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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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Th1 effector T-cell interactions with TNF-α–activated murine endothelium in an in vitro flow chamber. (A) The numbers of accumulated and (B) transmigrated T-cells in the videos were quantified by ImageJ software (National Institutes of Health, Bethesda, MD). Data are mean ± SEM. *p ≤ 0.05 and **p ≤0.01 vs. WT Th1 cell on WT MHEC. #p ≤ 0.05 are WT Th1 vs. CD47−/− on CD47−/− MHEC (Student's t test), n = 3 separate experiments. (C) MHECs were treated with medium or medium containing murine TNF-α (100 ng/ml) for 4 h, and CD47, VCAM-1, ICAM-1, E-selectin, ICAM-2, and PECAM-1 expression levels were detected by unlabeled primary mAb followed by staining with a PE-labeled goat anti-rat secondary mAb. Cell fluorescence was determined by FACSCalibur flow cytometry (BD, Franklin Lakes, NJ). Representative histograms of surface expression of molecules are shown from 10 separate experiments.
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Figure 2: Th1 effector T-cell interactions with TNF-α–activated murine endothelium in an in vitro flow chamber. (A) The numbers of accumulated and (B) transmigrated T-cells in the videos were quantified by ImageJ software (National Institutes of Health, Bethesda, MD). Data are mean ± SEM. *p ≤ 0.05 and **p ≤0.01 vs. WT Th1 cell on WT MHEC. #p ≤ 0.05 are WT Th1 vs. CD47−/− on CD47−/− MHEC (Student's t test), n = 3 separate experiments. (C) MHECs were treated with medium or medium containing murine TNF-α (100 ng/ml) for 4 h, and CD47, VCAM-1, ICAM-1, E-selectin, ICAM-2, and PECAM-1 expression levels were detected by unlabeled primary mAb followed by staining with a PE-labeled goat anti-rat secondary mAb. Cell fluorescence was determined by FACSCalibur flow cytometry (BD, Franklin Lakes, NJ). Representative histograms of surface expression of molecules are shown from 10 separate experiments.

Mentions: To further delineate the adhesion defect in CD47−/− T-cells, we monitored adhesion and TEM of WT and CD47−/− Th1 cells on WT and CD47−/− murine heart endothelial cell (MHEC) monolayers by live-cell videomicroscopy in an in vitro flow model (Alcaide et al., 2012). WT Th1 cells arrested, and subsequently ∼30% of adherent cells transmigrated across TNF-α–activated WT monolayers (Figure 2, A and B). As we would predict from the in vivo studies, CD47−/− Th1 cells exhibited reduced adhesion and TEM across WT endothelium compared with WT Th1 cells. CD47−/− T-cells also showed reduced adhesion and TEM across CD47−/− MHEC monolayers. Of interest, WT Th1 cells also exhibited significantly reduced adhesion and TEM across CD47−/− MHECs, indicating that endothelial cell CD47 also plays a role in TEM. Analysis of MHECs isolated from CD47−/− and WT mice showed essentially identical levels of surface-expressed PECAM-1 and ICAM-2 at baseline and similar levels of ICAM-1, VCAM-1, and E-selectin expression at baseline and after 4 h of TNF-α treatment (Figure 2C). These in vivo and in vitro studies indicate that expression of CD47 in both T-cells and endothelium is required for normal Th1 cell adhesion and TEM of TNF-α–inflamed endothelium. Here we focus on the role that CD47 expressed on T-cells plays in leukocyte recruitment.


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)

Th1 effector T-cell interactions with TNF-α–activated murine endothelium in an in vitro flow chamber. (A) The numbers of accumulated and (B) transmigrated T-cells in the videos were quantified by ImageJ software (National Institutes of Health, Bethesda, MD). Data are mean ± SEM. *p ≤ 0.05 and **p ≤0.01 vs. WT Th1 cell on WT MHEC. #p ≤ 0.05 are WT Th1 vs. CD47−/− on CD47−/− MHEC (Student's t test), n = 3 separate experiments. (C) MHECs were treated with medium or medium containing murine TNF-α (100 ng/ml) for 4 h, and CD47, VCAM-1, ICAM-1, E-selectin, ICAM-2, and PECAM-1 expression levels were detected by unlabeled primary mAb followed by staining with a PE-labeled goat anti-rat secondary mAb. Cell fluorescence was determined by FACSCalibur flow cytometry (BD, Franklin Lakes, NJ). Representative histograms of surface expression of molecules are shown from 10 separate experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Th1 effector T-cell interactions with TNF-α–activated murine endothelium in an in vitro flow chamber. (A) The numbers of accumulated and (B) transmigrated T-cells in the videos were quantified by ImageJ software (National Institutes of Health, Bethesda, MD). Data are mean ± SEM. *p ≤ 0.05 and **p ≤0.01 vs. WT Th1 cell on WT MHEC. #p ≤ 0.05 are WT Th1 vs. CD47−/− on CD47−/− MHEC (Student's t test), n = 3 separate experiments. (C) MHECs were treated with medium or medium containing murine TNF-α (100 ng/ml) for 4 h, and CD47, VCAM-1, ICAM-1, E-selectin, ICAM-2, and PECAM-1 expression levels were detected by unlabeled primary mAb followed by staining with a PE-labeled goat anti-rat secondary mAb. Cell fluorescence was determined by FACSCalibur flow cytometry (BD, Franklin Lakes, NJ). Representative histograms of surface expression of molecules are shown from 10 separate experiments.
Mentions: To further delineate the adhesion defect in CD47−/− T-cells, we monitored adhesion and TEM of WT and CD47−/− Th1 cells on WT and CD47−/− murine heart endothelial cell (MHEC) monolayers by live-cell videomicroscopy in an in vitro flow model (Alcaide et al., 2012). WT Th1 cells arrested, and subsequently ∼30% of adherent cells transmigrated across TNF-α–activated WT monolayers (Figure 2, A and B). As we would predict from the in vivo studies, CD47−/− Th1 cells exhibited reduced adhesion and TEM across WT endothelium compared with WT Th1 cells. CD47−/− T-cells also showed reduced adhesion and TEM across CD47−/− MHEC monolayers. Of interest, WT Th1 cells also exhibited significantly reduced adhesion and TEM across CD47−/− MHECs, indicating that endothelial cell CD47 also plays a role in TEM. Analysis of MHECs isolated from CD47−/− and WT mice showed essentially identical levels of surface-expressed PECAM-1 and ICAM-2 at baseline and similar levels of ICAM-1, VCAM-1, and E-selectin expression at baseline and after 4 h of TNF-α treatment (Figure 2C). These in vivo and in vitro studies indicate that expression of CD47 in both T-cells and endothelium is required for normal Th1 cell adhesion and TEM of TNF-α–inflamed endothelium. Here we focus on the role that CD47 expressed on T-cells plays in leukocyte recruitment.

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