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Departments of
*
Pediatric Research and
Pediatrics, The National Hospital, Oslo, Norway; and
Department of Infectious Diseases, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-stimulated PBMC, as determined by morphological
changes, phosphatidylserine exposure on the cell surface, uptake of
propidium iodide, and true counts by flow cytometry. In contrast,
apoptosis was not observed following culture with anti-CD47 mAbs
2D3 or B6H12 directed to a distant or closely adjacent region,
respectively. CD47-mediated cell death was independent of CD3, CD4,
CD45, or p56lck involvement as demonstrated by
studies with variant Jurkat cell lines deficient in these signaling
pathways. However, coligation of CD3 and CD47 enhanced
phosphatidylserine externalization on Jurkat cells with functional CD3.
Furthermore, normal T cells required preactivation to respond with
CD47-induced apoptosis. CD47-mediated cell death appeared to proceed
independent of Fas or TNF receptor signaling and did not involve
characteristic DNA fragmentation or requirement for IL-1ß-converting
enzyme-like proteases or CPP32. Taken together, our data demonstrate
that under appropriate conditions, CD47 activation results in very
rapid T cell death, apparently mediated by a novel apoptotic pathway.
Thus, CD47 may be critically involved in controlling the fate of
activated T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Negative regulators of T cell activation and molecules inducing cell death balance the level of mature T cells. Thus, mature peripheral T cells may respond with activation-induced apoptosis (AICD)3 following religation of the TCR/CD3 complex (16, 17). The Fas Ag is recognized as one of the major cell surface Ags involved in regulating the level of effector T cells by AICD (18, 19, 20). A critical role for Fas in the regulation of immune homeostasis is strongly supported by the development of generalized lymphoproliferative disorders in lpr and gld mice with mutations in the genes for Fas and Fas ligand (FasL), respectively (21, 22). However, although Fas expression is rapidly induced on activated T cells, they apparently require several days after initial Fas expression to develop susceptibility to CD95-mediated death signaling (18, 23, 24). Recent experiments demonstrate that activation of TNFRI/p55 and TNFRII/p75 also may contribute to deletion of mature peripheral T cells (25, 26, 27). Interestingly, CD4+ and CD8+ T cells have apparently different sensitivity to TNF- and FasL-mediated apoptosis (26).
The cytotoxic T lymphocyte-associated Ag CTLA-4 is further implicated as an important regulator of T cell responses. The Ag, which is expressed on both activated CD4+ and CD8+ T cells (28), is related to the CD28 molecule and also binds the B7 family members CD80 and CD86 (7). Several studies suggest a major role for CTLA-4 in controlling proliferation and even mediating apoptosis (29, 30, 31, 32). Thus, mice with a targeted mutation of CTLA-4 develop a spontaneous and fatal lymphoproliferative disease (32). Furthermore, studies of CTLA-4-/- mice shows that CTLA-4 Ig treatment prevents fatal lymphoproliferation and suggest a role for CTLA-4 in the induction and maintenance of tolerance (33). In contrast, CTLA-4 has also been implicated in coactivation (28). Notably, CD80 costimulation of CTLA-4 promotes clonal expansion of CD28-deficient T cells (34). In addition, B7-CTLA-4 interaction enhances the production of antitumor CTL and resistance to tumor challenge (35). Thus, the biologic role for CTLA-4 has not been clearly established (36).
Other signaling pathways may also influence T cell proliferation and survival, and recent reports suggest a role for CD47 in costimulation. The 50-kDa Ig gene superfamily member CD47 is expressed on most human cells, including immature and mature T cells. Structurally, CD47 is composed of an extracellular IgV domain, five transmembrane domains, and a short cytoplasmic domain (37, 38). As the molecule physically and functionally associates with ß3 integrins on several cell types, CD47 is frequently referred to as integrin-associated protein (IAP) (39). Specifically, recent studies demonstrate that coligation of CD3 and IAP (or CD47) may mobilize p56lck, induce IL-2 production, and initiate T cell proliferation independent of CD28 signaling (40, 41, 42). However, high concentrations of anti-CD47 mAbs may also interfere with allogeneic MLRs (42), thereby indicating a possible dual role for the Ig superfamily member in regulating T cell responses.
