By
From the * Laboratory of Cellular Physiology and Immunology, The Rockefeller University,
New York 10021; and the Division of Hematology-Oncology, Cornell University Medical
College, New York 10021
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Abstract |
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Dendritic cells, but not macrophages, efficiently phagocytose apoptotic cells and cross-present
viral, tumor, and self-antigens to CD8+ T cells. This in vitro pathway corresponds to the in
vivo phenomena of cross-priming and cross-tolerance. Here, we demonstrate that phagocytosis
of apoptotic cells is restricted to the immature stage of dendritic cell (DC) development, and
that this process is accompanied by the expression of a unique profile of receptors, in particular
the v
5 integrin and CD36. Upon maturation, these receptors and, in turn, the phagocytic capacity of DCs, are downmodulated. Macrophages engulf apoptotic cells more efficiently than
DCs, and although they express many receptors that mediate this uptake, they lack the
v
5 integrin. Furthermore, in contrast to DCs, macrophages fail to cross-present antigenic material
contained within the engulfed apoptotic cells. Thus, DCs use unique pathways for the phagocytosis, processing, and presentation of antigen derived from apoptotic cells on class I major
histocompatibility complex. We suggest that the
v
5 integrin plays a critical role in the trafficking of exogenous antigen by immature DCs in this cross-priming pathway.
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Introduction |
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poptosis triggers a distinct sequence of events characterized by the expression of phosphatidylserine (PS)1 on the cell surface (1), DNA fragmentation or laddering (2), and the release of membrane-bound cell fragments called apoptotic blebs and bodies (3, 4). Apoptotic cells and bodies are phagocytosed via various receptors that recognize PS and other undefined ligands unique to the surface of apoptotic material (1). In this way, dying cells, which contain potentially inflammatory factors, are rapidly cleared by neighboring cells, scavenger cells, or macrophages without inducing an inflammatory response (5, 6).
We have recently demonstrated that human dendritic cells (DCs) acquire antigens from apoptotic cells and stimulate antigen-specific class I-restricted CD8+ T cells (7). Apoptotic death is a critical trigger for this pathway, as antigen from necrotic cells is not presented on MHC I (7). This in vitro model for cross-priming may be akin to the in vivo phenomenon in which antigens derived from MHC-mismatched donor cells are cross-presented on host APC's class I MHC (8). This event is potentially significant in the maintenance of tolerance to tissue-specific antigens and the induction of immunity to antigens that may not access the endogenous MHC class I pathway of a professional APC.
Here, we investigate the developmental stage and receptors used by DCs for the efficient phagocytosis of apoptotic
cells in this exogenous pathway. DCs undergo a differentiation process that includes `immature' and `mature' stages
(11). Monocytes that have been cultured in GM-CSF and
IL-4 develop into immature DCs. These cells are analogous
to peripheral tissue DCs. Immature DCs are characterized by
high endocytic and macropinocytic activity (12) and low
expression of accessory signals for T cell activation (11, 17).
Maturation of DCs, induced by antigen, cytokines, or signaling molecules (e.g., LPS, monocyte-conditioned medium [MCM], ceramide, CD40L, TNF-, and PGE2 [12, 13, 18-
22]) is associated with the downregulation of antigen uptake,
but an enhancement of the T cell stimulatory capacity. By
using a modified FACS®-based method for detecting phagocytic uptake, we compared immature DCs, mature DCs, and
macrophages for their capacity to phagocytose apoptotic cells
and cross-present viral antigens to CTLs (23). We report that
immature DCs are specialized in their ability to cross-present
antigen and that the phagocytosis of apoptotic cells correlates
with the use of a unique receptor profile.
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Materials and Methods |
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Media.
RPMI 1640 supplemented with 20 µg/ml of gentamicin (GIBCO BRL, Gaithersburg, MD), 10 mM Hepes (Mediatech, Herndon, VA), and either 1% human plasma, 5% pooled human serum (c-Six Diagnostics, Mequon, WI), or 5% single donor human serum was used for DC preparation, cell isolation, and culture conditions (18, 24).Preparation of Cells.
