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Prepublished online as a Blood First Edition Paper on June 14, 2002; DOI 10.1182/blood-2001-12-0217.
IMMUNOBIOLOGY
From the Centre de Recherche du Centre Hospitalier
Université Montreal, Laboratoire d' Immunorégulation,
Université de Montréal, Canada; Program in Microbial
Pathogenesis and Host Defense, University of California, San Francisco;
Institut Pasteur, Paris, France; and Institut National Sante et
Recherches Médicales U429, Hôpital Necker, Paris,
France.
Dying cells, apoptotic or necrotic, are swiftly eliminated by
professional phagocytes. We previously reported that CD47 engagement by
CD47 mAb or thrombospondin induced caspase-independent cell death of
chronic lymphocytic leukemic B cells (B-CLL). Here we show that human
immature dendritic cells (iDCs) phagocytosed the CD47 mAb-killed
leukemic cells in the absence of caspases 3, 7, 8, and 9 activation in
the malignant lymphocytes. Yet the dead cells displayed the cytoplasmic
features of apoptosis, including cell shrinkage, phosphatidylserine
exposure, and decreased mitochondrial transmembrane potential
( Apoptosis is a noninflammatory destruction
process essential to the regulation of immune system and
homeostasis.1,2 It provides molecular basis for T- and
B-cell development,3 induction of immune tolerance, and
termination of normal immune response.4 By contrast,
necrosis is perceived by the immune system as a danger signal that
triggers inflammatory response and acquired immunity. Classically, the
activation and function of a set of proteinases, the caspases, is the
key event in apoptosis, with mitochondria playing a central
role.5,6 A critical event, besides the execution phase of
apoptosis, is the engulfment of dying cells by professional
antigen-presenting cells (APCs) before the dead cells disgorge
their toxic materials in the surrounding milieu.7
In recent years, cell death other than necrosis was
reported to occur in the absence of caspase activation in hematopoietic cells; it included glucocorticoid-induced death of thymocytes, Bax-mediated cell death, and death of cell lines induced by growth factor withdrawal.8-10 A growing number of surface
molecules were shown to be involved in the induction of this
nonclassical caspase-independent cell death. Engagement of CD2, CD45,
CD47, CD99, and MHC (major histocompatibility complex) class II
and I activated this death process.11-17
Caspase-independent cell death induced by CD47 ligation in B-chronic
lymphocytic leukemic (B-CLL) cells was characterized by cell shrinkage,
exposure of phosphatidylserine (PS), and mitochondrial matrix swelling
in complete absence of nuclear degradation.13
CD47 antigen is ubiquitously expressed on hematopoietic and
nonhematopoietic cells.18 It serves as a receptor for
thrombospondin (TSP) and as a ligand for transmembrane signal
regulatory protein (SIRP- The molecular basis for caspase-independent cell death remains elusive.
Apoptosis-inducing factor (AIF), released by mitochondria, induced
caspase-independent nuclear degradation.33 In most of the
studies, sole PS externalization in the presence of the broad caspase
inhibitor Z-VAD.fmk
(benzyloxycarbonyl-Val-Ala-Asp-OMe-fluoromethyl ketone) was used to
define caspase-independent cell death pathway. Coexistence of
caspase-dependent and -independent pathways occurred in various
systems, including CD2 and CD95 ligation.11,34,35 For
instance, in CD2-mediated apoptosis of T lymphocytes, the mitochondrial
release of AIF preceded the dissipation of transmembrane potential
( Our present findings confirm and extend to other cell populations our
previous observations that CD47 ligation exclusively induced the
cytoplasmic events of apoptosis in B-CLL cells. We here show that DCs