In this study we demonstrate that ligation of distinct epitopes on CD47 rapidly induces the death of activated T cells, apparently independently of Fas and TNFR signaling. Furthermore, our work suggests that CD47 may be involved in the regulation of immune responses by activation of a novel death pathway. Thus, expression of the proper CD47 ligand(s) on APC may represent an efficient strategy to clear preactivated T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The OKT3 hybridoma (anti-CD3, IgG2a) was obtained from
American Type Culture Collection (Manassas, VA). Apoptosis-inducing
anti-CD95 (CH11, IgM) was purchased from Immunotech (Marseille
Cedex, France). Anti-CD47 mAbs 2D3 (IgG1), B6H12 (IgG1), and 1F7 (IgG1)
have been described previously (37, 39). Murine IgG1 (MOPC-21) control
and recombinant human TNF-
and TNF-ß were purchased from Sigma
(St. Louis, MO). Murine IgM control (TEPC 183) was obtained from
BiosPacific (Emeryville, CA). Anti-CD120a (TNFRI/p55; IgG1-PE) and
anti-CD120b (TNFRII/p75; IgG1-PE) were obtained from Boehringer
Ingelheim (Heidelberg, Germany). Anti-CD4 (IgG1-FITC), anti-CD8
(IgG1-PE), IgG1-FITC, and IgG1-PE controls were purchased from Becton
Dickinson (San Jose, CA). Streptavidin-FITC was obtained from Dako
(Glostrup, Denmark), and streptavidin-PE was obtained from Southern
Biotechnology Associates (Birmingham, AL). Recombinant human TNFRI/Fc
and Fas-Fc chimera were obtained from R & D Systems (Abingdon, U.K.).
ICE inhibitor II (Ac-Tyr-Val-Ala-Asp-chloromethyl ketone; Ac-YVAD-CMK)
was purchased from Calbiochem (La Jolla, CA). ICE inhibitor
Z-Val-Ala-D,L-Asp-fluoromethylketone
(Z-VAD-FMK) and CPP32 inhibitor Ac-Asp-Glu-Val-Asp-aldehyde
(Ac-DEVD-CHO) were purchased from Bachem Feinchemikalien (Bubendorf,
Switzerland). Cytochalasin D was obtained from Calbiochem (La Jolla,
CA).
mAb production, cell culture, and transfectants
mAb Ad22 (IgG1
) was raised against the human T cell line
PFI-285 (43) as previously described (44). Human cells were cultured in
RPMI 1640 supplemented with 10% FCS, 1.5 mM glutamine, 100 U/ml
penicillin, and 100 µg/ml streptomycin. CD47-deficient human ovarian
carcinoma OV10 cells and CD47-expressing OV10 transfectants have been
described previously (45). The human promonocytic cell line U937, the
human T cell line Jurkat E6, and Jurkat derivatives J.RT3-T3.5
(TCRß/CD3 deficient), J.CaM1.6 (defective in
p56lck kinase expression), J45.01 (CD45
deficient), and D1.1 (CD4 negative) were obtained from American Type
Culture Collection. Cell lines were subcultured every 2–3 days to
maintain cell densities between 1 x 105 and
1 x 106 cells/ml. Experiments were performed on
5 x 105 cells/ml cultured in 24-well cell culture
cluster plates (3524, Costar, Cambridge, MA) unless otherwise
indicated. Human PBMC were isolated by Lymphoprep (Nycomed Pharma,
Oslo, Norway) centrifugation. All cultures were grown at 37°C in a
humidified 5% CO2 atmosphere.
Flow cytometry
Ag expression was determined with flow cytometry as previously described (46). Cells were finally washed twice in PBS and fixed with 1% paraformaldehyde in PBS. Samples were analyzed using a FACSort (Becton Dickinson), and data were collected for 10,000 cells.
Flow cytometric determination of apoptosis and cell death
Apoptosis was determined by monitoring changes in cell size and granularity by flow cytometry and assessment of phosphatidylserine (PS) exposure by annexin V-FLUOS (Boehringer Mannheim, Mannheim, Germany) binding according to the manufacturer’s instructions. DNA fragmentation was determined with the TUNEL assay kit from Boehringer Mannheim. Cell membrane permeability was assessed by determining uptake of the DNA-binding fluorescent dye propidium iodide (PI; 2.5 µg/ml) after incubation for 10 min. In each case data were collected for 10,000 cells. TruCount tubes (Becton Dickinson) containing fluorescent-dyed microbeads were used to assess relative cell numbers.
Proliferation assays with immobilized mAb
Proliferation assays were performed in flat-bottom 96-microwell plates (Costar) coated with mAb (1, 5, or 10 µg/ml) in PBS (100 µl/well) for 20–24 h at 4°C. Before the start of culture, the mAb solutions were aspirated, and each well was washed three times with 250 µl of PBS. Jurkat E6 cells (5 x 105 cells/ml) were cultured for 24 h with addition of 1 µCi of [3H]thymidine (Amersham, Aylesbury, U.K.) to each well for the last 6 h. Cells were harvested onto glass-fiber filters with a Skatron harvester (Skatron, Lierskogen, Norway), and [3H]thymidine incorporation was determined using a Betaplate scintillation counter (Wallac Oy, Turku, Finland).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The functional role(s) of CD47 on T cells is not clear. However,
recent studies suggest that CD47 may serve as a costimulatory molecule
in T cell activation (40, 41, 42). We produced a new mAb (Ad22, IgG1)
reacting with the IgV domain of CD47 (Fig. 1
) in proximity to epitopes defined by
mAb B6H12 and 1F7 (Fig. 2) and addressed
the functional role of this region on human T cells. Jurkat cells were
used as a model system, as these cells can respond with coactivation or
apoptosis following engagement of different integral membrane proteins
(40, 41, 42, 47, 48, 49). Surprisingly, microscopic examinations and
assessments with flow cytometry revealed that Jurkat E6 cells incubated
with Ad22 aggregated and showed morphological characteristics of
apoptotic cells (data not shown). To further determine whether
apoptosis could be signaled by activation of CD47, we examined the
impact of IgG1 anti-CD47 mAbs Ad22, B6H12, 1F7 (directed to
adjacent epitopes), and 2D3 (to a distant region) using flow cytometry.