PBMCs, DCs, macrophages, and T cells were prepared as previously described (18, 19, 24). In brief, peripheral blood was obtained from normal donors in heparinized syringes and PBMCs were isolated by sedimentation over Ficoll-Hypaque (Amersham Pharmacia Biotech, Piscataway, NJ). T cell-enriched and T cell-depleted fractions were prepared by rosetting with neuraminidase-treated sheep red blood cells (24). Immature DCs were prepared from the T cell-depleted fraction by culturing cells in the presence of GM-CSF and IL-4 for 7 d. 1,000 U/ml of GM-CSF (Immunex Corp., Seattle, WA) and 500-1,000 U/ml of IL-4 (Schering-Plough Corp., Kenilworth, NJ) were added to the cultures on days 0, 2, and 4. To generate mature DCs, the cultures were transferred to fresh wells on day 7 and MCM was added for an additional 3-4 d (18, 19). At day 7, >95% of the cells were CD14Antibodies.
Antibodies to the following proteins were used: CD8-PE, CD14-PE, HLA-DR-PE, HLA-DR-biotin (Becton Dickinson), IgG2b (clone 6603001; Coulter Corp., Hialeah, FL), CD8 (CRL 8014; American Type Culture Collection, Rockville, MD), CD83 (clone HB15a; Coulter Corp.), MHC I (W6/ 32, ATCC clone HB95), CD36 (clone FA6; obtained from the fifth international workshop on leukocyte differentiation antigens),Induction of Apoptotic Death.
Monocytes were infected with influenza virus in serum-free RPMI. These cells undergo viral-induced apoptotic death within 6-8 h. Cell death was confirmed using the Early Apoptosis Detection kit (Kayima Biomedical Co., Seattle, WA [7]). As previously described, cells are stained with Annexin V-FITC (Ann V) and propidium iodide (PI). Early apoptosis is defined by Ann V+/PIPhagocytosis of Apoptotic Cells.
Monocytes and HeLa cells were dyed red using PKH26-GL (Sigma Biosciences, St. Louis, MO), and induced to undergo apoptosis by influenza infection and UVB irradiation, respectively. After 6-8 h, allowing time for the cells to undergo apoptosis, they were cocultured with phagocytic cells that were dyed green using PKH67-GL (Sigma Biosciences), at a ratio of 1:1. Macrophages were used 3-6 d after isolation from peripheral blood; immature DCs were used on days 6-7 of culture; and mature DCs were used on days 10-11. Where direct comparison of cells was needed, cells were prepared from the same donor on different days. In blocking experiments, the immature DCs were preincubated in the presence of 50 µg/ml of various mAbs for 30 min before the establishment of cocultures. After 45-120 min, FACScan® analysis was performed and double positive cells were enumerated.Phagocytosis of Latex Beads.
Immature DCs were preincubated at 37°C with mAbs specific forImmunofluorescence.
Cells were adhered to Alcian blue (Sigma Chemical Co.) treated coverslips and fixed using 100% acetone. Cells were stained with antiinfluenza nucleoprotein antibody (HB85; American Type Culture Collection) and Texas red conjugated goat anti-mouse IgG (Jackson ImmunoResearch Labs., Inc., West Grove, PA). This was followed by staining with biotinylated anti-HLA-DR (Becton Dickinson) followed by FITC-conjugated streptavidin (Jackson ImmunoResearch). Cells were visualized using a Zeiss Axioplan2 microscope (Carl Zeiss, Inc., Thornwood, NY).Assay for Cross-priming of Apoptotic Cells.
Various APC populations were prepared from HLA-A2.1+ donors as described above. Mature DCs were further purified by labeling with the DC-restricted marker CD83, followed by cell sorting on the FACSort®. Immature DCs were CD14Reverse Transcriptase PCR.
