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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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Human Jurkat T-cell integrin expression and adhesion to HUVEC monolayers in an in vitro flow model. (A) Jurkat CD47+ clone E6 and CD47− () clone JINB8 express similar levels of LFA-1 and VLA-4 integrins. Data are representative of three separate experiments. (B) Jurkat CD47− T-cells were drawn across the TNF-α–activated HUVECs at various estimated shear stress levels as described in Materials and Methods. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 for indicated comparisons (Student's t test). (C) Both CD47+ and CD47− Jurkat T-cell adhesion (under shear stress of 0.76 dynes/cm2) to TNF-α–activated HUVECs is strongly dependent on VLA-4 integrins. The reduction in adhesion in CD47+ cells with blockade of LFA-1 is not observed in CD47− cells, suggesting that LFA-1–dependent adhesion requires CD47. Data are mean ± SEM of three experiments. *p ≤ 0.05, **p ≤ 0.01 vs. CD47+ with no mAb in medium; #p < 0.05, media CD47− vs. anti-VLA-4 mAb–treated CD47− cells (Student's t test).
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Figure 4: Human Jurkat T-cell integrin expression and adhesion to HUVEC monolayers in an in vitro flow model. (A) Jurkat CD47+ clone E6 and CD47− () clone JINB8 express similar levels of LFA-1 and VLA-4 integrins. Data are representative of three separate experiments. (B) Jurkat CD47− T-cells were drawn across the TNF-α–activated HUVECs at various estimated shear stress levels as described in Materials and Methods. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 for indicated comparisons (Student's t test). (C) Both CD47+ and CD47− Jurkat T-cell adhesion (under shear stress of 0.76 dynes/cm2) to TNF-α–activated HUVECs is strongly dependent on VLA-4 integrins. The reduction in adhesion in CD47+ cells with blockade of LFA-1 is not observed in CD47− cells, suggesting that LFA-1–dependent adhesion requires CD47. Data are mean ± SEM of three experiments. *p ≤ 0.05, **p ≤ 0.01 vs. CD47+ with no mAb in medium; #p < 0.05, media CD47− vs. anti-VLA-4 mAb–treated CD47− cells (Student's t test).

Mentions: An earlier study reported that human Jurkat T-cell clone JINB8 lacking CD47 (CD47−) showed reduced binding to a TNF-α–stimulated human endothelial-like cell line as compared with the parental clone expressing CD47 (CD47+; Ticchioni et al., 2001). Accordingly, we evaluated their adhesion to TNF-α–activated human umbilical vein endothelial cells (HUVECs) and immobilized ICAM-1 and VCAM-1. Both Jurkat clones express similar surface levels of LFA-1 and VLA-4 α-subunits and the respective common β2 and β1 integrin subunits (Figure 4A). CD47+ Jurkat cells showed significantly greater adhesion to 4-h TNF-α–activated HUVEC monolayers than did CD47− cells at each shear stress tested (Figure 4B). Of interest, the dominant integrin responsible for T-cell adhesion was VLA-4 as determined by function-blocking monoclonal antibody (mAb) studies (Figure 4C), which is consistent with earlier studies (Chen et al., 1999; Ticchioni et al., 2001). Blocking LFA-1 on CD47+ Jurkat cells reduced adhesion to similar levels as unblocked CD47− Jurkat cells. Blockade of LFA-1 on CD47− Jurkat cells did not further reduce adhesion to HUVECs. The lesser role of LFA-1 in Jurkat T-cell adhesion to HUVECs is likely the reason that Jurkat CD47− T-cell adhesion is not reduced to a greater level, as we would have predicted (Figure 4C).


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)

Human Jurkat T-cell integrin expression and adhesion to HUVEC monolayers in an in vitro flow model. (A) Jurkat CD47+ clone E6 and CD47− () clone JINB8 express similar levels of LFA-1 and VLA-4 integrins. Data are representative of three separate experiments. (B) Jurkat CD47− T-cells were drawn across the TNF-α–activated HUVECs at various estimated shear stress levels as described in Materials and Methods. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 for indicated comparisons (Student's t test). (C) Both CD47+ and CD47− Jurkat T-cell adhesion (under shear stress of 0.76 dynes/cm2) to TNF-α–activated HUVECs is strongly dependent on VLA-4 integrins. The reduction in adhesion in CD47+ cells with blockade of LFA-1 is not observed in CD47− cells, suggesting that LFA-1–dependent adhesion requires CD47. Data are mean ± SEM of three experiments. *p ≤ 0.05, **p ≤ 0.01 vs. CD47+ with no mAb in medium; #p < 0.05, media CD47− vs. anti-VLA-4 mAb–treated CD47− cells (Student's t test).
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Related In: Results  -  Collection

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Figure 4: Human Jurkat T-cell integrin expression and adhesion to HUVEC monolayers in an in vitro flow model. (A) Jurkat CD47+ clone E6 and CD47− () clone JINB8 express similar levels of LFA-1 and VLA-4 integrins. Data are representative of three separate experiments. (B) Jurkat CD47− T-cells were drawn across the TNF-α–activated HUVECs at various estimated shear stress levels as described in Materials and Methods. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 for indicated comparisons (Student's t test). (C) Both CD47+ and CD47− Jurkat T-cell adhesion (under shear stress of 0.76 dynes/cm2) to TNF-α–activated HUVECs is strongly dependent on VLA-4 integrins. The reduction in adhesion in CD47+ cells with blockade of LFA-1 is not observed in CD47− cells, suggesting that LFA-1–dependent adhesion requires CD47. Data are mean ± SEM of three experiments. *p ≤ 0.05, **p ≤ 0.01 vs. CD47+ with no mAb in medium; #p < 0.05, media CD47− vs. anti-VLA-4 mAb–treated CD47− cells (Student's t test).
Mentions: An earlier study reported that human Jurkat T-cell clone JINB8 lacking CD47 (CD47−) showed reduced binding to a TNF-α–stimulated human endothelial-like cell line as compared with the parental clone expressing CD47 (CD47+; Ticchioni et al., 2001). Accordingly, we evaluated their adhesion to TNF-α–activated human umbilical vein endothelial cells (HUVECs) and immobilized ICAM-1 and VCAM-1. Both Jurkat clones express similar surface levels of LFA-1 and VLA-4 α-subunits and the respective common β2 and β1 integrin subunits (Figure 4A). CD47+ Jurkat cells showed significantly greater adhesion to 4-h TNF-α–activated HUVEC monolayers than did CD47− cells at each shear stress tested (Figure 4B). Of interest, the dominant integrin responsible for T-cell adhesion was VLA-4 as determined by function-blocking monoclonal antibody (mAb) studies (Figure 4C), which is consistent with earlier studies (Chen et al., 1999; Ticchioni et al., 2001). Blocking LFA-1 on CD47+ Jurkat cells reduced adhesion to similar levels as unblocked CD47− Jurkat cells. Blockade of LFA-1 on CD47− Jurkat cells did not further reduce adhesion to HUVECs. The lesser role of LFA-1 in Jurkat T-cell adhesion to HUVECs is likely the reason that Jurkat CD47− T-cell adhesion is not reduced to a greater level, as we would have predicted (Figure 4C).

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