From the Center for Host/Pathogen Interactions,
University of California, San Francisco, California 94143 and the
Laboratoire Central d'Immunologie, Centre Hospitalier
Universitaire de Nice, Nice, 06202 France
Received for publication, September 28, 2000, and in revised form, December 6, 2000
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ABSTRACT |
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CD47 is a ubiquitously expressed membrane protein
with an extracellular Ig domain and a multiple
membrane-spanning domain that can synergize with antigen to
induce interleukin (IL)-2 secretion by T lymphocytes. Ligation of CD47
induced actin polymerization and increased protein kinase C CD47 (also known as integrin-associated protein) is a
50-kDa plasma membrane protein that was purified originally by
coimmunoprecipitation with Cytoskeletal components play critical roles in modulating T cell-APC
contact, early events of TCR activation, and assembly of signaling
complexes important to subsequent phenotypic responses (reviewed in
Refs. 13-15). Rapid cortical actin polymerization and reorganization
occurs with TCR ligation, and disruption of actin cytoskeletal dynamics
prevents effective T cell activation (16, 17). Cytoskeletal
rearrangements are known to be important in costimulatory CD28 and
LFA-1 receptor movements during cell activation and formation of
supramolecular activation clusters or immunological synapses (13, 14,
18). Moreover, TCR-stimulated alterations in the function of Vav, Rac,
Cdc42, and WASP can influence cytoskeletal architecture suggesting
bidirectional "cross-talk" between the cytoskeleton and
TCR-initiated signaling cascades.
Recently, membrane rafts, specialized domains in the plasma membrane
enriched in cholesterol and sphingolipids have been implicated in T
cell activation. During activation, TCR transiently associates with
membrane rafts, which are enriched in critical signaling components
such as Src family kinases, LAT, and G proteins. CD28 and several
raft-associated costimulatory molecules enhance association of TCR with
rafts during coligation (19-21). Disruption of raft integrity by a
variety of methods inhibits early activation events following
immunoreceptor ligation (22-25), supporting a critical role for these
domains in signaling. Multiple mechanisms for modulation of cortical
actin appear linked to rafts (26, 27), raising the possibility that
raft-dependent effects on actin may be critical for T cell activation.
The recent recognition that ligation of CD47 stimulates T cell
spreading (28) and that the structural requirements for spreading are
identical to those for synergy with the TCR to produce IL-2 suggested
that these two events may reflect signaling from CD47 to the
cytoskeleton important for T cell activation. Now we have shown that
CD47 ligation on surfaces stimulates an increase in F-actin and has a
similar structure activity profile to cell spreading and to T cell
activation. Association of CD47 with membrane rafts shares this
structure-function profile, and CD47 enhancement of cell spreading and
actin polymerization require its localization to membrane rafts.
Moreover, CD47 ligation induces association of PKC Cell Culture--
Jurkat (E6 clone), J.RT3-T3.5 (TCR-deficient)
(29), and JinB8 (CD47-deficient) cells were maintained in culture in
RPMI 1640 supplemented with 10% fetal calf serum, 2 mM
L-glutamine, 0.1 mM nonessential amino acids,
50 µM 2-mercaptoethanol, and 0.1% gentamicin.
Transfected Jurkat or JinB8 cells (6, 12) were maintained in the same
medium in the presence of 1-2 mg/ml Geneticin (Life Technologies,
Inc.). cDNA constructs and transfections were described
previously (6). The transfectants used in these studies included normal
form 2 CD47 (1), the CD47 Ig domain fused to the CD7 transmembrane
domain (CD47-CD7), the CD47 Ig domain with a GPI addition signal
(CD47-GPI), a molecule in which the CD47 Ig domain was replaced with an
8-amino acid epitope tag, FLAG (30) (FLAG-MMS), or the murine CD8
extracellular Ig domain (CD8-MMS) and have been described previously
(6, 12). Expression levels of the transfected constructs in Jurkat or
JinB8 cells were equivalent (6, 12). Endogenous expression levels of CD3, CD4, LFA-1, or HLA class I in the Jurkat and JinB8 cells used have
been described previously (12).
mAbs and Reagents--
The following mAbs were used in this
study: 2E11, 2D3, B6H12 (IgG1, murine anti-huCD47; Refs. 3 and 4);
W6/32 (IgG1, murine anti-HLA; Ref. 31); IB4 (IgG1, murine anti-huCD18;
Ref. 32); 15E8 (IgG1, murine anti-huCD28; Caltag, Burlingame, CA); 3D9
(IgG1, murine anti-huCD35; Ref. 33); M2 (IgG1, mouse anti-FLAG epitope;
Sigma), YTS105.18 (IgG2a, rat anti-mCD8; Serotec, Raleigh, NC); OKT3
(IgG2a, murine anti-huCD3; American Type Culture Collection, Manassas,
VA); anti-PLC Preparation of Antibody-coated Coverslips or Tissue Culture
Surfaces--
Coverslips were coated as previously described (6).
Briefly, 25-mm microscope coverslips were placed into 6-well plates and
precoated overnight at 4 °C with 5 µg/ml goat anti-mouse IgG Fc-specific antibodies (Cappel, Durham, NC) in 20 mM sodium
bicarbonate buffer, pH 9.0. Additional protein binding sites were
blocked by treatment with 2% BSA in RPMI 1640 overnight at 4 °C.
After washing wells three times with phosphate-buffered saline, the individual stimulating Abs were added in 1 ml of volume each and incubated overnight at 4 °C. OKT3 supernatant was used at various dilutions of supernatant with high = 10% and low = 0.03-0.1%. All other Abs were used at 1 µg/ml.
Tissue culture wells were coated similarly with goat anti-mouse IgG-Fc
or goat anti-human IgG-Fc and blocked with 1% BSA in phosphate-buffered saline. Monoclonal Abs were incubated at 1 µg/ml
in 1% BSA in phosphate-buffered saline in wells precoated with
anti-mouse Fc. In some cases the final mAb was omitted as a control.