RNA was purified from highly purified sorted cell populations of immature and mature DCs as described above. Messenger RNA for ![]() |
Results |
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Based on previous observations that immature DCs are the cells responsible for capturing antigen (11), we predicted that apoptotic cells would be engulfed best by immature DCs. To test this hypothesis, we established a phagocytosis assay that allowed us to visually detect the uptake of apoptotic cells, and compare the phagocytic capacity of immature DCs, mature DCs, and macrophages. In brief, immature DCs were prepared by culturing a T cell-depleted fraction from peripheral blood in the presence of IL-4 and GM-CSF. Mature DCs were generated with the addition of MCM and these cells expressed the cell surface DC-restricted maturation marker CD83 (18, 19, 30). Macrophages were prepared by culturing a plastic adherent cell population in Teflon beakers for 3-9 d. As a source of apoptotic cells, we used influenza-infected monocytes (7); virus infection induces apoptotic death in these cells within 6-10 h (7, 25, 26). Monocytes were first infected with influenza virus as previously described (24), then dyed red using PKH-26 (Sigma Biosciences). After 6-8 h, the various APCs were dyed green using the fluorescent cell linker compound PKH67-GL (Sigma Biosciences) and cocultured with the apoptotic cells at a ratio of 1:1. After 2 h at 37°C, cocultures of cells were analyzed by FACScan® analysis, allowing for quantification of phagocytic uptake as double positive cells. 80% of the macrophages, 50% of the immature DCs, and <10% of the mature DCs engulfed the apoptotic monocytes after 2 h of coculture (Fig. 1 A). The smear of double positive cells (PKH67-labeled APCs that engulfed the PKH26-labeled apoptotic cells) indicates that both apoptotic bodies and whole apoptotic cells served as `food' for the phagocytic cell (Fig. 1, iii, vi, and ix). Note that as the forward scatter of the APCs increased and the setting of the FACS® shifted, the dying monocytes were excluded from the established region (Fig. 1, ii, v, and viii). Maximal uptake by all APC populations was achieved within 2-4 h and partially depended upon the source of apoptotic cell used (Fig. 1 B and data not shown). Given this kinetic data, we believe that macrophages and DCs engage and internalize dying cells while still displaying features of early apoptotic cell death. This data also demonstrates that it is the immature DC that preferentially acquires apoptotic material compared with the mature DC. The source of apoptotic cells was not critical, since we obtained similar results with UVB-irradiated HeLa cells (see Fig. 7, and data not shown).
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To confirm that this FACS® assay was measuring phagocytosis, we carried out the assay at 4°C and in the presence of inhibitors of phagocytosis. Both low temperature (Fig. 2 A) and cytochalasin D, an inhibitor of cytoskeletal function, blocked uptake (Fig. 2 B). Phagocytosis by immature DCs also requires divalent cations as EDTA was inhibitory (Fig. 2 C). To visually confirm the uptake recorded by FACS®, we prepared cytospins of the dyed cocultures. The frequency of uptake correlated with that measured on FACS® (data not shown). We also performed immunofluorescence on cocultures of immature DCs labeled with anti- HLA-DR (DR) and apoptotic influenza-infected monocytes labeled with antiinfluenza nucleoprotein (NP) (Fig. 3). In the top panel an apoptotic cell is seen just prior to being engulfed by a DC (arrowhead). After phagocytosis, apoptotic cells were found in DR+ vesicles (arrows), but not in the cytoplasm.
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We next correlated the phagocytic
capability of macrophages and DCs with their ability to
cross-present antigenic material derived from apoptotic
cells. The cells were prepared from HLA-A2.1+ donors
(18, 19), cocultured with HLA-A2.1 influenza-infected
monocytes for 12 h, and then loaded with Na51CrO4 for
use as targets for influenza-specific CTLs (7, 24). Specific
lysis indicates that the APCs cross-presented antigenic material derived from the apoptotic cell by forming specific
peptide-MHC class I complexes on its surface (Fig. 4 A).
As a direct comparison with the endogenous pathway for
class I MHC presentation, the same APC populations were
infected with live influenza virus and used as targets (Fig. 4 B).
