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(The FASEB Journal. 2001;15:341-350.)
© 2001 FASEB

Integrin-associated protein (CD47/IAP) contributes to T cell arrest on inflammatory vascular endothelium under flow

MICHEL TICCHIONI, VINCENT RAIMONDI, LAURENCE LAMY, JOHN WIJDENES*, FREDERIK P. LINDBERG§, ERIC J. BROWN{ddagger} and ALAIN BERNARD1

Unité INSERM U343 et Laboratoire d’Immunologie, 06202 Nice cedex 3, France;
* Diaclone research, BP1985, F25020 Besançon cedex, France;
§ Division of Infectious Diseases, Washington University School of Medecine, Saint Louis, Missouri, 63110, USA; and
{ddagger} University of California, San Francisco, San Francisco, California 94143, USA

1Correspondence: Unité INSERM U343, Hôpital de l’Archet 1, Route de St. Antoine de Ginestiere-BP 3079, 06202 Nice cedex 3, France. E-mail: u343{at}hermes.unice.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrin-associated protein (CD47/IAP) is a pentaspan molecule that regulates integrin functions. We prepared a CD47-deficient Jurkat T cell line to assess its role in the arrest of T cells on inflammatory endothelium. Under flow conditions, constitutive arrest of CD47-deficient cells is strongly decreased as compared to the original cell line, whereas reexpression of CD47 reestablishes their ability to stop. Moreover, cells transfected with a chimera made with the extracellular portion of CD47 and the transmembrane domain of CD7 or several truncated forms of CD47 show that the first transmembrane domain and a short cytoplasmic loop are sufficient for this process. CD47 effect is indirect and depends mainly on the {alpha}4ß1/VCAM-1 pathway, as shown by blocking antibodies. We detected on endothelium the two CD47 counter receptors known to date: thrombospondin and SIRP1{alpha}. Blocking experiments show that both are involved. Overall, CD47 participates in the constitutive arrest of T lymphocytes on inflamed vascular endothelium by up-regulating {alpha} 4ß1 integrins.—Ticchioni, M., Raimondi, V., Lamy, L., Wijdenes, J., Lindberg, F. P., Brown, E. J., Bernard, A. Integrin-associated protein (CD47/IAP) contributes to T cell arrest on inflammatory vascular endothelium under flow.


Key Words: adhesion • T cell circulation • integrin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
INTERACTIONS BETWEEN LYMPHOCYTES and endothelial cells are essential for the recruitment of lymphocytes into inflammatory tissues and therefore the achievement of an efficient immune response. A multistep model involving tethering and rolling of lymphocytes on endothelial cells, rapid activation of integrins, followed by a firm arrest of the cells before diapedesis into the tissue, is now widely accepted (1 2 3) .

Therefore, one of the key issues in T cell extravasation is to determine the factors that induce and/or maintain integrins in an active form. Chemokines were shown to be particularly efficient in this process (4 5 6 ; see reviews, refs 7 , 8 ). Several surface molecules such as L-selectin (9 10 11 12 13) , P-selectin glycoprotein ligand 1 (14) , and other members of the integrin family (15 16 17) have also been shown to generate transmembrane signals that may enhance leukocyte–endothelial cells interactions. Even though it is likely that other cell surface receptors regulate T cell adhesion and transendothelial migration, their identities are not known, particularly those involved in the constitutive arrest of T cells on inflamed endothelium in the absence of exogenous chemokines (18 19 20) .

Integrin-associated protein (IAP, CD47) is a 50 kDa glycoprotein expressed on all mammalian cells (21) that is identical with the ovarian tumor marker OA3 (22) . It consists of an extracellular immunoglobulin-like domain, five membrane-spanning regions, and a short cytoplasmic tail (23) with four alternatively spliced forms differing only within the cytoplasmic tail (24) , the form 2 (with a 16 amino acid intra-cytoplasmic extension) being the predominant form in endothelial cells and bone marrow-derived cells (24) .

CD47 was first described as a molecule associated with the ß 3 integrin chain on placenta and platelets (21) . Indeed, CD47 is involved in several functions of ß3 such as RGD phagocytosis stimulation or generation of a respiratory burst in human neutrophils (polymorphonuclear cell, or PMN) (21 , 25 , 26) , vitronectin binding of erythroleukemia cells (23) , and calcium entry in endothelial cells induced by fibronectin (27) . Moreover, thrombospondin 1 (TSP-1), the first discovered ligand of CD47 (28 , 29) , modulates through CD47 the functions of {alpha}IIbß3 integrin in platelets (30) and {alpha}vß 3 in a melanoma cell line (31) . All these results reinforce the hypothesis that CD47 may regulate the functions of some integrins and acts as a transducer element in activation mediated via integrins (26) .

