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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 and β2 integrin interact on the cellular membrane of Jurkat T-cells. (A) Representation of interacting fraction τm by pseudocolor images of the FLIM-FRET analysis of the interaction between β2 integrins with CD47 in unstimulated conditions and upon Mg2+/EGTA activation, and the interaction between activated β2 integrin detected by mAb 24 and CD47 upon integrin activation with Mg2+/EGTA. The color scale for τm ranges from 10 to 3500 ps. The β2 integrin was identified with the donor fluorophore (Alexa Fluor 488) and CD47 with the acceptor fluorophore (Alexa Fluor 594). (B) Localization of β2 integrin and CD47 by epifluorescence. Fixed cells were stained with (a, c) anti-β2 integrin polyclonal antibody alone (Quinn et al., 2001), (b, d) anti-β2 integrin antibody and anti-CD47 (B6H12) antibody labeled with Alexa 594 (unstimulated or with Mg2+/EGTA stimulation, respectively), (e) anti–activated-β2 integrin (mAb 24) alone upon Mg2+/EGTA stimulation, and (f) activated-β2 integrin (mAb 24) and anti-CD47 antibody also upon Mg2+/EGTA stimulation. Nucleus stained with DAPI.
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Figure 7: CD47 and β2 integrin interact on the cellular membrane of Jurkat T-cells. (A) Representation of interacting fraction τm by pseudocolor images of the FLIM-FRET analysis of the interaction between β2 integrins with CD47 in unstimulated conditions and upon Mg2+/EGTA activation, and the interaction between activated β2 integrin detected by mAb 24 and CD47 upon integrin activation with Mg2+/EGTA. The color scale for τm ranges from 10 to 3500 ps. The β2 integrin was identified with the donor fluorophore (Alexa Fluor 488) and CD47 with the acceptor fluorophore (Alexa Fluor 594). (B) Localization of β2 integrin and CD47 by epifluorescence. Fixed cells were stained with (a, c) anti-β2 integrin polyclonal antibody alone (Quinn et al., 2001), (b, d) anti-β2 integrin antibody and anti-CD47 (B6H12) antibody labeled with Alexa 594 (unstimulated or with Mg2+/EGTA stimulation, respectively), (e) anti–activated-β2 integrin (mAb 24) alone upon Mg2+/EGTA stimulation, and (f) activated-β2 integrin (mAb 24) and anti-CD47 antibody also upon Mg2+/EGTA stimulation. Nucleus stained with DAPI.

Mentions: Prior studies reported that VLA-4 coimmunoprecipitated with CD47 in blood reticulocytes from sickle cell patients (Brittain et al., 2004). To evaluate whether CD47 associates with β2 integrins in T-cells, we applied fluorescence lifetime imaging microscopy (FLIM), a quantitative method for determining Förster resonance energy transfer (FRET; Table 2 and Figure 7A), to study whether β2 integrin and CD47 are sufficiently close to imply a physical interaction. Epifluorescence images were captured as quality control of the staining (Figure 7B). The lifetime of the donor molecule (τ1; picoseconds), in this case β2 integrin, labeled with Alexa Fluor 488–con­jugated secondary antibody was determined first in the absence of an acceptor fluorophore (Table 2 and Figure 7A, donor only). FRET between the donor fluorophore (β2 integrin) and acceptor (CD47 directly labeled with Alexa Fluor 594) was defined by the life­time of interacting molecules (τ1), with a1 (in percent) defining the frac­tion of interacting molecules. The significant decrease of τ1 (and also the mean lifetime, τm) for β2 integrin–CD47 indicates a close association between β2 integrin and CD47, and the a1 value indicates that 31.7 ± 4.1% of β2 integrin molecules interact with CD47 on the cellular membrane of Jurkat T-cells (Table 2). For comparison purposes the noninteracting molecules β2 integrin and PSGL-1 were costained as donor and acceptor molecules, respectively. A decrease of τ1 was found when PSGL-1 was used as acceptor, but the interacting fraction was only 13.3 ± 1.5% and approached the lower levels of sensitivity. Furthermore, the τm of the donor was unchanged in the presence of the acceptor, indicating that β2 integrin and PSGL-1 were not in sufficient proximity to support a physical interaction (Supplemental Table S1 and Supplemental Figure S2A). As a positive control a decrease in τ1 was observed between LFA-1 α-chain (αL integrin) and the common β2 chain (Supplemental Table S1 and Supplemental Figure 2A). Representative histograms illustrate the τm of the donor β2 integrin in the different conditions (Supplemental Figure S3, A–F). These results support a direct association of CD47 with β2 integrins.