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A and data not
shown) even at low mAb concentrations (Fig. 3B). The
response was readily observable within 30 min, and the effect, as
determined by annexin V-FLUOS binding, reached a maximum within the
first 3 h (Fig. 3C and data not shown). Furthermore,
mAb 1F7 also induced PS exposure and uptake of PI (Fig. 3, A
and B). In contrast, no significant impact on PS exposure or
uptake of PI was observed with control mAb MOPC-21 or the anti-CD47
mAbs B6H12 or 2D3 after 1, 3, or 5 h of cell culture (Fig. 3, A and B, and data not shown). Thus, CD47 can
signal apoptotic T cell death, and induction of a death response
apparently requires ligation of distinct epitopes on the IgV domain of
CD47.
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AICD of T cells plays a critical role in the regulation of immune
responses. During T cell activation, TCR signals are modulated by the
tyrosine kinase p56lck, which associates with
the cytoplasmic domain of CD4 and CD8 (50, 51, 52). CD45 expression is also
required for p56lck activation and TCR signaling
(50, 53, 54). Furthermore, recent studies demonstrate that ligation of
CD4 and CD45 can induce elimination of T cells by apoptosis (48, 55, 56, 57, 58), and that AICD of Jurkat T cells requires functional
p56lck (59, 60). Thus, as Ad22 rapidly induced
death of Jurkat T cells, we asked whether CD47-induced apoptosis was
related to AICD of T cells and/or required functional CD3, CD4, CD45,
or p56lck. To address these issues, Ad22
responses were assessed using established variant Jurkat T cell lines
selected for deficient expression of TCR/CD3 (clone J.RT3-T3.5), CD4
(clone D1.1), CD45 (clone J45.01), or deficient
p56lck signaling (clone J.CaM1.6). As shown in
Fig. 4, these experiments clearly
demonstrate that Ad22 rapidly induced apoptosis of all the variant
Jurkat clones as determined with annexin V binding and flow cytometry.
In contrast, no cell death was observed with control mAb MOPC-21 or
anti-CD47 mAb 2D3 or B6H12 after 1 or 3 h of culture (Fig. 4
and data not shown). Thus, CD47-induced apoptosis apparently proceeds
independent of the signaling pathways required to initiate AICD of T
cells.
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and data not shown). In contrast, no
significant induction of annexin V binding was observed with a
combination of OKT3 and MOPC-21 or OKT3 and 2D3 (Fig. 5 and data not
shown). Furthermore, OKT3 had no impact on PS exposure in the control
cell line J.RT3-T3.5 (data not shown). Taken together, CD47-induced
apoptosis is apparently independent of early TCR-related signaling, but
coactivation of these pathways enhances the apoptotic response.
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T cell death was observed with even low concentrations of soluble
Ad22, and soluble anti-CD47 mAb 1F7 also induced PS externalization
(Fig. 3
). To further confirm and understand the distinct requirements
for CD47 engagement in T cell death signaling we first examined the
responses of Jurkat E6 to Ad22 following preincubation with excess
anti-CD47 mAb B6H12 or 2D3. Similar experiments with control mAb
MOPC-21 were included. Based on assessments of annexin V binding with
flow cytometry, we found that mAb B6H12, which interferes with Ad22
binding to CD47 (Fig. 2), efficiently blocked Ad22-imposed death
responses (Fig. 6A). Notably,
preincubation with anti-CD47 mAb 2D3, which had no impact on
Ad22-CD47 interactions (Fig. 2), also profoundly reduced death
responses to Ad22 (Fig. 6A). In contrast, MOPC-21 had no
influence on Ad22-induced death of Jurkat E6 cells (Fig. 6A). Thus, restrictions in both Ad22-CD47 interactions and
Ag structure flexibility interfere with death signaling. Furthermore,
we examined the effect of immobilized Ad22, 2D3, and control mAb
MOPC-21 on Jurkat E6 cells cultured for 24 h. These experiments
were based on assessments of [3H]thymidine incorporation,
as immobilized anti-CD47 mAbs induced cell adherence to the plastic
surface of the microtiter wells (data not shown). At all concentrations
examined (1, 5, and 10 µg/ml) we found that immobilized mAb Ad22,
2D3, or MOPC-21 had no influence on Jurkat cell proliferation (data not
shown). Thus, the mode of Ad22 presentation is critical with respect to
functional responses.
