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JAM-L-mediated leukocyte adhesion to endothelial cells is regulated in cis by alpha4beta1 integrin activation.

Luissint AC, Lutz PG, Calderwood DA, Couraud PO, Bourdoulous S - J. Cell Biol. (2008)

Bottom Line: Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor).Interestingly, JAM-L expressed on neutrophils binds CAR independently of integrin activation.However, on resting monocytes and T lymphocytes, which express the integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form.

View Article: PubMed Central - PubMed

Affiliation: Institut Cochin, Université Paris Descartes, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, Paris F-75014, France.

ABSTRACT
Junctional adhesion molecules (JAMs) are endothelial and epithelial adhesion molecules involved in the recruitment of circulating leukocytes to inflammatory sites. We show here that JAM-L, a protein related to the JAM family, is restricted to leukocytes and promotes their adhesion to endothelial cells. Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor). Interestingly, JAM-L expressed on neutrophils binds CAR independently of integrin activation. However, on resting monocytes and T lymphocytes, which express the integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form. Integrin activation is required for the dissociation of JAM-L-VLA-4 complexes and the accumulation of functional JAM-L dimers, which indicates that the leukocyte integrin VLA-4 controls JAM-L function in cis by controlling its dimerization state. This provides a mechanism through which VLA-4 and JAM-L functions are coordinately regulated, allowing JAM-L to strengthen integrin-dependent adhesion of leukocytes to endothelial cells.

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JAM-L associates with VLA-4. (A) U937/pMT–JAM-L–HA cells were untreated (−) or treated (+) with Zn2+ to induce JAM-L–HA expression. After lysis, immunoprecipitations (IP) were performed with mouse IgG1 isotype antibodies directed against HA, α4, β1, or VSV-g epitope tag as a negative control. Immune complexes were immunoblotted (IB) with anti-HA or anti-β1 antibodies. Black lines indicate that intervening lanes have been spliced out. Numbers next to gel blots indicate molecular mass markers in kD. (B) Freshly isolated T lymphocytes were plated on poly-l-lysine and stimulated or not stimulated for 10 min at 37°C with 100 ng/ml SDF-1α in PBS Ca−/Mg− before fixation. Cells were triple stained for JAM-L (green), α4 subunit (red), and actin (blue), then analyzed by confocal microscopy. (right) Merged images (overlay) of the same fields. Bars, 10 μM.
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fig5: JAM-L associates with VLA-4. (A) U937/pMT–JAM-L–HA cells were untreated (−) or treated (+) with Zn2+ to induce JAM-L–HA expression. After lysis, immunoprecipitations (IP) were performed with mouse IgG1 isotype antibodies directed against HA, α4, β1, or VSV-g epitope tag as a negative control. Immune complexes were immunoblotted (IB) with anti-HA or anti-β1 antibodies. Black lines indicate that intervening lanes have been spliced out. Numbers next to gel blots indicate molecular mass markers in kD. (B) Freshly isolated T lymphocytes were plated on poly-l-lysine and stimulated or not stimulated for 10 min at 37°C with 100 ng/ml SDF-1α in PBS Ca−/Mg− before fixation. Cells were triple stained for JAM-L (green), α4 subunit (red), and actin (blue), then analyzed by confocal microscopy. (right) Merged images (overlay) of the same fields. Bars, 10 μM.

Mentions: VLA-4 (α4β1) is one of the main integrins involved in leukocyte interactions with endothelial cells, and is expressed in T lymphocytes and monocytes but not in K562 cells or neutrophils. As VLA-4 expression correlates with the regulation in cis of JAM-L function, we investigated whether JAM-L could interact with VLA-4. Immunoprecipitations were performed from cell lysates of the U937 cells that conditionally express JAM-L–HA. JAM-L–HA was detected both in α4 and β1 precipitates (Fig. 5 A). We therefore determined the cell-surface distribution of endogenous VLA-4 and JAM-L on T lymphocytes by confocal immunofluorescence analysis (Fig. 5 B). When plated on poly-l-lysine in PBS buffer, cells remained predominantly round, and both VLA-4 and JAM-L were evenly distributed over the cell surface, where they largely colocalized. Interestingly, after SDF-1α treatment for 10 min, T lymphocytes spread out and, while VLA-4 remained associated with polymerized cortical actin, JAM-L molecules accumulated at one pole of the cells, where they formed a cap structure. In addition, upon SDF-1α treatment, ∼30% of the cells acquired a polarized morphology, with the formation of a large actin-rich lamellipodia at the leading edge and a spherical membrane protrusion budding at the rear, called a “uropod.” In these polarized cells, both VLA-4 and JAM-L were present in the uropod; in addition, a fraction of VLA-4, but not of JAM-L, was present in the mid-hind region. These results indicate that JAM-L appears to colocalize with VLA-4 integrin in the absence of activating stimulus, whereas the two proteins partially overlapped upon SDF-1α treatment.


