Limits...
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.

Show MeSH

Related in: MedlinePlus

JAM-L function is regulated in cis by the activation state of VLA-4 integrin. (A) U937/pMT–JAM-L–HA cells were incubated with antibodies against α4 subunit (P4C2) or CD71 for 30 min at 37°C. Cells were washed and surface stained with 9EG7 antibody at 4°C for 30 min in the absence of cation (bold black lines). In addition, U937 cells were incubated with 9EG7 antibody in the presence of Mn2+ (gray broken lines) as a positive control for integrin activation or in the absence of cation (thin black lines) to indicate the basal level of integrin activation. Cells were labeled with R-phycoerythrin–conjugated secondary antibodies, then fixed and analyzed by flow cytometry. Gray areas represent staining with the secondary antibody alone. (B) U937/pMT–JAM-L–HA cells were untreated (white) or treated (gray) with Zn2+ to induce JAM-L–HA expression. Cells were labeled with CMFDA and incubated with either anti-α4 (P4C2) or anti-CD71, and adhesion to HBMECs prewashed with PBS Ca−/Mg− was assayed for 30 min at 37°C. Adherent cells were lysed and fluorescence intensity was measured. Basal adhesion was 2% of the total number of incubated cells. Results are means ± SD (n = 3) of a representative three independent experiments (**, P < 0.01). (C and D) K562 cells were transfected with vectors encoding JAM-L–GFP (gray) or GFP (white). Also, double transfectants were generated with human α4 subunit (+). K562 cells were added to wells containing immobilized VCAM1-Fc proteins (C) or CAR-Fc proteins (D) in PBS without cations or in the presence of Mn2+ for 40 min at 37°C. The number of adherent GFP-positive cells was determined by FACS analysis. Results are expressed as the quantity of adherent cells, normalized relative to the basal adhesion of K562/JAM-L and α4-negative cells. The basal adhesion level on CAR-Fc and VCAM1-Fc was 8% and 2% of the total number of incubated cells, respectively. Results are means ± SD (n = 4) of a representative three independent experiments (**, P < 0.01). (E) FACS analysis of K562 cells transfected with vectors encoding JAM-L-GFP alone, α4 subunit alone, or double transfected with both constructs. Lines demarcate the threshold for positive staining for both markers.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2600739&req=5

fig6: JAM-L function is regulated in cis by the activation state of VLA-4 integrin. (A) U937/pMT–JAM-L–HA cells were incubated with antibodies against α4 subunit (P4C2) or CD71 for 30 min at 37°C. Cells were washed and surface stained with 9EG7 antibody at 4°C for 30 min in the absence of cation (bold black lines). In addition, U937 cells were incubated with 9EG7 antibody in the presence of Mn2+ (gray broken lines) as a positive control for integrin activation or in the absence of cation (thin black lines) to indicate the basal level of integrin activation. Cells were labeled with R-phycoerythrin–conjugated secondary antibodies, then fixed and analyzed by flow cytometry. Gray areas represent staining with the secondary antibody alone. (B) U937/pMT–JAM-L–HA cells were untreated (white) or treated (gray) with Zn2+ to induce JAM-L–HA expression. Cells were labeled with CMFDA and incubated with either anti-α4 (P4C2) or anti-CD71, and adhesion to HBMECs prewashed with PBS Ca−/Mg− was assayed for 30 min at 37°C. Adherent cells were lysed and fluorescence intensity was measured. Basal adhesion was 2% of the total number of incubated cells. Results are means ± SD (n = 3) of a representative three independent experiments (**, P < 0.01). (C and D) K562 cells were transfected with vectors encoding JAM-L–GFP (gray) or GFP (white). Also, double transfectants were generated with human α4 subunit (+). K562 cells were added to wells containing immobilized VCAM1-Fc proteins (C) or CAR-Fc proteins (D) in PBS without cations or in the presence of Mn2+ for 40 min at 37°C. The number of adherent GFP-positive cells was determined by FACS analysis. Results are expressed as the quantity of adherent cells, normalized relative to the basal adhesion of K562/JAM-L and α4-negative cells. The basal adhesion level on CAR-Fc and VCAM1-Fc was 8% and 2% of the total number of incubated cells, respectively. Results are means ± SD (n = 4) of a representative three independent experiments (**, P < 0.01). (E) FACS analysis of K562 cells transfected with vectors encoding JAM-L-GFP alone, α4 subunit alone, or double transfected with both constructs. Lines demarcate the threshold for positive staining for both markers.

Mentions: To assess whether the activation of VLA-4 was sufficient to promote JAM-L function, the adhesion to HBMECs of U937 cells that conditionally express JAM-L–HA was assessed in PBS in the presence of P4C2, an antibody against α4. The addition of P4C2 antibody to U937 cells induced VLA-4 activation, as assessed by FACS analysis using antibodies directed against an activated β1 integrin subunit (Fig. 6 A). As a control, addition of an isotype-matched irrelevant antibody (anti-CD71, which binds to U937 cells) failed to activate VLA-4. Interestingly, addition of P4C2 antibody revealed JAM-L–mediated adhesion of U937 cells expressing JAM-L to resting HBMECs in the absence of Mn2+ (Fig. 6 B). Similar results were obtained using HP2/1, another antibody with VLA-4–activating activity (unpublished data; McGilvray et al., 1997). These results therefore strongly suggest that the activation of VLA-4 is required to unmask JAM-L function in cellular adhesion.


