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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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Adhesive interactions of Th1 effector cells with inflamed murine cremaster muscle microcirculation. (A) Impaired interaction of CD47−/− Th1 effector cells with TNFα-activated microvessels of the cremaster muscle. Tethered cells are defined as labeled Th1 cells that are stably bound to the inflamed vessel wall for more than three consecutive video frames (∼2 s). Data are means ± SEM from five WT and CD47−/− recipient mice (13 vessels in WT and 7 vessels in CD47−/− mice). *p < 0.05 vs. WT Th1 in CD47−/− recipient; **p < 0.01 vs. WT Th1 in WT recipient (Student's t test). (B) Rolling Th1 cells are defined as T-cells that interact with the vessel below Vcrit. Th1 cell rolling velocities were determined by measuring the length of time required to travel 200 μm. Velocities from three independent preparations of Th1 cells were analyzed in vessels from WT or CD47−/− mice. Tethered and rolling T-cells were identified in videos using Imaris software (Bitplane, Zurich, Switzerland). (C) WT and CD47−/− CD4+ Th1 effector cells have essentially the same surface expression levels of LFA-1, VLA-4, and PSGL-1. (D) Intracellular staining shows that IFN-γ production is similar in polarized WT (66% positive) and CD47−/− (68% positive) Th1 cells.
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Figure 1: Adhesive interactions of Th1 effector cells with inflamed murine cremaster muscle microcirculation. (A) Impaired interaction of CD47−/− Th1 effector cells with TNFα-activated microvessels of the cremaster muscle. Tethered cells are defined as labeled Th1 cells that are stably bound to the inflamed vessel wall for more than three consecutive video frames (∼2 s). Data are means ± SEM from five WT and CD47−/− recipient mice (13 vessels in WT and 7 vessels in CD47−/− mice). *p < 0.05 vs. WT Th1 in CD47−/− recipient; **p < 0.01 vs. WT Th1 in WT recipient (Student's t test). (B) Rolling Th1 cells are defined as T-cells that interact with the vessel below Vcrit. Th1 cell rolling velocities were determined by measuring the length of time required to travel 200 μm. Velocities from three independent preparations of Th1 cells were analyzed in vessels from WT or CD47−/− mice. Tethered and rolling T-cells were identified in videos using Imaris software (Bitplane, Zurich, Switzerland). (C) WT and CD47−/− CD4+ Th1 effector cells have essentially the same surface expression levels of LFA-1, VLA-4, and PSGL-1. (D) Intracellular staining shows that IFN-γ production is similar in polarized WT (66% positive) and CD47−/− (68% positive) Th1 cells.

Mentions: To evaluate the role of CD47 in leukocyte adhesive interactions with endothelium in vivo, we performed intravital microscopy studies in the murine cremaster microcirculation after intrascrotal injection of TNF as described earlier (Alcaide et al., 2012). Equal numbers of in vitro–generated wild-type (WT) and CD47−/− Th1 effector cells, each labeled with a different color fluorescent dye, were coinjected into WT recipient animals to enable comparison of WT and CD47−/− Th1 cells in the same postcapillary venules. The microvessel parameters for WT and CD47−/− mice are listed in Table 1. The behavior of transferred Th1 cells was monitored for 2–5 min postinjection. CD47−/− Th1 cells exhibited significantly reduced tethering adhesive interactions relative to WT Th1 cells (Figure 1A). The reciprocal experiment of simultaneous injection of differentially fluorescent-labeled WT and CD47−/− Th1 cells into CD47−/− recipient mice revealed that CD47−/− Th1 cells interacted less than did WT cells. Tethered Th1 cells become stably bound to the inflamed vessel wall for more than three consecutive video frames (∼2 s). Few, if any, of the transferred T-cells arrest for more than several seconds. These results indicate expression of CD47 in T-cells is required for optimal Th1 effector cell adhesive interactions in this model. There was no difference between WT and CD47−/− T-cells in the rolling velocities, indicating that CD47 was not involved in Th1 cell rolling on inflamed venules (Figure 1B). This adhesion defect is not explained by reduced expression of adhesion molecules in CD47−/− mice because WT and CD47−/− Th1 cells express identical levels of LFA-1, VLA-4, and P-selectin glycoprotein ligand-1 (PSGL-1) and similar levels of intracellular IFN-γ, the Th1 signature cytokine (Figure 1, C and D). We routinely verified the absence of CD47 expression in CD47−/− T-cells (Figure 1C).