SIRP Quantitation of F-actin--
For quantitation of changes in
F-actin content, 1 × 105 cells in RPMI with 0.1% BSA
were added to 96-well plate wells coated with various antibodies for
the indicated times. Cells were then fixed by addition of an equal
volume of 7.4% formaldehyde at 0 °C. After 30 min at 4 °C,
supernatants were removed, and wells were refilled with 50 µl of
staining solution including of Alexa-594 phalloidin and 2 µg/ml
Hoechst 33258. After 20 min at 25 °C, wells were washed three times
with phosphate-buffered saline and read on a Molecular Devices Disruption of Membrane Rafts with
Methyl- Cytoskeletal Fractionation--
Fractions were prepared
essentially as described by Villalba et al. (36). Cells
(5 × 106/sample) were applied to stimulatory
substrates in 6-well plates for 15-60 min in RPMI with 0.1% BSA. At
each time point, cells were harvested and suspended in 1.15 ml of
ice-cold hypotonic buffer (42 mM KCl, 5 mM
MgCl2, 10 mM HEPES, pH 7.4, 20 µg/ml
aprotinin, 20 µg/ml leupeptin) for 45 min. Samples were kept at
0-4 °C throughout the isolation. Cells were then passed through a
30-gauge needle 10 times and centrifuged at 200 × g to remove nucleii and large debris. Resulting supernatants
were centrifuged at 15,000 × g for 20 min. The pellet
was solubilized in 1 ml of 1% Nonidet P-40, 10 mM Tris, pH
7.4, 145 mM NaCl buffer for 30 min and recentrifuged at
15,000 × g for 20 min. Final pellets were
considered detergent insoluble cytoskeleton and were solubilized
with Laemmli sample buffer. PKC Isolation of Membrane Rafts--
The location of cell surface
proteins in sucrose density gradients was evaluated by Western blotting
of gradient fractions or by using tracer 125I-labeled
antibodies as described (28). In tracer studies, cells were incubated
with 5 µg/ml (saturating concentration) 125I-mAb
(Iodobeads; Pierce) in growth medium for 30 min at 4 °C and washed.
Cells were lysed in 20 mM Tris-HCl, pH 8.2, 140 mM NaCl, 2 mM EDTA, 25 µg/ml aprotinin, 25 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride,
0.1% Brij58 on ice. Sucrose was added to a final concentration of 40%
using 60% sucrose in 20 mM Tris-HCl, pH 8.2, 140 mM NaCl, 2 mM EDTA, and this was layered over a
volume of 60% sucrose. 25 and 5% sucrose layers were added to form a
step gradient, and these gradients were centrifuged at 170,000 × g for 18 h at 4 °C. Fractions of 0.5 ml were
collected from the top of the gradient, as well as the pellet, for
Western blotting or assessment of radioactivity in a Immunoprecipitations and Immunoblot--
Jurkat cells
(0.5-1.5 × 107 cells/point) were incubated on
Ab-coated surfaces at 37 °C for 5-15 min, as indicated in the text. For analysis of PLC Production of IL-2--
Production of IL-2 by adherent cells was
assessed as described previously (6). Briefly, cells (1 × 105) were plated in wells coated with anti-CD47, anti-CD8,
anti-FLAG, SIRP Single Cell Fluorescence Calcium Measurements--
Jurkat or
JinB8 cells at 2 × 107cells/ml in RPMI complete
medium were incubated with 3 µM fura-2/acetoxymethyl
ester at 37 °C for 20 min. The cell suspension was diluted
10-fold with complete medium and kept at 37 °C for another 20 min.
After the incubation periods, cells were washed three times in ice-cold
calcium buffer (25 mM HEPES, 125 mM NaCl, 5 mM KCL, 1 mM Na2HPO4,
0.5 mM MgCl2, 1 mM
CaCl2, pH 7.4) and resuspended at 2.5 × 106 cells/ml in the same buffer and kept on ice until use.
For evaluation of ligation of CD47 on a substrate with or without
coligation of CD3, fura-2-loaded cells were washed and resuspended in
calcium buffer and allowed to adhere to Ab-coated coverslips mounted in a Leiden coverslip dish in a PDMI-2 microincubator (Medical Systems Corp., Greenvale, NY) at 37 °C while viewed through a Zeiss Axiovert microscope. Samples were illuminated with light of alternating excitation wavelengths of 340 and 380 nm using a FL-4000 imaging system
(Georgia Instruments, Roswell, GA), and the emission images were
collected with a Dage MTI CCD72 camera and intensifier connected to a
Matrox MVP image processing card in a personal computer and stored on a
Panasonic TQ3031F optical memory disc recorder. Recordings were made
from the point of addition of cells to the coverslip with 340- and
380-nm images recorded every 10 s. The 10-min time point was used
for routine analysis based on evaluation of the kinetics of the CD47
response (see Fig. 6).
Statistical Analysis--
All experiments were repeated at least
three times. Error bars in graphs depict the S.E. The statistical
significance of each set of results was evaluated by performing a
one-way analysis of variance followed by Dunnett or individual
t tests as appropriate. A p value of <0.05 was
considered significant.
Ligation of CD47 Stimulates an Increase in F-actin--
Previous
studies showed that both Ig and MMS domains were required for
CD47-stimulated cell spreading and for synergy with TCR in IL-2
synthesis (6, 12). This suggested that similar signaling pathways might
activate both functions. Because the cytoskeleton is a critical
determinant of cell shape and spreading as well as T cell activation,
we hypothesized that CD47 ligation might signal actin cytoskeleton
rearrangement. As shown in Fig. 1A, ligation of CD47 caused
total cell F-actin content to increase more than when CD18
(
To determine whether a membrane-associated CD47 Ig domain was
sufficient for actin polymerization, the CD47-deficient Jurkat line
JinB8 (12) was studied (Fig. 1B). Whereas ligation of wild type CD47 transfected into JinB8-induced actin polymerization, ligation
of mutants in which the MMS domain was replaced by the CD7
transmembrane domain (CD47-CD7) or by a signal for addition of a
glycanphosphoinositol anchor (CD47-GPI) did not (Fig. 1B). Thus, both the Ig and MMS domains of CD47 are required for induction of
net actin polymerization.