recognized and eliminated B-CLL dead cells and present evidence that
cytoskeleton rearrangement was a triggering event in Patients
Cell lines and transfectants
Reagents Recombinant human IL-4, soluble CD40 ligand, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were kindly provided by Immunex (Seattle, WA) and Dr D. Bron (Institut Bordet, Brussels, Belgium), respectively. Dr R.-P. Sekaly (Université de Montréal, Montreal, Canada) kindly provided polyclonal anti-caspase-3 Ab. Anti-human CD3 (UCHT-1) was provided by P. Beverley (University College and Middlesex School of Medicine, London, United Kingdom). Isotype-matched negative control mAb (mouse IgG1) was prepared in our laboratory.The other antibodies and reagents used in this study were purchased
from manufacturers as indicated: anti-CD47 mAbs (clone B6H12),
Bioscience (Camarillo, CA); polyclonal anti-caspase-8, Upstate
Biotechnology (Lake Placid, NY); polyclonal antibody against caspase-9
and anti-mouse CD8- Cell preparation and culture conditions B cells were isolated from CLL patients or from tonsils by density gradient centrifugation of heparinized blood or cell suspension, respectively, using Lymphoprep (Nycomed, Olso, Norway) followed by one cycle of rosetting with S-(2 aminoethyl) isothiouronium bromide-treated (Aldrich, Milwaukee, WI) sheep red blood cells to deplete T cells. B-cell purity was shown to be > 98% by flow cytometry (FACSort, Becton Dickinson). Highly purified T cells were obtained from monocyte-depleted peripheral blood mononuclear cells (PBMCs) from healthy volunteers by rosetting with aminoethylisothiouronium bromide (AET)-treated sheep red blood cells (SRBC), followed by treatment of rosette-forming cells with Lympho-Kwik T (One Lambda, Los Angeles, CA), following manufacturer's recommendations. Cell purity was assessed by flow cytometry using phycoerythrin (PE)-conjugated anti-CD3, anti-CD4, or anti-CD8 mAbs (Ancell, London, ON, Canada) and was shown to be > 98%. CD34+ cells were obtained from heparinized cord blood using the Dynal CD34 progenitor cell selection system according to the manufacturer's instructions (Dynal Skøyen, Oslo, Norway). Human monocyte-derived iDCs were prepared exactly as described.28Highly purified lymphocytes were cultured at 4 × 106/mL,
iDC, lines, and transfectants at 2 × 106/mL in 100 µL
of HB101 serum-free synthetic medium (Irvine Scientific, Edmonton, AB,
Canada) or in special medium when indicated, on flat-bottomed
96-well plates (Nunc, Edmonton, AB, Canada) in the presence of
soluble or immobilized mAbs. Plates were precoated with anti-CD47 or
anti-CD8- For potassium efflux experiments, cells were cultured in 1% fetal calf serum (FCS) Na+K+-free RPMI medium, supplemented with normal or inverted [Na+]/[K+] ratio as described.38 Phagocytosis assay Freshly purified B-CLL cells were stained with PKH26 red fluorescent cell linker (Sigma) according to the manufacturer's instructions before induction of apoptosis by culture for 16 hours on immobilized CD47 mAb, hydrocortisone (HC) (5 × 10 4 M), or soluble CD47 mAb as negative control.
Cells were washed and given to iDCs (2 × 105/mL) as a
phagocytic meal (10 apoptotic cells per iDC) for 3 hours at 37°C.
Endocytosis of PKH26-stained B cells was determined by fluorescence-activated cell-sorter scanner (FACS) after gating on live iDCs.
Flow cytometry analysis CD47 and constructs expression was assessed using a 2-step procedure. Briefly, cells were incubated for 1 hour at 4°C with biotinylated CD47 mAb (B6H12) or unconjugated anti-CD8 (clone no.
53-6.7) or isotype-matched control mAb (5 µg/mL). After
washing, cells were incubated with PE-labeled streptavidin or
fluorescein isothiocyanate (FITC)-conjugated antirat mAb
(Ancell) and analyzed by flow cytometry (FACSort, Becton Dickinson).