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Although mature DCs were efficient targets when infected with influenza, they were unable to cross-present antigens, presumably because they had downregulated the ability to phagocytose the apoptotic monocytes. However, the immature DCs did cross-present antigens from apoptotic cells. Furthermore, if the immature DCs were cocultured with the apoptotic cells in the presence of MCM, a maturation stimulus, they were even better targets. This is possibly due to the upregulation of costimulator and adhesion molecules (13, 31), or to the increased stability of peptide-MHC I complexes. Given that maximal uptake of apoptotic cells by immature DCs occurs between 2 and 4 h (Fig. 1 D), we believe that cross-presentation of apoptotic material reflects the phagocytosis and processing of early apoptotic cells rather than secondary necrotic cells (see Materials and Methods). With respect to this issue, it is important to recognize that the influenza-infected monocytes require 24 h to undergo secondary necrosis (Albert, M.L., and N. Bhardwaj, unpublished data; references 25, 26).
Notably, macrophages that efficiently phagocytose apoptotic cells (Fig. 1 A) did not cross-present antigens to CTLs (Fig. 4 B). Presumably, the engulfed material is degraded, not cross-presented, on MHC I. This profound difference between the DC and macrophage populations is supported by our previous findings that macrophages do not cross-present antigens from apoptotic cells during the induction phase of a class I-restricted antigen-specific T cell response. In fact, when put into culture with DCs in a competition assay, they sequester the apoptotic material and abrogate the CTL response (7).
Immature DCs Can Be Distinguished from Macrophages by Intracellular Expression of CD83 and a Unique Profile of Phagocytic Receptors.We investigated the possibility that immature DCs might phagocytose apoptotic cells via pathways distinct from macrophages. To clearly distinguish these cells, we characterized them phenotypically. Immature DCs are distinguished by the absence of both CD14, a macrophage restricted marker, and CD83, a maturation marker for DCs (30). We have extended the use of CD83, finding that immature DCs can be distinguished from both macrophages and mature DCs by their intracellular expression of CD83. Macrophages do not express CD83 intra- or extracellularly, whereas mature DCs express CD83 both intra- and extracellularly (Fig. 5).
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These APC populations were examined for surface expression of receptors involved in phagocytosing apoptotic
material (Table 1). These include: v
3 and CD36, which
act as coreceptors for engulfment of apoptotic neutrophils
and lymphocytes by macrophages (32, 33); and CD14,
which has been implicated in the uptake of apoptotic cells
by macrophages (34). While studying the immature DC
populations, we identified a discrepancy in the expression of the
v and
3 integrin chains and investigated the possibility that
v was binding an alternate
chain. Using antibodies that recognize combined epitopes of the
v
3 and
the
v
5 heterodimers, we noted the selective expression of
v
5 on immature DCs (Fig. 6 A). As is true for most
receptors involved in antigen uptake (11, 12), the expression of CD36,
v
5, and mannose receptor on DCs is
downregulated with maturation (Fig. 6 B, Table 1).
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To evaluate whether this downregulation could be observed on the level of mRNA expression, we performed
reverse transcriptase PCR using primers specific for 3,
5,
and CD36 (Fig. 6 C). Immature DCs (lane 1) showed amplified DNA of the appropriate size for
3,
5, and CD36.
In contrast, in mature DCs (lane 2), no
5 and much fewer
CD36 sequences were seen, whereas
3 sequences were comparable to those in immature cells. These data, although not quantitative, are consistent with the levels of
protein expression observed by FACS® and suggest that
phagocytic receptor expression in DCs may be regulated at
a transcriptional level as mRNA expression of CD36 and
5 is downregulated during maturation.
To demonstrate a direct role for v
5 in
the recognition of apoptotic cells by immature DCs, we
performed the phagocytosis FACS® assay in the presence of
antibodies specific for
v
5 (Fig. 7 A). In addition to the
blocking observed using the mAb to
v
5, blocking was
also detected when using mAbs to
v,
5, and CD36. Blocking was not observed when isotype-matched mAbs
were specific for
1,
3, or the transferrin receptor CD71.
