CD47 and the 19 kDa Interacting Protein-3 (BNIP3) in T Cell Apoptosis

CD47 is a surface receptor that induces either coactivation or apoptosis in lymphocytes, depending on the ligand(s) bound. Interestingly, the apoptotic pathway is independent of caspase activation and cytochrome c release and is accompanied by early mitochondrial dysfunction with suppression of mitochondrial membrane potential (Δψm). Using CD47 as bait in a yeast two-hybrid system, we identified the Bcl-2 homology 3 (BH3)-only protein 19 kDa interacting protein-3 (BNIP3), a pro-apoptotic member of the Bcl-2 family, as a novel partner. Interaction between CD47 and the BH3-only protein was confirmed by immunoprecipitation analysis, and CD47-induced apoptosis was inhibited by attenuating BNIP3 expression with antisense oligonucleotides. Finally, we showed that the C-terminal domain of thrombospondin-1 (TSP-1), but not signal-regulatory protein (SIRPα1), is the ligand for CD47 involved in inducing cell death. Immunofluorescence analysis of CD47 and BNIP3 revealed a partial colocalization of both molecules under basal conditions. After T cell stimulation via CD47, BNIP3 translocates to the mitochondria to induce apoptosis. These results show that the BH3-dependent apoptotic pathways, previously shown to be activated by intracellular pro-apoptotic events, can also be turned on by surface receptors. This new pathway results in a fast induction of cell death resembling necrosis, which is likely to play an important role in lymphocyte regulation at inflammatory sites and/or in the vicinity of thrombosis. CD47 is a surface receptor that induces either coactivation or apoptosis in lymphocytes, depending on the ligand(s) bound. Interestingly, the apoptotic pathway is independent of caspase activation and cytochrome c release and is accompanied by early mitochondrial dysfunction with suppression of mitochondrial membrane potential (Δψm). Using CD47 as bait in a yeast two-hybrid system, we identified the Bcl-2 homology 3 (BH3)-only protein 19 kDa interacting protein-3 (BNIP3), a pro-apoptotic member of the Bcl-2 family, as a novel partner. Interaction between CD47 and the BH3-only protein was confirmed by immunoprecipitation analysis, and CD47-induced apoptosis was inhibited by attenuating BNIP3 expression with antisense oligonucleotides. Finally, we showed that the C-terminal domain of thrombospondin-1 (TSP-1), but not signal-regulatory protein (SIRPα1), is the ligand for CD47 involved in inducing cell death. Immunofluorescence analysis of CD47 and BNIP3 revealed a partial colocalization of both molecules under basal conditions. After T cell stimulation via CD47, BNIP3 translocates to the mitochondria to induce apoptosis. These results show that the BH3-dependent apoptotic pathways, previously shown to be activated by intracellular pro-apoptotic events, can also be turned on by surface receptors. This new pathway results in a fast induction of cell death resembling necrosis, which is likely to play an important role in lymphocyte regulation at inflammatory sites and/or in the vicinity of thrombosis. Multicellular organisms eliminate excess, damaged or infected cells by stereotypic programs of cell death (PCD). 1The abbreviations used are: PCD, programmed cell death; TSP-1, thrombospondin-1; SIRPα1, signal-regulatory protein; IAP, integrin-associated protein; TM, transmembrane; BNIP3, 19 kDa interacting protein-3; BH3, Bcl-2 homology 3; GFP, green fluorescent protein; JIN, Jurkat IAP-negative cells; Δψm, mitochondrial membrane potential; DiOC6, 3,3′-dihexyloxacarbocyanine iodide; TOM, translocase of outer membrane; MMS, multiply membrane spanning; CD47ec, CD47 extracellular; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate. 1The abbreviations used are: PCD, programmed cell death; TSP-1, thrombospondin-1; SIRPα1, signal-regulatory protein; IAP, integrin-associated protein; TM, transmembrane; BNIP3, 19 kDa interacting protein-3; BH3, Bcl-2 