To determine whether F-actin increased when CD47 and CD3 were ligated
simultaneously, F-actin was assessed when either wt CD47 or FLAG-MMS
was coligated with CD3. Ligation of CD3 caused a
concentration-dependent increase in F-actin content (Fig.
1C). In the absence of CD3 ligation, there was increased
actin when CD47 was ligated compared with FLAG-MMS, as expected (Fig.
1A). The effects of CD47 and CD3 on F-actin were additive at
low anti-CD3 concentrations, suggesting that this could contribute to
CD47 synergy in T cell activation.
Ligation of CD47 Stimulates PKC SIRP Association of CD47 with Membrane Rafts Correlates with Its Ability
to Stimulate Cell Spreading and to Increase F-actin Content--
These
data demonstrated that ligation of CD47 induces both actin
polymerization and PKC FLAG-MMS and CD8-MMS Can Synergize with CD3 for PLC FLAG-MMS Costimulates [Ca2+]i
Flux--
To determine whether PLC We and others previously showed that CD47, a ubiquitously
expressed plasma membrane molecule, can cooperate with suboptimal engagement of the TCR to activate normal T cells and continuous T cell
lines (6, 8, 12, 40). Although the molecular mechanisms for this
synergy are not known, cross-linking CD47 can cause a
TCR-dependent increase in [Ca2+]i,
known to be a necessary component of signaling for activation of IL-2
gene transcription. Thus, prior to this work, the data pointed to
cooperation in Ca2+ signaling as a likely mechanism to
explain CD47 synergy with TCR. No TCR-independent effect of CD47
ligation had been demonstrated, raising the possibility that ligation
of CD47 simply increased the efficiency of antigen presentation to TCR
rather than generating an independent signal. In the current studies,
we have shown that both these postulates Our previous studies had demonstrated that both the Ig and MMS domains
of CD47 are necessary for synergy with TCR for activation of IL-2
synthesis (6, 12) and had shown similar requirements for CD47-induced
cell spreading (12). Now we have now shown that ligation of CD47
induces net actin polymerization and PKC The TCR-independent signals that we have demonstrated do not occur
outside rafts. However, the CD47 MMS domain can cooperate with TCR to
increase [Ca2+]i while apparently outside
membrane rafts. Although the minimal amount of FLAG-MMS detected in
rafts might be enough to effectively cooperate with CD3 to activate
PLC These discoveries have two implications. First, CD47-TCR cooperation
has at least two discrete effects on T cell signaling pathways.
Although synergy for Ca2+ elevation does not require CD47
cytoskeletal effects, JNK activation and IL-2 synthesis do. We suggest
that this actin- and raft-independent PLC CD47 synergy with TCR is distinct from CD28 costimulation in several
respects. First, CD28 costimulation does not require cell adhesion,
whereas CD47 effects on T cell activation do. Consistent with this,
CD47 but not CD28 induces T cell spreading in response to cell
adhesion, and ligation of CD47 but not CD28 increases F-actin. Although
recent reports indicate that in some T cells CD28 influences Rac
activity and consequent cytoskeletal rearrangement (45, 46), this was
not reflected by changes in F-actin in Jurkat cells. CD47 and TCR must
both be ligated on the adherent T cell surface for effective synergy,
which is another difference from CD28 costimulation. It is likely that
by inducing translocation of PKC Structure-function studies demonstrated a requirement for both the MMS
and the Ig domains of CD47 for effective TCR-independent signaling. The
requirement for the extracellular Ig domain in signal transduction is
quite unusual, because signaling is thought to be a property of the
cytoplasmic or intramembrane sequences of most receptors. The
explanation for this unusual requirement appears to be that the Ig
domain is required for appropriate localization of CD47 to membrane
rafts. Why this should be so is unclear, because most signals for raft
localization in transmembrane molecules lie within the transmembrane
domain or the juxtamembrane cytoplasmic tail. The requirement for the
Ig domain suggests that either it interacts with another membrane
molecule to stabilize CD47 raft association or the presence of the Ig
domain influences the conformation of the MMS domain for its
association with rafts. If lateral association of intact CD47 with
another membrane protein mediates raft association, this protein must
be ubiquitous, because CD47 localizes to rafts in ovarian carcinoma
(OV10), melanoma (C32), and neutrophils as well as Jurkat cells.
Although we cannot rule this out, we favor the hypothesis that the CD47
Ig domain directly influences the ability of the MMS domain to
associate with rafts. The reduced localization of CD47 to rafts in
JinB8 versus Jurkat cells may represent a reduced general
stability of raft domains from JinB8s under the detergent conditions
used, because even CD47-GPI shows some extra-raft distribution in these
cells. Reduced raft organization in JinB8 cells may also contribute to
the reduced CD3 responsiveness of this
line.3 The MMS domain
certainly is required for CD47 signaling, because its deletion
abolishes both raft-dependent and raft-independent functions of CD47. The primacy of this domain in CD47 signaling is
logical, because the C-terminal cytoplasmic tails of CD47 are quite
short and have no known signaling motifs (1). How the MMS domain
signals, either in or out of rafts, is entirely unknown. Initial
mutagenesis experiments have not revealed sequences within the
predicted transmembrane domains or intracytoplasmic loops that have
affected CD47 function in Jurkat cells.3 It is interesting
that in an assay of T cell adhesion to endothelium, CD47 lacking most
of its MMS domain still functioned
normally.4
A potentially significant role for CD47 in T cell response to antigen
presentation in vivo is suggested by the finding that SIRP Several adhesion molecules may synergize with TCR in a manner similar
to that of CD47. For example, in some T cells, LFA-1 also synergizes
with TCR in activation, and this requires cell adhesion to an APC or
experimental surface presenting both TCR ligand and ICAM-1 (9).
Reorganization of the cortical actin cytoskeleton is stimulated by
engagement of LFA-1, and this plays an important role in the spatial
organization of Ag receptors during T cell activation (18, 47). We
postulate that adhesion molecules in general may contribute to T cell
activation through similar rearrangement of the cytoskeleton and
signaling factor localization. Because many signaling molecules
interact directly or indirectly with cytoskeletal elements, the role of
adhesion molecules can be seen as concentrating the elements of
specific signaling cascades at the sites of TCR ligation.
