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

In monocytic cells, JAM-L function requires integrin activation in cis to enhance cell adhesion. (A) K562 cells transfected with an empty vector or a vector encoding JAM-L (left) or U937/pMT–JAM-L–HA cells untreated or treated with Zn2+ to induce JAM-L–HA expression (right) were labeled using anti–JAM-L, then fixed and analyzed by flow cytometry. (B) K562 cells were transfected with JAM-L–GFP (gray) or GFP alone (white). Cells were preincubated for 5 min at 37°C in PBS with EDTA or Mn2+, and adhesion to HBMECs was assayed in the same buffer for 30 min at 37°C. The number of adherent GFP-positive cells was determined by FACS analysis. The basal adhesion level was 2% of the total number of incubated cells (**, P < 0.01). (C and D) U937 cells stably transfected with pMT–JAM-L–HA were untreated (white) or treated (gray) with Zn2+ to induce JAM-L expression and then loaded with CMFDA. (C) U937 cells were preincubated for 5 min at 37°C in PBS with EDTA or Mn2+. Adhesion to HBMECs was assayed in the same buffers for 30 min at 37°C. The basal adhesion level was 5% (***, P < 0.001). (D) U937 cells were left untreated (NT) or stimulated for 5 min at 37°C with 100 ng/ml SDF-1α in PBS Ca−/Mg−. Cells were then subjected to adhesion to HBMECs in the same buffers for 30 min at 37°C. The basal adhesion level was 5% (**, P < 0.01). (E) FACS analysis of U937/pMT–JAM-L–HA transfected with Talin-F23–GFP (bold black lines) or with GFP alone (broken lines). Cells were stained with 9EG7 or MEM-148 antibodies, which recognize active β1 or β2 integrin subunits, respectively, in PBS (no cation, top) or PBS with Mn2+ (manganese, bottom). Only GFP-positives cells were analyzed by flow cytometry. Gray areas represent staining profiles with secondary antibodies alone. (F) U937/pMT–JAM-L–HA cells were untreated (white) or treated (gray) with Zn2+ and transfected with Talin-F23–GFP or GFP for 8 h. Adhesion to HBMECs was assayed in PBS Ca−/Mg− for 30 min at 37°C. The number of adherent GFP-positive cells was determined by FACS analysis. The basal adhesion level was 6% (***, P < 0.001). Results in B, C, D, and F are means ± SD (n = 3) of a representative three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: In monocytic cells, JAM-L function requires integrin activation in cis to enhance cell adhesion. (A) K562 cells transfected with an empty vector or a vector encoding JAM-L (left) or U937/pMT–JAM-L–HA cells untreated or treated with Zn2+ to induce JAM-L–HA expression (right) were labeled using anti–JAM-L, then fixed and analyzed by flow cytometry. (B) K562 cells were transfected with JAM-L–GFP (gray) or GFP alone (white). Cells were preincubated for 5 min at 37°C in PBS with EDTA or Mn2+, and adhesion to HBMECs was assayed in the same buffer for 30 min at 37°C. The number of adherent GFP-positive cells was determined by FACS analysis. The basal adhesion level was 2% of the total number of incubated cells (**, P < 0.01). (C and D) U937 cells stably transfected with pMT–JAM-L–HA were untreated (white) or treated (gray) with Zn2+ to induce JAM-L expression and then loaded with CMFDA. (C) U937 cells were preincubated for 5 min at 37°C in PBS with EDTA or Mn2+. Adhesion to HBMECs was assayed in the same buffers for 30 min at 37°C. The basal adhesion level was 5% (***, P < 0.001). (D) U937 cells were left untreated (NT) or stimulated for 5 min at 37°C with 100 ng/ml SDF-1α in PBS Ca−/Mg−. Cells were then subjected to adhesion to HBMECs in the same buffers for 30 min at 37°C. The basal adhesion level was 5% (**, P < 0.01). (E) FACS analysis of U937/pMT–JAM-L–HA transfected with Talin-F23–GFP (bold black lines) or with GFP alone (broken lines). Cells were stained with 9EG7 or MEM-148 antibodies, which recognize active β1 or β2 integrin subunits, respectively, in PBS (no cation, top) or PBS with Mn2+ (manganese, bottom). Only GFP-positives cells were analyzed by flow cytometry. Gray areas represent staining profiles with secondary antibodies alone. (F) U937/pMT–JAM-L–HA cells were untreated (white) or treated (gray) with Zn2+ and transfected with Talin-F23–GFP or GFP for 8 h. Adhesion to HBMECs was assayed in PBS Ca−/Mg− for 30 min at 37°C. The number of adherent GFP-positive cells was determined by FACS analysis. The basal adhesion level was 6% (***, P < 0.001). Results in B, C, D, and F are means ± SD (n = 3) of a representative three independent experiments.