On peripheral T cells, however, CD47 is present at high densities, although the ß3 integrin chain is barely detectable (32) and no association with integrins has been shown. Yet CD47 transduces within T lymphocytes a powerful comitogenic signal (33 34 35) , activates an apoptosis pathway (36) or transduces signals leading to T cell spreading (37) suggesting that some important functions of CD47 might be independent of its association with the ß3 integrin chain.

Given the regulatory role of CD47 on integrins, we investigated whether CD47 could play a role in the adhesion events occurring between T lymphocytes and inflamed vascular endothelium under physiological shear stress. Here we report that CD47 is involved in the arrest of T cells on inflammatory endothelium.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and antibodies
Tumor necrosis factor {alpha} (TNF-{alpha}) and recombinant human vascular cellular adhesion molecule 1 (VCAM-1 the seven-domain form minus the transmembrane and cytoplasmic domains) were obtained from R&D Systems (Abingdon, U.K.). Peptide corresponding to the cell binding domain (CBD) of TSP-1 (KRFYVVMWKK) was purchased from Bachem (Voisins-le-Bretonneux, France). CD47 monoclonal antibodies (mAbs), B6H12 was obtained from the American Type Culture Collection (ATCC) (Rockville, Md.), BRIC126 from the International Blood Group Reference Laboratory (Bristol, U.K.) and FITC-BRIC126 from Serotec (Oxford, U.K.). Anti-intercellular adhesion molecule 1 (anti-ICAM-1; B-H17) and VCAM-1 (B-K9) mAbs were kindly obtained from Dr J. Widjenes (Diaclone, Besançon, France); anti-very late activation antigen 4 (anti-VLA-4; HP2/1) mAb was from Dr. F. Sanchez-Madrid (Hospital de la Princesa, Madrid, Spain). Rabbit anti-TSP R1 Ab was a gift from Dr J. Lawler (Beth Israel Hospital, Boston, Mass.), murine anti-TSP mAbs C6.7, and C6.9, directed respectively against the binding site of CD47 or the binding site of heparin on TSP-1, were purchased from NeoMarkers (Union City, Calif.). mAbs SE5A5 directed against SIRP{alpha}1 and SIRP{alpha} 2 and SE7C2 directed against SIRP{alpha} 1 were a kind gift from Dr. H.-J. Büring (University of Tübingen, Germany).

Cells
The Jurkat T cell line (JE6.1) was obtained from ATCC and cultured in RPMI 1640 (Gibco Laboratories, Cergy Pontoise, France) supplemented with 5% fetal calf serum (FCS) (Dutcher, Brumath, France), 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine, and 1 mM pyruvate (Merck, Darmstadt, Germany); The transformed human umbilical vein endothelial cell line EA.hy926 was kindly provided by Dr. Edgell (University of North Carolina, Chapel Hill, N.C.) (38) and cultured in DMEM (Gibco Laboratories) supplemented with 20% FCS. CD47-deficient Jurkat T cell line (JIN for Jurkat IAP-negative cells) was obtained as follow: cells were cultured for 18 h at the concentration of 0.5 x 106/ml in the presence of ethyl-methanesulfonate (EMS, 200 µg/ml) (Sigma, St. Louis, Mo.), washed three times, and allowed to recover for 5 days. CD47-expressing cells were next eliminated by seven rounds of negative selection using the mAb BRIC126 (1 µg/ml) and nontoxic rabbit complement (Behring, Marburg, Germany). Cells were then sorted using cell sorting (FACStar, Becton-Dickinson, Mountain View, Calif.) after labeling with BRIC126 and FITC-rabbit anti-mouse F(ab')2 (Dako, Glostrup, Denmark), before cloning by limiting dilution.

Human PHA-activated T cells and CD4 T cells were obtained from healthy volunteers. Briefly, whole blood cells were centrifuged on a Ficoll-Hypaque gradient. Mononuclear cells were cultured for 2 days with PHA, then interleukin 2 was added for 1 wk. CD4 T cells were purified from peripheral mononuclear cells in one step using positive selection with CD4-recovered microbeads and Detach-a-Bead (Dynal, Oslo, Norway). Either CD4RO or CD4RA Microbeads (Miltenyi Biotec, Paris, France) were then used to purify respectively memory or naive CD4-T cells. Typically, CD4-T cells were more than 95% pure.