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B). Taken together, death signaling by
CD47 apparently requires an unrestricted and flexible Ag structure and
involvement of the actin cytoskeleton. CD47-induced apoptosis is independent of TNFR and Fas signaling
TCR-induced apoptosis can result from TNFRI/p55, TNFRII/p75, and
Fas activation (25, 26, 27). Thus, to assess whether these TNFR superfamily
members and related signaling pathways were involved in CD47-mediated
apoptosis, we first examined the impact of recombinant human TNF-
and TNF-ß (from 10 pg/ml to 1.0 µg/ml in increments of 10-fold) on
Jurkat E6 cells in short term cultures. The TNF-sensitive human
promonocytic cell line U937 was included as a positive control to
confirm cytokine activity. These studies revealed that the cytokines
had no effect on PS exposure on Jurkat E6 cells following 3- and 5-h
cultures (data not shown). In comparison, control cultures with Ad22
responded with extensive PS exposure as previously observed (data not
shown). Cultures with U937 confirmed cytokine activity and revealed
significant death responses with concentrations as low as 10 pg/ml for
TNF- and 100 pg/ml for TNF-ß after 18 h (data not shown).
Flow cytometric assessments revealed low expression of CD120a and
undetectable expression of CD120b on Jurkat E6, while both receptors
were clearly expressed on U937 (data no shown). To further examine
whether Ad22 imposed increased TNF sensitivity to T cells, we performed
experiments to assess the impact of Ad22 on Jurkat E6 cells
preincubated with soluble human recombinant TNFRI/Fc to block TNFR-TNF
interactions or with control IgG (MOPC-21). Control cultures with U937
incubated with recombinant TNFRI/Fc or MOPC-21 and TNF- or TNF-ß
(10 and 100 ng/ml) were also included. We found no interference of
soluble human recombinant TNFRI/Fc with the ability of Ad22 to induce
PS exposure on Jurkat E6 cells after 1 or 3 h of culture (Fig. 7). In contrast, TNFRI/Fc pretreatment of
U937 cells profoundly interfered with TNF-- and TNF-ß-induced cell
death (Fig. 7 and data not shown). Thus, CD47-induced apoptosis
apparently did not proceed by promoting TNF- or TNF-ß interactions
with TNFRs and subsequent activation of the TNFR signaling pathways.
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). Thus, CD47-mediated apoptosis
apparently proceeds without Fas activation.
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and data not shown).
Furthermore, as combined OKT3 and Ad22 treatment showed enhanced
annexin V binding compared with Ad22 alone (Fig. 5), we similarly
examined the impact of CD3 and CD47 costimulation with the TUNEL assay.
These experiments revealed no significant increase in staining
intensity in cultures with both OKT3 and Ad22 compared with that in
cultures with Ad22 alone or in control cultures (Fig. 9 and data not
shown). However, in contrast to Ad22, CH11 clearly induced DNA
fragmentation (Fig. 9). Taken together, CD47-mediated apoptosis
apparently proceeds independently of TNFRs and Fas involvement and does
not involve DNA fragmentation.
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Activation of ICE- and CPP32-like proteases is required as a
downstream event to accomplish programmed cell death following TNFRs
and Fas ligation (62, 63). To address whether ICE and CPP32 activities
also were involved in CD47-mediated apoptosis we assessed the impact of
specific ICE and CPP32 peptide inhibitors on Ad22-induced responses of
Jurkat E6 cells. Control cultures with CH11 (anti-Fas) were also
included. Jurkat E6 cells were preincubated for 1 h with the ICE
inhibitors Ac-Tyr-Val-Ala-Asp-chloromethyl ketone and
Z-Val-Ala-D,L-Asp-fluoromethylketone or the
CPP32 inhibitor Ac-Asp-Glu-Val-Asp-aldehyde and further cultured for 1
or 3 h with Ad22 or MOPC-21 or for 6 h with CH11 or isotype
control mAb TEPC 183. Apoptosis was determined by assessment of annexin
V-FLUOS binding with flow cytometry. These experiments showed that
whereas Fas-mediated apoptosis was greatly suppressed by peptide
inhibitors of ICE and CPP32, the apoptotic response induced by Ad22 was
not significantly influenced by blocking ICE- or CPP32-like activity
(Table I). Thus, CD47-mediated death of T
cells does apparently not require ICE and CPP32 involvement.
