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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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Attachment and transmigration of T cells. (A) Attachment of T cells to MBEC4 endothelial cells under flow. T cells infected with adenoviruses encoding either GFP alone (cont), Spa1, or Rap1V12 were incubated with or without either 100 nM SLC or 10 ng/ml PMA for 1, 5, and 10 min on MBEC4 monolayers pretreated with TNFα for 24 h. Shear stress was then applied at 2 dyne/cm2 for 20 min. In the absence of TNFα pretreatment, lymphocyte adherence to MBEC4 endothelial cells was <10% of the input cells (not depicted). Loaded cell numbers were 2 × 106 and ∼100–120 GFP-positive cells per microscopic field (440 × 440 μm) were accumulated on the MBEC monolayers at each time point. Shear-resistant attachment to the endothelial cells was reduced to less than a half of normal levels after blocking with mAb specific for LFA-1 or VLA-4. Treatment with both antibodies further reduced cell numbers to ∼15% of normal levels. The mean and SE of triplicate experiments are shown. *, P < 0.005 (1 min), 0.002 (5 min), and 0.003 (10 min), compared with unstimulated control adenovirus-infected lymphocytes. **, P < 0.006 (1 min), 0.003 (5 min), and 0.002 (10 min), compared with SLC-stimulated control lymphocytes. ***, P < 0.001 (1 min), 0.001 (5 min), and 0.001 (10 min), compared with unstimulated control lymphocytes. (B) Transmigration through MBEC4 endothelial cells under flow. T cells infected with the control, Spa1, or Rap1V12 adenovirus were incubated with or without 100 nM SLC, or with 10 ng/ml PMA as indicated, for 1, 5, and 10 min on the MBEC4 monolayers. Shear stress was then applied at 2 dyne/cm2 for 20 min. The mean and SE of triplicate experiments are shown. *, P < 0.002 (1 min), 0.001 (5 min), and 0.001 (10 min), compared with unstimulated control adenovirus-infected lymphocytes. **, P < 0.003 (1 min), 0.001 (5 min), and 0.001 (10 min), compared with SLC-stimulated control lymphocytes. ***, P < 0.01 (1 min), 0.001 (5 min), and 0.001 (10 min), compared with unstimulated control lymphocytes. (C) PTX had little effect on Rap1V12-induced adhesion and transmigration. Lymphocytes infected with the control or Rap1V12 adenovirus were cultured for 2 h with 50 ng/ml PTX, and were then incubated with or without 100 nM SLC, as indicated, for 10 min on the MBEC4 monolayers. Shear stress was then applied at 2 dyne/cm2 for 20 min *, P < 0.005, compared with adhesion of SLC-stimulated lymphocytes without PTX pretreatment. **, P < 0.002, compared with transmigration of SLC-stimulated lymphocytes without PTX pretreatment.
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fig6: Attachment and transmigration of T cells. (A) Attachment of T cells to MBEC4 endothelial cells under flow. T cells infected with adenoviruses encoding either GFP alone (cont), Spa1, or Rap1V12 were incubated with or without either 100 nM SLC or 10 ng/ml PMA for 1, 5, and 10 min on MBEC4 monolayers pretreated with TNFα for 24 h. Shear stress was then applied at 2 dyne/cm2 for 20 min. In the absence of TNFα pretreatment, lymphocyte adherence to MBEC4 endothelial cells was <10% of the input cells (not depicted). Loaded cell numbers were 2 × 106 and ∼100–120 GFP-positive cells per microscopic field (440 × 440 μm) were accumulated on the MBEC monolayers at each time point. Shear-resistant attachment to the endothelial cells was reduced to less than a half of normal levels after blocking with mAb specific for LFA-1 or VLA-4. Treatment with both antibodies further reduced cell numbers to ∼15% of normal levels. The mean and SE of triplicate experiments are shown. *, P < 0.005 (1 min), 0.002 (5 min), and 0.003 (10 min), compared with unstimulated control adenovirus-infected lymphocytes. **, P < 0.006 (1 min), 0.003 (5 min), and 0.002 (10 min), compared with SLC-stimulated control lymphocytes. ***, P < 0.001 (1 min), 0.001 (5 min), and 0.001 (10 min), compared with unstimulated control lymphocytes. (B) Transmigration through MBEC4 endothelial cells under flow. T cells infected with the control, Spa1, or Rap1V12 adenovirus were incubated with or without 100 nM SLC, or with 10 ng/ml PMA as indicated, for 1, 5, and 10 min on the MBEC4 monolayers. Shear stress was then applied at 2 dyne/cm2 for 20 min. The mean and SE of triplicate experiments are shown. *, P < 0.002 (1 min), 0.001 (5 min), and 0.001 (10 min), compared with unstimulated control adenovirus-infected lymphocytes. **, P < 0.003 (1 min), 0.001 (5 min), and 0.001 (10 min), compared with SLC-stimulated control lymphocytes. ***, P < 0.01 (1 min), 0.001 (5 min), and 0.001 (10 min), compared with unstimulated control lymphocytes. (C) PTX had little effect on Rap1V12-induced adhesion and transmigration. Lymphocytes infected with the control or Rap1V12 adenovirus were cultured for 2 h with 50 ng/ml PTX, and were then incubated with or without 100 nM SLC, as indicated, for 10 min on the MBEC4 monolayers. Shear stress was then applied at 2 dyne/cm2 for 20 min *, P < 0.005, compared with adhesion of SLC-stimulated lymphocytes without PTX pretreatment. **, P < 0.002, compared with transmigration of SLC-stimulated lymphocytes without PTX pretreatment.

