Skip to main page content (V体育官网入口)
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official VSports app下载. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2008 Aug 15;112(4):1280-9.
doi: 10.1182/blood-2008-01-134429. Epub 2008 Jun 4.

Endothelial CD47 interaction with SIRPgamma is required for human T-cell transendothelial migration under shear flow conditions in vitro

Michael Stefanidakis  1 , Gail Newton, Winston Y Lee, Charles A Parkos, Francis W Luscinskas
Affiliations

Endothelial CD47 interaction with SIRPgamma is required for human T-cell transendothelial migration under shear flow conditions in vitro

"V体育平台登录" Michael Stefanidakis et al. Blood. .

"V体育平台登录" Abstract

Leukocyte transendothelial migration (TEM) is a critical event during inflammation. CD47 has been implicated in myeloid cell migration across endothelium and epithelium. CD47 binds to signal regulatory protein (SIRP), SIRPalpha and SIRPgamma. So far, little is known about the role of endothelial CD47 in T-cell TEM in vivo or under flow conditions in vitro. Fluorescence-activated cell sorting and biochemical analysis show that CD3(+) T cells express SIRPgamma but not SIRPalpha, and fluorescence microscopy showed that CD47 was enriched at endothelial junctions. These expression patterns suggested that CD47 plays a role in T-cell TEM through binding interactions with SIRPgamma VSports手机版. We tested, therefore, whether CD47-SIRPgamma interactions affect T-cell transmigration using blocking mAb against CD47 or SIRPgamma in an in vitro flow model. These antibodies inhibited T-cell TEM by 70% plus or minus 6% and 82% plus or minus 1%, respectively, but had no effect on adhesion. In agreement with human mAb studies, transmigration of murine wild-type T helper type 1 cells across TNF-alpha-activated murine CD47(-/-) endothelium was reduced by 75% plus or minus 2% even though murine T cells appear to lack SIRPgamma. Nonetheless, these findings suggest endothelial cell CD47 interacting with T-cell ligands, such as SIRPgamma, play an important role in T-cell transendothelial migration. .