Immunofluorescence analysis
Endothelial cells were incubated at 4°C for 30 min in the dark in 100 µl phosphate-buffered saline (PBS), 0.1% NaN3, 0.1% bovine serum albumin (BSA) with saturating concentrations of mAbs, then stained with a PE-RAM F(ab')2 (Dako, Dakopatts). Between each step, cells were washed twice with PBS, 0.1% NaN3, 0.1% BSA. Controls included cells incubated with CD3 mAb plus PE-conjugated RAM-F(ab')2. Forward and right-angle scatter gatings were set in order to include endothelial cells. Analysis were performed on a FACScan (Becton-Dickinson, Mountain View, Calif.).

Fluorescence dyes
We used different dyes, all purchased from Molecular Probes (Eugene, Oreg.). Blue fluorescent aminocoumarin (CellTracker Blue CMAC) is a blue fluorescent tracer with excitation at 372 nm and emission at 470 nm; green fluorescent fluorescein diacetate (CellTracker Green CMFDA) is a green fluorescent tracer with excitation at 522 nm and emission at 529 nm; orange fluorescent tetramethylrhodamine (CellTracker Orange CMTMR) is a red fluorescent tracer with excitation at 541 nm and emission at 567 nm. T cells were labeled in medium with the different probes (1 µg/ml) for 30 min at 37°C in the dark, then washed three times before using.

cDNA, transfections, and expression
DNA constructs were obtained from Dr. E. Brown and described in detail elsewhere (34) . Briefly, human CD47 form 2 was amplified by polymerase chain reaction (PCR) in PBS. CD16-CD7-Syk construct was used to generate the CD47-CD7 construct. cDNAs were cloned into the expression vector BSR{alpha}EN (Dr. A. Shaw, Washington University School of Medicine, St. Louis, Mo.). Standard techniques were used for nucleic acid manipulations and preparation of DNA constructs. Three CD47 glycoprotein mutations were performed, corresponding to transmembrane domain deletions (Fig. 1 ). The extracellular domain plus the first, the two first, or the four first transmembrane domains (respectively TM1-, TM2-, and TM4-CD47) were amplified, using the Expand high-fidelity PCR System (Boehringer Mannheim, Germany). Human CD47 cDNA form 2 was used as a template for PCR amplification. Primers used to generate the DNA fragments were 5'-CAGTCTCGAGATGTGGCCCCTGGTAGCGGCG-3' (sense oligonucleotide, XhoI site underlined), and 5'-GCTCTAGACTACTCATCCATACCACCGGATCT-3' (defining a 504 pb product, TM1), 5'-GTTCTAGACTAACCAAGGCCAGTAGCATTCT-3' (633 pb, TM2), 5'-AGTCTAGACTACGCCGCAATACAGAGACTCAG-3' (786 pb, TM4) (antisense oligonucleotides, XbaI site underlined, mutation introducing a stop codon in boldface). These fragments were cloned into the expression vector BSR{alpha}EN using the XhoI and XbaI sites.



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  		Figure 1. Structure of CD47/IAP and the different constructions used in this study. From left to right, all forms express the extracellular domain of CD47 plus the transmembrane domain of CD7 (CD7-CD47), the first, the two first, or the four first transmembrane domains of CD47 (respectively TM1, TM2, and TM4), and the whole CD47 isoform 2.

CD47-deficient cells in log phase growth were washed twice in RPMI. 5 x 106 were resuspended in 500 µl of RPMI and transferred into 4 mm electroporation cuvettes (Eurogentec, Seraing, Belgium). Stable transfections were performed using the BSR{alpha}EN constructs (20 µg) at 900 µF, 250 V, 14 W (Cellject, Eurogentec). Cells were placed for 10 min in ice before and after electroporation, then transferred in a culture flask. Selection was performed 48 h after the electroporation using Geneticin (1 mg/ml) (Sigma). Stably transfected clones with CD47 expression at the same level as the wild-type Jurkat cells were selected by fluorescence-activated cell sorting (FACS).