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)

Attachment and transmigration of T cells. (A) Attachment of T cells to MBEC4 endothelial cells under flow. T cells infected with adenoviruses encoding either GFP alone (cont), Spa1, or Rap1V12 were incubated with or without either 100 nM SLC or 10 ng/ml PMA for 1, 5, and 10 min on MBEC4 monolayers pretreated with TNFα for 24 h. Shear stress was then applied at 2 dyne/cm2 for 20 min. In the absence of TNFα pretreatment, lymphocyte adherence to MBEC4 endothelial cells was <10% of the input cells (not depicted). Loaded cell numbers were 2 × 106 and ∼100–120 GFP-positive cells per microscopic field (440 × 440 μm) were accumulated on the MBEC monolayers at each time point. Shear-resistant attachment to the endothelial cells was reduced to less than a half of normal levels after blocking with mAb specific for LFA-1 or VLA-4. Treatment with both antibodies further reduced cell numbers to ∼15% of normal levels. The mean and SE of triplicate experiments are shown. *, P < 0.005 (1 min), 0.002 (5 min), and 0.003 (10 min), compared with unstimulated control adenovirus-infected lymphocytes. **, P < 0.006 (1 min), 0.003 (5 min), and 0.002 (10 min), compared with SLC-stimulated control lymphocytes. ***, P < 0.001 (1 min), 0.001 (5 min), and 0.001 (10 min), compared with unstimulated control lymphocytes. (B) Transmigration through MBEC4 endothelial cells under flow. T cells infected with the control, Spa1, or Rap1V12 adenovirus were incubated with or without 100 nM SLC, or with 10 ng/ml PMA as indicated, for 1, 5, and 10 min on the MBEC4 monolayers. Shear stress was then applied at 2 dyne/cm2 for 20 min. The mean and SE of triplicate experiments are shown. *, P < 0.002 (1 min), 0.001 (5 min), and 0.001 (10 min), compared with unstimulated control adenovirus-infected lymphocytes. **, P < 0.003 (1 min), 0.001 (5 min), and 0.001 (10 min), compared with SLC-stimulated control lymphocytes. ***, P < 0.01 (1 min), 0.001 (5 min), and 0.001 (10 min), compared with unstimulated control lymphocytes. (C) PTX had little effect on Rap1V12-induced adhesion and transmigration. Lymphocytes infected with the control or Rap1V12 adenovirus were cultured for 2 h with 50 ng/ml PTX, and were then incubated with or without 100 nM SLC, as indicated, for 10 min on the MBEC4 monolayers. Shear stress was then applied at 2 dyne/cm2 for 20 min *, P < 0.005, compared with adhesion of SLC-stimulated lymphocytes without PTX pretreatment. **, P < 0.002, compared with transmigration of SLC-stimulated lymphocytes without PTX pretreatment.
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fig6: Attachment and transmigration of T cells. (A) Attachment of T cells to MBEC4 endothelial cells under flow. T cells infected with adenoviruses encoding either GFP alone (cont), Spa1, or Rap1V12 were incubated with or without either 100 nM SLC or 10 ng/ml PMA for 1, 5, and 10 min on MBEC4 monolayers pretreated with TNFα for 24 h. Shear stress was then applied at 2 dyne/cm2 for 20 min. In the absence of TNFα pretreatment, lymphocyte adherence to MBEC4 endothelial cells was <10% of the input cells (not depicted). Loaded cell numbers were 2 × 106 and ∼100–120 GFP-positive cells per microscopic field (440 × 440 μm) were accumulated on the MBEC monolayers at each time point. Shear-resistant attachment to the endothelial cells was reduced to less than a half of normal levels after blocking with mAb specific for LFA-1 or VLA-4. Treatment with both antibodies further reduced cell numbers to ∼15% of normal levels. The mean and SE of triplicate experiments are shown. *, P < 0.005 (1 min), 0.002 (5 min), and 0.003 (10 min), compared with unstimulated control adenovirus-infected lymphocytes. **, P < 0.006 (1 min), 0.003 (5 min), and 0.002 (10 min), compared with SLC-stimulated control lymphocytes. ***, P < 0.001 (1 min), 0.001 (5 min), and 0.001 (10 min), compared with unstimulated control lymphocytes. (B) Transmigration through MBEC4 endothelial cells under flow. T cells infected with the control, Spa1, or Rap1V12 adenovirus were incubated with or without 100 nM SLC, or with 10 ng/ml PMA as indicated, for 1, 5, and 10 min on the MBEC4 monolayers. Shear stress was then applied at 2 dyne/cm2 for 20 min. The mean and SE of triplicate experiments are shown. *, P < 0.002 (1 min), 0.001 (5 min), and 0.001 (10 min), compared with unstimulated control adenovirus-infected lymphocytes. **, P < 0.003 (1 min), 0.001 (5 min), and 0.001 (10 min), compared with SLC-stimulated control lymphocytes. ***, P < 0.01 (1 min), 0.001 (5 min), and 0.001 (10 min), compared with unstimulated control lymphocytes. (C) PTX had little effect on Rap1V12-induced adhesion and transmigration. Lymphocytes infected with the control or Rap1V12 adenovirus were cultured for 2 h with 50 ng/ml PTX, and were then incubated with or without 100 nM SLC, as indicated, for 10 min on the MBEC4 monolayers. Shear stress was then applied at 2 dyne/cm2 for 20 min *, P < 0.005, compared with adhesion of SLC-stimulated lymphocytes without PTX pretreatment. **, P < 0.002, compared with transmigration of SLC-stimulated lymphocytes without PTX pretreatment.
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