Mentions: In characterizing the JAM-L–dependent adhesion of leukocytes to endothelial cells, we routinely performed adhesion in RPMI medium containing the cations Ca2+, Mg2+, and Mn2+. The dependence of integrin function on divalent cations is well established (Luo et al., 2007); therefore, we examined the possible role of integrins in JAM-L–mediated adhesion by analyzing the dependence of K562 cells or U937 cells (expressing or not expressing JAM-L to a similar extent; Fig. 3 A) on divalent cations for adhesion to endothelial cells using PBS buffer containing EDTA (which inhibits integrin function) or Mn2+ (which activates integrins). Intriguingly, JAM-L–mediated adhesion of K562 cells to HBMECs was observed both in PBS buffer containing EDTA or Mn2+ (Fig. 3 B), whereas JAM-L–mediated adhesion of U937 cells, which express zinc-inducible JAM-L–HA, was strictly dependent on the presence of Mn2+ (Fig. 3 C), which suggests the involvement of an integrin in JAM-L–mediated function in U937 cells.


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)

In monocytic cells, JAM-L function requires integrin activation in cis to enhance cell adhesion. (A) K562 cells transfected with an empty vector or a vector encoding JAM-L (left) or U937/pMT–JAM-L–HA cells untreated or treated with Zn2+ to induce JAM-L–HA expression (right) were labeled using anti–JAM-L, then fixed and analyzed by flow cytometry. (B) K562 cells were transfected with JAM-L–GFP (gray) or GFP alone (white). Cells were preincubated for 5 min at 37°C in PBS with EDTA or Mn2+, and adhesion to HBMECs was assayed in the same buffer for 30 min at 37°C. The number of adherent GFP-positive cells was determined by FACS analysis. The basal adhesion level was 2% of the total number of incubated cells (**, P < 0.01). (C and D) U937 cells stably transfected with pMT–JAM-L–HA were untreated (white) or treated (gray) with Zn2+ to induce JAM-L expression and then loaded with CMFDA. (C) U937 cells were preincubated for 5 min at 37°C in PBS with EDTA or Mn2+. Adhesion to HBMECs was assayed in the same buffers for 30 min at 37°C. The basal adhesion level was 5% (***, P < 0.001). (D) U937 cells were left untreated (NT) or stimulated for 5 min at 37°C with 100 ng/ml SDF-1α in PBS Ca−/Mg−. Cells were then subjected to adhesion to HBMECs in the same buffers for 30 min at 37°C. The basal adhesion level was 5% (**, P < 0.01). (E) FACS analysis of U937/pMT–JAM-L–HA transfected with Talin-F23–GFP (bold black lines) or with GFP alone (broken lines). Cells were stained with 9EG7 or MEM-148 antibodies, which recognize active β1 or β2 integrin subunits, respectively, in PBS (no cation, top) or PBS with Mn2+ (manganese, bottom). Only GFP-positives cells were analyzed by flow cytometry. Gray areas represent staining profiles with secondary antibodies alone. (F) U937/pMT–JAM-L–HA cells were untreated (white) or treated (gray) with Zn2+ and transfected with Talin-F23–GFP or GFP for 8 h. Adhesion to HBMECs was assayed in PBS Ca−/Mg− for 30 min at 37°C. The number of adherent GFP-positive cells was determined by FACS analysis. The basal adhesion level was 6% (***, P < 0.001). Results in B, C, D, and F are means ± SD (n = 3) of a representative three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: