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CD47 plays a critical role in T-cell recruitment by regulation of LFA-1 and VLA-4 integrin adhesive functions.

Azcutia V, Routledge M, Williams MR, Newton G, Frazier WA, Manica A, Croce KJ, Parkos CA, Schmider AB, Turman MV, Soberman RJ, Luscinskas FW - Mol. Biol. Cell (2013)

Bottom Line: CD47, also called integrin-associated protein, has been demonstrated to associate in cis with β1 and β3 integrins.In cis interactions between Jurkat T-cell β2 integrins and CD47 were detected by fluorescence lifetime imaging microscopy.Unexpectedly, Jurkat CD47 cells exhibited a striking defect in β1 and β2 integrin activation in response to Mn(2+) or Mg(2+)/ethylene glycol tetraacetic acid treatment.

View Article: PubMed Central - PubMed

Affiliation: Center for Excellence in Vascular Biology, Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115 Harvard Medical School, Boston, MA 02115 Department of Biochemistry and Molecular Biophysics, Washington University, St. Louis, MO 63130 Instituto de Cardiologia do Rio Grande do Sul, Fundação Universitária de Cardiologia, Porto Alegre 90010-395, Brazil Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115 Division of Gastrointestinal Pathology, Emory University School of Medicine, Atlanta, GA 30322 Division of Nephrology, Department of Medicine, Massachusetts General Hospital, Boston, MA 02114.

ABSTRACT
CD47 plays an important but incompletely understood role in the innate and adaptive immune responses. CD47, also called integrin-associated protein, has been demonstrated to associate in cis with β1 and β3 integrins. Here we test the hypothesis that CD47 regulates adhesive functions of T-cell α4β1 (VLA-4) and αLβ2 (LFA-1) in in vivo and in vitro models of inflammation. Intravital microscopy studies reveal that CD47(-/-) Th1 cells exhibit reduced interactions with wild-type (WT) inflamed cremaster muscle microvessels. Similarly, murine CD47(-/-) Th1 cells, as compared with WT, showed defects in adhesion and transmigration across tumor necrosis factor-α (TNF-α)-activated murine endothelium and in adhesion to immobilized intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1) under flow conditions. Human Jurkat T-cells lacking CD47 also showed reduced adhesion to TNF-α-activated endothelium and ICAM-1 and VCAM-1. In cis interactions between Jurkat T-cell β2 integrins and CD47 were detected by fluorescence lifetime imaging microscopy. Unexpectedly, Jurkat CD47 cells exhibited a striking defect in β1 and β2 integrin activation in response to Mn(2+) or Mg(2+)/ethylene glycol tetraacetic acid treatment. Our results demonstrate that CD47 associates with β2 integrins and is necessary to induce high-affinity conformations of LFA-1 and VLA-4 that recognize their endothelial cell ligands and support leukocyte adhesion and transendothelial migration.

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CD47−/− Th1 cells have impaired adhesion to immobilized ICAM-1 and VCAM-1 but not E-selectin in an in vitro flow chamber model. WT and CD47−/− Th1 cells were drawn across immobilized ICAM-1-Fc (A), VCAM-1-Fc (B), and E-selectin-Fc chimeric proteins (C) at the shear stress levels indicated, and cell adhesion was determined as detailed in Materials and Methods. Data are mean ± SEM, n = 3. *p ≤ 0.05, **p ≤ 0.01 (Student's t test). (D) Shear flow–mediated detachment of Th1 cells prebound to immobilized ICAM-1 + CXCL12 is not altered in CD47−/− Th1 cells. Data are mean ± SEM, n = 3 separate experiments.
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Figure 3: CD47−/− Th1 cells have impaired adhesion to immobilized ICAM-1 and VCAM-1 but not E-selectin in an in vitro flow chamber model. WT and CD47−/− Th1 cells were drawn across immobilized ICAM-1-Fc (A), VCAM-1-Fc (B), and E-selectin-Fc chimeric proteins (C) at the shear stress levels indicated, and cell adhesion was determined as detailed in Materials and Methods. Data are mean ± SEM, n = 3. *p ≤ 0.05, **p ≤ 0.01 (Student's t test). (D) Shear flow–mediated detachment of Th1 cells prebound to immobilized ICAM-1 + CXCL12 is not altered in CD47−/− Th1 cells. Data are mean ± SEM, n = 3 separate experiments.

Mentions: CD47 associates with and regulates the adhesive functions of reticulocyte-expressed α4β1 integrins (Brittain et al., 2004), but no study examined whether CD47 regulates αLβ2 (LFA-1) integrin adhesive interactions with its endothelial ligand ICAM-1. We therefore studied the adhesion of WT and CD47−/− Th1 cells to ICAM-1-Fc coimmobilized with CXCL12 (SDF-1α) chemokine under flow conditions (Alcaide et al., 2012). WT Th1 cells arrested on ICAM-1, whereas CD47−/− Th1 cells showed a significant reduction in arrest (Figure 3A). Of note, the greatest relative reduction occurred at the highest shear stress level examined. This assay was repeated using immobilized VCAM-1-Fc molecules without chemokine because T-cells constitutively express VLA-4 integrins capable of binding VCAM-1 under flow conditions (Chen et al., 1999). A significant defect in CD47−/− Th1 cell arrest to VCAM-1 was observed at high shear stress levels but not at the lowest shear stress tested (Figure 3B). There were no differences between WT and CD47−/− Th1 cell accumulation on immobilized E-selectin, suggesting that induction of Th1 selectin ligand expression was not affected (Figure 3C). Taken together, the data suggest that CD47 in Th1 cells plays an important role in the function of LFA-1 and VLA-4 integrins required for adhesion to their endothelial cell ligands.


