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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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Rap1 is involved in lymphocyte polarization. (A) Asymmetrical morphology and distribution of CXCR4 (green) and CD44 (red) in chemokine-stimulated T cells. T cells were stimulated with 100 nM SLC or 100 nM SDF-1 for 10 min, fixed in suspension, and stained as in Fig. 8. (B) Quantitative analysis of lymphocyte polarization. T cells infected with adenoviruses encoding either GFP alone (control), Spa1, or Rap1V12 adenovirus were incubated without or with 100 nM SLC, as indicated, for 10 min and fixed in suspension. GFP-positive lymphocytes with CD44-marked uropods were quantified (100–150 cells), and are expressed as a percentage of the total GFP-positive cells. The mean and SE of triplicate experiments are shown. (C) Effects of Rap1V12 and Spa1 expression on lymphocyte polarization. T cells infected with the control, Spa1, or Rap1V12 adenovirus were incubated without or with 100 nM SLC as indicated, for 10 min, and fixed in suspension. Images detail GFP fluorescence (top), morphology by differential interference contrast (middle), and CD44 (bottom). Control adenovirus-infected lymphocytes exhibited a homogeneous distribution of CD44 without typical uropods. Polarized cell shape and CD44 redistribution were induced in control lymphocytes by SLC treatment and Rap1V12 expression. Spa1-expressing T cells failed to develop polarized morphology and redistribute CD44 when stimulated with SLC. Asterisks indicate adenovirus-infected cells. Bars, 10 μm.
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fig9: Rap1 is involved in lymphocyte polarization. (A) Asymmetrical morphology and distribution of CXCR4 (green) and CD44 (red) in chemokine-stimulated T cells. T cells were stimulated with 100 nM SLC or 100 nM SDF-1 for 10 min, fixed in suspension, and stained as in Fig. 8. (B) Quantitative analysis of lymphocyte polarization. T cells infected with adenoviruses encoding either GFP alone (control), Spa1, or Rap1V12 adenovirus were incubated without or with 100 nM SLC, as indicated, for 10 min and fixed in suspension. GFP-positive lymphocytes with CD44-marked uropods were quantified (100–150 cells), and are expressed as a percentage of the total GFP-positive cells. The mean and SE of triplicate experiments are shown. (C) Effects of Rap1V12 and Spa1 expression on lymphocyte polarization. T cells infected with the control, Spa1, or Rap1V12 adenovirus were incubated without or with 100 nM SLC as indicated, for 10 min, and fixed in suspension. Images detail GFP fluorescence (top), morphology by differential interference contrast (middle), and CD44 (bottom). Control adenovirus-infected lymphocytes exhibited a homogeneous distribution of CD44 without typical uropods. Polarized cell shape and CD44 redistribution were induced in control lymphocytes by SLC treatment and Rap1V12 expression. Spa1-expressing T cells failed to develop polarized morphology and redistribute CD44 when stimulated with SLC. Asterisks indicate adenovirus-infected cells. Bars, 10 μm.

Mentions: To confirm whether Rap1 is also involved in lymphocyte polarization, we examined the effects of Rap1V12 and Spa1 after their introduction into lymphocytes via adenoviral infection. When stimulated with SLC or SDF-1, lymphocytes were transformed into polarized cell shapes with well-developed leading edges and uropods marked by CXCR4 and CD44, respectively (Fig. 9 A). Control adenovirus-infected lymphocytes, indicated by GFP expression, were similarly transformed by SLC into a polarized cell shape with the development of CD44-marked uropods (Fig. 9 C). Quantitative analysis indicated that ∼60% of stimulated cells exhibited this polarized phenotype (Fig. 9 B). Rap1V12 expression, also indicated by GFP expression, markedly increased the number of polarized lymphocytes with CD44-marked uropods (Fig. 9, B and C), comparable to the levels induced by SLC. Conversely, Spa1 expression reduced the number of SLC-stimulated polarized lymphocytes exhibiting an asymmetric redistribution of CD44 to basal levels (Fig. 9, B and C). Collectively, these results indicate that Rap1 activation triggers lymphocyte polarization even in nonadherent lymphocytes. Thus, Rap1-mediated cell polarization triggered by chemokines does not depend on integrin-mediated adhesion.