Note that control antibodies chosen recognized surface receptors present on the immature DCs (Fig. 7 A, Table 1).
mAbs were tested in doses ranging from 10 to 80 µg/ml
(data not shown). Maximal inhibition of phagocytosis of
apoptotic cells was seen with mAbs specific for CD36,
v,
and
5 at 50 µg/ml. The inhibition of phagocytosis of apoptotic cells by DCs was specific. We were unable to block
the uptake of red fluorescent latex beads, a control particle,
by DCs in the presence of these mAbs (Fig. 7 B). By histogram analysis, DCs phagocytose 1-6 particles per cell.
mAbs to
v
5 or
v did not alter the profile of these histogram plots (data not shown).
Although some inhibition of phagocytosis was observed
when using v
3 this may be due in part to transdominance
and/or the effect on the pool of free
v (35). For example,
anti-
v
3 antibodies suppress the intracellular signaling of
the
5
1 integrin (36). Alternatively,
v
3 and
v
5 may be
working cooperatively in the immature DCs. We therefore
tested combinations of anti-
v
3 and anti-
v
5 but did not
observe an increase in the inhibition of phagocytosis. The
low receptor density of
v
3 on DCs (average mean fluorescence intensity of 7 ± 2; Table 1) also makes it unlikely
that this integrin heterodimer is involved in the engulfment of apoptotic cells by immature DCs.
Our data do not exclude a role for other receptors in the
phagocytosis of apoptotic cells, e.g., the putative PS receptor or the lectin receptor (5). In fact, other receptors are
probably involved, as blocking observed did not exceed
60% even when combinations of all relevant mAbs were
tested (data not shown). CD14 is unlikely to be involved in
the engulfment of apoptotic cells by DCs, as DCs do not
express this receptor (Table 1). In macrophages, phagocytosis of apoptotic cells was inhibited by antibodies to v,
3,
v
3, and CD36 but not by antibodies to
1,
5, or
v
5 (data not shown). This correlates with published data
(6, 33).
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Discussion |
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Cross-presentation of antigens to CTLs appears to have two critical features: (a) it is mediated by DCs, and (b) apoptotic cells are the preferred source of antigen (7). The requisite stage of DC development for the acquisition of apoptotic cells is the immature phase. In fact, immature DCs are four to five times more efficient than mature DCs in phagocytosis, a feature that also correlates with their ability to cross-present antigen. This exogenous pathway for class I MHC loading is highly effective: relatively few apoptotic cells (ratio of 1:10 DCs) are needed to charge the DCs as efficiently as the live replicating virus; exposure of 3-12 h is sufficient for generating a peptide-MHC complex that is capable of activating CTLs; and it is relatively indiscriminate, as the cellular source can be allogeneic or xenogeneic cells (7). We believe our earlier studies with mature DCs are explained by the fact that our cell populations were asynchronous and that only by sorting these cells have the differences become apparent. Based on the findings presented here, we suggest that the peripheral tissue DC, exemplified by the immature DC, has an additional important role. It is responsible for phagocytosing cells within tissues that undergo apoptosis (e.g., secondary to viral infection; during normal cell turnover) and migrating to the draining lymph nodes where appropriate T cells are engaged. This pathway may be used for stimulating or tolerizing CTLs and can account for the in vivo observations of cross-priming of tumor and viral antigens (9, 37) and cross-tolerance of self-proteins (10, 38) in their requirement for a bone marrow-derived APC.
A sharp distinction was also demonstrated between immature DCs and macrophages in the handling of apoptotic material. Although macrophages are more efficient at phagocytosing apoptotic cells than immature DCs, they fail to induce virus-specific CTLs (7). In fact, they cannot even generate effective levels of peptide-MHC I complexes. In a short-term assay, influenza-specific CTLs could not kill macrophages cocultured with apoptotic cells. Therefore, macrophages degrade rather than cross-present the ingested apoptotic cells. Our findings probably do not conflict with the report of Bellone et al. (39), as their `macrophages' were prepared from bone marrow-derived precursors by culturing the cells in GM-CSF for 7 d. This method is traditionally used to generate DCs from bone marrow (40). We believe that contaminating DCs account for the cross-presentation observed in their studies.