JAM-L-mediated leukocyte adhesion to endothelial cells is regulated in cis by alpha4beta1 integrin activation.

Luissint AC, Lutz PG, Calderwood DA, Couraud PO, Bourdoulous S - J. Cell Biol. (2008)

JAM-L associates with VLA-4. (A) U937/pMT–JAM-L–HA cells were untreated (−) or treated (+) with Zn2+ to induce JAM-L–HA expression. After lysis, immunoprecipitations (IP) were performed with mouse IgG1 isotype antibodies directed against HA, α4, β1, or VSV-g epitope tag as a negative control. Immune complexes were immunoblotted (IB) with anti-HA or anti-β1 antibodies. Black lines indicate that intervening lanes have been spliced out. Numbers next to gel blots indicate molecular mass markers in kD. (B) Freshly isolated T lymphocytes were plated on poly-l-lysine and stimulated or not stimulated for 10 min at 37°C with 100 ng/ml SDF-1α in PBS Ca−/Mg− before fixation. Cells were triple stained for JAM-L (green), α4 subunit (red), and actin (blue), then analyzed by confocal microscopy. (right) Merged images (overlay) of the same fields. Bars, 10 μM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2600739&req=5

fig5: JAM-L associates with VLA-4. (A) U937/pMT–JAM-L–HA cells were untreated (−) or treated (+) with Zn2+ to induce JAM-L–HA expression. After lysis, immunoprecipitations (IP) were performed with mouse IgG1 isotype antibodies directed against HA, α4, β1, or VSV-g epitope tag as a negative control. Immune complexes were immunoblotted (IB) with anti-HA or anti-β1 antibodies. Black lines indicate that intervening lanes have been spliced out. Numbers next to gel blots indicate molecular mass markers in kD. (B) Freshly isolated T lymphocytes were plated on poly-l-lysine and stimulated or not stimulated for 10 min at 37°C with 100 ng/ml SDF-1α in PBS Ca−/Mg− before fixation. Cells were triple stained for JAM-L (green), α4 subunit (red), and actin (blue), then analyzed by confocal microscopy. (right) Merged images (overlay) of the same fields. Bars, 10 μM.
Mentions: VLA-4 (α4β1) is one of the main integrins involved in leukocyte interactions with endothelial cells, and is expressed in T lymphocytes and monocytes but not in K562 cells or neutrophils. As VLA-4 expression correlates with the regulation in cis of JAM-L function, we investigated whether JAM-L could interact with VLA-4. Immunoprecipitations were performed from cell lysates of the U937 cells that conditionally express JAM-L–HA. JAM-L–HA was detected both in α4 and β1 precipitates (Fig. 5 A). We therefore determined the cell-surface distribution of endogenous VLA-4 and JAM-L on T lymphocytes by confocal immunofluorescence analysis (Fig. 5 B). When plated on poly-l-lysine in PBS buffer, cells remained predominantly round, and both VLA-4 and JAM-L were evenly distributed over the cell surface, where they largely colocalized. Interestingly, after SDF-1α treatment for 10 min, T lymphocytes spread out and, while VLA-4 remained associated with polymerized cortical actin, JAM-L molecules accumulated at one pole of the cells, where they formed a cap structure. In addition, upon SDF-1α treatment, ∼30% of the cells acquired a polarized morphology, with the formation of a large actin-rich lamellipodia at the leading edge and a spherical membrane protrusion budding at the rear, called a “uropod.” In these polarized cells, both VLA-4 and JAM-L were present in the uropod; in addition, a fraction of VLA-4, but not of JAM-L, was present in the mid-hind region. These results indicate that JAM-L appears to colocalize with VLA-4 integrin in the absence of activating stimulus, whereas the two proteins partially overlapped upon SDF-1α treatment.

Bottom Line: Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor).Interestingly, JAM-L expressed on neutrophils binds CAR independently of integrin activation.However, on resting monocytes and T lymphocytes, which express the integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form.

View Article: PubMed Central - PubMed

Affiliation: Institut Cochin, Université Paris Descartes, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, Paris F-75014, France.

ABSTRACT
Junctional adhesion molecules (JAMs) are endothelial and epithelial adhesion molecules involved in the recruitment of circulating leukocytes to inflammatory sites. We show here that JAM-L, a protein related to the JAM family, is restricted to leukocytes and promotes their adhesion to endothelial cells. Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor). Interestingly, JAM-L expressed on neutrophils binds CAR independently of integrin activation. However, on resting monocytes and T lymphocytes, which express the integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form. Integrin activation is required for the dissociation of JAM-L-VLA-4 complexes and the accumulation of functional JAM-L dimers, which indicates that the leukocyte integrin VLA-4 controls JAM-L function in cis by controlling its dimerization state. This provides a mechanism through which VLA-4 and JAM-L functions are coordinately regulated, allowing JAM-L to strengthen integrin-dependent adhesion of leukocytes to endothelial cells.

Show MeSH
Related in: MedlinePlus