Assays for apoptosis Detection of PS exposure, caspase 3 activity, and decrease in  m were performed by flow cytometry using a FACSort (Lysys II
Software, Becton Dickinson). For detection of PS exposure, cells were double-stained with FITC-labeled annexin-V (R&D Systems, Minneapolis, MN) and propidium iodide (PI) at 2 µg/mL
(Sigma). Decrease of m was assessed using
3,3'-dihexyloxacarbocyanine iodide (DiOC6) (Molecular
Probes, Hornby, ON, Canada). Caspase 3 activity was detected by
flow cytometry in unfixed cells using a fluorogenic substrate
(PhiPhilux, OncoImmunium, Gaithersburg, MD). Caspases 3, 7, and 9 cleavage products and caspase 8 expression were analyzed by Western
blotting. Briefly, 5 × 106 cells were directly lysed in
hot sample buffer containing 10%  -mercaptoethanol. Samples were
then boiled for 5 minutes, electrophorezed on 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and
transferred onto polyvinylidenefluoride (PVDF) membranes
(Millipore, Mississauga, MN). Membranes were probed with
indicated antibodies, and immunoreactive products were revealed using enhanced chemiluminescence (ECL; Amersham, Baie d'Urfe, QC, Canada).
CD47-induced cell death was calculated as follows:
Statistical analysis The Student paired t test was used for statistical analysis.
Engulfment by professional phagocytes of CD47 mAb-treated B-CLL cells dying in the absence of caspase activation We previously reported that CD47 ligation by immobilized anti-CD47 monoclonal antibodies (CD47 mAbs) or its natural ligand, TSP,13 induced caspase-independent cell death in B-CLL cells. This cell death was characterized by the cytoplasmic features of apoptosis. They included cell shrinking, exposure of PS to the outer leaflet membrane and, as shown in Figure 1A, a drop in mitochondrial membrane potential (m) as demonstrated by decreased 3,3'-dihexyloxacarbocyanine iodide (DiOC6) staining. This cell death
was considered to be caspase independent since the nucleus of the
leukemic dead cells remained intact and PS exposure was not prevented
by the presence of the broad caspase inhibitor
z-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk).13 To
formally demonstrate that the caspase remained inactive during this
process, we performed Western blot analysis (Figure 1B) to search for
the cleavage products of caspases in CD47 mAb-treated leukemic cells.
We failed to detect any cleavage products of caspases 3, 7, and 9, and
similar amounts of procaspase-8 were found in immobilized CD47 mAb and
control mAb-treated B-CLL cells. The absence of caspases 3/7 activity
in individual CD47-treated cells was confirmed by flow cytometry
using a fluorogenic caspase substrate (Figure 1C). HC-treated B-CLL
cells displayed caspase 3 activity. Note that B-CLL cells with a low
level of spontaneous apoptosis were selected for these experiments.
Both caspase activation and PS exposure generally have been considered as prerequisites for recognition and phagocytosis of cells dying by apoptosis.39,40 It was therefore important to determine whether CD47 mAb-killed B-CLL cells could signal their death to phagocytes and be eliminated. To test this hypothesis, we performed a phagocytosis assay using human monocyte-derived iDCs as professional phagocytes.41 B-CLL cells were stained by PKH26, a nontoxic red fluorescent cell-linker, and exposed for 16 hours to immobilized CD47 mAb or HC, a drug that induced caspase-dependent apoptosis in the leukemic cells.42 Soluble CD47 mAb did not induce cell death13 nor Fc-mediated phagocytosis23 and was used as negative control. Treated samples were cocultured with iDCs for 3 hours. Phagocytosis was assessed by flow cytometry by quantifying FL2 fluorescence after gating on iDCs (by size scatter). Results indicated that B-CLL killed by immobilized CD47 mAb were phagocytosed as efficiently as HC-treated cells (39% vs 46% FL2-positive cells) (Figure 1D). Note that each cell preparation was stained separately with FITC-labeled annexin-V to evaluate their percentage of annexin-V-positive cells (39%, 75%, and 83% for soluble, immobilized CD47 mAb, and HC, respectively). Taken together, these results indicate that the caspase-independent death signal delivered by CD47 ligation to B-CLL cells is sufficient to allow their elimination by iDCs. Blood cell susceptibility to CD47-induced cell death Since CD47 Ag is ubiquitously expressed on hematopoietic cells, we assessed the in vitro sensitivity of normal blood cells to CD47-induced cell death. From the perspective of a potential therapeutic use of this mAb, we first examined CD34+ cells since they represent the pool of hematopoietic progenitors.43,44 As depicted in Figure 2A, CD34+ cells remained totally insensitive to CD47 mAb-induced killing (0% of cell-death induction, n = 6), despite their high level of CD47 expression. Immature DCs are major players in the clearance of apoptotic cells. Like CD34+ cells, they were resistant to CD47-induced cell death (0%, n = 4) (Figure 2A).