In conclusion, we have shown that CD47 in Jurkat cells can generate
TCR-independent signals upon ligation with antibody or its known
intercellular ligand, SIRP (PKC
)
association with the cytoskeleton independent of antigen receptor
ligation, but ligation of mutant forms of the molecule missing either
the Ig domain or the multiple membrane-spanning domain did not.
Simultaneous ligation of CD47 and CD3 led to additive effects on
F-actin and synergistic effects on PKC
cytoskeletal association.
Disruption of membrane rafts by removal of cholesterol with
cyclodextrin blocked CD47-induced actin polymerization, and mutant
forms of CD47 that localized poorly to rafts failed to effect
cytoskeletal rearrangement. However, raft association alone was not
sufficient, because a raft-localized CD47 Ig domain bound to the
membrane by a glycan phosphoinositol anchor was unable to induce actin
polymerization. A mutant form of CD47 without its Ig domain that did
not induce actin polymerization or localize to rafts still enhanced T
cell receptor (TCR)-dependent tyrosine phosphorylation of
PLC
and associated Ca2+ signaling but did not
augment IL-2 secretion. Thus, CD47 synergy with TCR to increase
[Ca2+]i is independent of actin and rafts but is
insufficient to explain CD47 cooperation with TCR in IL-2 synthesis.
Full synergy with TCR requires CD47 localization to membrane rafts
where ligation leads to TCR-independent signals causing actin
polymerization and PKC
translocation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 integrins from placenta.
Molecular cloning of CD47 cDNA revealed that it is an unusual Ig
family member, with an Ig variable-like extracellular domain, a domain
with five putative membrane spanning segments (MMS
domain)1 and a short
cytoplasmic tail (1). Antibodies against CD47 modulate several
3 integrin functions, such as adhesion, chemotaxis, and
phagocytosis in neutrophils (2-4), and CD47-deficient animals have a
defect in host defense that results from lack of phagocyte activation
at the site of infection (5). In addition, CD47 can synergize with
antigen to activate thymocytes and T cells, a function that appears to
be integrin-independent (6, 7). Although the potential significance of
this synergy is suggested by the decreased number of circulating T
cells in CD47-deficient mice (5), its molecular mechanism is unknown.
Cross-linking CD47 on T cells can induce a TCR-dependent
rise in [Ca2+]i (8), and synergy between CD47 and
TCR ligation is found in early events in T cell activation, such as
chain phosphorylation and ZAP-70 activation. These data suggest the possibility that CD47 cooperation with TCR in these immediate consequences of TCR ligation accounts for its synergistic effect (6),
in contrast to costimulation by CD28, which cooperates at later steps
in the signaling cascade leading to IL-2 synthesis (9-11). These and
other differences between CD28 and CD47 (6, 12) suggest that these two
cell surface molecules may cooperate with TCR by quite distinct
pathways. Indeed, it has been proposed that the major effect of CD47
ligation may be simply to more efficiently present antigens to TCR.
with the
cytoskeleton, and CD47 synergizes with TCR for translocation of this
important component of signaling for T cell activation. In contrast,
CD47 participation in PLC
and Ca2+ signaling is not
dependent on its raft localization or its ability to induce actin
polymerization and cell spreading. Thus, the effects of CD47 on
tyrosine phosphorylation and Ca2+ are distinct from its
effects on PKC
and not sufficient to explain its synergy with TCR in
T cell activation. Our data lead to the conclusion that there are two
distinct effects of CD47 in T cell signaling. Raft association and
actin polymerization are not required for CD47 synergy in the early
activation events of TCR signaling such as increase in
[Ca2+]i but are required for synergy in induction
of PKC
translocation, JNK activation, and IL-2 synthesis. We propose that CD47 can interact with signaling cascades both in and out of
membrane rafts and that signaling within rafts is required for its
synergy with TCR in T cell activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (rabbit antiserum; Upstate Biotechnology, Inc., Lake
Placid, NY). Cytochalasin D was purchased from Sigma. Fura-2/acetoxymethyl ester, Hoechst 33258, and Alexa-488/594
phalloidins were obtained from Molecular Probes (Eugene, OR).
Methyl-
-cyclodextrin was from Aldrich. To construct SIRP
1-Fc,
cDNA encoding the extracellular three Ig domains of SIRP
1
(SHPS-1) was obtained as IMAGE clone 2017171. It was modified and
transferred by standard molecular techniques into pIAP412 (34), which
encodes the hinge and constant regions of the heavy chain of human IgG1
(35). SIRP
1-Fc fusion protein was produced by transient transfection
of COS cells using LipofectAMINE PLUS reagent (Life Technologies,
Inc.). Protein was harvested from culture supernatants by standard
methods using protein A-Sepharose and confirmed to exist as a dimer by
Western blot with anti-human IgG-Fc (molecular mass, 130 kDa).
1-Fc fusion protein or hIgG (as control) were incubated at 1 µg/ml in medium with 10% fetal bovine serum in wells precoated with
anti-human Fc.
Max
fluorescence microplate reader at dual ex/em wavelengths for Alexa-594
and Hoechst. Average cell F-actin content was determined by comparing
phalloidin and DNA staining in individual wells. Standard curves of
cell number versus Hoechst or Alexa-594 phalloidin staining
prepared from serial dilutions of cells added to
poly-L-lysine-coated plates showed linear, specific
staining over at least a 20-fold range from 5-100 × 103 cells/well. Results were expressed as phalloidin
fluorescence/Hoechst fluorescence. All measurements were performed in
quadruplicate wells.
-cyclodextrin--
Cells were suspended at 2.5 × 105/ml in RPMI with 0.1% fatty acid-free BSA and 0-10
mM methyl-
-cyclodextrin for 10 min at 37 °C and then
washed. For reconstitution of membrane cholesterol content, cells were
subsequently incubated with 1.33 mg/ml
cholesterol-methyl-
-cyclodextrin inclusion complexes in RPMI with
0.1% fatty acid-free BSA and then washed. Cells were then applied to
prepared 96-well plates for assessment of spreading activity and
F-actin content.
content was determined by Western
blot using goat anti-PKC
(Santa Cruz Biotechnology, Santa Cruz, CA).
