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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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Mn2+ or Mg2+/EGTA fails to induce strong LFA-1 and β1 high-affinity conformation expression or binding of soluble ICAM-1-Fc chimera in CD47− () Jurkat T-cells. (A–D) Mn2+ or Mg2+/EGTA–induced expression of LFA-1 extended and open “activated” conformations of LFA-1 were detected by the reporter mAb KIM127 (A) and mAb24 (B). Results are normalized to isotype-matched, control nonbinding mAb. Expression of these active conformations of integrins by 0.5 mM Mn2+ or 10 mM Mg2+/1 mM EGTA treatments was significantly reduced in CD47− Jurkat T-cells. (C) Total levels of β2 and β1 integrins detected with TS1/18 and LIA1/2.1 mAb, respectively, did not change after Mn2+ or Mg2+/EGTA stimulation. No change was detected in CD47 (data not shown). (D) High-affinity β1 integrins induced by Mn2+ or Mg2+/EGTA buffer were measured by mAb HUTS21. (E) CD47− T-cells exhibited little binding of soluble ICAM-1 as compared with CD47+ T-cells induced by Mn2+ and Mg2+/EGTA treatments. Results are normalized to incubation buffer alone. Data are mean ± SEM, n = 3. *p ≤ 0.05, **p ≤ 0.01 (Student's t test). (F) Mn2+ activates solubilized β2 integrins from CD47+ (lane 3) and CD47- (lane 6) Jurkat T-cells. Lysates of Jurkat cells were subjected to immunoprecipitation with anti-β2 integrin mAb 24 (lanes 2, 3 and 5, 6) in the absence (–) or presence (+) of 1.0 mM Mn2+, followed by SDS–PAGE and immunoblotting with anti-β2 integrin polyclonal Ab (R&D Systems). Immunoprecipitation with mAb TS1/18 (lanes 1 and 4) served as a positive control to ensure the presence of β2 integrin in the lysate.
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Figure 6: Mn2+ or Mg2+/EGTA fails to induce strong LFA-1 and β1 high-affinity conformation expression or binding of soluble ICAM-1-Fc chimera in CD47− () Jurkat T-cells. (A–D) Mn2+ or Mg2+/EGTA–induced expression of LFA-1 extended and open “activated” conformations of LFA-1 were detected by the reporter mAb KIM127 (A) and mAb24 (B). Results are normalized to isotype-matched, control nonbinding mAb. Expression of these active conformations of integrins by 0.5 mM Mn2+ or 10 mM Mg2+/1 mM EGTA treatments was significantly reduced in CD47− Jurkat T-cells. (C) Total levels of β2 and β1 integrins detected with TS1/18 and LIA1/2.1 mAb, respectively, did not change after Mn2+ or Mg2+/EGTA stimulation. No change was detected in CD47 (data not shown). (D) High-affinity β1 integrins induced by Mn2+ or Mg2+/EGTA buffer were measured by mAb HUTS21. (E) CD47− T-cells exhibited little binding of soluble ICAM-1 as compared with CD47+ T-cells induced by Mn2+ and Mg2+/EGTA treatments. Results are normalized to incubation buffer alone. Data are mean ± SEM, n = 3. *p ≤ 0.05, **p ≤ 0.01 (Student's t test). (F) Mn2+ activates solubilized β2 integrins from CD47+ (lane 3) and CD47- (lane 6) Jurkat T-cells. Lysates of Jurkat cells were subjected to immunoprecipitation with anti-β2 integrin mAb 24 (lanes 2, 3 and 5, 6) in the absence (–) or presence (+) of 1.0 mM Mn2+, followed by SDS–PAGE and immunoblotting with anti-β2 integrin polyclonal Ab (R&D Systems). Immunoprecipitation with mAb TS1/18 (lanes 1 and 4) served as a positive control to ensure the presence of β2 integrin in the lysate.