We next observed a differential induction of cell death in normal resting and activated B and T lymphocytes. The percentage of CD47-induced cell death in normal resting B and T cells was 36% (n = 7, P < .01) and 49% (n = 14, P < .01), respectively (Figure 2B). B-CLL cells displayed the highest susceptibility (64%, n = 10). Unexpectedly, anti-CD3-stimulated T lymphocytes became almost insensitive to CD47 mAb-killing, whereas T-cell-dependent B-cell activation (sCD40L + IL-4) significantly increased the level of death induction (P < .01). Activation did not modulate CD47 expression as detected by CD47 mAb (clone B6H12) (Figure 2B). Human cell lines represent the malignant counterpart of lymphoid and
myeloid cells at different stages of maturation. In this context, we
examined 4 lymphoid cell lines (RAJI, RPMI-8226, 8866, and Jurkat), 3 myeloid cell lines (U937, THP-1, and K-562), one granulocytic cell line
(KU812), as well as the ovarian carcinoma cell line,
OV10,37 transfected with CD47 cDNA. As shown in Figure 3, these cell lines displayed
differential sensitivity to CD47 mAb, with no correlation, as in
untransformed cells, with the level of CD47 expression. In addition,
the level of CD47-induced cell death induction did not discriminate
between specific cell lineage, RAJI, THP-1, and K562 being resistant.
Of interest, OV10 CD47 transfectants were efficiently killed by CD47
mAb (44% of death induction, n = 5, P < .001),
indicating that CD47 expression was sufficient to confer cell death
susceptibility in a cell that did not primarily express the CD47
molecule.
From this data, we conclude that normal as well as transformed cells display differential susceptibility to CD47-induced cell death. CD34+ cells and immature DCs are resistant, whereas cell activation differentially modulates the intensity of the response, regardless of the level of CD47 expression. CD47 extracellular and multiply membrane-spanning domains are necessary to signal cell death As indicated above, Jurkat and U937 cell lines were sensitive to CD47-induced cell death. These cell lines were therefore suitable to perform structure-function studies to determine which portion(s) of the CD47 molecule was dispensable to mediate the cell death signaling. CD47 molecule is made of an extracellular immunoglobulinlike domain (IgV), a 5-membrane spanning domain (multiply membrane spanning [MMS]), and a cytoplasmic tail displaying 4 alternatively spliced isoforms (form 1 to 4 in order of increasing length).18,45 Form 2 of CD47 is predominant in the hematopoietic lineage, while the nearly tailless form 1 (only 3 amino acids) is expressed in keratinocytes and endothelial cells.We first examined whether the cytoplasmic tail of CD47 was
required. We used JinB8 cells, a CD47-negative Jurkat cell
line,46 transfected with either of the 2 native splice
forms (hIAP-form 1 and 2) or a chimera, CD8C2, made of the
extracellular membrane domain of mouse CD8
Previous studies on the CD47 molecule have demonstrated that IgV and
MMS domains are generally necessary for CD47-induced cell spreading and
costimulation.26,27,46,47 We therefore explored the
requirement of the IgV or the MMS domains in CD47-induced cell death.
To this end, we used 2 constructs: IAP/CD7, made of the CD47 IgV domain
fused to the membrane and cytoplasmic domains of human CD7, and CD8MC2,
made of the extracellular domain of mouse CD8 PS externalization in CD47-induced cell death is down-regulated by K+ efflux impairment and antimycin A treatment Cell shrinkage, PS externalization, and drop inm are major
hallmarks of apoptosis. Cell volume loss has been demonstrated to
result in potassium [K+] and sodium [Na+]
efflux.38,48 Furthermore, K+ efflux and drop
in m are tightly coupled.49,50 To examine whether
CD47-induced cell death was dependent on K+ efflux, we
performed cell cultures in RPMI medium in which
Na+/K+ concentrations had been inverted,
resulting in K+ efflux impairment by osmotic
forces.38 Under these conditions, CD47-induced PS
externalization, together with cell shrinkage, were delayed following
CD47 ligation in B-CLL cells and the U937 cell line when compared to
that observed in cultures with medium reconstituted with a normal
[Na+]/[K+] ratio (Figure
5A and not shown). m was also
slightly decreased (V.M. and M.S., unpublished observations, May
2000).