Quantitation was performed on scanned blots using Adobe Photoshop.
counter.
1 phosphorylation, cells were lysed in 1% Nonidet P-40, 0.5% deoxycholate, 50 mM HEPES, pH
7.5, 150 mM NaCl, 20 mM NaF, 1 mM
EDTA, 10 µg/ml leupeptin and aprotinin, 10 mM
-glycerophosphate, 50 nM calyculin, and 250 µM sodium vanadate. Insoluble material was removed by
centrifugation at 13,000 × g for 5 min. PLC
1 was
immunoprecipitated overnight by incubation at 4 °C with rabbit
anti-PLC
1 Ab (Upstate Biotechnology, Inc.) and GammaBind
Plus-Sepharose (Amersham Pharmacia Biotech). Immunoprecipitates were
Western blotted with 4G10 antiphosphotyrosine mAb (Upstate Biotechnology, Inc.) as previously described (6). For the JNK assays,
samples were processed according to the kit manufacturer's instructions (New England Biolab, Beverly, MA). Briefly, lysate from
2 × 106 cells/sample was mixed with glutathione
S-transferase-c-Jun-bound glutathione-Sepharose beads, and a
kinase reaction was performed with the bound material. Products were
resolved by SDS-polyacrylamide gel electrophoresis on a 12.5% gel,
transferred to polyvinylidene difluoride, and probed with a
phospho-Ser63-specific rabbit anti-c-Jun polyclonal
antibody. Equal loading of samples was confirmed by Coomassie staining
prior to probing. The positive control for PLC
phosphorylation was
0.5 mM pervanadate and for JNK activation was 20 ng/ml
PMA/2 µM ionomycin for the last 15 min of treatment.
1-Fc, or control mAbs together with various
concentrations of anti-CD3 in RPMI medium. Anti-CD3 dilutions are
indicated as percentages of hybridoma supernatant. After 18-24 h, IL-2
in harvested supernatants was measured by enzyme-linked immunosorbent
assay using mAbs 5344.111 and B33-2 and recombinant human IL-2
(Pharmingen, San Diego, CA) to construct a standard curve or by
incorporation of [3H] thymidine (0.4 µCi/well; specific
activity, 6.7 Ci/mmol; ICN) by CTLL-2 cells as previously described
(6).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 integrin LFA-1) was engaged. Because Jurkat cells do
not spread on anti-CD18-coated surfaces (12), the F-actin content of
these cells was used as a nonactivated control. CD47-induced increase
in F-actin was less than that induced by optimal stimulation of CD3
(Fig. 1A). However, CD47 ligation increased F-actin content in both TCR-expressing and TCR-deficient Jurkat cells to the same extent (Fig. 1A) showing that, in contrast to CD47-induced
Ca2+ flux (8), this response is not
TCR-dependent. Ligation of the costimulatory molecule CD28,
which does not stimulate spreading on its own, failed to stimulate a
significant elevation of F-actin above the level of CD18-adherent
cells. Ligation of CD47 mutants in which the Ig domain had been
replaced either with the CD8 extracellular Ig domain (CD8-MMS) or the
FLAG epitope (FLAG-MMS) using anti-CD8 or anti-FLAG failed to increase
F-actin content (Fig. 1A), demonstrating that the Ig domain
was necessary for this effect of CD47 ligation.
View larger version (21K):
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Fig. 1.
Increase in F-actin by ligation of CD47.
A, Jurkat cells expressing wt CD47, FLAG-MMS, or CD8-MMS
were applied to surfaces coated with anti-CD18, anti-CD47, anti-FLAG,
anti-CD8, or anti-CD28 antibodies for 1 h at 37 °C and fixed,
and F-actin content was assessed as described under "Experimental
Procedures." TCR-deficient Jurkat cells were allowed to adhere to
anti-CD18- or anti-CD47-coated surfaces and assessed similarly. In all
cases, F-actin content was normalized to the number of adherent cells
by measuring DNA content; the F-actin in cells adherent to anti-CD18
was set at 100. Results indicate the means ± S.E. for five
experiments. The diagrams above FLAG-MMS and CD8-MMS depict the form of
the molecule ligated. Only CD47 and CD3 ligation resulted in increased
F-actin (p < 0.05). B, CD47-deficient
Jurkats transfected with wt CD47, CD47-CD7, or CD47-GPI were similarly
treated and F-actin content in cells adherent via CD47 and CD18
compared. Actin content in CD18-adherent cells was 100. Results
indicate the means ± S.E. for five experiments. Above each
bar is a diagram of the mechanism for the association of
each molecule with the plasma membrane. Only ligation of wt CD47 led to
increased F-actin (p < 0.01). C, Jurkat
cells expressing wt CD47 and FLAG-MMS were applied to surfaces cocoated
with anti-CD47 or anti-FLAG along with varying amounts of anti-CD3.
F-actin content was determined as previously described and is shown as
fluorescence ratios without standardization to CD18 levels. Results are
from a experiment (performed in triplicate) representative of four
experiments with similar results.
Association with the
Cytoskeleton--
Because PKC
association with the actin
cytoskeleton may be important for its function (36), we evaluated the
ability of CD47 to stimulate PKC
cytoskeletal association. Ligation
of CD47 stimulated PKC
cytoskeletal association by 15 min after
adhesion in the absence of CD3 ligation (Fig.
2A). In contrast, FLAG-MMS, CD8-MMS, and CD28 failed to stimulate PKC
translocation (Fig. 2A and data not shown). However, CD47-induced translocation
was transient, because cytoskeleton-associated PKC
returned to base line by 30-40 min after stimulation (Fig. 2B and data not
shown). These kinetics contrast with CD47-induced actin polymerization, which remained elevated for at least 1 h (Fig. 1 and data not shown). However, coligation of CD47 and subthreshold CD3 led to sustained PKC
translocation (Fig. 2B). This was not an
effect simply of CD3 signaling, because its ligation together with CD28 or FLAG-MMS showed no PKC
association with cytoskeleton above the
basal (BSA) level. The CD47 Ig domain was required for both its
transient and sustained effects on PKC
, as shown by the failure of
FLAG-MMS to induce PKC translocation on its own (Fig. 2A) or in synergy with CD3 (Fig. 2B).