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Mature T cells can be eliminated by FasL- or TNF-induced apoptosis
following reactivation of the TCR/CD3 complex (16, 17). Although
CD47-mediated apoptosis seems to proceed independently of these death
pathways, we asked whether resting or anti-CD3-stimulated normal
T cells also responded with cell death when challenged with the Ad22
mAb. To address the role of CD47 signaling in T cells, we determined
the impact of Ad22, 2D3, and control mAb MOPC-21 on unstimulated and
OKT3-stimulated PBMC in short term cultures. In these experiments we
used TruCount tubes containing a known number of fluorescent-dyed
microbeads (counts) to determine relative numbers of lymphocytes
following mAb treatment, with flow cytometry. The region representing
living lymphocytes (R1) was defined, and the number of cells in R1 was
determined following sampling of 2000 reference beads. With seven
different donors, we found comparable numbers of lymphocytes in R1 when
unstimulated PBMC were incubated with Ad22, 2D3, or isotype control mAb
MOPC-21 for 24 h (Fig. 10 and data
not shown). On the average, we found 91.1% (range, 78.0–101.1%) of
the number of cells in R1 with Ad22 and 99.4% (range, 87.9–124.7%)
of the number of cells in R1 with 2D3 compared with similar cultures
with MOPC-21. In contrast, when cells were prestimulated for 18 h
with OKT3 and then further incubated for 6 h with Ad22, we
observed a marked decrease in the number of living lymphocytes compared
with that in similar cultures with MOPC-21 or 2D3 (Fig. 10 and data not
shown). Of 12 different samples of OKT3-stimulated PBMC, 11 responded
to Ad22 with a reduction in the number of lymphocytes compared with
control cultures with MOPC-21. On the average, these donors had 66.9%
(range, 52.1–93.8%) of the number of lymphocytes compared with
similar cultures with MOPC-21. With 2D3, we observed an average of
99.9% (range, 76.1–124%) of the number of cells in R1 compared with
MOPC-21. Student’s t test showed significant responses to
Ad22 compared with MOPC-21 and 2D3 (p < 0.001;
no data overlap) and no impact of 2D3 compared with MOPC-21
(p > 0.5). Furthermore, both activated
CD4+ and CD8+ cells appeared to be sensitive to
Ad22 treatment (data not shown). Thus, activated, but not resting,
normal T cells were sensitive to death signaling by CD47.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies implicate CD47 in diverse biological functions. In this context, a critical role for CD47 in regulating ß3 integrin-dependent migration and activation of PMN has been demonstrated using CD47-deficient mice (64). CD47 may also execute its functions independent of ß3 integrins. Thus, it has recently been shown that coligation of CD3 and CD47 on T cells, which do not express ß3 integrins, may enhance IL-2 production and T cell proliferation independent of CD28 signaling (40, 41, 42). Our studies apparently add induction of T cell apoptosis to the ß3 integrin-independent biologic repertoire of CD47.
The anti-CD47 mAbs B6H12 and 2D3 recognize distinct, nonoverlapping regions of CD47 associated with functional ß3 integrin interactions and T cell activation, respectively (39, 40). Assessments with competitive mAb binding and flow cytometry revealed that Ad22, B6H12, and 1F7 recognize overlapping or adjacent epitopes on the extracellular IgV domain. Thus, these mAbs apparently define a unique region on CD47 associated with functional ß3 integrin association and induction of T cell apoptosis, respectively. However, assessments with Jurkat T cells revealed that Ad22 and 1F7 were unique among the anti-CD47 mAbs tested and were the only mAbs capable of inducing T cell death. Distinct death signaling by CD47 was directly confirmed, as both B6H12 and 2D3 interfered with Ad22 responses. The fact that 2D3, in contrast to B6H12, noncompetitively inhibits the death response to Ad22 suggests that a distinct conformation or conformational change in CD47 is required for death signaling. Furthermore, CD47 signaling appeared to be easily activated by proper stimulation, as low concentrations of Ad22 could initiate death responses, and moreover, the kinetics of PS exposure and PI uptake suggested a rapid transition through the apoptotic (annexin V+/PI-) stage.
T cell susceptibility to different forms of negative signaling depends on the differentiation stage. Developing thymocytes may respond with apoptosis following activation of CD45 (58) and CD99 (49), suggesting a role for these molecules in T cell selection. However, as CD47-deficient mice show normal development of CD4+ and CD8+ T cells (Frederik P. Lindberg, unpublished observations), CD47 has apparently no function in the negative selection of T cells.
CD3, CD45, and the Src kinase p56lck control early events in TCR signaling. Furthermore, CD4 signaling may also be involved in regulating AICD of T cells (55, 56, 57). However, in the context of CD47-mediated apoptosis, we found no requirements for functional CD3, CD4, CD45, or p56lck. These findings suggest that Ad22-induced apoptosis is not controlled by early signaling events associated with TCR signaling. Interestingly, we also excluded the possibility that CD47-induced apoptosis proceeds by functional engagement of CD45, which recently has been implicated in T and B cell apoptosis (48, 58). Notably, coligation of CD47 and CD3 induced a significant increase in Jurkat T cell apoptosis, as judged by annexin V binding. Thus, coactivation of distinct CD47 epitopes and the TCR/CD3 complex may represent a dramatic challenge to proliferating T cells, resulting in unusual rapid elimination of the cells.