Flow chamber and in vitro flow studies
The flow chamber was purchased from Immunetics (Cambridge, Mass.) and has been described elsewhere (39) . It was designed to allow stabilized laminar flow between 0.1 and 2 dyn/cm2. T cells at the concentration of 1 x 106/ml in HBSS supplemented with 1 mM of CaCl2 and MgCl2 were perfused through the chamber on a monolayer of endothelial cells, immobilized VCAM-1, or peptide using a withdrawal syringe pump (Harvard Apparatus, Boston, Mass.). Immobilized VCAM-1 (1 µg/ml) or peptide (50 µM) was performed by coating Lab-TeK 1 chamber slides (Poly Labo, Strasbourg, France) overnight at 4°C. In experiments using coimmobilized VCAM-1 and peptide, VCAM-1 was incubated overnight first, then peptide was added and incubated overnight. Between each step, the Lab-Tek were washed three times with PBS. Then PBS with 1% BSA was added for 2 h and slides were washed three more times before using. In most experiments, the different T cell lines were fluorescently labeled, washed three times, then mixed and perfused on the endothelial cell line EA.hy926 for 5 min. Medium was perfused to remove non-firmly adherent lymphocytes before quantification on six random fields of 0.65 mm2 each, using a laser scanning confocal microscope (Ultima Meridian, DGL Bioscience) with 10x objective. In rolling experiments, lymphocyte–endothelial interactions in different fields were videotaped for 30 s using a CCD video camera and a video recorder. Images were then digitized using a Power Macintosh G3 (Apple Computer, Cupertino, Calif.) with video card (miroMOTION DC30, Pinnacle systems, Germany) and commercial software (Adobe Premiere and Abobe Photoshop, Adobe Systems, San Jose, Calif.). Rolling velocities were measured by determining the number of frames (each 1/30th s length of time) it took 25 cells to cross a 350 µm length field.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Constitutive arrest of T cells on TNF-{alpha}-activated endothelial cells requires the presence of CD47 and its transmembrane and/or intracellular domains.
We have investigated whether or not CD47 is involved in the interactions between T and endothelial cells under flow conditions by preparing a CD47-deficient T cell line (derived from Jurkat cells by EMS mutagenesis) we have named the JIN T cell line. Cells were obtained after chemical mutagenesis and complement-dependent negative selection as described in Materials and Methods. No CD47 could be detected by immunofluorescence at their surface, in marked contrast with Jurkat cells (Fig. 2A , B ). We transfected in these cells the cDNAs encoding for the wild-type isoform 2 of CD47 (wt-CD47POS cells) or a chimeric form of CD47 devoid of the multispan portion (CD7-CD47POS cells) (Fig. 1) . This chimera comprises the extracellular domain of CD47 and the transmembrane portion of CD7, and is unable to transduce a comitogenic signal within CD3-activated cells (34) . We selected stable transfectants that expressed similar CD47 levels as the Jurkat cells (Fig. 2C , D ).



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  		Figure 2. Expression of CD47/IAP on Jurkat cells (A), CD47-deficient JIN cells (B), CD47-deficient cells transfected with the wild-type form 2 of CD47 (wt-CD47POS cells) (C), CD47-deficient cells transfected with the chimera CD7-CD47 (D). Cells were labeled with a CD47-FITC mAb (full line) on ice for 30 min, washed 3 times, then analyzed by flow cytometry. Cells labeled with a control CD20-FITC mAb are shown in dotted line.

In a first set of experiments, we used three different fluorescent dyes, orange fluorescent tetramethylrhodamine, green fluorescent fluorescein diacetate, and blue fluorescent aminocoumarin to label respectively Jurkat T cells in green, CD47-deficient cells in blue, and wt-CD47POS cells in red. Then cells were extensively washed, mixed, and perfused for 5 min on a monolayer of endothelial cells at a flow rate of 40 ml/h, corresponding to a wall shear stress of 2 dyn/cm2. Medium alone supplemented with 1 mM of CaCl2 and MgCl2 was perfused at the same flow rate. The number of firmly arrested cells was next quantified on six independent areas of 0.65 mm2 each.

As expected, we did not observe any arrest of the cells on unactivated EA.hy926 monolayers, even at very low shear stresses (data not shown). Strikingly, on TNF-{alpha}-activated-endothelial cells, CD47-deficient Jurkat-derived cell line showed a decreased ability (~50%) to stop on activated endothelium as compared to the original cell line. Reexpression of CD47 reestablished the ability of cells to firmly stop (Fig. 3 ).