homology 3; GFP, green fluorescent protein; JIN, Jurkat IAP-negative cells; Δψm, mitochondrial membrane potential; DiOC6, 3,3′-dihexyloxacarbocyanine iodide; TOM, translocase of outer membrane; MMS, multiply membrane spanning; CD47ec, CD47 extracellular; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate. In its classic form, apoptosis is characterized by well defined ultrastructural changes including cell shrinkage, exposure of phosphatidylserine at the outer leaflet of the cytoplasmic membrane, changes in mitochondrial permeability, membrane blebbing, caspases activation, and DNA degradation. Lymphocyte PCD plays an important role in controlling immune responses and occurs both in central and peripheral lymphoid organs. Disturbed PCD may contribute to multiple immune disorders such as cancer and autoimmune and degenerative diseases. Upon signaling, pathways that influence T cell proliferation and survival, CD95/CD95L and tumor necrosis factor receptor pathways have been extensively studied over the past few years (1Siegel R.M. Chan F.K. Chun H.J. Lenardo M.J. Nat. Immunol. 2000; 1: 469-474Google Scholar). However, a number of others T cell surface receptors such as major histocompatibility complex class I and II (2Skov S. Klausen P. Claesson M.H. J. Cell Biol. 1997; 139: 1523-1531Google Scholar), CD2 (3Deas O. Dumont C. MacFarlane M. Rouleau M. Hebib C. Harper F. Hirsch F. Charpentier B. Cohen G.M. Senik A. J. Immunol. 1998; 161: 3375-3383Google Scholar), CD4 (4Berndt C. Mopps B. Angermuller S. Gierschik P. Krammer P.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12556-12561Google Scholar), CD45 (5Lesage S. Steff A.M. Philippoussis F. Page M. Trop S. Mateo V. Hugo P. J. Immunol. 1997; 159: 4762-4771Google Scholar, 6Klaus S.J. Sidorenko S.P. Clark E.A. J. Immunol. 1996; 156: 2743-2753Google Scholar), or CD99 (7Bernard G. Breittmayer J.P. de Matteis M. Trampont P. Hofman P. Senik A. Bernard A. J. Immunol. 1997; 158: 2543-2550Google Scholar) can also trigger PCD. In contrast to the former pathways, they have all been described to act independently of any of the known caspases (8Scaffidi C. Kirchhoff S. Krammer P.H. Peter M.E. Curr. Opin. Immunol. 1999; 11: 277-285Google Scholar), and the molecular mechanisms and the physiological and/or the pathological relevance of these death pathways remains to be established. Among these molecules, recent reports implicate CD47 in the triggering of atypical cell death (9Pettersen R.D. Hestdal K. Olafsen M.K. Lie S.O. Lindberg F.P. J. Immunol. 1999; 162: 7031-7040Google Scholar, 10Mateo V. Lagneaux L. Bron D. Biron G. Armant M. Delespesse G. Sarfati M. Nat. Med. 1999; 5: 1277-1284Google Scholar). CD47 (also known as IAP for integrin-associated protein), expressed on all mammalian cells (11Brown E. Hooper L. Ho T. Gresham H. J. Cell Biol. 1990; 111: 2785-2794Google Scholar), displays an extracellular Ig-like domain with five transmembrane (TM) segments and a short C-terminal cytoplasmic tail. CD47 is associated with β3 integrins on several cell types. However, on other cell types such as lymphocytes, no association with integrins has been documented. More generally, CD47 has been shown to activate integrins, either through direct interaction or at a distance (11Brown E. Hooper L. Ho T. Gresham H. J. Cell Biol. 1990; 111: 2785-2794Google Scholar, 12Lindberg F.P. Gresham H.D. Schwarz E. Brown E.J. J. Cell Biol. 1993; 123: 485-496Google Scholar, 13Brittain J.E. Mlinar K.J. Anderson C.S. Orringer E.P. Parise L.V. J. Clin. Invest. 2001; 107: 1555-1562Google Scholar). The two natural ligands currently known for CD47 are thrombospondin-1 (TSP-1), a protein found in extracellular matrix and released in large amounts by platelets upon activation, and the signal-regulatory protein (SIRPα1), expressed on the surface of macrophages and endothelial and dendritic cells. Moreover, we and others have shown that CD47 can trigger T cell activation and proliferation (14Ticchioni M. Deckert M. Mary F. Bernard G. Brown E.J. Bernard A. J. Immunol. 