Mentions: Integrin affinity regulation was evaluated on Jurkat CD47− and CD47+ cells by reporter mAbs that detect activated conformations of β1 and β2 integrins. KIM127 mAb detects and stabilizes an extended and closed conformation of β2 integrins (intermediate affinity), and mAb 24 recognizes an extended and open “active” conformation of β2 integrins (high affinity; (Salas et al., 2004). Jurkat T-cells express predominantly LFA-1 (αLβ2) and not the other α-subunit chains that associate with β2 integrins, and hence these mAbs to β2 integrins detect primarily the intermediate- and high-affinity conformations of LFA-1 (Salas et al., 2004). HUTS21 detects and stabilizes activated β1 integrins (Luque et al., 1996), and we showed previously that expression of this epitope correlated with robust adhesion of human memory T-cells to VCAM-1 (Lim et al., 2000). As expected, incubation of CD47+ T-cells with Mn2+ or Mg2+/ethylene glycol tetraacetic acid (EGTA), global activators of integrins, triggered robust expression of both intermediate- and high-affinity LFA-1 (Figure 6, A and B). In contrast, CD47− T-cells showed a significantly reduced expression of intermediate- and high-affinity conformations of LFA-1 in response to Mn2+ or Mg2+/EGTA. It is unlikely that this assay did not detect transient LFA-1 activation because the mAbs stabilize the active conformation and were present throughout the assay. In addition, failure to detect mAb expression was not due to a change (loss) in total surface expression of β1 or β2 integrins upon exposure to Mn2+ or Mg2+/EGTA (Figure 6C). CD47− T-cells also showed reduced induction of high-affinity β1 integrins versus CD47+ T-cells after treatment with Mn2+ or Mg2+/EGTA buffers (Figure 6D). Consistent with the failure of Mn2+ or Mg2+/EGTA treatments to induce intermediate- and high-affinity LFA-1 conformations in the absence of CD47, CD47− T-cells exhibited little binding of soluble ICAM-1-Fc versus CD47+ T-cells (Figure 6E). In addition, treatment of CD47− cells with Mn2+ did not restore binding comparable to CD47+ T-cell adhesion to either immobilized ICAM-1 or VCAM-1 under flow (Supplemental Figure S1, A and B). Taken together, the results indicate that CD47 expression is required for expression of intermediate- and high-affinity conformations of LFA-1 and for binding soluble ICAM-1, as well as for induction of activated β1 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)

Mn2+ or Mg2+/EGTA fails to induce strong LFA-1 and β1 high-affinity conformation expression or binding of soluble ICAM-1-Fc chimera in CD47− () Jurkat T-cells. (A–D) Mn2+ or Mg2+/EGTA–induced expression of LFA-1 extended and open “activated” conformations of LFA-1 were detected by the reporter mAb KIM127 (A) and mAb24 (B). Results are normalized to isotype-matched, control nonbinding mAb. Expression of these active conformations of integrins by 0.5 mM Mn2+ or 10 mM Mg2+/1 mM EGTA treatments was significantly reduced in CD47− Jurkat T-cells. (C) Total levels of β2 and β1 integrins detected with TS1/18 and LIA1/2.1 mAb, respectively, did not change after Mn2+ or Mg2+/EGTA stimulation. No change was detected in CD47 (data not shown). (D) High-affinity β1 integrins induced by Mn2+ or Mg2+/EGTA buffer were measured by mAb HUTS21. (E) CD47− T-cells exhibited little binding of soluble ICAM-1 as compared with CD47+ T-cells induced by Mn2+ and Mg2+/EGTA treatments. Results are normalized to incubation buffer alone. Data are mean ± SEM, n = 3. *p ≤ 0.05, **p ≤ 0.01 (Student's t test). (F) Mn2+ activates solubilized β2 integrins from CD47+ (lane 3) and CD47- (lane 6) Jurkat T-cells. Lysates of Jurkat cells were subjected to immunoprecipitation with anti-β2 integrin mAb 24 (lanes 2, 3 and 5, 6) in the absence (–) or presence (+) of 1.0 mM Mn2+, followed by SDS–PAGE and immunoblotting with anti-β2 integrin polyclonal Ab (R&D Systems). Immunoprecipitation with mAb TS1/18 (lanes 1 and 4) served as a positive control to ensure the presence of β2 integrin in the lysate.