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  		Figure 3. Arrest of Jurkat T cells (JE 6.1), CD47-deficient cells (CD47NEG), and CD47-deficient cells transfected with the wild-type form of CD47 (wt-CD47POS cells) on TNF-{alpha}-activated endothelium. We used 3 different fluorescent dyes (green fluorescent fluorescein diacetate, blue fluorescent aminocoumarin, and orange fluorescent tetramethylrhodamine) to label respectively Jurkat T cells in green, CD47-deficient cells in blue, and wt-CD47POS cells in red. Cells were perfused in a flow chamber at a flow rate of 2 dyn/cm2 for 6 min, then medium was perfused. The number of firmly arrested cells was next quantified by scanning 6 independent areas with a confocal microscope.

In a second set of experiments we mixed CD47-deficient cells, wt-CD47POS cells, and CD7-CD47POS cells. Results show that transfection of the chimera did not restore the ability to stop (Fig. 4 ). Thus, CD47 appears to be required for T cell arrest on TNF-{alpha}-activated endothelial cells. The multispan domain and/or the intracytoplasmic portion of CD47 is also required for T cell arrest. The role of CD47 was clearly critical for shear stress values below 3.5 dyn/cm2. Beyond this value, the number of CD47-expressing cells able to stop was not different from the number of CD47-deficient cells (Fig. 5 ).



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  		Figure 4. Arrest of CD47-deficient cells (CD47NEG) and CD47-deficient cells transfected with the wild-type form of CD47 (wt-CD47POS cells) or the transmembrane deleted chimera (CD7-CD47) on TNF-{alpha}-activated endothelium. As detailed in the legend of Fig. 3 , we used 3 different fluorescent dyes, to labeled CD47NEG, wt-CD47POS and CD7-CD47. Cells were perfused in a flow chamber at a flow rate of 2 dyn/cm2 for 6 min, then medium was perfused. The number of firmly arrested cells was next quantified by scanning 6 independent areas with a confocal microscope. A scanned area where the three cell lines are arrested is shown on the right panel. Arrow indicates the direction of flow. This experiment is representative of at least 3 different experiments.



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  		Figure 5. Arrest of CD47-deficient cells (CD47NEG) and CD47-reconstituted cells (wt-CD47POS cells) on inflammatory endothelium at different shear stresses. Cells were labeled as described in Materials and Methods and perfused at different flow rates on inflammatory endothelium. The number of firmly arrested cells was quantified as above.

CD47-dependent arrest is blocked by anti-VCAM-1 or anti-{alpha} 4 mAbs
Since the arrest of the cells was observed only on activated endothelium, we investigated the role of VCAM-1 in this process by preincubating endothelial monolayers with blocking anti-VCAM-1 mAb before washing. As above, a mixture of CD47-deficient cells, wt-CD47POS cells, and CD7-CD47POS cells were perfused at a shear stress of 2 dyn/cm2. As shown in Fig. 6 , the arrest was almost completely blocked by endothelial preincubation with anti-VCAM-1 mAb. Inhibition was also observed when we used the blocking anti-{alpha} 4 HP2/1 mAb (not shown). Thus, it is likely that CD47 by itself is not involved in the firm arrest of T cells, but might transduce an activating signal mainly to integrin VLA-4, inducing the arrest of the cells.



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  		Figure 6. Effects of blocking anti-VCAM-1 mAb on arrest of CD47-reconstituted cells with the wild-type CD47 (wt-CD47POS cells) or the multispan transmembrane domain-deficient construction (CD7-CD47 cells) and CD47-deficient cells (CD47NEG cells) on inflammatory endothelium. Endothelial cells were preincubated or not with the mAb, then T cells were perfused in the flow chamber at a flow rate of 2 dyn/cm2 for 6 min. The number of firmly arrested cells was quantified as above. This experiment is representative of at least 3 different experiments.

The arrest of T cells requires only the first transmembrane domain of CD47
The role of the different portions of the transmembrane domain of CD47 was assessed using mutated forms of CD47. We generated different forms deleted with the last four, three, and one transmembrane domains of CD47. These forms, respectively named TM1- (since it possesses only the first transmembrane domain and a short intracytoplasmic loop of 12 AA), TM2-, and TM4-CD47, were transfected in CD47-deficient cells (Fig. 1) . All the transfected cells expressed CD47 at the same level (data not shown). Cells transfected with the TM1-CD47 construction show an ability to stop on endothelium higher than CD47-deficient cells. These data suggest that the first transmembrane and a short intracytoplasmic portion of 12 AA length are still sufficient to transduce an activating signal within T cells (Fig. 7 ).