1997; 158: 677-684Google Scholar, 15Reinhold M.I. Lindberg F.P. Kersh G.J. Allen P.M. Brown E.J. J. Exp. Med. 1997; 185: 1-11Google Scholar, 16Waclavicek M. Majdic O. Stulnig T. Berger M. Baumruker T. Knapp W. Pickl W.F. J. Immunol. 1997; 159: 5345-5354Google Scholar) or induce T cell spreading (17Reinhold M.I. Green J.M. Lindberg F.P. Ticchioni M. Brown E.J. Int. Immunol. 1999; 11: 707-718Google Scholar). Therefore an important task has been to determine under which condition(s) CD47 induces T cell death and/or survival. Indeed, in addition to the cytoplasmic proteins Gi (18Frazier W.A. Gao A.G. Dimitry J. Chung J. Brown E.J. Lindberg F.P. Linder M.E. J. Biol. Chem. 1999; 274: 8554-8560Google Scholar, 19Gao A.G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Google Scholar) and proteins linking IAP with cytoskeleton (PLIC) (20Wu A.L. Wang J. Zheleznyak A. Brown E.J. Mol. Cell. 1999; 4: 619-625Google Scholar), we show in the present study that CD47 associates with the pro-apoptotic molecule 19 kDa interacting protein-3 (BNIP3). BNIP3 belongs to the Bcl-2 homology 3 (BH3)-only family, a Bcl-2-related family possessing an atypical Bcl-2 homology 3 (BH3) domain, which regulates PCD from mitochondrial sites by selective Bcl-2/Bcl-XL interactions (21Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Google Scholar, 22Yasuda M. Theodorakis P. Subramanian T. Chinnadurai G. J. Biol. Chem. 1998; 273: 12415-12421Google Scholar). BNIP3 family members contain a C-terminal transmembrane domain that is required for their mitochondrial localization, homodimerization, as well as regulation of their pro-apoptotic activities (23Chen G. Ray R. Dubik D. Shi L. Cizeau J. Bleackley R.C. Saxena S. Gietz R.D. Greenberg A.H. J. Exp. Med. 1997; 186: 1975-1983Google Scholar). BNIP3-mediated apoptosis has been reported to be independent of caspase activation and cytochrome c release and is characterized by early plasma membrane and mitochondrial damage, prior to the appearance of chromatin condensation or DNA fragmentation (24Van de Velde C. Cizeau J. Dubik D. Alimonti J. Brown T. Israels S. Hakem R. Greenberg A.H. Mol. Cell. Biol. 2000; 20: 5454-5468Google Scholar). In the present work, we describe an original mechanism by which the BH3-only protein BNIP3 could be an important mediator of CD47-induced cell death. This pathway would be likely to act at sites where large amounts of soluble TSP-1 are available and could play an important role in T cell death in the vicinity of thrombotic events. Reagents—The CD47 mAb Ad22 was kindly provided by Dr. Rolf D. Pettersen (Departement of Pediatric Research and Pediatrics, The National Hospital, Oslo, Norway) and has been described elsewhere (9Pettersen R.D. Hestdal K. Olafsen M.K. Lie S.O. Lindberg F.P. J. Immunol. 1999; 162: 7031-7040Google Scholar). CD47 mAb B6H12 was from the American Type Culture Collection (ATCC, Rockville, MD). Rabbit polyclonal anti-BNIP3 and mouse monoclonal anti-BNIP3 Ana40 antibodies have been described elsewhere (21Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Google Scholar). MAb against Fas CH11 was from Immunotech (BD Biosciences), mAb against Bcl-2 was from Santa Cruz Biotechnology, and mAb against V5 was from Invitrogen. SIRPα1-Fc fusion proteins have previously been described (25Rebres R.A. Green J.M. Reinhold M.I. Ticchioni M. Brown E.J. J. Biol. Chem. 2001; 276: 7672-7680Google Scholar). The peptides 4N1K (KRFYVVMWKK) and 4NGG (KRFYGGMWWKK) were from Genosys Biotechnologies (The Woodlands, TX). Plasmids encoding green fluorescent protein (GFP)-tagged cytochrome c and Bcl-2 (pCMV-Bcl-2) were kindly provided by Dr. A. Galmiche (INSERM U462, Nice, France). Cells—The Jurkat T cell line (JE6.1) was obtained from ATCC and in with and The Jurkat T cell line for Jurkat IAP-negative has been previously described M. V. L. J. Lindberg F.P. Brown