PubMed Disclaimer

Figures

Figure 1
Figure 1
Cell-surface expression and localization of CD47 in HUVECs. (A) HUVECs were treated with medium or medium containing TNF-α (left), IL-1β (middle), or IL-1β and IFN-γ (right) for 4 hours and 24 hours, and CD47 expression was detected by flow cytometry using anti-CD47 (B6H12) mAb and PE-labeled anti–mouse secondary mAb. An isotype-matched mAb was used as a negative control. (B) Untreated (top) and TNF-α treated (bottom) nonpermeabilized HUVECs were fixed with 2% paraformaldehyde and stained with anti-CD47 (B6H12; left; 10 μg/mL) or anti–VE-cadherin (Hec 1; right) mAb as described in “Immunofluorescence microscopy.” Bars, 40 μm.
Figure 2
Figure 2
Endothelial cell CD47 mediates TEM of T cells. (A) HUVEC monolayers stimulated with TNF-α for 4 hours were preincubated with function blocking mAb to CD47 (B6H12), ICAM-1 (HU5/3), or a nonblocking control mAb to MHC class I (W6/32) at 37°C for 30 minutes (30 μg/mL each). Before perfusion of T cells, TNF-α–stimulated HUVECs were treated with SDF-1α at 37°C for 15 minutes. The total number of accumulated T cells was determined as detailed in “Methods.” NS indicates P > .05. (B) TNF-α–stimulated HUVEC monolayers were preincubated with a CD47 (B6H12), ICAM-1 (HU5/3) mAb, medium alone (no additions), or MHC-class I mAb at 37°C for 30 minutes. The percentage of T cells that transmigrated after 10 minutes was determined by live cell imaging as described earlier. Data are means (± SEM) of 4 separate experiments; * indicates statistically significant difference (P = .001). (C) DIC images of live cells in the TEM assay after 10 minutes were produced as described in “T-cell locomotion and transmigration assay under flow.” The polarity of T cells on untreated (no Ab; left, formula image), B6H12 (middle, ▶), and control W6/32 (right) mAbs are shown. Arrows show migrated CD3+ cells in medium-treated monolayers. In contrast, T cells (▶) on B6H12-treated monolayers exhibit a rounded-up morphology and defects in migratory behavior. (Inset) A higher magnification of a single T cell during TEM is shown in the right upper corner of each DIC image to better show the differences in morphology in the presence or absence of a blocking CD47 antibody. (D) The migration velocity of T cells perfused on a HUVEC monolayer treated with B6H12 or W6/32 mAb or left untreated was determined using Image J software. *P = .01 versus W6/32 mAb.
Figure 3
Figure 3
SIRPγ and SIRPα expression in T cells. (A) FACS analysis of resting human CD3+ T cells and PMNs stained with anti-SIRPα, anti-SIRPγ, or anti-MHC class I mAbs (solid histograms). The black solid lines represent a nonbinding isotype control mAb. (B) Equal amounts of total protein from lysates of resting PMNs and T cells were separated by SDS-PAGE under reducing conditions and analyzed by Western blotting for SIRPα expression (top). The membrane was stripped and reprobed with a SIRPγ mAb. The positions of SIRPα and SIRPγ proteins are marked. In addition, PMNs or T-cell lysates were loaded under reducing (lanes 1 and 3 from left) or nonreducing conditions (lanes 2 and 4 from left), electrophoresed, transferred to nitrocellulose membranes, and probed with a rabbit polyclonal Ab, which specifically recognizes the cytoplasmic tail of SIRPα (bottom). (C) FACS analysis of resting (black solid line) and 4 hours of TNF-α (black dotted line) treated HUVECs stained with anti-SIRPα (SE7C2; left), anti-SIRPγ (LSB2.20; right), or an isotype IgG control mAb (gray solid lines). These data are representative of 3 different preparations of cells.
Figure 4
Figure 4
T-cell SIRPγ binds immobilized CD47-AP. Adhesion of resting CD3+ T cells to immobilized CD47-AP fusion protein under static conditions was performed as described in “Methods.” Data are means (± SD) of triplicate wells from 2 independent experiments; *P = .001.
Figure 5
Figure 5
SIRPγ mediates TEM of HUVECs under flow. (A) HUVEC monolayers were prepared as described in Figure 2. Isolated CD3+ T cells were preincubated with SIRPα (SE7C2), SIRPγ (LSB2.20), or a nonblocking mAb to MHC class I and then cells were drawn across TNF-α–stimulated endothelium. The total number of accumulated T cells was determined 10 minutes after perfusion as described in “Methods.” Data are means (± SEM) of 4 separate experiments. (B) T cells were treated with listed mAb (30 μg/mL; SIRPα, SE7C2; SIRPγ, LSB2.20; nonblocking control mAb MHC class I, W6/32) and then were drawn across TNF-α–stimulated endothelium for 10 minutes. The percentage of T cells that transmigrated the endothelium after 10 minutes was determined