Classically, PS exposure is dependent on caspase activation, and
PS externalization can be blocked by antimycin A, an inhibitor of the
mitochondrial respiratory chain. As depicted in Figure 5B, antimycin A
totally prevented PS exposure in CD47-treated cells, whereas it had no
effect on decreased These results support the hypothesis that CD47-induced cell death
involves cell volume dysregulation, which is partly associated with
K+ efflux, and that PS exposure is intimately linked to
cell shrinkage but may be uncoupled from decreased CD47-induced cell death involves cytoskeleton rearrangement It was previously reported that T- and B-cell lines46,52 as well as B-CLL cells13 attach to and spread on CD47 mAb-coated surfaces, suggesting that CD47 ligation induces a change in cytoskeleton organization. Moreover, immobilized CD47 mAb reportedly induced F-actin polymerization in activated T cells.27 To assess the role of cytoskeleton in CD47-triggered cell death, we examined the effect of cytochalasin D, an inhibitor of actin polymerization. Treatment with cytochalasin D prevented both PS exposure andm loss in CD47 mAb-treated U937
and B-CLL cells (Figure 6A). These data
confirm and extend a previous report showing that cytochalasin D
inhibited CD47-induced caspase-independent cell death in normal TCR-activated T cells.14
The motility of B-cell lines plated on CD47 mAb-coated surfaces
requires the GTPase Cdc42.52 Since the Cdc42/WASP pathway is defective in cells from patients with WAS, we examined the effect of
CD47 ligation in PBMCs isolated from WAS patients. We tested PBMCs of 4 characterized patients carrying WAS gene mutations, all leading to
impaired WASP expression. As depicted in Figure 6B,D, we consistently
failed to observe PS exposure and Taken together, these data indicate that cytoskeleton rearrangement is
a key event in the triggering of CD47-mediated PS externalization and
The present findings indicate that caspase-independent cell death induced by CD47 stimulation is sufficient to trigger a signal for phagocytosis in human DCs. The CD47-induced cell death involves PS exposure, disruption of mitochondrial function, and cytoskeleton rearrangement possibly linked to the Cdc42/WAS protein (WASP) signaling pathway. This observation challenges the classical concept that both caspase activation and PS exposure triggered by caspases are prerequisites for phagocytosis. Elimination of dead cells in the absence of caspase activation and nuclear degradation has been previously reported. For instance, constitutive but caspase-independent death of platelets, anucleated blood cells, leads to PS exposure and clearance by phagocytes.53 However, caspase-independent or -dependent PS exposure appears to be a critical event to allow engulfment of dead cells by phagocytic cells.54 A PS receptor was recently identified and cloned, and anti-PS receptor mAb inhibited elimination of apoptotic cells.55 Blocking PS exposure without suppression of mitochondrial collapse and nuclear degradation prevented phagocytosis.56 But cells dying by delayed necrosis upon exposure to staurosporine and z-VAD-fmK were eliminated without PS externalization, underlying the potential role of other candidates for recognition and elimination of dead cells by APCs.57 Caspases play a central role in the execution of cell death. These enzymes are recruited and activated by either receptors of the tumor necrosis factor receptor (TNFR) family or by apoptogenic molecules released from the intermembrane space of the mitochondria.58 Nevertheless, apart from CD47, stimulation of several surface antigens resulted in the activation of caspase-independent PS exposure in human cells. This includes CD2, MHC class I, and MHC class II.11,16,17 In contrast to CD47, ligation of these antigens may induce either caspase-dependent or -independent death according to the epitope triggered. So far, CD47 ligation by soluble, immobilized, or cross-linked mAbs recognizing at least 3 different epitopes exclusively induced caspase-independent cell death (Mateo et al,13 Pettersen et al,14 and data not shown). During apoptosis, mitochondrial changes result in the dissipation of
the mitochondrial transmembrane potential ( We also provide evidence that CD47-induced PS exposure may be
dissociated from As described for CD47-mediated cell spreading and costimulation in