Additional evidence exists that macrophages process apoptotic cells differently from DCs and prevent an immune response. Two groups have demonstrated that phagocytosis
of apoptotic cells suppresses a subsequent inflammatory response to LPS stimulation. The macrophage's cytokine
profile is skewed toward the synthesis of IL-10, IL-13, and
TGF-, whereas the production of proinflammatory cytokines such as TNF-
, IL-1
, and IL-12 is downmodulated (41, 42). Therefore, the resolution of inflammation is
dependent on at least two pathways for removal of apoptotic cells: via (a) macrophages, which subvert and suppress
proinflammatory responses, and (b) DCs, which stimulate
T cell responses that clear pathogens responsible for the induction of the apoptotic death.
The v
5 integrin receptor may be pivotal in the distinctive handling of apoptotic cells by immature DCs versus
macrophages, in that its expression is restricted to the
former. We suggest that the unique profile of receptors expressed by immature DCs affects trafficking of phagocytosed apoptotic cells, and consequently facilitates cross-presentation. We have previously shown that NH4Cl inhibits
the ability of DCs to process antigen derived from apoptotic cells, suggesting that processing in an acidic vesicle (e.g., CIIVs or MIICs) is required (7). Indeed, class I MHC may interact with processed antigens in such a compartment, as MHC I molecules have been described in association with invariant chain (43) and can recycle from the cell
surface to class II vesicles (44). Additionally, there may be
as yet undescribed routes whereby antigens within vesicles
can enter the classical endogenous pathway as described recently for antigens derived from the endoplasmic reticulum
(45).
In contrast to our studies, Rubartelli et al. (46) have
shown that immature DCs express high levels of the v
3
integrin but lack CD36. Their observations are hard to reconcile with data that indicates that
v
3 and CD36 are both
required for the engulfment of apoptotic cells by macrophages (33, 47). Furthermore, although they demonstrated
inhibition of phagocytosis by DCs with anti-
v antibodies,
they did not present phagocytosis data relevant to
3 or the
v
3 heterodimer. However, it is possible that a different process is being studied, as phagocytosis in their hands is
dependent on late stage Ann V+/PI+ apoptotic cells. Here,
we discuss data relevant to the phagocytosis of early apoptotic cells with intact plasma membranes.
v
5 and
v
3 have both been described as important in
angiogenesis, cell adhesion, migration, and now in their
ability to phagocytose apoptotic cells.
v
3 is critical in the
phagocytosis of apoptotic cells in macrophages, where it
acts in a cooperative way with CD36 and thrombospondin,
collectively forming a `molecular bridge' (47). Recently, it
was reported that
v
5 but not
v
3 is critical for the engulfment of rod outer segments by CD36+ retinal pigment
epithelial cells (48, 49). This phagocytic system is also inhibited by anti-CD36 antibodies, suggesting that
v
5, like
v
3, might cooperate with CD36. Taken together with our observations, thrombospondin, or possibly other soluble factors, may serve to bridge CD36,
v
5, and the apoptotic cell.
Although similarities in function exist, v
5 can be distinguished from
v
3 in its use of various ligands (e.g., vascular endothelial growth factor [VEGF] vs. basic fibroblast
growth factor [bFGF]), by the requirements for activation,
and by the intracellular signaling pathways (e.g., indirect
activation of protein kinase C) (50). Also significant is the
fact that the cytoplasmic domains of the two
chains are
the portions that show the most considerable diversity (51).
Thus, it is possible that the distinct use of the
v
5 versus
v
3 integrin receptors might account for the specialized
functions of DCs in the route by which apoptotic material is trafficked and presented. In other words, differential expression of
v
5 may be responsible for the ability of DCs
to cross-present antigenic material derived from apoptotic
cells, whereas macrophages scavenge and degrade such material.
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Footnotes |
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Address correspondence to Nina Bhardwaj, Laboratory of Cellular Physiology and Immunology, The Rockefeller University, 1230 York Ave., New York, NY 10021. Phone: 212-327-7597; Fax: 212-327-8875; E-mail: bhardwn{at}rockvax.rockefeller.edu
Received for publication 23 June 1998 and in revised form 11 August 1998.
We thank Drs. R. Steinman and R. Darnell for advice and critical review of the manuscript; M. