ERM-deficient T cells were then placed on top of each monolayer, and diapesesis was imaged using DIC microscopy. As shown in Figure 6A and Video S2, T cells migrating on top of the endothelial monolayer are highly refractile, while cells that have passed beneath the monolayer are readily identifiable based on loss of refractility. Quantitative analysis showed that diapedesis of both control and ERM-deficient T cells was infrequent on untreated monolayers (Figure 6B). In contrast, approximately 50% of T cells applied to TNF-α treated monolayers passed through during the 2 hour imaging period. No difference in rate or frequency of diapedesis was observed between wild-type and ERM-deficient T cells.

Bottom Line: Members of the ezrin, radixin and moesin (ERM) family of actin-binding proteins have been implicated in several aspects of this process, but studies have yielded conflicting results.In vivo, ERM-deficient T cells showed defects in homing to lymphoid organs.Taken together, these results show that ERM proteins are largely dispensable for T cell chemotaxis, but are important for β1 integrin function and homing to lymphoid organs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Pennsylvania, USA.

ABSTRACT
T cell trafficking between the blood and lymphoid organs is a complex, multistep process that requires several highly dynamic and coordinated changes in cyto-architecture. Members of the ezrin, radixin and moesin (ERM) family of actin-binding proteins have been implicated in several aspects of this process, but studies have yielded conflicting results. Using mice with a conditional deletion of ezrin in CD4+ cells and moesin-specific siRNA, we generated T cells lacking ERM proteins, and investigated the effect on specific events required for T cell trafficking. ERM-deficient T cells migrated normally in multiple in vitro and in vivo assays, and could undergo efficient diapedesis in vitro. However, these cells were impaired in their ability to adhere to the β1 integrin ligand fibronectin, and to polarize appropriately in response to fibronectin and VCAM-1 binding. This defect was specific for β1 integrins, as adhesion and polarization in response to ICAM-1 were normal. In vivo, ERM-deficient T cells showed defects in homing to lymphoid organs. Taken together, these results show that ERM proteins are largely dispensable for T cell chemotaxis, but are important for β1 integrin function and homing to lymphoid organs.

Show MeSH
Related in: MedlinePlus