as detailed in “Methods.” Data are means ± SEM, n = 4 separate experiments; *P = .001. (C) DIC images of live cells in the TEM assay after 10 minutes were prepared as described in “T-cell locomotion and transmigration under flow.” The polarity of T cells on control (W6/32), SIRPγ (LSB2.20), and SIPRα (SEC7C2) mAbs are shown. formula image show normal, migrated CD3+ cells in W6/32-treated monolayers. In contrast, T cells (▶) on SIRPγ-treated T cells exhibit a rounded-up morphology and defects in migratory behavior and show reduced transmigration. (D) The apical migration velocities of T cells on HUVEC monolayers treated with SIRPα (SE7C2), SIRPγ (LSB2.20), or nonblocking control mAb MHC class I mAbs was determined by the Image J software. *P = .01. (E) TNF-α–stimulated HUVEC monolayers were preincubated for 30 minutes with anti-CD47 (B6H12) or SIRPα (SE7C2) mAbs. Medium alone (no additions) or MHC-class I (W6/32) mAb served as controls. The percentage of T cells that transmigrated after 10 minutes was determined as described in “Methods.” Data are means (± SEM) of 4 separate experiments; *P = .001.
Figure 6
Figure 6
CD47−/− MHECs have impaired T-cell transmigration. (A) Expression of mouse CD47 in WT and CD47−/− MHECs before (left) or after TNF-α (right). The gray solid lines represent a nonbinding isotype control mAb. (B) Isolated mouse Th1 cells prepared from naive WT mice (2 × 106 cells/200 μL) were added as a bolus, and the total number of accumulated T cells was determined 10 minutes after perfusion as detailed in “Methods.” Data are means (± SEM) of 4 separate experiments; *indicates statistical significant difference (P < .001). (C) Mouse WT Th1 cell transmigration of MHECs from WT or CD47−/− mice was performed as described in “Methods.” Data are means (± SEM) of 4 separate experiments; * indicates statistical significant difference (P = .001) were prepared as described in “T-cell locomotion and transmigration under flow.” (D) DIC images from live cell imaging of T-cell TEM were prepared as described in “T-cell locomotion and transmigration under flow.” The images show differences in the morphology of T cells on WT (left) versus CD47−/− MHEC monolayers. formula image identify migrated CD3+ cells (left), and ▶ show CD3+ cells with a rounded-up morphology and impaired apical migration on TNF-α stimulated CD47−/− MHEC (right). (E,F) WT and CD47−/− MHECs were stained for listed adhesion molecules in the presence (right) or absence (left) of TNF-α and analyzed by flow cytometry. Expression levels were shown in histograms and quantitated as mean fluorescence intensity. Data are means (± SD), n = 3 independent MHEC preparations; indicates statistical significant difference (P = .001).
Figure 7
Figure 7
Schematic summary of the endothelial CD47 association with SIRPγ in T cells. SIRPγ is selectively expressed and localized on the surface of CD3+ T cells and binds to endothelial CD47 in trans. Because SIRPγ contains a short cytoplasmic tail and is believed not to signal to the T cells, we propose a model in which CD47 engagement by SIRPγ promotes CD47-induced signaling pathways in endothelial cells (unidirectional). These signals could facilitate TEM by Gi-coupled signaling and/or through association with integrins in cis that may occur in endothelial cells. For example, CD47 ligation by SIRPγ could induce actin cytoskeleton remodeling and disassembly of the adherent junctions in endothelial cells, processes that are required for a successful T-cell transendothelial migration., Disruption of the CD47-SIRYγ complex by CD47- and SIRPγ-specific blocking monoclonal antibodies strongly reduced T-cell transmigration under flow conditions in vitro.
See this image and copyright information in PMC

References

    1. Butcher EC. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell. 1991;67:1033–1036. - PubMed (V体育官网入口)
    1. Rao RM, Yang L, Garcia-Cardena G, Luscinskas FW. Endothelial-dependent mechanisms of leukocyte recruitment to the vascular wall. Circ Res. 2007;101:234–247. - PubMed
    1. Lindberg FP, Gresham HD, Schwarz E, Brown EJ. Molecular cloning of integrin-associated protein: an immunoglobulin family member with multiple membrane-spanning domains implicated in alpha v beta 3-dependent ligand binding. J Cell Biol. 1993;123:485–496. - PMC - PubMed
    1. Brown EJ, Frazier WA. Integrin-associated protein (CD47) and its ligands. Trends Cell Biol. 2001;11:130–135. - "VSports app下载" PubMed
    1. Barclay AN, Brown MH. The SIRP family of receptors and immune regulation. Nat Rev Immunol. 2006;6:457–464. - PubMed

Publication types