E.J. Bernard A. J. 2001; Scholar). for exposure and in mitochondrial membrane potential were by a (BD phosphatidylserine cells were with and as described by the The in was by Jurkat cells with 3,3′-dihexyloxacarbocyanine for at or not with the pro-apoptotic 4N1K for were on mitochondrial was to the to a of for were with with and for with and were for CD47 SIRPα1-Fc and mouse at and for BNIP3 the mouse monoclonal anti-BNIP3 (21Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Google Scholar) by a were in and with a and were in and in with Cell was by and were for at with was with protein Biosciences), for at The were in and proteins were on and with polyclonal anti-BNIP3 (21Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Google Scholar) or B6H12 The immune were by antibodies and c were by Jurkat cells in mitochondrial and were and by through an cells and were by The was at for at and the was as the were on and to membrane were with mouse c (BD and with and In a of Jurkat cells were with cytochrome via CD47 or Fas with as described by the were with a cells were with the mAb against CD47 Ad22 or in and in and were a with a The cell was at for The was at for The the mitochondrial was in and on a B. E.A. 1990; Scholar). The in the was by Cell were by polyclonal anti-BNIP3 a of the outer mitochondrial membrane and of 3 was in with of protein as described previously J.E. L. V. C. F. B. Hofman P. P. 2001; Scholar). cells were in with were in a with and of as for at was by the release of at in either the or of an of The caspase the and is expressed as of and of BNIP3 of BNIP3 number or were to the cells at the of and the was C-terminal of CD47 to the and cytoplasmic of the molecule were to the domain in multiply membrane and used as a bait for a M. S. C. T. A. 1996; 5: Scholar, Bernard A. Deckert M. J. Biol. Chem. Scholar). were used for and of DNA used to the DNA were and in of was a as a CD47 domain and BNIP3 interactions were for on the of on for was for BNIP3 in were previously described (21Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Google and were the sites of the yeast two-hybrid expression CD47 a and previously described (9Pettersen R.D. Hestdal K. Olafsen M.K. Lie S.O. Lindberg F.P. J. Immunol. 1999; 162: 7031-7040Google Scholar, 10Mateo V. Lagneaux L. Bron D. Biron G. Armant M. Delespesse G. Sarfati M. Nat. Med. 1999; 5: 1277-1284Google Scholar), CD47 Ad22 in a soluble induces a fast cell death with an early phosphatidylserine exposure and mitochondrial Indeed, to cell stimulation via CD47 we that the mitochondrial dysfunction as early as cell death. CD47 stimulation was as as at Δψm, as early mitochondrial dysfunction CD47 we a and release of cytochrome c which was 3 in contrast with the of stimulation cytochrome c release to be a that cytochrome c release occurs cell we Jurkat cells with cytochrome c and its mitochondrial release CD47 stimulation mitochondrial release was a role for caspases could be by to PCD with the caspase not Moreover, no activation of caspase 3 could be PCD in the in contrast with the of a CD47 with the determine the apoptotic pathway by CD47 we for CD47 interacting in the yeast two-hybrid used the CD47 domain to as a bait for a lymphocyte the for the domain of CD47 to induce apoptosis by a of CD47, of the extracellular domain of CD47 and the membrane and cytoplasmic of M.I. Lindberg F.P. Kersh G.J. Allen P.M. Brown E.J. J. Exp. Med. 1997; 185: 1-11Google Scholar). This was to an apoptotic in the line which we previously M. V. L. J. Lindberg F.P. Brown E.J. Bernard A. J. 2001; Scholar), and CD47 cells showed of apoptosis not that the of domain as bait to the two-hybrid has been Shi P. D. J. Biol. Chem. 1999; 274: Scholar, A. D. L. L. R. D. M. J.H. R.M. G. J. Biol. Chem. 1999; 274: Scholar, L. P. L. J. Biol. Chem. 1999; 274: Scholar, T. M. S. M. S. S. M. H. J. Cell Biol. 1999; Scholar, Shi Gao W. G. G. S. G.M. D. J. Biol. Chem. Scholar, C. T. H. G. Scholar). In