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  		Figure 7. Arrest of CD47-deficient cells (CD47NEG), TM1-, TM2-, and TM4-CD47-transfected cells on TNF-{alpha}-activated endothelium. As detailed in the legend of Fig. 3 , we used two different fluorescent dyes to labeled CD47-deficient cells and the different transfected cells. A) CD47NEG and TM1-CD47 were mixed and perfused in the flow chamber at a flow rate of 2 dyn/cm2 for 6 min. B, C) CD47-deficient cells were mixed respectively with TM2- or TM4-CD47. The number of firmly arrested cells was quantified as above.

Influence of TSP and SIRP{alpha} 1 on CD47-dependent T cell arrest
To date, two natural ligands of CD47—thrombospondin 1 (28 , 29) and the P84 neural adhesion molecule (also known as SHPS-1, BIT, and SIRP) (40 , 41) —have been described. We first checked their expression by flow cytometry on resting or TNF-{alpha} activated endothelium. As shown in Fig. 8 , both are well expressed on endothelium. Then we tested whether they could be involved in this process.



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  		Figure 8. Expression of TSP and SIRP on endothelial cells. Endothelial cells were labeled with different mAbs—namely, anti-CD47 (B6H12, A), anti-TSP (C6.7, B), anti-SIRP{alpha} 1 and SIRP{alpha} 2 (SE5A5, C), and anti-SIRP{alpha}1 (SE7C2, D)—washed twice, then stained with a PE-RAM F(ab')2. Negative control included cells labeled with CD3 mAb and are shown in dotted line.

The peptide 4N1K (KRFYVVMWKK) is derived from the carboxyl-terminal CBD of TSP-1 and corresponds to a sequence highly conserved in all TS family members (42 , 43) . We tested the effects of this peptide either immobilized on plastic or in a soluble form on T cell arrest.

In a first set of experiments, we immobilized the 4N1K peptide on plastic. As represented in Fig. 9 , no cell stopped on immobilized peptide alone or on BSA. By contrast, both cell lines were able to stop on immobilized VCAM-1 at the same level. The arrest was increased on wt-CD47POS cells, but not CD47-deficient cells, when we coimmobilized the peptide and VCAM-1 on plastic, suggesting a specific effect via CD47. The 4N1K peptide was also quite efficient in increasing arrest of human CD4-T cells and PHA-activated T cells on VCAM-1 (Table 1 ). Moreover, as expected, arrest involved mainly memory T cells as observed after purification by microbeads (not shown).



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  		Figure 9. Effects of immobilized 4N1K peptide on the arrest of CD47-reconstituted and CD47-deficient cells. We immobilized BSA, the 4N1K peptide (50 µM), VCAM-1 (1 µg/ml), or the association of 4N1K peptide and VCAM-1 on plastic. CD47-reconstituted cells (wt-CD47POS cells, open histograms) and CD47-deficient cells (CD47NEG, full histograms) were labeled with the different fluorescent dyes, mixed, and perfused in the flow chamber at a flow rate of 2 dyn/cm2. The number of firmly arrested cells was quantified as above.


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  		Table 1. Effects of immobilized 4N1K peptide on the arrest of purified CD4- or PHA-activated T cellsa

Next, peptide was used in a soluble form at a concentration of 50 µM, which has been widely used in the literature (29 , 43 , 70 , 81) . Both cell lines labeled with the different dyes were mixed and incubated either with the peptide 4N1K or kept in medium alone for 30 min at 37°C before being used in flow experiments. Figure 10 shows that the 4N1K peptide reduces the ability of wt-CD47POS cells to arrest down to almost the level of CD47NEG cells. By contrast, the arrest of CD47-deficient cells was not significantly decreased in the same conditions. On human peripheral blood T cells, arrest of T cells was also decreased in the presence of 4N1K peptide (not shown).



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  		Figure 10. Effects of soluble 4N1K peptide on arrest of CD47-reconstituted cells and CD47-deficient cells on inflammatory endothelium. Cells were labeled with the fluorescent dyes, mixed, and incubated in medium only or with the 4N1K peptide (50 µM) for 30 min before perfusion in the flow chamber at a flow rate of 2 dyn/cm2. The number of firmly arrested cells was quantified as above.

By this time we had collected evidence for a role of TSP in CD47-mediated arrest. Therefore, we tried to block T cell arrest with Abs directed against the binding site of TSP-1 (C6.7 mAb) (44) on CD47. We did not observe under the conditions tested any decrease in T cell arrest, even when we used high concentration of mAbs (data not shown). By contrast, preincubation of endothelium with both anti-TSP and anti-SIRP mAbs decreased the arrest of wt-CD47POS cells as compared to CD47NEG cells (Fig. 11 ). These results suggest that both ligands are involved in this process.