TNF- and FasL-mediated apoptosis are recognized as major pathways in AICD of mature T cells (18, 19, 20, 25, 26, 27). However, CD47-induced T cell death signaling proceeded independently of TNFR or Fas ligation, apparently by a novel signaling pathway. In this context, programmed cell death may proceed with or without DNA fragmentation. Thus, Fas-mediated apoptosis proceeds with DNA fragmentation into oligonucleosomes (47), whereas CD45, CD99, and HLA class I-mediated apoptosis do not involve observable DNA fragmentation (48, 49, 58, 65, 66). Comparison between Ad22- and CH11-induced apoptosis using the TUNEL assay further demonstrated distinctions between the CD47- and Fas-mediated apoptotic pathways, as Ad22 imposed no characteristic DNA fragmentation even when cells were costimulated with OKT3. Another distinct feature of Fas-mediated apoptosis is the requirement for ICE- and CPP32-like proteases (62), which were not needed in CD47-induced T cell death. In contrast, Ad22-induced T cell death revealed close resemblance to the characteristic apoptosis associated with distinct CD45 and HLA class I signaling (48, 65, 66).
Whereas substantial information has recently been achieved on how Fas and TNFR signaling activate the cell’s death program, the basis for apoptosis proceeding without caspase or DNA endonuclease activation is not clear, but these novel pathways may represent optional, fast death strategies for efficient clearance of activated cells. Notably, T cells require days to develop susceptibility to Fas and TNFR-mediated cell death (18, 23, 24, 26). Thus, alternative death pathways, as represented by CD45 (48, 58) and CD47, may be required to control FasL- and TNF-insensitive T cells. Furthermore, as some virus may interfere with caspase activation and thereby the induction of conventional programmed cell death (67, 68, 69), the immune system may also depend on optional caspase-independent apoptotic pathways to challenge these infections (66). Notably, activation of Fas-independent and nonclassical apoptosis has also been implicated in HIV-mediated depletion of CD4+ T cells (70, 71).
In three recent studies purified T cells and immobilized anti-CD3 and anti-CD47 mAbs were used to address the role of CD47 on normal T cells (40, 41, 42). The reports demonstrate the ability of CD47 to costimulate T cell activation provided that the mAbs are presented immobilized on the same surface. In contrast, addition of soluble anti-CD47 mAbs had no stimulatory effect even if they were cross-linked. Furthermore, high doses of soluble anti-CD47 mAb 1/1A4 interfered with T cell proliferation after stimulation with suboptimal numbers of dendritic cells (42). However, 1/1A4 did not influence allogeneic MLR induced by optimal numbers of dendritic cells (42). Our studies with Jurkat cells revealed that CD47-mediated death signaling apparently requires an unrestricted and flexible Ag structure. The impacts of Ad22 and CD47 signaling on normal T cells were addressed using PBMC, as these conditions include cosignaling provided by the normal, hemopoietic cellular environment, including CD28 costimulation. While Ad22 disclosed no impact on unstimulated T cells, preactivation with OKT3 induced a marked T lymphocyte sensitivity to short term Ad22 treatment. Thus, CD47, like Fas and TNFRs, seem to share the feature that expression of the respective Ags is not sufficient to trigger apoptosis, but depend on preactivation to signal cell death. Taken together, the outcome of CD47 engagement on normal T cells is apparently determined by their activation status and the specific CD47 region activated.
CD47 ligands inducing T cell activation or apoptosis have not been identified. However, the Ad22 and 1F7 binding site seems to be exposed at the IgV domain of CD47 near the B6H12 epitopes, suggesting easy access for ligands presented or released by other cells as well as interactions with molecules expressed on the same T cell. Activated T cells induced to express a functional CD47L or encountering CD47L presented on APC would expectedly be efficiently eliminated. Recent reports demonstrate that transformed cells may use a similar strategy for immune escape by expressing FasL (72, 73).
In conclusion, we have found that ligation of distinct CD47 epitopes rapidly induces TNFR- and Fas-independent death of activated T cells. This may indicate that CD47 has a physiological role in clearance of activated T cells by a novel apoptotic pathway. Thus, it will be important to provide further understanding of the involvement of CD47 in the regulation of immune responses.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rolf D. Pettersen, Department of Pediatric Research, Rikshospitalet, The National Hospital, N-0027 Oslo, Norway. E-mail address:
3 Abbreviations used in this paper: AICD, activation-induced apoptosis; FasL, Fas ligand; TNFRI, TNF receptor type I; TNFRII, TNF receptor type II; IgV, Ig variable; IAP, integrin-associated protein; ICE, IL-1ß-converting enzyme; PS, phosphatidylserine; PI, propidium iodide.