the present five were of an for their to a Among we found a encoding the pro-apoptotic BH3-only family protein BNIP3 determine the domain of the BNIP3 molecule involved in to CD47, we a of BNIP3 (21Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Google Scholar) by the two-hybrid BNIP3 showed a interaction with CD47, was to with CD47 that the transmembrane domain of BNIP3 is required for interaction with the the domain, or the not to CD47 that other in the of BNIP3 are for the interaction with the association between CD47 and BNIP3 in we BNIP3 was with CD47 from Jurkat T cells. as no was from cells or cells the BNIP3 with the domain of CD47 both in yeast and mammalian cells. we by BNIP3 with antisense the apoptotic could be Jurkat cells were with antisense and apoptosis in to soluble CD47 Ad22 or CH11 was to BNIP3 CD47-induced cell a in cell a of the no Moreover, no on either of cells or on apoptosis by the analysis confirmed a of BNIP3 expression from cells with the BNIP3 antisense of BNIP3 expression to a of cell death via Finally, has been previously shown that BNIP3 Bcl-2 at the mitochondrial membrane, an likely to play a role in PCD (24Van de Velde C. Cizeau J. Dubik D. Alimonti J. Brown T. Israels S. Hakem R. Greenberg A.H. Mol. Cell. Biol. 2000; 20: 5454-5468Google Scholar), we of Bcl-2 in Jurkat cells. cells were to PCD either via CD47 their of BNIP3 expression or of Bcl-2 to a of cell death via CD47, a interaction between CD47 and TSP-1 CD47-induced and BNIP3 to as a receptor both for the C-terminal domain of TSP-1 and for A.G. Lindberg F.P. Brown E.J. Frazier W.A. J. Biol. Chem. 1996; Scholar, P. V. J. Biol. Chem. 1999; 274: Scholar). TSP-1 is a protein expressed on endothelial found in large amounts in extracellular matrix and released by activated is expressed on the surface of macrophages and endothelial and dendritic cells. determine which natural ligand induces cell we Jurkat cells or cells with the from TSP-1 4N1K A.G. Lindberg F.P. Brown E.J. Frazier W.A. J. Biol. Chem. 1996; Scholar) or with a SIRPα1-Fc fusion protein (25Rebres R.A. Green J.M. Reinhold M.I. Ticchioni M. Brown E.J. J. Biol. Chem. 2001; 276: 7672-7680Google Scholar) soluble 4N1K a death of Jurkat T but no on cells. 4NGG to induce cell death in the two cell types. the soluble SIRPα1-Fc fusion which CD47 not no apoptotic of the of protein or the we is that of 4N1K are to induce cell have been reported to of inflammatory T cells J. Immunol. 2000; Scholar) or to induce of T cells M. M. Delespesse G. Sarfati M. J. Immunol. 2000; Scholar, L. K. D. J. Immunol. 2001; Scholar). This apoptotic of 4N1K on T cells is not to a cells are to cell death by BNIP3 has been described to at and to act on the mitochondria (21Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Google Scholar, G. Ray R. Dubik D. Shi L. Cizeau J. Bleackley R.C. Saxena S. Gietz R.D. Greenberg A.H. J. Exp. Med. 1997; 186: 1975-1983Google Scholar, J.M. S. Subramanian T. U. B. C. Chinnadurai G. Cell. Scholar). The of a potential association between the cell surface CD47 molecule and BNIP3 to the of BNIP3 at the leaflet of the plasma membrane in addition to mitochondrial not was shown that BNIP3 is in the outer membrane of the we have the plasma BNIP3 molecule would to the we by of Jurkat cells with the CD47 Ad22 or in In cells in BNIP3 was both in the mitochondrial as by the of a protein on the outer mitochondrial membrane, and in the including the plasma membrane, as by the of that is not to the and the mitochondrial the of these be Jurkat cells were by the BNIP3 was no in the its in the mitochondrial to be that BNIP3 translocates from the plasma membrane to the mitochondria upon CD47 we in which T cells were or not with the pro-apoptotic 4N1K and for either CD47 or BNIP3, and mitochondria were revealed with the T cells were with the pro-apoptotic 4N1K BNIP3 was to to amounts of BNIP3 at the cell membrane in basal that a BH3-only protein