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 and β2 integrin interact on the cellular membrane of Jurkat T-cells. (A) Representation of interacting fraction τm by pseudocolor images of the FLIM-FRET analysis of the interaction between β2 integrins with CD47 in unstimulated conditions and upon Mg2+/EGTA activation, and the interaction between activated β2 integrin detected by mAb 24 and CD47 upon integrin activation with Mg2+/EGTA. The color scale for τm ranges from 10 to 3500 ps. The β2 integrin was identified with the donor fluorophore (Alexa Fluor 488) and CD47 with the acceptor fluorophore (Alexa Fluor 594). (B) Localization of β2 integrin and CD47 by epifluorescence. Fixed cells were stained with (a, c) anti-β2 integrin polyclonal antibody alone (Quinn et al., 2001), (b, d) anti-β2 integrin antibody and anti-CD47 (B6H12) antibody labeled with Alexa 594 (unstimulated or with Mg2+/EGTA stimulation, respectively), (e) anti–activated-β2 integrin (mAb 24) alone upon Mg2+/EGTA stimulation, and (f) activated-β2 integrin (mAb 24) and anti-CD47 antibody also upon Mg2+/EGTA stimulation. Nucleus stained with DAPI.
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Figure 7: CD47 and β2 integrin interact on the cellular membrane of Jurkat T-cells. (A) Representation of interacting fraction τm by pseudocolor images of the FLIM-FRET analysis of the interaction between β2 integrins with CD47 in unstimulated conditions and upon Mg2+/EGTA activation, and the interaction between activated β2 integrin detected by mAb 24 and CD47 upon integrin activation with Mg2+/EGTA. The color scale for τm ranges from 10 to 3500 ps. The β2 integrin was identified with the donor fluorophore (Alexa Fluor 488) and CD47 with the acceptor fluorophore (Alexa Fluor 594). (B) Localization of β2 integrin and CD47 by epifluorescence. Fixed cells were stained with (a, c) anti-β2 integrin polyclonal antibody alone (Quinn et al., 2001), (b, d) anti-β2 integrin antibody and anti-CD47 (B6H12) antibody labeled with Alexa 594 (unstimulated or with Mg2+/EGTA stimulation, respectively), (e) anti–activated-β2 integrin (mAb 24) alone upon Mg2+/EGTA stimulation, and (f) activated-β2 integrin (mAb 24) and anti-CD47 antibody also upon Mg2+/EGTA stimulation. Nucleus stained with DAPI.
Mentions: Prior studies reported that VLA-4 coimmunoprecipitated with CD47 in blood reticulocytes from sickle cell patients (Brittain et al., 2004). To evaluate whether CD47 associates with β2 integrins in T-cells, we applied fluorescence lifetime imaging microscopy (FLIM), a quantitative method for determining Förster resonance energy transfer (FRET; Table 2 and Figure 7A), to study whether β2 integrin and CD47 are sufficiently close to imply a physical interaction. Epifluorescence images were captured as quality control of the staining (Figure 7B). The lifetime of the donor molecule (τ1; picoseconds), in this case β2 integrin, labeled with Alexa Fluor 488–con­jugated secondary antibody was determined first in the absence of an acceptor fluorophore (Table 2 and Figure 7A, donor only). FRET between the donor fluorophore (β2 integrin) and acceptor (CD47 directly labeled with Alexa Fluor 594) was defined by the life­time of interacting molecules (τ1), with a1 (in percent) defining the frac­tion of interacting molecules. The significant decrease of τ1 (and also the mean lifetime, τm) for β2 integrin–CD47 indicates a close association between β2 integrin and CD47, and the a1 value indicates that 31.7 ± 4.1% of β2 integrin molecules interact with CD47 on the cellular membrane of Jurkat T-cells (Table 2). For comparison purposes the noninteracting molecules β2 integrin and PSGL-1 were costained as donor and acceptor molecules, respectively. A decrease of τ1 was found when PSGL-1 was used as acceptor, but the interacting fraction was only 13.3 ± 1.5% and approached the lower levels of sensitivity. Furthermore, the τm of the donor was unchanged in the presence of the acceptor, indicating that β2 integrin and PSGL-1 were not in sufficient proximity to support a physical interaction (Supplemental Table S1 and Supplemental Figure S2A). As a positive control a decrease in τ1 was observed between LFA-1 α-chain (αL integrin) and the common β2 chain (Supplemental Table S1 and Supplemental Figure 2A). Representative histograms illustrate the τm of the donor β2 integrin in the different conditions (Supplemental Figure S3, A–F). These results support a direct association of CD47 with β2 integrins.

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