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)

Adhesive interactions of Th1 effector cells with inflamed murine cremaster muscle microcirculation. (A) Impaired interaction of CD47−/− Th1 effector cells with TNFα-activated microvessels of the cremaster muscle. Tethered cells are defined as labeled Th1 cells that are stably bound to the inflamed vessel wall for more than three consecutive video frames (∼2 s). Data are means ± SEM from five WT and CD47−/− recipient mice (13 vessels in WT and 7 vessels in CD47−/− mice). *p < 0.05 vs. WT Th1 in CD47−/− recipient; **p < 0.01 vs. WT Th1 in WT recipient (Student's t test). (B) Rolling Th1 cells are defined as T-cells that interact with the vessel below Vcrit. Th1 cell rolling velocities were determined by measuring the length of time required to travel 200 μm. Velocities from three independent preparations of Th1 cells were analyzed in vessels from WT or CD47−/− mice. Tethered and rolling T-cells were identified in videos using Imaris software (Bitplane, Zurich, Switzerland). (C) WT and CD47−/− CD4+ Th1 effector cells have essentially the same surface expression levels of LFA-1, VLA-4, and PSGL-1. (D) Intracellular staining shows that IFN-γ production is similar in polarized WT (66% positive) and CD47−/− (68% positive) Th1 cells.
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Related In: Results  -  Collection

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Figure 1: Adhesive interactions of Th1 effector cells with inflamed murine cremaster muscle microcirculation. (A) Impaired interaction of CD47−/− Th1 effector cells with TNFα-activated microvessels of the cremaster muscle. Tethered cells are defined as labeled Th1 cells that are stably bound to the inflamed vessel wall for more than three consecutive video frames (∼2 s). Data are means ± SEM from five WT and CD47−/− recipient mice (13 vessels in WT and 7 vessels in CD47−/− mice). *p < 0.05 vs. WT Th1 in CD47−/− recipient; **p < 0.01 vs. WT Th1 in WT recipient (Student's t test). (B) Rolling Th1 cells are defined as T-cells that interact with the vessel below Vcrit. Th1 cell rolling velocities were determined by measuring the length of time required to travel 200 μm. Velocities from three independent preparations of Th1 cells were analyzed in vessels from WT or CD47−/− mice. Tethered and rolling T-cells were identified in videos using Imaris software (Bitplane, Zurich, Switzerland). (C) WT and CD47−/− CD4+ Th1 effector cells have essentially the same surface expression levels of LFA-1, VLA-4, and PSGL-1. (D) Intracellular staining shows that IFN-γ production is similar in polarized WT (66% positive) and CD47−/− (68% positive) Th1 cells.
Mentions: To evaluate the role of CD47 in leukocyte adhesive interactions with endothelium in vivo, we performed intravital microscopy studies in the murine cremaster microcirculation after intrascrotal injection of TNF as described earlier (Alcaide et al., 2012). Equal numbers of in vitro–generated wild-type (WT) and CD47−/− Th1 effector cells, each labeled with a different color fluorescent dye, were coinjected into WT recipient animals to enable comparison of WT and CD47−/− Th1 cells in the same postcapillary venules. The microvessel parameters for WT and CD47−/− mice are listed in Table 1. The behavior of transferred Th1 cells was monitored for 2–5 min postinjection. CD47−/− Th1 cells exhibited significantly reduced tethering adhesive interactions relative to WT Th1 cells (Figure 1A). The reciprocal experiment of simultaneous injection of differentially fluorescent-labeled WT and CD47−/− Th1 cells into CD47−/− recipient mice revealed that CD47−/− Th1 cells interacted less than did WT cells. Tethered Th1 cells become stably bound to the inflamed vessel wall for more than three consecutive video frames (∼2 s). Few, if any, of the transferred T-cells arrest for more than several seconds. These results indicate expression of CD47 in T-cells is required for optimal Th1 effector cell adhesive interactions in this model. There was no difference between WT and CD47−/− T-cells in the rolling velocities, indicating that CD47 was not involved in Th1 cell rolling on inflamed venules (Figure 1B). This adhesion defect is not explained by reduced expression of adhesion molecules in CD47−/− mice because WT and CD47−/− Th1 cells express identical levels of LFA-1, VLA-4, and P-selectin glycoprotein ligand-1 (PSGL-1) and similar levels of intracellular IFN-γ, the Th1 signature cytokine (Figure 1, C and D). We routinely verified the absence of CD47 expression in CD47−/− T-cells (Figure 1C).

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