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Fig. 2.
CD47 ligation induces association of
PKC with the cytoskeleton. A,
Jurkat cells expressing wt CD47 and FLAG-MMS were applied to surfaces
coated with anti-CD47, anti-FLAG, anti-CD28, and anti-CD3. After 15 min
at 37 °C, cytoskeletal fractions were prepared, and
cytoskeleton-associated PKC
was determined as described under
"Experimental Procedures." Cytoskeleton-associated PKC
in cells
incubated in wells without primary antibody is set to 1. Results
indicate the means ± S.E. for four experiments. Incubation with
an optimal amount of anti-CD3 (10%) led to a 9 ± 3.8-fold
increase in cytoskeleton-associated PKC
. Only CD47 ligation induced
translocation of PKC
to the cytoskeleton (p < 0.05)
B, the experiment was performed as in A except
that in some cases cells were incubated on surfaces cocoated with
substimulatory anti-CD3 (0.01%), and cytoskeleton-associated PKC
was determined after a 45 min incubation. BSA, wells without
primary mAb (density set to 1). hi CD3, 10% anti-CD3 alone.
Results are from a experiment representative of three experiments
performed with similar results.
1 Ligation of CD47 Synergizes with CD3 for IL-2 Synthesis
and Stimulates PKC
Cytoskeletal Association--
SIRP
1 is a
recently described cell surface ligand for CD47 that is highly
expressed on macrophages and dendritic cells but is not present on T
cells (37, 38). CD47 interaction with SIRP
1 therefore has the
potential to modulate antigen presentation to T cells. To determine
whether SIRP
1 could induce CD47 signaling, Jurkat cells were allowed
to adhere to surfaces coated with a SIRP
1-Fc fusion protein.
Compared with the normal human IgG control, SIRP
1-Fc synergized with
anti-CD3 for IL-2 synthesis, shifting the EC50 for CD3
ligation about 25-fold (Fig.
3A). SIRP
1 ligation of CD47
also stimulated an increase in PKC
association with cytoskeleton in
the absence of CD3 ligation (Fig. 3B). Thus, SIRP
1
ligation of CD47 induces TCR-independent PKC
translocation and
synergizes with TCR ligation for T cell activation, suggesting the
possibility of a physiologic role for CD47/SIRP
1 binding during T
cell/APC interactions.
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Fig. 3.
SIRP 1 ligation of
CD47 synergizes with CD3 for T cell activation and induces
PKC
translocation. A, Jurkat
cells were applied to substrates coated with varied concentrations of
anti-CD3 (OKT3, percentage of supernatant) in the presence of
SIRP
1-Fc or normal human IgG (hIgG) and incubated for
24 h. Supernatants were collected, and IL-2 content (pg/ml) was
determined by enzyme-linked immunosorbent assay. B, Jurkat
cells were applied to surfaces coated with SIRP
1-Fc fusion protein
or hIgG as control. After 15 min at 37 °C, cytoskeleton-associated
PKC
was quantitated. PKC
in cells adherent to hIgG was set to 1. Results are the mean ± S.E. from four experiments. SIRP
1-Fc
induced PKC
translocation (p < 0.01).
translocation to the cytoskeleton. These
signals from CD47 required the Ig domain as well as the MMS domain.
Because the ligation of CD8 or FLAG could not induce similar effects,
the data demonstrate a function for the Ig domain independent of ligand
binding. Thus, the Ig domain has a fundamental role in CD47 signaling
in addition to its role in ligand recognition. Because the Ig domain is
required for CD47 localization to membrane rafts in ovarian carcinoma
cells (28), we hypothesized that a similar role for the Ig domain in T
cells might account for its necessary contribution to CD47 signaling.
To test this hypothesis, we first determined whether CD47 is
concentrated in membrane rafts in Jurkat T cells. CD47 localized to
rafts in both normal Jurkat cells and JinB8 cells transfected with wild
type CD47 (Fig. 4). We also examined the
structural requirements for CD47 association with membrane rafts in
Jurkat cells. Although >65% of wild type plasma membrane CD47
associated with membrane raft fractions, CD47-CD7 failed to localize
significantly to rafts, and the FLAG-MMS and CD8-MMS mutants also were
markedly defective in raft localization (Fig. 4). Thus, both the MMS
and Ig domains of CD47 were necessary for its enrichment in plasma
membrane rafts in Jurkat cells, identical to the requirements for
spreading, actin polymerization, and TCR synergy. Furthermore,
methyl-
-cyclodextrin, which disrupts raft organization (39), blocked
CD47-induced cell spreading and the increase in F-actin in both
TCR
and TCR+ Jurkat cells, and these effects
were reversed by reconstitution of membrane cholesterol (data not
shown). These data suggested that only CD47 in rafts could generate the
signal for actin polymerization. To test whether raft localization was
sufficient for CD47-induced actin polymerization, CD47-GPI was studied.
This mutant localized even better to rafts than wild type CD47 (Fig.
4). However, the failure of CD47-GPI to increase F-actin (Fig.
1B) demonstrates that raft localization of the CD47 Ig
domain is not a sufficient signal for actin polymerization. Altogether
these data are consistent with the hypothesis that CD47-induced actin
polymerization and PKC
translocation are necessary for its effects
on T cell activation and that CD47 localization to rafts is necessary
but insufficient for these consequences of CD47
ligation.
View larger version (20K):
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Fig. 4.
Localization of CD47 to membrane rafts.
Normal Jurkat cells transfected with FLAG-MMS or CD8-MMS or
CD47-deficient JinB8 transfected with wt CD47, CD47-CD7, or CD47-GPI
were incubated with 125I-labeled anti-CD47, anti-FLAG, or
anti-CD8. After cell lysis and equilibrium sucrose density gradient
fractionation, distribution of labeled protein was determined. The
lightest gradient fractions are at the right (lanes
8-10) of each graph. Lane P, pellet. Distribution of
proteins was confirmed by Western blotting of sucrose gradient
fractions from unlabeled cells with appropriate antibodies (data not
shown). Results are representative of three or more experiments.