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  		Figure 11. Effects of mAbs anti-TSP and SIRP on arrest of CD47-reconstituted cells and CD47-deficient cells on inflammatory endothelium. Cells were labeled with the fluorescent dyes, mixed, and incubated in medium only or with mAbs against TSP and SIRP for 15 min before perfusion in the flow chamber at a flow rate of 2 dyn/cm2. The number of firmly arrested cells was quantified as above.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we show that CD47 is involved in the constitutive arrest of T cells on inflammatory endothelium under physiological shear stress. The CD47-deficient, Jurkat-derived cell line we prepared exhibits a decreased ability to stop on activated endothelium as compared to the original cell line, whereas reexpression of wild CD47 reestablished their ability to firmly stop. CD47 appears to influence the number of Jurkat T cells arrested on endothelium by transducing an activating signal within T cells rather than by direct adhesive interactions since 1) arrest of cells on inflammatory endothelium is inhibited by blocking Abs against VCAM-1 or {alpha}4ß 1, and 2) cells transfected with a chimera that does not express the multispan domain of CD47 are not able to recover the arrest. These results raise a question about the role of CD47 in the multistep model of lymphocyte emigration.

T cells that adhere to and migrate through endothelium of inflamed areas are mainly of memory/effector phenotype (45 46 47) , characterized by an increased expression of adhesion molecules as compared to naive T cells (48) . Jurkat T cells carry large amount of {alpha}4ß1 integrins and can be compared to the memory subpopulation of mature T cells (49) . Note that whereas T cells expressed high levels of CD47, no difference in CD47 expression could be found between resting vs. activated and naive vs. memory T cells (data not shown). In our study, cell arrest on inflammatory endothelium is strongly inhibited by blocking Abs against VCAM-1 or {alpha}4ß1 whereas on unactivated endothelium, a circumstance where VCAM-1 is not expressed (50) , no arrest of Jurkat T cells could be observed. Blocking ICAM-1 alone does not induce a significant decrease in adhesion, which suggests that ß 2 integrins play a minor role in arrest of Jurkat T cells (not shown).

Integrins present multiple activation states (51 , 52) . Oscillation between inactive, partially, and fully active states is critical for biological effects (53 54 55) . On peripheral T cells, for instance, VLA-4 is in a partially active state allowing a spontaneous and constitutive arrest on VCAM-1 (18 , 19) . Jurkat cells also display {alpha}4ß1 integrins in various states of activation (49) . Without further activation signal, a small subset of integrins is in a high-affinity/avidity state, accounting for the basal level of cell arrest on immobilized VCAM-1. Enhancing the general avidity/affinity of {alpha}4 integrins with external stimuli (like PMA or Mn2+) increases the firm adhesion of cells on immobilized VCAM-1, but not the number of tethering cells or the rolling adhesion, which are mediated by {alpha}4 integrins in a lower affinity state (49) . Our results are fully consistent with these previous observations. Whereas CD47 expression strongly influences the number of firmly arrested cells on endothelium, there is still residual VLA-4-dependent arrest of CD47-deficient Jurkat cells on activated endothelium. Furthermore, we observed no difference in the number of tethering cells or in the speed of rolling between CD47-expressing and CD47-deficient Jurkat cells (data not shown). These data suggest that CD47 is involved in the regulation of the high-affinity/avidity state of {alpha}4ß1 integrins. This cross-talk between CD47 and VLA-4 is consistent with the previous work of Imhof et al. who showed that CD47 regulates a cross-talk between the vitronectin receptor and {alpha}4ß1 integrin in a model of T cell locomotion on VCAM-1 (56) . Moreover, gene-targeted knockout mice deficient in CD47 expression suffer from a defect in PMN accumulation at the site of infection that is linked to an impairment in PMN migration (57) . Whether impairment in neutrophil arrest on inflammatory endothelium, in addition to specific defect of migration, could also account for the reduced number of neutrophils to the site of infection in these mice remains to be tested.