can be in an via a surface BH3-only proteins have been as of intracellular or and BNIP3 was shown to apoptosis Proc. Natl. Acad. Sci. U. S. A. 2000; Scholar). However, the to be likely to a for de of BNIP3, the molecule is by the G. Cizeau J. Vande Velde C. Park J.H. G. J. Shi L. Dubik D. Greenberg A. J. Biol. Chem. 1999; 274: Scholar, J. Ray R. Chen G. Gietz R.D. Greenberg A.H. 2000; Scholar). has been previously (21Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Google Scholar, J.M. S. Subramanian T. U. B. C. Chinnadurai G. Cell. Scholar) BNIP3 to we the we also several of that under basal a of BNIP3 is at the plasma membrane where with In a yeast two-hybrid the domain of CD47 as we identified immunoprecipitation of CD47 was accompanied by of Finally, of CD47 and BNIP3 and revealed that a of both molecules The of of BNIP3 to the transmembrane domain of BNIP3 as the for interaction with the of of showed that BNIP3 is required for the pro-apoptotic of we a in CD47 pro-apoptotic BNIP3 expression was an antisense of BNIP3, or the molecule BNIP3 was has been shown that BNIP3 its pro-apoptotic at the mitochondria membrane M. Theodorakis P. Subramanian T. Chinnadurai G. J. Biol. Chem. 1998; 273: 12415-12421Google Scholar, G. Ray R. Dubik D. Shi L. Cizeau J. Bleackley R.C. Saxena S. Gietz R.D. Greenberg A.H. J. Exp. Med. 1997; 186: 1975-1983Google Scholar, G. Cizeau J. Vande Velde C. Park J.H. G. J. Shi L. Dubik D. Greenberg A. J. Biol. Chem. 1999; 274: Scholar), a on the of BNIP3 from the surface of the cell membrane to the with we showed a pro-apoptotic via CD47 upon of a mAb or a TSP-1, BNIP3 translocates from the plasma membrane to the mitochondria as both by and This for a fast apoptosis and is with the by the Bcl-2 family proteins A. M. L. M. S. S. Cell. Scholar, S. R.A. T. S.J. Mol. Cell. 2001; Scholar, J.M. S. 1998; Scholar, C. Green 2001; Scholar). is that several other BH3-only proteins were reported to also be to fast and A. Cell. 2000; Scholar). be that BNIP3, with a and J. Ray R. Chen G. Gietz R.D. Greenberg A.H. 2000; Scholar, M. C. Chinnadurai G. 1998; Scholar) the BH3-only domain was found to be in of association with the proteins Bcl-2 and was that their transmembrane C-terminal as well as are for these a of have shown that BH3-only protein associates with and to induce cell death A. M. L. M. S. S. Cell. Scholar, J.C. Green Nat. Mol. Cell. Biol. 2001; Scholar), the mitochondrial and mechanisms to cell death with the BNIP3 to be established. Moreover, other BH3-only proteins would apoptotic via transmembrane receptors remains to be T cell PCD via CD47 can be by TSP-1 and not by its other natural ligand The that amounts of TSP-1 can trigger apoptosis also with the amounts of TSP-1 and 4N1K can of T cells (14Ticchioni M. Deckert M. Mary F. Bernard G. Brown E.J. Bernard A. J. Immunol. 1997; 158: 677-684Google Scholar, 15Reinhold M.I. Lindberg F.P. Kersh G.J. Allen P.M. Brown E.J. J. Exp. Med. 1997; 185: 1-11Google Scholar, 16Waclavicek M. Majdic O. Stulnig T. Berger M. Baumruker T. Knapp W. Pickl W.F. J. Immunol. 1997; 159: 5345-5354Google Scholar). can be that pro-apoptotic pathway could the inflammatory which to thrombosis. have in in an for such a role of M. A. E. J. and A. in with be in that the CD47 pro-apoptotic pathway on but not T cells (9Pettersen R.D. Hestdal K. Olafsen M.K. Lie S.O. Lindberg F.P. J. Immunol. 1999; 162: 7031-7040Google Scholar). Interestingly, an of TSP-1 has been a G. P. J. Scholar). Moreover, cell types other T cells could be to an apoptotic regulation via the as both TSP-1 and CD47 have been reported to be on endothelial cells upon of and can induce their apoptosis D. R. P. P. 2000; Scholar, D. R. J. P. 2001; Scholar). E. and K. E. for of the and we members of the for are to R. D. Pettersen for kindly Ad22 mAb, R. A. for SIRPα1-Fc fusion D. Dubik for antibodies against BNIP3 and for for BNIP3, A. Galmiche for Bcl-2 and cytochrome and P. for caspase