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Rap1 translates chemokine signals to integrin activation, cell polarization, and motility across vascular endothelium under flow.

Shimonaka M, Katagiri K, Nakayama T, Fujita N, Tsuruo T, Yoshie O, Kinashi T - J. Cell Biol. (2003)

Bottom Line: However, the key regulatory molecules regulating this process have remained elusive.Here, we demonstrate that Rap1 plays a pivotal role in chemokine-induced integrin activation and migration.Spa1 effectively suppressed this polarization after SLC treatment.

View Article: PubMed Central - PubMed

Affiliation: Bayer-chair, Dept. of Molecular Immunology and Allergy, Graduate School of Medicine, Kyoto University, Yoshida-konoe, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
Chemokines arrest circulating lymphocytes within the vasculature through the rapid up-regulation of leukocyte integrin adhesive activity, promoting subsequent lymphocyte transmigration. However, the key regulatory molecules regulating this process have remained elusive. Here, we demonstrate that Rap1 plays a pivotal role in chemokine-induced integrin activation and migration. Rap1 was activated by secondary lymphoid tissue chemokine (SLC; CCL21) and stromal-derived factor 1 (CXCL4) treatment in lymphocytes within seconds. Inhibition of Rap1 by Spa1, a Rap1-specific GTPase-activating protein, abrogated chemokine-stimulated lymphocyte rapid adhesion to endothelial cells under flow via intercellular adhesion molecule 1. Expression of a dominant active Rap1V12 in lymphocytes stimulated shear-resistant adhesion, robust cell migration on immobilized intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, and transendothelial migration under flow. We also demonstrated that Rap1V12 expression in lymphocytes induced a polarized morphology, accompanied by the redistribution of CXCR4 and CD44 to the leading edge and uropod, respectively. Spa1 effectively suppressed this polarization after SLC treatment. This unique characteristic of Rap1 may control chemokine-induced lymphocyte extravasation.

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Time-lapse images of transendothelial migration under flow. The transmigration of SLC-stimulated (top) or PMA-stimulated (bottom) T cells infected with the control adenovirus and Rap1V12-expressing T cells (middle), was recorded at the indicated times after the induction of shear flow. Composite images of phase-contrast and GFP fluo-rescence are shown. Both control T cells stimulated with SLC and Rap1V12- expressing cells transmigrated through the endothelial monolayer (became phase-dark) within 10 min. Bars, 20 μm.
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fig7: Time-lapse images of transendothelial migration under flow. The transmigration of SLC-stimulated (top) or PMA-stimulated (bottom) T cells infected with the control adenovirus and Rap1V12-expressing T cells (middle), was recorded at the indicated times after the induction of shear flow. Composite images of phase-contrast and GFP fluo-rescence are shown. Both control T cells stimulated with SLC and Rap1V12- expressing cells transmigrated through the endothelial monolayer (became phase-dark) within 10 min. Bars, 20 μm.

Mentions: Next, we examined the role of the promigratory effect of Rap1 on transendothelial migration under shear stress using an MBEC4 endothelial cell line. LN cells infected with either control or Spa1-encoding adenovirus were incubated with MBEC4 monolayers in the presence or absence of SLC for the indicated times (1, 5, and 10 min). 20 min of shear stress (2 dyne/cm2) was then applied. SLC stimulated shear-resistant adhesion of T cells infected with control adenovirus, enhancing transmigration as early as 1 min (Fig. 6, A and B) . The adhesion and transmigration levels were augmented by increasing the period of SLC incubation, with the maximal transmigration level (55% of input cells) at the 10-min time point. The transmigration efficiency reached ∼70% of the attached cells under these conditions (Fig. 6, A and B). However, in the absence of shear flow, no lymphocytes transmigrated through the MBEC4 monolayer, demonstrating shear-stress dependency of lymphocyte transmigration, as seen for HUVECs (Cinamon et al., 2001). Experiments using soluble or immobilized SDF-1 stimulation demonstrated similar results, but possessed very low efficiencies of adhesion and transmigration through the MBEC4 monolayer (unpublished data). Spa1 expression in lymphocytes reduced SLC-induced adhesion and transmigration to basal levels (Fig. 6, A and B). Conversely, Rap1V12 expression in T cells augmented both adhesion and transmigration under flow in the absence of SLC (Fig. 6, A and B). The rate of transmigration without shear flow was <10% at any time points measured (unpublished data), indicating that Rap1V12-expressing T cells still requires shear stress for efficient transmigration. The time course and efficiency of Rap1V12-expressing T cell transmigration was similar to those of SLC-stimulated cells (Fig. 6, A and B). These results indicate that Rap1 rapidly induces firm attachment and enhances transmigration, which is consistent with the Rap1 effect on integrin-dependent adhesion and migration (Fig. 3 and Fig. 5). PMA stimulated attachment to endothelial cells, but failed to induce transmigration under flow (Fig. 6, A and B). Although treatment with PTX reduced SLC-stimulated adhesion and transmigration to basal levels, the transmigration induced by Rap1V12 was unaffected (Fig. 6 C). Time-lapse images exhibit the active migration of SLC-stimulated or Rap1V12-expressing lymphocytes over the endothelium before transmigration under shear stress (Fig. 7) . In contrast, PMA-stimulated lymphocytes adhered to the endothelium were not motile (Fig. 7). These results paralleled those obtained for adhesion and migration on immobilized ICAM-1 and VCAM-1 (Fig. 3 and Fig. 5), suggesting that cell migration enhancement by Rap1 is crucial for transmigration.