CD47 plays a critical role in T-cell recruitment by regulation of LFA-1 and VLA-4 integrin adhesive functions.

Azcutia V, Routledge M, Williams MR, Newton G, Frazier WA, Manica A, Croce KJ, Parkos CA, Schmider AB, Turman MV, Soberman RJ, Luscinskas FW - Mol. Biol. Cell (2013)

CD47−/− Th1 cells have impaired adhesion to immobilized ICAM-1 and VCAM-1 but not E-selectin in an in vitro flow chamber model. WT and CD47−/− Th1 cells were drawn across immobilized ICAM-1-Fc (A), VCAM-1-Fc (B), and E-selectin-Fc chimeric proteins (C) at the shear stress levels indicated, and cell adhesion was determined as detailed in Materials and Methods. Data are mean ± SEM, n = 3. *p ≤ 0.05, **p ≤ 0.01 (Student's t test). (D) Shear flow–mediated detachment of Th1 cells prebound to immobilized ICAM-1 + CXCL12 is not altered in CD47−/− Th1 cells. Data are mean ± SEM, n = 3 separate experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814154&req=5

Figure 3: CD47−/− Th1 cells have impaired adhesion to immobilized ICAM-1 and VCAM-1 but not E-selectin in an in vitro flow chamber model. WT and CD47−/− Th1 cells were drawn across immobilized ICAM-1-Fc (A), VCAM-1-Fc (B), and E-selectin-Fc chimeric proteins (C) at the shear stress levels indicated, and cell adhesion was determined as detailed in Materials and Methods. Data are mean ± SEM, n = 3. *p ≤ 0.05, **p ≤ 0.01 (Student's t test). (D) Shear flow–mediated detachment of Th1 cells prebound to immobilized ICAM-1 + CXCL12 is not altered in CD47−/− Th1 cells. Data are mean ± SEM, n = 3 separate experiments.
Mentions: CD47 associates with and regulates the adhesive functions of reticulocyte-expressed α4β1 integrins (Brittain et al., 2004), but no study examined whether CD47 regulates αLβ2 (LFA-1) integrin adhesive interactions with its endothelial ligand ICAM-1. We therefore studied the adhesion of WT and CD47−/− Th1 cells to ICAM-1-Fc coimmobilized with CXCL12 (SDF-1α) chemokine under flow conditions (Alcaide et al., 2012). WT Th1 cells arrested on ICAM-1, whereas CD47−/− Th1 cells showed a significant reduction in arrest (Figure 3A). Of note, the greatest relative reduction occurred at the highest shear stress level examined. This assay was repeated using immobilized VCAM-1-Fc molecules without chemokine because T-cells constitutively express VLA-4 integrins capable of binding VCAM-1 under flow conditions (Chen et al., 1999). A significant defect in CD47−/− Th1 cell arrest to VCAM-1 was observed at high shear stress levels but not at the lowest shear stress tested (Figure 3B). There were no differences between WT and CD47−/− Th1 cell accumulation on immobilized E-selectin, suggesting that induction of Th1 selectin ligand expression was not affected (Figure 3C). Taken together, the data suggest that CD47 in Th1 cells plays an important role in the function of LFA-1 and VLA-4 integrins required for adhesion to their endothelial cell ligands.

Bottom Line: CD47, also called integrin-associated protein, has been demonstrated to associate in cis with β1 and β3 integrins.In cis interactions between Jurkat T-cell β2 integrins and CD47 were detected by fluorescence lifetime imaging microscopy.Unexpectedly, Jurkat CD47 cells exhibited a striking defect in β1 and β2 integrin activation in response to Mn(2+) or Mg(2+)/ethylene glycol tetraacetic acid treatment.

View Article: PubMed Central - PubMed

Affiliation: Center for Excellence in Vascular Biology, Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115 Harvard Medical School, Boston, MA 02115 Department of Biochemistry and Molecular Biophysics, Washington University, St. Louis, MO 63130 Instituto de Cardiologia do Rio Grande do Sul, Fundação Universitária de Cardiologia, Porto Alegre 90010-395, Brazil Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115 Division of Gastrointestinal Pathology, Emory University School of Medicine, Atlanta, GA 30322 Division of Nephrology, Department of Medicine, Massachusetts General Hospital, Boston, MA 02114.

ABSTRACT
CD47 plays an important but incompletely understood role in the innate and adaptive immune responses. CD47, also called integrin-associated protein, has been demonstrated to associate in cis with β1 and β3 integrins. Here we test the hypothesis that CD47 regulates adhesive functions of T-cell α4β1 (VLA-4) and αLβ2 (LFA-1) in in vivo and in vitro models of inflammation. Intravital microscopy studies reveal that CD47(-/-) Th1 cells exhibit reduced interactions with wild-type (WT) inflamed cremaster muscle microvessels. Similarly, murine CD47(-/-) Th1 cells, as compared with WT, showed defects in adhesion and transmigration across tumor necrosis factor-α (TNF-α)-activated murine endothelium and in adhesion to immobilized intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1) under flow conditions. Human Jurkat T-cells lacking CD47 also showed reduced adhesion to TNF-α-activated endothelium and ICAM-1 and VCAM-1. In cis interactions between Jurkat T-cell β2 integrins and CD47 were detected by fluorescence lifetime imaging microscopy. Unexpectedly, Jurkat CD47 cells exhibited a striking defect in β1 and β2 integrin activation in response to Mn(2+) or Mg(2+)/ethylene glycol tetraacetic acid treatment. Our results demonstrate that CD47 associates with β2 integrins and is necessary to induce high-affinity conformations of LFA-1 and VLA-4 that recognize their endothelial cell ligands and support leukocyte adhesion and transendothelial migration.

Show MeSH
Related in: MedlinePlus