Received for publication July 6, 1998. Accepted for publication March 29, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3-dependent ligand binding. J. Cell Biol. 123:485.2 domain in regulation of cell adhesion and proliferation. J. Immunol. 156:1415.[Abstract]
-induced apoptosis in STAT1-null cells due to low constitutive levels of caspases. Science 278:1630.2 domain signals rapid Fas-independent cell death: a direct pathway for T cell-mediated killing of target cells?. J. Immunol. 160:4343.This article has been cited by other articles:
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  F. Guimont-Desrochers, C. Beauchamp, G. Chabot-Roy, V. Dugas, E. E. Hillhouse, J. Dusseault, G. Langlois, P. Gautier-Ethier, J. Darwiche, M. Sarfati, et al. Absence of CD47 in vivo influences thymic dendritic cell subset proportions but not negative selection of thymocytes Int. Immunol., February 1, 2009; 21(2): 167 - 177. [Abstract] [Full Text] [PDF]
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 T. Tretter, R. K. C. Venigalla, V. Eckstein, R. Saffrich, S. Sertel, A. D. Ho, and H.-M. Lorenz Induction of CD4+ T-cell anergy and apoptosis by activated human B cells Blood, December 1, 2008; 112(12): 4555 - 4564. [Abstract] [Full Text] [PDF]
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 J. S. Isenberg, D. D. Roberts, and W. A. Frazier CD47: A New Target in Cardiovascular Therapy Arterioscler. Thromb. Vasc. Biol., April 1, 2008; 28(4): 615 - 621. [Abstract] [Full Text] [PDF]
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 I. Can, S. Tahara-Hanaoka, K. Hitomi, T. Nakano, C. Nakahashi-Oda, N. Kurita, S.-i. Honda, K. Shibuya, and A. Shibuya Caspase-Independent Cell Death by CD300LF (MAIR-V), an Inhibitory Immunoglobulin-Like Receptor on Myeloid Cells J. Immunol., January 1, 2008; 180(1): 207 - 213. [Abstract] [Full Text] [PDF]
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 M. Bras, V. J. Yuste, G. Roue, S. Barbier, P. Sancho, C. Virely, M. Rubio, S. Baudet, J. E. Esquerda, H. Merle-Beral, et al. Drp1 Mediates Caspase-Independent Type III Cell Death in Normal and Leukemic Cells Mol. Cell. Biol., October 15, 2007; 27(20): 7073 - 7088. [Abstract] [Full Text] [PDF]
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L. Lamy, A. Foussat, E. J. Brown, P. Bornstein, M. Ticchioni, and A. Bernard Interactions between CD47 and Thrombospondin Reduce Inflammation J. Immunol., May 1, 2007; 178(9): 5930 - 5939. [Abstract] [Full Text] [PDF]
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 P. P. Manna, J. Dimitry, P.-A. Oldenborg, and W. A. Frazier CD47 Augments Fas/CD95-mediated Apoptosis J. Biol. Chem., August 19, 2005; 280(33): 29637 - 29644. [Abstract] [Full Text] [PDF]
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A. Saumet, M. B. Slimane, M. Lanotte, J. Lawler, and V. Dubernard Type 3 repeat/C-terminal domain of thrombospondin-1 triggers caspase-independent cell death through CD47/{alpha}v{beta}3 in promyelocytic leukemia NB4 cells Blood, July 15, 2005; 106(2): 658 - 667. [Abstract] [Full Text] [PDF]
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G. Brooke, J. D. Holbrook, M. H. Brown, and A. N. Barclay Human Lymphocytes Interact Directly with CD47 through a Novel Member of the Signal Regulatory Protein (SIRP) Family J. Immunol., August 15, 2004; 173(4): 2562 - 2570. [Abstract] [Full Text] [PDF]
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 M. Miyashita, H. Ohnishi, H. Okazawa, H. Tomonaga, A. Hayashi, T.-T. Fujimoto, N. Furuya, and T. Matozaki Promotion of Neurite and Filopodium Formation by CD47: Roles of Integrins, Rac, and Cdc42 Mol. Biol. Cell, August 1, 2004; 15(8): 3950 - 3963. [Abstract] [Full Text] [PDF]
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 P. P. Manna and W. A. Frazier CD47 Mediates Killing of Breast Tumor Cells via Gi-Dependent Inhibition of Protein Kinase A Cancer Res., February 1, 2004; 64(3): 1026 - 1036. [Abstract] [Full Text] [PDF]
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K. C. Jung, W. S. Park, H. J. Kim, E. Y. Choi, M.-C. Kook, H.-W. Lee, and Y. Bae TCR-Independent and Caspase-Independent Apoptosis of Murine Thymocytes by CD24 Cross-Linking J. Immunol., January 15, 2004; 172(2): 795 - 802. [Abstract] [Full Text] [PDF]