Rap1 translates chemokine signals to integrin activation, cell polarization, and motility across vascular endothelium under flow.

Shimonaka M, Katagiri K, Nakayama T, Fujita N, Tsuruo T, Yoshie O, Kinashi T - J. Cell Biol. (2003)

Time-lapse images of transendothelial migration under flow. The transmigration of SLC-stimulated (top) or PMA-stimulated (bottom) T cells infected with the control adenovirus and Rap1V12-expressing T cells (middle), was recorded at the indicated times after the induction of shear flow. Composite images of phase-contrast and GFP fluo-rescence are shown. Both control T cells stimulated with SLC and Rap1V12- expressing cells transmigrated through the endothelial monolayer (became phase-dark) within 10 min. Bars, 20 μm.
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Related In: Results  -  Collection

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fig7: Time-lapse images of transendothelial migration under flow. The transmigration of SLC-stimulated (top) or PMA-stimulated (bottom) T cells infected with the control adenovirus and Rap1V12-expressing T cells (middle), was recorded at the indicated times after the induction of shear flow. Composite images of phase-contrast and GFP fluo-rescence are shown. Both control T cells stimulated with SLC and Rap1V12- expressing cells transmigrated through the endothelial monolayer (became phase-dark) within 10 min. Bars, 20 μm.
Mentions: Next, we examined the role of the promigratory effect of Rap1 on transendothelial migration under shear stress using an MBEC4 endothelial cell line. LN cells infected with either control or Spa1-encoding adenovirus were incubated with MBEC4 monolayers in the presence or absence of SLC for the indicated times (1, 5, and 10 min). 20 min of shear stress (2 dyne/cm2) was then applied. SLC stimulated shear-resistant adhesion of T cells infected with control adenovirus, enhancing transmigration as early as 1 min (Fig. 6, A and B) . The adhesion and transmigration levels were augmented by increasing the period of SLC incubation, with the maximal transmigration level (55% of input cells) at the 10-min time point. The transmigration efficiency reached ∼70% of the attached cells under these conditions (Fig. 6, A and B). However, in the absence of shear flow, no lymphocytes transmigrated through the MBEC4 monolayer, demonstrating shear-stress dependency of lymphocyte transmigration, as seen for HUVECs (Cinamon et al., 2001). Experiments using soluble or immobilized SDF-1 stimulation demonstrated similar results, but possessed very low efficiencies of adhesion and transmigration through the MBEC4 monolayer (unpublished data). Spa1 expression in lymphocytes reduced SLC-induced adhesion and transmigration to basal levels (Fig. 6, A and B). Conversely, Rap1V12 expression in T cells augmented both adhesion and transmigration under flow in the absence of SLC (Fig. 6, A and B). The rate of transmigration without shear flow was <10% at any time points measured (unpublished data), indicating that Rap1V12-expressing T cells still requires shear stress for efficient transmigration. The time course and efficiency of Rap1V12-expressing T cell transmigration was similar to those of SLC-stimulated cells (Fig. 6, A and B). These results indicate that Rap1 rapidly induces firm attachment and enhances transmigration, which is consistent with the Rap1 effect on integrin-dependent adhesion and migration (Fig. 3 and Fig. 5). PMA stimulated attachment to endothelial cells, but failed to induce transmigration under flow (Fig. 6, A and B). Although treatment with PTX reduced SLC-stimulated adhesion and transmigration to basal levels, the transmigration induced by Rap1V12 was unaffected (Fig. 6 C). Time-lapse images exhibit the active migration of SLC-stimulated or Rap1V12-expressing lymphocytes over the endothelium before transmigration under shear stress (Fig. 7) . In contrast, PMA-stimulated lymphocytes adhered to the endothelium were not motile (Fig. 7). These results paralleled those obtained for adhesion and migration on immobilized ICAM-1 and VCAM-1 (Fig. 3 and Fig. 5), suggesting that cell migration enhancement by Rap1 is crucial for transmigration.

Bottom Line: However, the key regulatory molecules regulating this process have remained elusive.Here, we demonstrate that Rap1 plays a pivotal role in chemokine-induced integrin activation and migration.Spa1 effectively suppressed this polarization after SLC treatment.

View Article: PubMed Central - PubMed

Affiliation: Bayer-chair, Dept. of Molecular Immunology and Allergy, Graduate School of Medicine, Kyoto University, Yoshida-konoe, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
Chemokines arrest circulating lymphocytes within the vasculature through the rapid up-regulation of leukocyte integrin adhesive activity, promoting subsequent lymphocyte transmigration. However, the key regulatory molecules regulating this process have remained elusive. Here, we demonstrate that Rap1 plays a pivotal role in chemokine-induced integrin activation and migration. Rap1 was activated by secondary lymphoid tissue chemokine (SLC; CCL21) and stromal-derived factor 1 (CXCL4) treatment in lymphocytes within seconds. Inhibition of Rap1 by Spa1, a Rap1-specific GTPase-activating protein, abrogated chemokine-stimulated lymphocyte rapid adhesion to endothelial cells under flow via intercellular adhesion molecule 1. Expression of a dominant active Rap1V12 in lymphocytes stimulated shear-resistant adhesion, robust cell migration on immobilized intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, and transendothelial migration under flow. We also demonstrated that Rap1V12 expression in lymphocytes induced a polarized morphology, accompanied by the redistribution of CXCR4 and CD44 to the leading edge and uropod, respectively. Spa1 effectively suppressed this polarization after SLC treatment. This unique characteristic of Rap1 may control chemokine-induced lymphocyte extravasation.

Show MeSH
Related in: MedlinePlus