CD47 plays a critical role in T-cell recruitment by regulation of LFA-1 and VLA-4 integrin adhesive functions

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 null 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.

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 V_crit. 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.

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.

FIGURE 3:

CD47^−/− Th1 cells have impaired adhesion to immobilized ICAM-1 and VCAM-1 but not E-selectin in an in vitro flow chamber model. WT and CD47^−/− Th1 cells were drawn across immobilized ICAM-1-Fc (A), VCAM-1-Fc (B), and E-selectin-Fc chimeric proteins (C) at the shear stress levels indicated, and cell adhesion was determined as detailed in Materials and Methods. Data are mean ± SEM, n = 3. *p ≤ 0.05, **p ≤ 0.01 (Student's t test). (D) Shear flow–mediated detachment of Th1 cells prebound to immobilized ICAM-1 + CXCL12 is not altered in CD47^−/− Th1 cells. Data are mean ± SEM, n = 3 separate experiments.

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⁻ (null) 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).

FIGURE 5:

Jurkat CD47⁻ (null) T-cells have impaired adhesion to immobilized ICAM-1 and VCAM-1 but not E-selectin under shear flow conditions. (A–C) Jurkat T-cells were drawn across immobilized ICAM-1 (A), VCAM-1 (B), or E-selectin (C) proteins at various shear stress levels, and adhesion was measured as described in Materials and Methods. (D) Transfection of CD47⁻ Jurkat cells with a plasmid containing CD47 tagged with GFP (CD47 + GFP) rescued cell adhesion to ICAM-1. In contrast, transfection of CD47⁻ cells with a plasmid containing only GFP (CD47-GFPcontrol) did not. (E) Detachment of Jurkat T-cells prebound to immobilized ICAM-1 is not affected by the absence of CD47. Data are means ± SEM of at least three independent experiments. *p ≤ 0.05, **p ≤ 0.01 (Student's t test).

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⁻ (null) 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-null (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.

FIGURE 7:

CD47 and β2 integrin interact on the cellular membrane of Jurkat T-cells. (A) Representation of interacting fraction τ_m 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 τ_m 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.