T-cell lines, both extracellular and multispan transmembrane domains of
CD47 molecule were required to induce killing. The short cytoplasmic
domain was dispensable. In T cells, ~ 65% of the CD47 molecule is
localized in membrane rafts, where it regulates TCR-dependent and
-independent T-cell activation. Immobilized CD47 mAb reportedly
controlled the activation of heterotrimeric G proteins and triggered
F-actin polymerization and PKC Yoshida et al previously reported that CD47 regulated human B-cell motility through Cdc42.52 We observed that engagement of CD47 induced spreading of B-CLL cells. Cdc42 belongs to the small Rho GTPases family, which includes Rho and Rac, known to regulate formation of actin structures in many cell types.68 Among others, Cdc42 appears to have a unique role in actin remodeling during T-cell activation and endocytosis of immature DCs. WASP, uniquely expressed on hematopoietic cells,69 is the specific effector of Cdc42. WASP binds Cdc42 and controls actin polymerization by distinct domains.70 WAS-immunodeficiency syndrome is characterized by abnormalities in cytoskeletal function, resulting in abnormal chemotaxis, phagocytosis, and T-cell responses.69 Two observations strongly suggest a direct role of cytoskeleton
rearrangement in CD47-induced cell death: (1) inhibition of both PS
exposure and
When blood cell susceptibility was examined, immature DCs, activated T cells, and CD34+ precursors appeared to be much less sensitive and virtually resistant to CD47-induced killing. Similar differences in APC susceptibility have been reported in HLA-DR-induced caspase-independent cell death.71 Pettersen et al14 reported that CD47 mAb preferentially induces killing in normal activated but not resting T cells, and this was inhibited by cytochalasin D. We failed to induce cell death in anti-CD3-activated T cells. The use of different mAbs in the 2 studies and the regulation of CD47 conformation during T-cell activation (M.S. and V.M., unpublished data, January 2000) may provide one explanation for this discrepancy. The role of CD47-induced killing in the regulation of immune response
remains poorly understood. One may envision that it at least
participates in the maintenance of homeostasis. Indeed, apoptosis is a
self-destruction process which, in contrast to necrosis, does not lead
to inflammatory response and may be involved in the induction of
tolerance. Thrombospondin, the natural ligand of CD47, establishes a
molecular bridge between apoptotic cells and APCs, facilitating
phagocytosis of apoptotic cells.72 We reported that TSP,
like CD47 mAb, induced caspase-independent cell death via CD47 binding
but also acted on APCs to down-regulate the production of the
proinflammatory molecule IL-12 and prevent DC
maturation.23 It is important to mention that additional mechanisms inhibit inflammation during phagocytosis of apoptotic cells.
They include induction of transforming growth factor (TGF)- CD47 was recently reported to be a marker of self on murine cells
that prevented clearance of intact red blood and lymphoid cells by CD47+ APCs through the engagement of its
counterstructure SIRP- Taken together, we propose that TSP-induced/enhanced PS exposure via CD47 on lymphoid cells, followed by their elimination by professional phagocytes, represent a process that continuously takes place in peripheral tissues to ensure the maintenance of tissue and host homeostasis. Additionally, the anti-inflammatory activity of TSP, combined with its ability to facilitate the clearance of apoptotic cells, may further contribute to homeostasis and induction of tolerance by avoiding inappropriate immune response to self-antigens.
Submitted December 14, 2001; accepted May 23, 2002.
Prepublished online as Blood First Edition Paper, June 14, 2002; DOI 10.1182/blood-2001-12-0217.
Supported by grants from Fondation Medic and the Leukemia Research Foundation of Canada.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Marika Sarfati, Centre de Recherche du CHUM, Hôpital Notre-Dame, Laboratoire d'Immunorégulation (M4211-K), 1560 Sherbrooke St East, Montréal, QC H2L 4M1, Canada; e-mail: m.sarfati{at}umontreal.ca.
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Top
Abstract
Introduction
reportedly activated
scramblase in apoptotic cells.
in T
cells.
, 
, 
can't live without it.
Cell.
1997;91:559-562