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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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The dependence of JAM-L–mediated adhesion of human leukocytes on integrin activation is cell type specific. Freshly isolated human neutrophils (A) or monocytes (C) were loaded with CMFDA, incubated with 20 μg/ml of soluble JAM-L–Fc or CD25-Fc, or no soluble proteins (−), as indicated, for 30 min at 37°C, then added to wells containing immobilized chimeric CD25-Fc, ICAM-1-Fc, or CAR-Fc proteins for 40 min at 37°C in PBS Ca−/Mg− and with EDTA. Adherent cells were lysed and fluorescence intensity was measured. Results are expressed as the amount of fluorescence released from adherent cells, normalized relative to the adhesion of the cells to CD25-Fc, to which 7% of neutrophils or 14% of monocytes adhered. Results are means ± SD (n = 4) of a representative four independent experiments (***, P < 0.001). (B) K562 cells were transfected with vectors encoding JAM-L–GFP (gray) or GFP alone (white), and added to wells containing immobilized chimeric CD25-Fc or CAR-Fc proteins for 40 min at 37°C in PBS Ca−/Mg− with EDTA. The number of adherent GFP-positive cells was determined by FACS analysis. The basal adhesion level was 7%. Results are means ± SD (n = 3) of a representative three independent experiments (**, P < 0.01). (D) U937 cells stably transfected with pMT–JAM-L–HA were untreated (white) or treated (gray) with Zn2+ to induce JAM-L expression. Cells were loaded with CMFDA and added to wells containing immobilized chimeric CD25-Fc or CAR-Fc proteins for 40 min at 37°C in PBS Ca−/Mg− with EDTA. The basal adhesion level was 26%. Results are means ± SD (n = 3) of a representative three independent experiments.
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fig4: The dependence of JAM-L–mediated adhesion of human leukocytes on integrin activation is cell type specific. Freshly isolated human neutrophils (A) or monocytes (C) were loaded with CMFDA, incubated with 20 μg/ml of soluble JAM-L–Fc or CD25-Fc, or no soluble proteins (−), as indicated, for 30 min at 37°C, then added to wells containing immobilized chimeric CD25-Fc, ICAM-1-Fc, or CAR-Fc proteins for 40 min at 37°C in PBS Ca−/Mg− and with EDTA. Adherent cells were lysed and fluorescence intensity was measured. Results are expressed as the amount of fluorescence released from adherent cells, normalized relative to the adhesion of the cells to CD25-Fc, to which 7% of neutrophils or 14% of monocytes adhered. Results are means ± SD (n = 4) of a representative four independent experiments (***, P < 0.001). (B) K562 cells were transfected with vectors encoding JAM-L–GFP (gray) or GFP alone (white), and added to wells containing immobilized chimeric CD25-Fc or CAR-Fc proteins for 40 min at 37°C in PBS Ca−/Mg− with EDTA. The number of adherent GFP-positive cells was determined by FACS analysis. The basal adhesion level was 7%. Results are means ± SD (n = 3) of a representative three independent experiments (**, P < 0.01). (D) U937 cells stably transfected with pMT–JAM-L–HA were untreated (white) or treated (gray) with Zn2+ to induce JAM-L expression. Cells were loaded with CMFDA and added to wells containing immobilized chimeric CD25-Fc or CAR-Fc proteins for 40 min at 37°C in PBS Ca−/Mg− with EDTA. The basal adhesion level was 26%. Results are means ± SD (n = 3) of a representative three independent experiments.

Mentions: Because previous work indicated that neutrophil adhesion to CAR can occur in a cation-independent manner (Zen et al., 2005), we then analyzed the contribution of the endogenous JAM-L to the adhesion of freshly isolated neutrophils and monocytes to immobilized CAR in PBS containing EDTA. We observed that the adhesion of neutrophils to CAR was abolished in the presence of soluble JAM-L–Fc, whereas the addition of soluble CD25-Fc at the same concentration as a control had no effect (Fig. 4 A). These results indicate that JAM-L can mediate the adhesion of human neutrophils to CAR independently of integrin activation, as observed with K562 cells, in which JAM-L expression promoted their cation-independent adhesion to CAR (Fig. 4 B). However, the addition of soluble JAM-L–Fc did not affect the adhesion of freshly isolated monocytes to CAR (Fig. 4 C) or of T lymphocytes (not depicted), which indicates that JAM-L does not support their adhesion in the absence of integrin activation. Interestingly, these results are similar to what was observed in the monocytic U937 cell line, in which JAM-L expression did not promote adhesion to CAR in PBS containing EDTA (Fig. 4 D).