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)

Rap1 is involved in lymphocyte polarization. (A) Asymmetrical morphology and distribution of CXCR4 (green) and CD44 (red) in chemokine-stimulated T cells. T cells were stimulated with 100 nM SLC or 100 nM SDF-1 for 10 min, fixed in suspension, and stained as in Fig. 8. (B) Quantitative analysis of lymphocyte polarization. T cells infected with adenoviruses encoding either GFP alone (control), Spa1, or Rap1V12 adenovirus were incubated without or with 100 nM SLC, as indicated, for 10 min and fixed in suspension. GFP-positive lymphocytes with CD44-marked uropods were quantified (100–150 cells), and are expressed as a percentage of the total GFP-positive cells. The mean and SE of triplicate experiments are shown. (C) Effects of Rap1V12 and Spa1 expression on lymphocyte polarization. T cells infected with the control, Spa1, or Rap1V12 adenovirus were incubated without or with 100 nM SLC as indicated, for 10 min, and fixed in suspension. Images detail GFP fluorescence (top), morphology by differential interference contrast (middle), and CD44 (bottom). Control adenovirus-infected lymphocytes exhibited a homogeneous distribution of CD44 without typical uropods. Polarized cell shape and CD44 redistribution were induced in control lymphocytes by SLC treatment and Rap1V12 expression. Spa1-expressing T cells failed to develop polarized morphology and redistribute CD44 when stimulated with SLC. Asterisks indicate adenovirus-infected cells. Bars, 10 μm.
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fig9: Rap1 is involved in lymphocyte polarization. (A) Asymmetrical morphology and distribution of CXCR4 (green) and CD44 (red) in chemokine-stimulated T cells. T cells were stimulated with 100 nM SLC or 100 nM SDF-1 for 10 min, fixed in suspension, and stained as in Fig. 8. (B) Quantitative analysis of lymphocyte polarization. T cells infected with adenoviruses encoding either GFP alone (control), Spa1, or Rap1V12 adenovirus were incubated without or with 100 nM SLC, as indicated, for 10 min and fixed in suspension. GFP-positive lymphocytes with CD44-marked uropods were quantified (100–150 cells), and are expressed as a percentage of the total GFP-positive cells. The mean and SE of triplicate experiments are shown. (C) Effects of Rap1V12 and Spa1 expression on lymphocyte polarization. T cells infected with the control, Spa1, or Rap1V12 adenovirus were incubated without or with 100 nM SLC as indicated, for 10 min, and fixed in suspension. Images detail GFP fluorescence (top), morphology by differential interference contrast (middle), and CD44 (bottom). Control adenovirus-infected lymphocytes exhibited a homogeneous distribution of CD44 without typical uropods. Polarized cell shape and CD44 redistribution were induced in control lymphocytes by SLC treatment and Rap1V12 expression. Spa1-expressing T cells failed to develop polarized morphology and redistribute CD44 when stimulated with SLC. Asterisks indicate adenovirus-infected cells. Bars, 10 μm.
Mentions: To confirm whether Rap1 is also involved in lymphocyte polarization, we examined the effects of Rap1V12 and Spa1 after their introduction into lymphocytes via adenoviral infection. When stimulated with SLC or SDF-1, lymphocytes were transformed into polarized cell shapes with well-developed leading edges and uropods marked by CXCR4 and CD44, respectively (Fig. 9 A). Control adenovirus-infected lymphocytes, indicated by GFP expression, were similarly transformed by SLC into a polarized cell shape with the development of CD44-marked uropods (Fig. 9 C). Quantitative analysis indicated that ∼60% of stimulated cells exhibited this polarized phenotype (Fig. 9 B). Rap1V12 expression, also indicated by GFP expression, markedly increased the number of polarized lymphocytes with CD44-marked uropods (Fig. 9, B and C), comparable to the levels induced by SLC. Conversely, Spa1 expression reduced the number of SLC-stimulated polarized lymphocytes exhibiting an asymmetric redistribution of CD44 to basal levels (Fig. 9, B and C). Collectively, these results indicate that Rap1 activation triggers lymphocyte polarization even in nonadherent lymphocytes. Thus, Rap1-mediated cell polarization triggered by chemokines does not depend on integrin-mediated adhesion.

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