Phosphorylation but Not JNK Activation--
CD47 has been shown to
synergize with TCR for early steps in T cell activation and to
stimulate a TCR-dependent Ca2+ flux (7, 8). To
determine whether this synergy also required membrane raft localization
of CD47, we assayed PLC
activation by wt CD47 and the Ig and MMS
mutants that failed to associate with membrane rafts, induce actin
changes, or costimulate T cell activation (6, 12). Surprisingly,
FLAG-MMS and CD8-MMS synergized to activate PLC
1 as well as wt CD47
(Fig. 5A and data not shown). In contrast, neither FLAG-MMS or CD8-MMS could synergize with CD3 to
activate JNK (Fig. 5A and data not shown), a required step in synergistic signaling for IL-2 synthesis. Wild type CD47 and CD28
costimulate JNK activation similarly (12). Neither the CD47-CD7 nor the
CD47-GPI mutants could synergize with CD3 either for PLC
phosphorylation (Fig. 5B) or JNK activation (data not shown).
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Fig. 5.
Synergistic JNK activation by CD47 requires
the Ig domain of CD47, but PLC phosphorylation
does not. A, cells were incubated with anti-CD3 (0.1%)
coimmobilized with 1 µg/ml anti-CD47, anti-FLAG, or anti-CD18 control
mAbs on plates for 10 or 30 min at 37 °C. JNK activity was
determined after 30 min of incubation and PLC
1 phosphorylation after
10 min of incubation. FLAG-MMS does not mediate JNK activation but does
induce PLC
1 phosphorylation. Results are representative of three
experiments. GST, glutathione S-transferase. B,
the transmembrane domain of CD47 is required for synergistic PLC
1
phosphorylation. CD47-deficient JinB8 cells transfected with CD47,
CD47-CD7, or CD47-GPI (5 × 106 cells/sample) were
stimulated with anti-CD3 (0.1%) coimmobilized with 1 µg/ml anti-CD47
or anti-CD18 control mAbs on plates for 10 min at 37 °C. PLC
1
phosphorylation was assessed as in A and quantitated by
densitometry. PLC
1 phosphorylation in cells in which CD3 and CD18
were simultaneously ligated was set to 1. Results are the means ± S.E. from three experiments. Only intact CD47 shows synergy with CD3 in
PLC
1 phosphorylation (p < 0.05).
phosphorylation by the FLAG-MMS
mutant was capable of continuing the signaling cascade, single cell [Ca2+]i recordings were made on cells plated onto
surfaces coated with substimulatory anti-CD3 together with anti-CD47,
anti-FLAG, or control mAbs. In cells expressing wt CD47, this led to a
slow but sustained rise in [Ca2+]i that lasted
>40 min (Fig. 6 and data not shown).
There was no increase in [Ca2+]i when anti-CD18
(Fig. 6, A and B) or anti-CD35 (isotype matched
nonbinding control; Fig. 6B) were substituted for anti-CD47. Ligation of CD47 alone did not induce a Ca2+ flux,
demonstrating that synergy with TCR signaling was required (Fig.
6B). Consistent with its synergy in PLC
phosphorylation, ligation of FLAG-MMS costimulated Ca2+ flux (Fig.
6C) that was sustained for >40 min.
FLAG-MMS-dependent [Ca2+]i flux
occurred in JinB8 as well as Jurkat cells (data not shown), ruling out
interaction between FLAG-MMS and endogenous CD47 as a cause of the
Ca2+ signaling. Thus, failure of FLAG-MMS to synergize for
JNK activation and IL-2 synthesis is not due to the inability of
activated PLC
to continue the signaling cascade. The ability of
FLAG-MMS to costimulate Ca2+ demonstrates that neither
efficient raft localization nor actin polymerization is required for
CD47 synergy in PLC
activation. Consistent with its inability to
induce PLC
phosphorylation, CD47-CD7 did not synergize with anti-CD3
to stimulate a [Ca2+]i rise (Fig.
6D).
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Fig. 6.
The MMS domain is necessary and sufficient
for synergy with CD3 to increase [Ca2+]i.
Fura-2/acetoxymethyl ester-loaded Jurkat cells (A and
B), Jurkat cells transfected with FLAG-MMS (C),
and JinB8 cells transfected with CD47 or CD47-CD7 (D) were
allowed to settle onto coverslips coated with high concentration
anti-CD3; low concentration anti-CD3 alone or in combination with
anti-CD47, anti-FLAG, anti-CD18, or anti-CD35; anti-CD47 alone; or
anti-FLAG alone. A, kinetics of
[Ca2+]i responses. 340/380 ratios were measured
in individual cells at 10-s intervals over 10 min. Simultaneous
ligation of CD47 with a substimulatory CD3 signal led to a slow but
sustained rise in [Ca2+]i. B,
[Ca2+]i in Jurkat cells adherent to a variety of
surfaces. In B-D cells were assayed at 10 min. Although two
different anti-CD47 antibodies synergized with low anti-CD3 (0.01%) to
stimulate an increase in [Ca2+]i
(p < 0.01), anti-CD18 and anti-CD35 did not. Neither
anti-CD3 nor anti-CD47 induced a rise in [Ca2+]i
on its own, and the synergistic rise required extracellular
Ca2+. The +EDTA sample represents cells applied
to low CD3 + CD47 substrates in the presence of 5 mM EDTA.
C, the CD47 MMS domain is sufficient to synergize with CD3
to increase [Ca2+]i (p < 0.05),
because ligation of FLAG-MMS was as effective as ligation of wt CD47.
