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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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Random cell migration on ICAM-1. (A) Adhesion of T cells on immobilized mouse ICAM-1–Fc. The adhesion of T cells infected with control adenovirus (control) stimulated with either 100 nM SLC or 10 ng/ml PMA and Rap1V12-expressing T cells (Rap1V12) are shown. Adhesion to ICAM-1 was <10% in the presence of anti-LFA-1 or anti-ICAM-1 antibodies. The mean and SE of triplicate determinations are shown. (B) Migration velocity of T cells on immobilized mouse ICAM-1–Fc. Migration velocity of T cells infected with control adenovirus (control) stimulated with either 100 nM SLC or 10 ng/ml PMA and Rap1V12-expressing T cells (Rap1V12) are shown. The mean velocity of GFP-positive cells (n = 20) was calculated and indicated with SE.
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fig3: Random cell migration on ICAM-1. (A) Adhesion of T cells on immobilized mouse ICAM-1–Fc. The adhesion of T cells infected with control adenovirus (control) stimulated with either 100 nM SLC or 10 ng/ml PMA and Rap1V12-expressing T cells (Rap1V12) are shown. Adhesion to ICAM-1 was <10% in the presence of anti-LFA-1 or anti-ICAM-1 antibodies. The mean and SE of triplicate determinations are shown. (B) Migration velocity of T cells on immobilized mouse ICAM-1–Fc. Migration velocity of T cells infected with control adenovirus (control) stimulated with either 100 nM SLC or 10 ng/ml PMA and Rap1V12-expressing T cells (Rap1V12) are shown. The mean velocity of GFP-positive cells (n = 20) was calculated and indicated with SE.

Mentions: Next, we examined the effect of Rap1V12 on T cell adhesion and migration. Rap1V12 expression in primary T cells increased adhesion to ICAM-1 (Fig. 3 A), as previously seen for T cell clones (Katagiri et al., 2002) or lymphocytes derived from Rap1V12-transgenic mice (Sebzda et al., 2002). Surprisingly, Rap1V12 also stimulated robust cell migration on ICAM-1, comparable to that seen after SLC stimulation (Fig. 3 B). Rap1V12-expressing cells moved on ICAM-1 as fast as 25 μm/min (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200301133/DC1). PMA treatment increased adhesion levels, but did not stimulate migration (Fig. 3, A and B), in contrast to the stimulatory effect of Rap1 on both adhesion and migration.


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)

Random cell migration on ICAM-1. (A) Adhesion of T cells on immobilized mouse ICAM-1–Fc. The adhesion of T cells infected with control adenovirus (control) stimulated with either 100 nM SLC or 10 ng/ml PMA and Rap1V12-expressing T cells (Rap1V12) are shown. Adhesion to ICAM-1 was <10% in the presence of anti-LFA-1 or anti-ICAM-1 antibodies. The mean and SE of triplicate determinations are shown. (B) Migration velocity of T cells on immobilized mouse ICAM-1–Fc. Migration velocity of T cells infected with control adenovirus (control) stimulated with either 100 nM SLC or 10 ng/ml PMA and Rap1V12-expressing T cells (Rap1V12) are shown. The mean velocity of GFP-positive cells (n = 20) was calculated and indicated with SE.
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Related In: Results  -  Collection

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fig3: Random cell migration on ICAM-1. (A) Adhesion of T cells on immobilized mouse ICAM-1–Fc. The adhesion of T cells infected with control adenovirus (control) stimulated with either 100 nM SLC or 10 ng/ml PMA and Rap1V12-expressing T cells (Rap1V12) are shown. Adhesion to ICAM-1 was <10% in the presence of anti-LFA-1 or anti-ICAM-1 antibodies. The mean and SE of triplicate determinations are shown. (B) Migration velocity of T cells on immobilized mouse ICAM-1–Fc. Migration velocity of T cells infected with control adenovirus (control) stimulated with either 100 nM SLC or 10 ng/ml PMA and Rap1V12-expressing T cells (Rap1V12) are shown. The mean velocity of GFP-positive cells (n = 20) was calculated and indicated with SE.
Mentions: Next, we examined the effect of Rap1V12 on T cell adhesion and migration. Rap1V12 expression in primary T cells increased adhesion to ICAM-1 (Fig. 3 A), as previously seen for T cell clones (Katagiri et al., 2002) or lymphocytes derived from Rap1V12-transgenic mice (Sebzda et al., 2002). Surprisingly, Rap1V12 also stimulated robust cell migration on ICAM-1, comparable to that seen after SLC stimulation (Fig. 3 B). Rap1V12-expressing cells moved on ICAM-1 as fast as 25 μm/min (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200301133/DC1). PMA treatment increased adhesion levels, but did not stimulate migration (Fig. 3, A and B), in contrast to the stimulatory effect of Rap1 on both adhesion and migration.

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