The next question was whether endothelial cells expressed counter receptor(s) for CD47. Two ligands of CD47—TSP-1 (28 , 29) and the P84 neural adhesion molecule (also known as SHPS-1, BIT, and SIRP)—(40) have so far been described. P84 is a phosphatase binding protein (58) that has been described on the membrane of myeloid cells and neurons (59) . Our data show that it is also expressed by endothelial cells. TSP-1 is a multifunctional matrix glycoprotein with major roles in tumor growth, angiogenesis, and metastasis (42 , 60 , 61) that is synthesized and secreted by several cell types, including endothelial cells (62 , 63) . Thrombospondin secreted by endothelial cells is organized with heparan sulfate proteoglycans in spherical granules at the cell surface (64 65 66) and can mediate interactions with blood cells under flow conditions (67 68 69) . Recent papers highlight the importance of CD47 interactions with TSP or SIRP. For instance, a trimolecular complex CD47-TSP-CD36 might be involved in activation and infiltration of T cells into chronically inflamed tissues (70) . Moreover, SIRP-1 induces aggregation of a murine pro-B cell line through interactions with CD47 (71) , whereas interaction of SIRP with CD47 appears to be important for its synaptic localization in mouse retina (72) . Several lines of evidences suggest a role for TSP and SIRP in our model. A peptide derived from the carboxyl-terminal CBD of TSP-1, a region directly involved in interaction with CD47 (30 , 31) , markedly enhanced VLA-4-mediated arrest when it was coimmobilized with VCAM-1 on plastic. Moreover, the TSP peptide enhanced only the arrest of wt-CD47POS, but not CD47-deficient cells on VCAM-1. Conversely, preincubation of wt-CD47POS T cells with soluble peptide decreased their arrest on endothelium whereas CD47-deficient cells arrest was not significantly decreased. Since CD47 on T cells requires cross-linking in order to transduce an activating signal for mitogenesis (33 34 35) , these data are consistent with a requirement for CD47 aggregation or cross-linking to induce VLA-4 activation, a condition not fulfilled by soluble peptide. Although we were unable to modify the arrest of T cells by preincubation with mAb against TSP alone, no significant difference between CD47POS and CD47-deficient cells arrest was observed when we blocked both TSP-1 and SIRP-1{alpha}. These data suggest strongly that both ligands are involved in T cell arrest.

Next we assessed the role of the multispan domain of CD47 in the transmission of activating signals within Jurkat cells to integrin {alpha}4ß1. Transfection of a chimeric CD47 that possesses only the extracellular portion of CD47 does not allow cells to recover their ability to stop suggesting a critical role of the pentaspan domain of CD47. Although some important functions of CD47 are assigned to the extracellular domain only (73) , the membrane-spanning domain is required for the transduction of comitogenic signals in a murine T cell hybridoma (34) and in JIN cells (37) . To better assess the region of the pentaspan domain involved in signal transduction, we transfected different CD47 mutants with different portions of the multiply membrane-spanning domain deleted. A construct that possessed only the first transmembrane segment and a short intracytoplasmic loop of 12 AA was sufficient to allowed arrest of transfected cells. Since the multiply membrane-spanning domain binds cholesterol and directs CD47 in glycosphingolipid-enriched membrane domains (74) , where it associates with Gi protein (75) , one could suggest an important role of the first transmembrane domain and/or the first intracytoplasmic loop of CD47 in such association. Whether the different functions of CD47 described so far in different systems such as stroma-induced erythropoiesis (76) , memory formation in rats (77) , transendothelial and transepithelial migration of neutrophils (78 , 79) , sCD23-mediated secretion of inflammatory cytokines (80) , and platelet activation (30 , 81) depend on precise transmembrane segments of CD47 remains to be determined. That different functions of CD47 might be linked to different portions of the molecule would explain, at least in part, the diversity of functional effects mediated by CD47.

In conclusion, our results demonstrate that CD47 contributes to the arrest of T cells on inflammatory endothelium under shear flow. Moreover, CD47 on T cells is a comitogenic molecule (33 34 35) and interacts with the P84 neural adhesion molecule (40) , which is expressed by dendritic cells (59) . This may indicate that CD47 plays a critical role in the immune response.


   ACKNOWLEDGMENTS
 
We thank Dr. Jean-Philippe Breittmayer for his help in cell sorting and Drs. G. Bernard, C. Legrand and J. Hatton for helpful discussions and Dr. H.-J. Büring for anti-SIRP mAbs. This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale, the Association pour la Recherche contre le Cancer, the Program Hospitalier de Recherche Clinique, the FEGEFLUC, the Etablissement Français des Greffes, the Federation de la Recherche Mèdicale, and National Institutes of Health grant GM57573–01.

Received for publication September 13, 1999. Revision received July 12, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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