In monocytic cells, JAM-L function requires integrin activation in cis to enhance cell adhesion. (A) K562 cells transfected with an empty vector or a vector encoding JAM-L (left) or U937/pMT–JAM-L–HA cells untreated or treated with Zn2+ to induce JAM-L–HA expression (right) were labeled using anti–JAM-L, then fixed and analyzed by flow cytometry. (B) K562 cells were transfected with JAM-L–GFP (gray) or GFP alone (white). Cells were preincubated for 5 min at 37°C in PBS with EDTA or Mn2+, and adhesion to HBMECs was assayed in the same buffer for 30 min at 37°C. The number of adherent GFP-positive cells was determined by FACS analysis. The basal adhesion level was 2% of the total number of incubated cells (**, P < 0.01). (C and D) U937 cells stably transfected with pMT–JAM-L–HA were untreated (white) or treated (gray) with Zn2+ to induce JAM-L expression and then loaded with CMFDA. (C) U937 cells were preincubated for 5 min at 37°C in PBS with EDTA or Mn2+. Adhesion to HBMECs was assayed in the same buffers for 30 min at 37°C. The basal adhesion level was 5% (***, P < 0.001). (D) U937 cells were left untreated (NT) or stimulated for 5 min at 37°C with 100 ng/ml SDF-1α in PBS Ca−/Mg−. Cells were then subjected to adhesion to HBMECs in the same buffers for 30 min at 37°C. The basal adhesion level was 5% (**, P < 0.01). (E) FACS analysis of U937/pMT–JAM-L–HA transfected with Talin-F23–GFP (bold black lines) or with GFP alone (broken lines). Cells were stained with 9EG7 or MEM-148 antibodies, which recognize active β1 or β2 integrin subunits, respectively, in PBS (no cation, top) or PBS with Mn2+ (manganese, bottom). Only GFP-positives cells were analyzed by flow cytometry. Gray areas represent staining profiles with secondary antibodies alone. (F) U937/pMT–JAM-L–HA cells were untreated (white) or treated (gray) with Zn2+ and transfected with Talin-F23–GFP or GFP for 8 h. Adhesion to HBMECs was assayed in PBS Ca−/Mg− for 30 min at 37°C. The number of adherent GFP-positive cells was determined by FACS analysis. The basal adhesion level was 6% (***, P < 0.001). Results in B, C, D, and F are means ± SD (n = 3) of a representative three independent experiments.
Mentions: In characterizing the JAM-L–dependent adhesion of leukocytes to endothelial cells, we routinely performed adhesion in RPMI medium containing the cations Ca2+, Mg2+, and Mn2+. The dependence of integrin function on divalent cations is well established (Luo et al., 2007); therefore, we examined the possible role of integrins in JAM-L–mediated adhesion by analyzing the dependence of K562 cells or U937 cells (expressing or not expressing JAM-L to a similar extent; Fig. 3 A) on divalent cations for adhesion to endothelial cells using PBS buffer containing EDTA (which inhibits integrin function) or Mn2+ (which activates integrins). Intriguingly, JAM-L–mediated adhesion of K562 cells to HBMECs was observed both in PBS buffer containing EDTA or Mn2+ (Fig. 3 B), whereas JAM-L–mediated adhesion of U937 cells, which express zinc-inducible JAM-L–HA, was strictly dependent on the presence of Mn2+ (Fig. 3 C), which suggests the involvement of an integrin in JAM-L–mediated function in U937 cells.

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