Anti-FLAG did not induce a [Ca2+]i rise in the
absence of CD3 ligation. D, the MMS domain is required for
synergy with CD3. [Ca2+]i was increased by
coligation of CD47 and CD3 in JinB8 transfected with CD47
(+CD47) (p < 0.01) but not JinB8
transfected with CD47-CD7 (+CD47-CD7). Expression of the wt
CD47 and CD47-CD7 constructs was equivalent. The mean ± S.E. of
12 representative cells was calculated for each sample. Data are from
experiments representative of at least three experiments for each
condition with similar results. lo, low concentration;
hi, high concentration.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
that CD47 cooperates simply
in Ca2+ signaling and that its role is only to increase the
efficiency of antigen presentation
are incorrect.
translocation to the
cytoskeleton independent of any TCR signals. This signaling by CD47
appears to require its targeting to plasma membrane rafts. These data
suggest the hypothesis that only within these membrane rafts can CD47
engage a signaling cascade leading to actin polymerization and PKC
translocation. Because CD47 synergy for IL-2 synthesis has an identical
structure-function profile as association with membrane rafts, actin
polymerization, and PKC
translocation, it is tempting to speculate
that these events are related. Certainly, PKC
plays an important
role in activation of IL-2 synthesis in T cells (36, 41); both membrane
rafts and actin polymerization have been implicated in this process as
well (16, 42, 43). CD47 is known to cooperate with integrins in
membrane rafts to activate heterotrimeric G proteins (28). However, we
do not believe this is the mechanism by which the relevant signaling
cascades are activated in T cells because (a) CD47
cooperation with TCR is independent of all integrins with which it is
known to interact (6, 7) and (b) pertussis toxin does not
inhibit synergy with TCR nor CD47-induced cell
spreading.2 Whereas some
heterotrimeric G proteins are pertussis toxin-independent, G protein
signaling in Jurkat cells is potently inhibited by this toxin (44).
Hence, we suggest that the most proximate steps in CD47 signaling in T
cell membrane rafts do not involve heterotrimeric G proteins and remain
to be discovered.
, it is clearly insufficient to activate actin polymerization or
PKC
translocation or to synergize in JNK activation or IL-2
synthesis. Thus, we believe it most likely that the MMS domain outside
rafts can engage signaling cascades important in PLC
activation, and
only the MMS domain inside rafts can induce the additional actin and
PKC
signals required in T cell activation.
activation results from
the previously reported synergy between CD47 and TCR for
chain
phosphorylation and zap-70 activation (6). The second implication of
this work is that CD47 synergy in the early events of T cell activation
are not sufficient to account for its effects on IL-2 synthesis. Thus,
it is unlikely that the role of CD47 is simply to increase the
efficiency of antigen presentation. Instead, our data support the
hypothesis that CD47 cooperation with TCR for IL-2 synthesis results
from its independent cytoskeletal signaling within raft domains. It is
likely that the CD47-related F-actin and PKC
are both closely associated with the raft domains where CD47 localizes. Because TCR
signaling occurs most effectively within membrane rafts (22), this
could promote a molecular mechanism for CD47/TCR synergy.
, CD47 increases the concentration
of this critical enzyme in the vicinity of ligated TCR. This could
enhance TCR activation of PKC, and downstream events, like JNK
activation, could proceed more efficiently. In Jurkat cells, at least,
CD28 does not increase cytoskeletal association of PKC
, implying
that its synergy of IL-2 secretion proceeds by an entirely distinct mechanism.
1, a CD47 ligand highly expressed on dendritic cells (38), can
activate CD47 signaling. It is likely that SIRP ligation of CD47 can
increase the sensitivity of T cells to limiting doses of antigen.
Although the B7 ligands for CD28 are not constitutively expressed on
APC but require some cell activation to achieve a significant level of
expression, SIRP expression appears constitutive. Thus, SIRP-CD47
interaction is likely to have its greatest effect on T cell activation
during initial antigen presentation prior to generation of an
inflammatory response that would activate B7 expression. Perhaps this
is the reason that CD47-deficient mice have approximately half the
number of peripheral T cells as their wild type littermates (5).
1. Most prominently, CD47 induces the
translocation of PKC
to the cytoskeleton, an effect that in turn
requires an increase in actin polymerization. This signaling, but not
synergy with TCR for Ca2+ flux, requires CD47 localization
to membrane rafts, presumably for this complex molecule to engage the
appropriate signaling cascades. CD47 and TCR cooperate to sustain the
cytoskeletal association of PKC
, perhaps because TCR signaling
generates the lipid mediators necessary to activate the enzyme, leading
to more stable association with the plasma membrane and the associated
cortical actin cytoskeleton. This synergy likely is responsible for the
cooperation between CD47 and TCR in JNK activation and IL-2
transcription. Although much has been written about how signal
transduction cascades affect the cytoskeleton, this may be an example
of how modulation of the cytoskeleton can affect signal transduction.
This could be general mechanism by which effects on actin
polymerization can modulate signaling cues from many receptors and may
be the mechanism by which many adhesion receptors modulate inputs from
the TCR.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health grants (to E. J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Protein Design Labs, Fremont, CA 94555.
¶ Present address: Dept. of Pathology, University of Texas Health Sciences Center, San Antonio, TX 78229.
** To whom correspondence should be addressed: Program in Host-Pathogen Interactions, University of California, San Francisco, Box 0654, 513 Parnassus Ave., San Francisco, CA 94143. Tel.: 415-514-0167; Fax: 415-514-0169; E-mail: ebrown@medicine.ucsf.edu.
Published, JBC Papers in Press, December 12, 2000, DOI 10.1074/jbc.M008858200
2 R. A. Rebres, M. I. Reinhold, and E. J. Brown, unpublished data.
3 R. A. Rebres and E. J. Brown, unpublished observations.
4 M. Ticchioni, V. Raimondi, L. Lamy, J. Wijdenes, F. P. Lindberg, E. J. Brown, and A. Bernard. (2001) FASEB J., in press.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MMS, multiple
membrane-spanning;
TCR, T cell receptor;
PLC, phospholipase C
;
JNK, Jun N-terminal kinase;
IL, interleukin;
PKC
, protein kinase
C
;
GPI, glycan phosphoinositol;
Ab, antibody;
mAb, monoclonal
antibody;
BSA, bovine serum albumin;
wt, wild type.
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