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L. Lamy, M. Ticchioni, A. K. Rouquette-Jazdanian, M. Samson, M. Deckert, A. H. Greenberg, and A. Bernard CD47 and the 19 kDa Interacting Protein-3 (BNIP3) in T Cell Apoptosis J. Biol. Chem., June 20, 2003; 278(26): 23915 - 23921. [Abstract] [Full Text] [PDF]
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 B. Murphy, J. Yu, Q. Jiao, M. Lin, T. Chitnis, and M. H. Sayegh A Novel Mechanism for the Immunomodulatory Functions of Class II MHC-Derived Peptides J. Am. Soc. Nephrol., April 1, 2003; 14(4): 1053 - 1065. [Abstract] [Full Text] [PDF]
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P. P. Manna and W. A. Frazier The Mechanism of CD47-Dependent Killing of T Cells: Heterotrimeric Gi-Dependent Inhibition of Protein Kinase A J. Immunol., April 1, 2003; 170(7): 3544 - 3553. [Abstract] [Full Text] [PDF]
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K. N. Dahl, C. M. Westhoff, and D. E. Discher Fractional attachment of CD47 (IAP) to the erythrocyte cytoskeleton and visual colocalization with Rh protein complexes Blood, February 1, 2003; 101(3): 1194 - 1199. [Abstract] [Full Text] [PDF]
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H. O. Barazi, Z. Li, J. A. Cashel, H. C. Krutzsch, D. S. Annis, D. F. Mosher, and D. D. Roberts Regulation of Integrin Function by CD47 Ligands. DIFFERENTIAL EFFECTS ON alpha vbeta 3 AND alpha 4beta 1 INTEGRIN-MEDIATED ADHESION J. Biol. Chem., November 1, 2002; 277(45): 42859 - 42866. [Abstract] [Full Text] [PDF]
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V. Mateo, E. J. Brown, G. Biron, M. Rubio, A. Fischer, F. L. Deist, and M. Sarfati Mechanisms of CD47-induced caspase-independent cell death in normal and leukemic cells: link between phosphatidylserine exposure and cytoskeleton organization Blood, September 26, 2002; 100(8): 2882 - 2890. [Abstract] [Full Text] [PDF]
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Y. Liu, H.-J. Buhring, K. Zen, S. L. Burst, F. J. Schnell, I. R. Williams, and C. A. Parkos Signal Regulatory Protein (SIRPalpha ), a Cellular Ligand for CD47, Regulates Neutrophil Transmigration J. Biol. Chem., March 15, 2002; 277(12): 10028 - 10036. [Abstract] [Full Text] [PDF]
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Y. Liu, D. Merlin, S. L. Burst, M. Pochet, J. L. Madara, and C. A. Parkos The Role of CD47 in Neutrophil Transmigration. INCREASED RATE OF MIGRATION CORRELATES WITH INCREASED CELL SURFACE EXPRESSION OF CD47 J. Biol. Chem., October 19, 2001; 276(43): 40156 - 40166. [Abstract] [Full Text] [PDF]
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M.-N. Avice, M. Rubio, M. Sergerie, G. Delespesse, and M. Sarfati Role of CD47 in the Induction of Human Naive T Cell Anergy J. Immunol., September 1, 2001; 167(5): 2459 - 2468. [Abstract] [Full Text] [PDF]
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 B. R. Blazar, F. P. Lindberg, E. Ingulli, A. Panoskaltsis-Mortari, P.-A. Oldenborg, K. Iizuka, W. M. Yokoyama, and P. A. Taylor CD47 (Integrin-associated Protein) Engagement of Dendritic Cell and Macrophage Counterreceptors Is Required to Prevent the Clearance of Donor Lymphohematopoietic Cells J. Exp. Med., August 20, 2001; 194(4): 541 - 550. [Abstract] [Full Text] [PDF]
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J. E. Brittain, K. J. Mlinar, C. S. Anderson, E. P. Orringer, and L. V. Parise Integrin-associated protein is an adhesion receptor on sickle red blood cells for immobilized thrombospondin Blood, April 1, 2001; 97(7): 2159 - 2164. [Abstract] [Full Text] [PDF]
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 S. R. Goth and R. S. Stephens Rapid, Transient Phosphatidylserine Externalization Induced in Host Cells by Infection with Chlamydia spp. Infect. Immun., February 1, 2001; 69(2): 1109 - 1119. [Abstract] [Full Text] [PDF]
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 M. TICCHIONI, V. RAIMONDI, L. LAMY, J. WIJDENES, F. P. LINDBERG, E. J. BROWN, and A. BERNARD Integrin-associated protein (CD47/IAP) contributes to T cell arrest on inflammatory vascular endothelium under flow FASEB J, February 1, 2001; 15(2): 341 - 350. [Abstract] [Full Text] [PDF]
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M.-N. Avice, M. Rubio, M. Sergerie, G. Delespesse, and M. Sarfati CD47 Ligation Selectively Inhibits the Development of Human Naive T Cells into Th1 Effectors J. Immunol., October 15, 2000; 165(8): 4624 - 4631. [Abstract] [Full Text] [PDF]
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