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 function is regulated in cis by the activation state of VLA-4 integrin. (A) U937/pMT–JAM-L–HA cells were incubated with antibodies against α4 subunit (P4C2) or CD71 for 30 min at 37°C. Cells were washed and surface stained with 9EG7 antibody at 4°C for 30 min in the absence of cation (bold black lines). In addition, U937 cells were incubated with 9EG7 antibody in the presence of Mn2+ (gray broken lines) as a positive control for integrin activation or in the absence of cation (thin black lines) to indicate the basal level of integrin activation. Cells were labeled with R-phycoerythrin–conjugated secondary antibodies, then fixed and analyzed by flow cytometry. Gray areas represent staining with the secondary antibody alone. (B) U937/pMT–JAM-L–HA cells were untreated (white) or treated (gray) with Zn2+ to induce JAM-L–HA expression. Cells were labeled with CMFDA and incubated with either anti-α4 (P4C2) or anti-CD71, and adhesion to HBMECs prewashed with PBS Ca−/Mg− was assayed for 30 min at 37°C. Adherent cells were lysed and fluorescence intensity was measured. Basal adhesion was 2% of the total number of incubated cells. Results are means ± SD (n = 3) of a representative three independent experiments (**, P < 0.01). (C and D) K562 cells were transfected with vectors encoding JAM-L–GFP (gray) or GFP (white). Also, double transfectants were generated with human α4 subunit (+). K562 cells were added to wells containing immobilized VCAM1-Fc proteins (C) or CAR-Fc proteins (D) in PBS without cations or in the presence of Mn2+ for 40 min at 37°C. The number of adherent GFP-positive cells was determined by FACS analysis. Results are expressed as the quantity of adherent cells, normalized relative to the basal adhesion of K562/JAM-L and α4-negative cells. The basal adhesion level on CAR-Fc and VCAM1-Fc was 8% and 2% of the total number of incubated cells, respectively. Results are means ± SD (n = 4) of a representative three independent experiments (**, P < 0.01). (E) FACS analysis of K562 cells transfected with vectors encoding JAM-L-GFP alone, α4 subunit alone, or double transfected with both constructs. Lines demarcate the threshold for positive staining for both markers.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2600739&req=5

fig6: JAM-L function is regulated in cis by the activation state of VLA-4 integrin. (A) U937/pMT–JAM-L–HA cells were incubated with antibodies against α4 subunit (P4C2) or CD71 for 30 min at 37°C. Cells were washed and surface stained with 9EG7 antibody at 4°C for 30 min in the absence of cation (bold black lines). In addition, U937 cells were incubated with 9EG7 antibody in the presence of Mn2+ (gray broken lines) as a positive control for integrin activation or in the absence of cation (thin black lines) to indicate the basal level of integrin activation. Cells were labeled with R-phycoerythrin–conjugated secondary antibodies, then fixed and analyzed by flow cytometry. Gray areas represent staining with the secondary antibody alone. (B) U937/pMT–JAM-L–HA cells were untreated (white) or treated (gray) with Zn2+ to induce JAM-L–HA expression. Cells were labeled with CMFDA and incubated with either anti-α4 (P4C2) or anti-CD71, and adhesion to HBMECs prewashed with PBS Ca−/Mg− was assayed for 30 min at 37°C. Adherent cells were lysed and fluorescence intensity was measured. Basal adhesion was 2% of the total number of incubated cells. Results are means ± SD (n = 3) of a representative three independent experiments (**, P < 0.01). (C and D) K562 cells were transfected with vectors encoding JAM-L–GFP (gray) or GFP (white). Also, double transfectants were generated with human α4 subunit (+). K562 cells were added to wells containing immobilized VCAM1-Fc proteins (C) or CAR-Fc proteins (D) in PBS without cations or in the presence of Mn2+ for 40 min at 37°C. The number of adherent GFP-positive cells was determined by FACS analysis. Results are expressed as the quantity of adherent cells, normalized relative to the basal adhesion of K562/JAM-L and α4-negative cells. The basal adhesion level on CAR-Fc and VCAM1-Fc was 8% and 2% of the total number of incubated cells, respectively. Results are means ± SD (n = 4) of a representative three independent experiments (**, P < 0.01). (E) FACS analysis of K562 cells transfected with vectors encoding JAM-L-GFP alone, α4 subunit alone, or double transfected with both constructs. Lines demarcate the threshold for positive staining for both markers.
Mentions: To assess whether the activation of VLA-4 was sufficient to promote JAM-L function, the adhesion to HBMECs of U937 cells that conditionally express JAM-L–HA was assessed in PBS in the presence of P4C2, an antibody against α4. The addition of P4C2 antibody to U937 cells induced VLA-4 activation, as assessed by FACS analysis using antibodies directed against an activated β1 integrin subunit (Fig. 6 A). As a control, addition of an isotype-matched irrelevant antibody (anti-CD71, which binds to U937 cells) failed to activate VLA-4. Interestingly, addition of P4C2 antibody revealed JAM-L–mediated adhesion of U937 cells expressing JAM-L to resting HBMECs in the absence of Mn2+ (Fig. 6 B). Similar results were obtained using HP2/1, another antibody with VLA-4–activating activity (unpublished data; McGilvray et al., 1997). These results therefore strongly suggest that the activation of VLA-4 is required to unmask JAM-L function in cellular adhesion.

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