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Figure 6: Mn2+ or Mg2+/EGTA fails to induce strong LFA-1 and β1 high-affinity conformation expression or binding of soluble ICAM-1-Fc chimera in CD47− () Jurkat T-cells. (A–D) Mn2+ or Mg2+/EGTA–induced expression of LFA-1 extended and open “activated” conformations of LFA-1 were detected by the reporter mAb KIM127 (A) and mAb24 (B). Results are normalized to isotype-matched, control nonbinding mAb. Expression of these active conformations of integrins by 0.5 mM Mn2+ or 10 mM Mg2+/1 mM EGTA treatments was significantly reduced in CD47− Jurkat T-cells. (C) Total levels of β2 and β1 integrins detected with TS1/18 and LIA1/2.1 mAb, respectively, did not change after Mn2+ or Mg2+/EGTA stimulation. No change was detected in CD47 (data not shown). (D) High-affinity β1 integrins induced by Mn2+ or Mg2+/EGTA buffer were measured by mAb HUTS21. (E) CD47− T-cells exhibited little binding of soluble ICAM-1 as compared with CD47+ T-cells induced by Mn2+ and Mg2+/EGTA treatments. Results are normalized to incubation buffer alone. Data are mean ± SEM, n = 3. *p ≤ 0.05, **p ≤ 0.01 (Student's t test). (F) Mn2+ activates solubilized β2 integrins from CD47+ (lane 3) and CD47- (lane 6) Jurkat T-cells. Lysates of Jurkat cells were subjected to immunoprecipitation with anti-β2 integrin mAb 24 (lanes 2, 3 and 5, 6) in the absence (–) or presence (+) of 1.0 mM Mn2+, followed by SDS–PAGE and immunoblotting with anti-β2 integrin polyclonal Ab (R&D Systems). Immunoprecipitation with mAb TS1/18 (lanes 1 and 4) served as a positive control to ensure the presence of β2 integrin in the lysate.
Mentions: Integrin affinity regulation was evaluated on Jurkat CD47− and CD47+ cells by reporter mAbs that detect activated conformations of β1 and β2 integrins. KIM127 mAb detects and stabilizes an extended and closed conformation of β2 integrins (intermediate affinity), and mAb 24 recognizes an extended and open “active” conformation of β2 integrins (high affinity; (Salas et al., 2004). Jurkat T-cells express predominantly LFA-1 (αLβ2) and not the other α-subunit chains that associate with β2 integrins, and hence these mAbs to β2 integrins detect primarily the intermediate- and high-affinity conformations of LFA-1 (Salas et al., 2004). HUTS21 detects and stabilizes activated β1 integrins (Luque et al., 1996), and we showed previously that expression of this epitope correlated with robust adhesion of human memory T-cells to VCAM-1 (Lim et al., 2000). As expected, incubation of CD47+ T-cells with Mn2+ or Mg2+/ethylene glycol tetraacetic acid (EGTA), global activators of integrins, triggered robust expression of both intermediate- and high-affinity LFA-1 (Figure 6, A and B). In contrast, CD47− T-cells showed a significantly reduced expression of intermediate- and high-affinity conformations of LFA-1 in response to Mn2+ or Mg2+/EGTA. It is unlikely that this assay did not detect transient LFA-1 activation because the mAbs stabilize the active conformation and were present throughout the assay. In addition, failure to detect mAb expression was not due to a change (loss) in total surface expression of β1 or β2 integrins upon exposure to Mn2+ or Mg2+/EGTA (Figure 6C). CD47− T-cells also showed reduced induction of high-affinity β1 integrins versus CD47+ T-cells after treatment with Mn2+ or Mg2+/EGTA buffers (Figure 6D). Consistent with the failure of Mn2+ or Mg2+/EGTA treatments to induce intermediate- and high-affinity LFA-1 conformations in the absence of CD47, CD47− T-cells exhibited little binding of soluble ICAM-1-Fc versus CD47+ T-cells (Figure 6E). In addition, treatment of CD47− cells with Mn2+ did not restore binding comparable to CD47+ T-cell adhesion to either immobilized ICAM-1 or VCAM-1 under flow (Supplemental Figure S1, A and B). Taken together, the results indicate that CD47 expression is required for expression of intermediate- and high-affinity conformations of LFA-1 and for binding soluble ICAM-1, as well as for induction of activated β1 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