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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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Lymphocyte adhesion under shear stress. (A) SLC-stimulated adhesion to immobilized ICAM-1. Lymphocytes infected with the control or Spa1-encoding adenovirus were incubated in the absence or presence of either 100nM SLC or 10 ng/ml PMA, at 37°C for the indicated time on ICAM-1, and then washed with shear stress at 2 dyne/cm2 for 1 min. Attachment of SLC-stimulated control cells treated with anti-LFA-1 or anti-ICAM-1 antibody is also indicated at the 10-min time point. Quantitated numbers of GFP-positive attached cells were counted and expressed as a percentage of the input GFP-positive cells. PMA-stimulated attachment is indicated for the 10- and 20-min time points. The data are representative of three independent experiments. (B) Spa-1–inhibited lymphocyte adhesion triggered by immobilized SLC. ICAM-1–coated disks were pretreated with 100 nM SLC. Unbound SLC was removed by washing. Lymphocytes infected with either the control or Spa1 adenovirus were incubated on uncoated or SLC-coated ICAM-1 discs at 37°C for the indicated time and then washed with shear stress at 2 dyne/cm2 for 1 min. GFP-positive attached cells were counted and expressed as a percentage of the input GFP-positive cells. The data are representative of three independent experiments. (C) Attachment of lymphocytes to endothelial cells under flow. Lymphocytes infected with adenoviruses encoding GFP alone (control), Spa1, or Rap1V12 adenovirus were perfused over a BC1 endothelial monolayer overlaid with 100 nM SDF-1 under shear stress (0.1 dyne/cm2) for 10 min. The effect of antibody blocking of LFA-1 or ICAM-1 was shown for lymphocytes infected with the control adenovirus. Lymphocytes that rolled away or attached throughout the shear stress phase were considered as “roll-away” (open bar) and “firm attachment” (closed bar), respectively. These categories were expressed as a percentage of total interacting cells, including rolling, transiently attached, and firmly attached cells. Adenovirus-infected cells, suspended at 2 × 106/ml were perfused into the flow chamber. Approximately 60–70 GFP-positive cells were captured by the BC1 monolayer during a 10-min perfusion (per high power field, 440 × 440 μm, magnification 20). Data means and SE were determined from four independent experiments with SE. Statistical significance was determined by t test. *, P < 0.002, compared with unstimulated lymphocytes. **, P < 0.002, compared with SLC-stimulated control adenoviruses-infected lymphocytes. ***, P < 0.003, compared with unstimulated control lymphocytes.
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fig2: Lymphocyte adhesion under shear stress. (A) SLC-stimulated adhesion to immobilized ICAM-1. Lymphocytes infected with the control or Spa1-encoding adenovirus were incubated in the absence or presence of either 100nM SLC or 10 ng/ml PMA, at 37°C for the indicated time on ICAM-1, and then washed with shear stress at 2 dyne/cm2 for 1 min. Attachment of SLC-stimulated control cells treated with anti-LFA-1 or anti-ICAM-1 antibody is also indicated at the 10-min time point. Quantitated numbers of GFP-positive attached cells were counted and expressed as a percentage of the input GFP-positive cells. PMA-stimulated attachment is indicated for the 10- and 20-min time points. The data are representative of three independent experiments. (B) Spa-1–inhibited lymphocyte adhesion triggered by immobilized SLC. ICAM-1–coated disks were pretreated with 100 nM SLC. Unbound SLC was removed by washing. Lymphocytes infected with either the control or Spa1 adenovirus were incubated on uncoated or SLC-coated ICAM-1 discs at 37°C for the indicated time and then washed with shear stress at 2 dyne/cm2 for 1 min. GFP-positive attached cells were counted and expressed as a percentage of the input GFP-positive cells. The data are representative of three independent experiments. (C) Attachment of lymphocytes to endothelial cells under flow. Lymphocytes infected with adenoviruses encoding GFP alone (control), Spa1, or Rap1V12 adenovirus were perfused over a BC1 endothelial monolayer overlaid with 100 nM SDF-1 under shear stress (0.1 dyne/cm2) for 10 min. The effect of antibody blocking of LFA-1 or ICAM-1 was shown for lymphocytes infected with the control adenovirus. Lymphocytes that rolled away or attached throughout the shear stress phase were considered as “roll-away” (open bar) and “firm attachment” (closed bar), respectively. These categories were expressed as a percentage of total interacting cells, including rolling, transiently attached, and firmly attached cells. Adenovirus-infected cells, suspended at 2 × 106/ml were perfused into the flow chamber. Approximately 60–70 GFP-positive cells were captured by the BC1 monolayer during a 10-min perfusion (per high power field, 440 × 440 μm, magnification 20). Data means and SE were determined from four independent experiments with SE. Statistical significance was determined by t test. *, P < 0.002, compared with unstimulated lymphocytes. **, P < 0.002, compared with SLC-stimulated control adenoviruses-infected lymphocytes. ***, P < 0.003, compared with unstimulated control lymphocytes.