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)

The dependence of JAM-L–mediated adhesion of human leukocytes on integrin activation is cell type specific. Freshly isolated human neutrophils (A) or monocytes (C) were loaded with CMFDA, incubated with 20 μg/ml of soluble JAM-L–Fc or CD25-Fc, or no soluble proteins (−), as indicated, for 30 min at 37°C, then added to wells containing immobilized chimeric CD25-Fc, ICAM-1-Fc, or CAR-Fc proteins for 40 min at 37°C in PBS Ca−/Mg− and with EDTA. Adherent cells were lysed and fluorescence intensity was measured. Results are expressed as the amount of fluorescence released from adherent cells, normalized relative to the adhesion of the cells to CD25-Fc, to which 7% of neutrophils or 14% of monocytes adhered. Results are means ± SD (n = 4) of a representative four independent experiments (***, P < 0.001). (B) K562 cells were transfected with vectors encoding JAM-L–GFP (gray) or GFP alone (white), and added to wells containing immobilized chimeric CD25-Fc or CAR-Fc proteins for 40 min at 37°C in PBS Ca−/Mg− with EDTA. The number of adherent GFP-positive cells was determined by FACS analysis. The basal adhesion level was 7%. Results are means ± SD (n = 3) of a representative three independent experiments (**, P < 0.01). (D) U937 cells stably transfected with pMT–JAM-L–HA were untreated (white) or treated (gray) with Zn2+ to induce JAM-L expression. Cells were loaded with CMFDA and added to wells containing immobilized chimeric CD25-Fc or CAR-Fc proteins for 40 min at 37°C in PBS Ca−/Mg− with EDTA. The basal adhesion level was 26%. Results are means ± SD (n = 3) of a representative three independent experiments.
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fig4: The dependence of JAM-L–mediated adhesion of human leukocytes on integrin activation is cell type specific. Freshly isolated human neutrophils (A) or monocytes (C) were loaded with CMFDA, incubated with 20 μg/ml of soluble JAM-L–Fc or CD25-Fc, or no soluble proteins (−), as indicated, for 30 min at 37°C, then added to wells containing immobilized chimeric CD25-Fc, ICAM-1-Fc, or CAR-Fc proteins for 40 min at 37°C in PBS Ca−/Mg− and with EDTA. Adherent cells were lysed and fluorescence intensity was measured. Results are expressed as the amount of fluorescence released from adherent cells, normalized relative to the adhesion of the cells to CD25-Fc, to which 7% of neutrophils or 14% of monocytes adhered. Results are means ± SD (n = 4) of a representative four independent experiments (***, P < 0.001). (B) K562 cells were transfected with vectors encoding JAM-L–GFP (gray) or GFP alone (white), and added to wells containing immobilized chimeric CD25-Fc or CAR-Fc proteins for 40 min at 37°C in PBS Ca−/Mg− with EDTA. The number of adherent GFP-positive cells was determined by FACS analysis. The basal adhesion level was 7%. Results are means ± SD (n = 3) of a representative three independent experiments (**, P < 0.01). (D) U937 cells stably transfected with pMT–JAM-L–HA were untreated (white) or treated (gray) with Zn2+ to induce JAM-L expression. Cells were loaded with CMFDA and added to wells containing immobilized chimeric CD25-Fc or CAR-Fc proteins for 40 min at 37°C in PBS Ca−/Mg− with EDTA. The basal adhesion level was 26%. Results are means ± SD (n = 3) of a representative three independent experiments.
Mentions: Because previous work indicated that neutrophil adhesion to CAR can occur in a cation-independent manner (Zen et al., 2005), we then analyzed the contribution of the endogenous JAM-L to the adhesion of freshly isolated neutrophils and monocytes to immobilized CAR in PBS containing EDTA. We observed that the adhesion of neutrophils to CAR was abolished in the presence of soluble JAM-L–Fc, whereas the addition of soluble CD25-Fc at the same concentration as a control had no effect (Fig. 4 A). These results indicate that JAM-L can mediate the adhesion of human neutrophils to CAR independently of integrin activation, as observed with K562 cells, in which JAM-L expression promoted their cation-independent adhesion to CAR (Fig. 4 B). However, the addition of soluble JAM-L–Fc did not affect the adhesion of freshly isolated monocytes to CAR (Fig. 4 C) or of T lymphocytes (not depicted), which indicates that JAM-L does not support their adhesion in the absence of integrin activation. Interestingly, these results are similar to what was observed in the monocytic U937 cell line, in which JAM-L expression did not promote adhesion to CAR in PBS containing EDTA (Fig. 4 D).

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