| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
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
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 INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
4ß1/VCAM-1
pathway, as shown by blocking antibodies. We detected on endothelium
the two CD47 counter receptors known to date: thrombospondin and
SIRP1. Blocking experiments show that both are involved. Overall,
CD47 participates in the constitutive arrest of T lymphocytes on
inflamed vascular endothelium by up-regulating 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 |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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
IIbß3 integrin in platelets (30)
and 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 |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(TNF-) 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 SIRP1 and SIRP
2 and SE7C2 directed against SIRP 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 BSREN (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 BSREN using the XhoI
and XbaI sites.
|
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
BSREN 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 |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-activated endothelial
cells requires the presence of CD47 and its transmembrane and/or
intracellular domains.
, 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
).
|
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--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
).
|
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--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
).
|
|
CD47-dependent arrest is blocked by anti-VCAM-1 or anti- 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- 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.
|
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
).
|
Influence of TSP and SIRP 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-
activated endothelium. As shown in Fig. 8
, both are well expressed on endothelium. Then we tested whether they
could be involved in this process.
|
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).
|
|
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).
|
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.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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 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
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 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 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 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 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 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. 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
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 |
|---|
Received for publication September 13, 1999.
Revision received July 12, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
v/ß 3 differentially regulates adhesive and phagocytic functions of the fibronectin receptor vß 1. J. Cell Biol. 127,1129-1137 4ß 1 and 5ß 1-mediated function. J. Cell Biol. 138,1437-1447 4 integrin-dependent adhesion under static and flow conditions are differentially regulated. J. Immunol. 157,5061-5069[Abstract]
vß3-dependent ligand binding. J. Cell Biol. 123,485-496IIbß 3. J. Biol. Chem. 272,14740-14746vß3 function through integrin-associated protein. J. Cell Biol. 135,533-544This article has been cited by other articles:
|
|
 |
|
  J. S. Isenberg, D. S. Annis, M. L. Pendrak, M. Ptaszynska, W. A. Frazier, D. F. Mosher, and D. D. Roberts Differential Interactions of Thrombospondin-1, -2, and -4 with CD47 and Effects on cGMP Signaling and Ischemic Injury Responses J. Biol. Chem., January 9, 2009; 284(2): 1116 - 1125. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Stefanidakis, G. Newton, W. Y. Lee, C. A. Parkos, and F. W. Luscinskas Endothelial CD47 interaction with SIRP{gamma} is required for human T-cell transendothelial migration under shear flow conditions in vitro Blood, August 15, 2008; 112(4): 1280 - 1289. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Bouguermouh, V. Q. Van, J. Martel, P. Gautier, M. Rubio, and M. Sarfati CD47 Expression on T Cell Is a Self-Control Negative Regulator of Type 1 Immune Response J. Immunol., June 15, 2008; 180(12): 8073 - 8082. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Isenberg, D. D. Roberts, and W. A. Frazier CD47: A New Target in Cardiovascular Therapy Arterioscler. Thromb. Vasc. Biol., April 1, 2008; 28(4): 615 - 621. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Rao, L. Yang, G. Garcia-Cardena, and F. W. Luscinskas Endothelial-Dependent Mechanisms of Leukocyte Recruitment to the Vascular Wall Circ. Res., August 3, 2007; 101(3): 234 - 247. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. de Leval, D. S. Rickman, C. Thielen, A. d. Reynies, Y.-L. Huang, G. Delsol, L. Lamant, K. Leroy, J. Briere, T. Molina, et al. The gene expression profile of nodal peripheral T-cell lymphoma demonstrates a molecular link between angioimmunoblastic T-cell lymphoma (AITL) and follicular helper T (TFH) cells Blood, June 1, 2007; 109(11): 4952 - 4963. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Li, Z. Liu, M. Uzunel, and K.-G. Sundqvist Endogenous thrombospondin-1 is a cell-surface ligand for regulation of integrin-dependent T-lymphocyte adhesion Blood, November 1, 2006; 108(9): 3112 - 3120. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. P. Manna, J. Dimitry, P.-A. Oldenborg, and W. A. Frazier CD47 Augments Fas/CD95-mediated Apoptosis J. Biol. Chem., August 19, 2005; 280(33): 29637 - 29644. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Richter, R. Bistrian, S. Escher, W.-G. Forssmann, J. Vakili, R. Henschler, N. Spodsberg, A. Frimpong-Boateng, and U. Forssmann Quantum Proteolytic Activation of Chemokine CCL15 by Neutrophil Granulocytes Modulates Mononuclear Cell Adhesiveness J. Immunol., August 1, 2005; 175(3): 1599 - 1608. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Rosenthal-Allieri, M. Ticchioni, J. P. Breittmayer, Y. Shimizu, and A. Bernard Influence of {beta}1 Integrin Intracytoplasmic Domains in the Regulation of VLA-4-Mediated Adhesion of Human T Cells to VCAM-1 under Flow Conditions J. Immunol., July 15, 2005; 175(2): 1214 - 1223. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. V. Kolesnikova, C. S. Stipp, R. M. Rao, W. S. Lane, F. W. Luscinskas, and M. E. Hemler EWI-2 modulates lymphocyte integrin {alpha}4{beta}1 functions Blood, April 15, 2004; 103(8): 3013 - 3019. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. T. Chen, E. J. Brown, E. J. Huang, and W. E. Seaman Expression and Activation of Signal Regulatory Protein {alpha} on Astrocytomas Cancer Res., January 1, 2004; 64(1): 117 - 127. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. W. Feigelson, V. Grabovsky, R. Shamri, S. Levy, and R. Alon The CD81 Tetraspanin Facilitates Instantaneous Leukocyte VLA-4 Adhesion Strengthening to Vascular Cell Adhesion Molecule 1 (VCAM-1) under Shear Flow J. Biol. Chem., December 19, 2003; 278(51): 51203 - 51212. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. N. Vallejo, H. Yang, P. A. Klimiuk, C. M. Weyand, and J. J. Goronzy Synoviocyte-Mediated Expansion of Inflammatory T Cells in Rheumatoid Synovitis Is Dependent on CD47-Thrombospondin 1 Interaction J. Immunol., August 15, 2003; 171(4): 1732 - 1740. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Lamy, M. Ticchioni, A. K. Rouquette-Jazdanian, M. Samson, M. Deckert, A. H. Greenberg, and A. Bernard CD47 and the 19 kDa Interacting Protein-3 (BNIP3) in T Cell Apoptosis J. Biol. Chem., June 20, 2003; 278(26): 23915 - 23921. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Lagadec, O. Dejoux, M. Ticchioni, F. Cottrez, M. Johansen, E. J. Brown, and A. Bernard Involvement of a CD47-dependent pathway in platelet adhesion on inflamed vascular endothelium under flow Blood, June 15, 2003; 101(12): 4836 - 4843. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. O. Barazi, Z. Li, J. A. Cashel, H. C. Krutzsch, D. S. Annis, D. F. Mosher, and D. D. Roberts Regulation of Integrin Function by CD47 Ligands. DIFFERENTIAL EFFECTS ON alpha vbeta 3 AND alpha 4beta 1 INTEGRIN-MEDIATED ADHESION J. Biol. Chem., November 1, 2002; 277(45): 42859 - 42866. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Yamao, T. Noguchi, O. Takeuchi, U. Nishiyama, H. Morita, T. Hagiwara, H. Akahori, T. Kato, K. Inagaki, H. Okazawa, et al. Negative Regulation of Platelet Clearance and of the Macrophage Phagocytic Response by the Transmembrane Glycoprotein SHPS-1 J. Biol. Chem., October 11, 2002; 277(42): 39833 - 39839. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. E. de Vries, J. J. A. Hendriks, H. Honing, C. R. de Lavalette, S. M. A. van der Pol, E. Hooijberg, C. D. Dijkstra, and T. K. van den Berg Signal-Regulatory Protein {alpha}-CD47 Interactions Are Required for the Transmigration of Monocytes Across Cerebral Endothelium J. Immunol., June 1, 2002; 168(11): 5832 - 5839. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ticchioni, C. Charvet, N. Noraz, L. Lamy, M. Steinberg, A. Bernard, and M. Deckert Signaling through ZAP-70 is required for CXCL12-mediated T-cell transendothelial migration Blood, May 1, 2002; 99(9): 3111 - 3118. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Li, M. J. Calzada, J. M. Sipes, J. A. Cashel, H. C. Krutzsch, D. S. Annis, D. F. Mosher, and D. D. Roberts Interactions of thrombospondins with {alpha}4{beta}1 integrin and CD47 differentially modulate T cell behavior J. Cell Biol., April 29, 2002; 157(3): 509 - 519. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Yoshida, Y. Tomiyama, K. Oritani, Y. Murayama, J. Ishikawa, H. Kato, J.-i. Miyagawa, N. Honma, T. Nishiura, and Y. Matsuzawa Interaction Between Src Homology 2 Domain Bearing Protein Tyrosine Phosphatase Substrate-1 and CD47 Mediates the Adhesion of Human B Lymphocytes to Nonactivated Endothelial Cells J. Immunol., April 1, 2002; 168(7): 3213 - 3220. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Li, M. J. Calzada, J. M. Sipes, J. A. Cashel, H. C. Krutzsch, D. S. Annis, D. F. Mosher, and D. D. Roberts Interactions of thrombospondins with {alpha}4{beta}1 integrin and CD47 differentially modulate T cell behavior J. Cell Biol., April 29, 2002; 157(3): 509 - 519. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