Mentions: We examined the requirement of Rap1 activation for integrin-dependent adhesion. Soluble SLC induced the transient shear-resistant adhesion of LN cells to immobilized intercellular adhesion molecule 1 (ICAM-1; Fig. 2 A), an effect that was blocked by treatment with either anti-LFA-1 or anti-ICAM-1 antibodies. Increased adhesion was first detected at 1 min and peaked at 10 min, but was quickly down-regulated to basal levels after 15 min. To examine the induction of adhesion after Rap1 activation, we inhibited Rap1 activation by overexpressing Spa1, a Rap1-specific GTPase-activating protein (Kurachi et al., 1997), in lymphocytes. LN cells derived from transgenic mice in which the adenovirus receptor (coxsackie adenovirus receptor) is only expressed in T cells (Wan et al., 2000) were infected with adenovirus expressing either GFP alone or Spa1. Approximately 80% of the infected cells, primarily Thy-1–positive T cells, expressed GFP (unpublished data). Rap1 activation after a 30-s SLC stimulation in Spa1-expressing cells was reduced to 20% of the levels observed in control cells (Fig. 1 C). The degree of Rap1 suppression correlated with the infection efficiency, suggesting that Rap1 activation is markedly inhibited in Spa1-expressing cells. Then, we examined the effect of Spa1 expression on lymphocyte adhesion to ICAM-1 after SLC stimulation (Fig. 2 A). Spa1-expressing T cells did not adhere in response to soluble SLC treatment. PMA-stimulated adhesion was not affected in Spa1-expressing cells, indicating that Spa1 specifically inhibits chemokine-induced adhesion (Fig. 2 A).


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)

Lymphocyte adhesion under shear stress. (A) SLC-stimulated adhesion to immobilized ICAM-1. Lymphocytes infected with the control or Spa1-encoding adenovirus were incubated in the absence or presence of either 100nM SLC or 10 ng/ml PMA, at 37°C for the indicated time on ICAM-1, and then washed with shear stress at 2 dyne/cm2 for 1 min. Attachment of SLC-stimulated control cells treated with anti-LFA-1 or anti-ICAM-1 antibody is also indicated at the 10-min time point. Quantitated numbers of GFP-positive attached cells were counted and expressed as a percentage of the input GFP-positive cells. PMA-stimulated attachment is indicated for the 10- and 20-min time points. The data are representative of three independent experiments. (B) Spa-1–inhibited lymphocyte adhesion triggered by immobilized SLC. ICAM-1–coated disks were pretreated with 100 nM SLC. Unbound SLC was removed by washing. Lymphocytes infected with either the control or Spa1 adenovirus were incubated on uncoated or SLC-coated ICAM-1 discs at 37°C for the indicated time and then washed with shear stress at 2 dyne/cm2 for 1 min. GFP-positive attached cells were counted and expressed as a percentage of the input GFP-positive cells. The data are representative of three independent experiments. (C) Attachment of lymphocytes to endothelial cells under flow. Lymphocytes infected with adenoviruses encoding GFP alone (control), Spa1, or Rap1V12 adenovirus were perfused over a BC1 endothelial monolayer overlaid with 100 nM SDF-1 under shear stress (0.1 dyne/cm2) for 10 min. The effect of antibody blocking of LFA-1 or ICAM-1 was shown for lymphocytes infected with the control adenovirus. Lymphocytes that rolled away or attached throughout the shear stress phase were considered as “roll-away” (open bar) and “firm attachment” (closed bar), respectively. These categories were expressed as a percentage of total interacting cells, including rolling, transiently attached, and firmly attached cells. Adenovirus-infected cells, suspended at 2 × 106/ml were perfused into the flow chamber. Approximately 60–70 GFP-positive cells were captured by the BC1 monolayer during a 10-min perfusion (per high power field, 440 × 440 μm, magnification 20). Data means and SE were determined from four independent experiments with SE. Statistical significance was determined by t test. *, P < 0.002, compared with unstimulated lymphocytes. **, P < 0.002, compared with SLC-stimulated control adenoviruses-infected lymphocytes. ***, P < 0.003, compared with unstimulated control lymphocytes.
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fig2: Lymphocyte adhesion under shear stress. (A) SLC-stimulated adhesion to immobilized ICAM-1. Lymphocytes infected with the control or Spa1-encoding adenovirus were incubated in the absence or presence of either 100nM SLC or 10 ng/ml PMA, at 37°C for the indicated time on ICAM-1, and then washed with shear stress at 2 dyne/cm2 for 1 min. Attachment of SLC-stimulated control cells treated with anti-LFA-1 or anti-ICAM-1 antibody is also indicated at the 10-min time point. Quantitated numbers of GFP-positive attached cells were counted and expressed as a percentage of the input GFP-positive cells. PMA-stimulated attachment is indicated for the 10- and 20-min time points. The data are representative of three independent experiments. (B) Spa-1–inhibited lymphocyte adhesion triggered by immobilized SLC. ICAM-1–coated disks were pretreated with 100 nM SLC. Unbound SLC was removed by washing. Lymphocytes infected with either the control or Spa1 adenovirus were incubated on uncoated or SLC-coated ICAM-1 discs at 37°C for the indicated time and then washed with shear stress at 2 dyne/cm2 for 1 min. GFP-positive attached cells were counted and expressed as a percentage of the input GFP-positive cells. The data are representative of three independent experiments. (C) Attachment of lymphocytes to endothelial cells under flow. Lymphocytes infected with adenoviruses encoding GFP alone (control), Spa1, or Rap1V12 adenovirus were perfused over a BC1 endothelial monolayer overlaid with 100 nM SDF-1 under shear stress (0.1 dyne/cm2) for 10 min. The effect of antibody blocking of LFA-1 or ICAM-1 was shown for lymphocytes infected with the control adenovirus. Lymphocytes that rolled away or attached throughout the shear stress phase were considered as “roll-away” (open bar) and “firm attachment” (closed bar), respectively. These categories were expressed as a percentage of total interacting cells, including rolling, transiently attached, and firmly attached cells. Adenovirus-infected cells, suspended at 2 × 106/ml were perfused into the flow chamber. Approximately 60–70 GFP-positive cells were captured by the BC1 monolayer during a 10-min perfusion (per high power field, 440 × 440 μm, magnification 20). Data means and SE were determined from four independent experiments with SE. Statistical significance was determined by t test. *, P < 0.002, compared with unstimulated lymphocytes. **, P < 0.002, compared with SLC-stimulated control adenoviruses-infected lymphocytes. ***, P < 0.003, compared with unstimulated control lymphocytes.
Mentions: We examined the requirement of Rap1 activation for integrin-dependent adhesion. Soluble SLC induced the transient shear-resistant adhesion of LN cells to immobilized intercellular adhesion molecule 1 (ICAM-1; Fig. 2 A), an effect that was blocked by treatment with either anti-LFA-1 or anti-ICAM-1 antibodies. Increased adhesion was first detected at 1 min and peaked at 10 min, but was quickly down-regulated to basal levels after 15 min. To examine the induction of adhesion after Rap1 activation, we inhibited Rap1 activation by overexpressing Spa1, a Rap1-specific GTPase-activating protein (Kurachi et al., 1997), in lymphocytes. LN cells derived from transgenic mice in which the adenovirus receptor (coxsackie adenovirus receptor) is only expressed in T cells (Wan et al., 2000) were infected with adenovirus expressing either GFP alone or Spa1. Approximately 80% of the infected cells, primarily Thy-1–positive T cells, expressed GFP (unpublished data). Rap1 activation after a 30-s SLC stimulation in Spa1-expressing cells was reduced to 20% of the levels observed in control cells (Fig. 1 C). The degree of Rap1 suppression correlated with the infection efficiency, suggesting that Rap1 activation is markedly inhibited in Spa1-expressing cells. Then, we examined the effect of Spa1 expression on lymphocyte adhesion to ICAM-1 after SLC stimulation (Fig. 2 A). Spa1-expressing T cells did not adhere in response to soluble SLC treatment. PMA-stimulated adhesion was not affected in Spa1-expressing cells, indicating that Spa1 specifically inhibits chemokine-induced adhesion (Fig. 2 A).

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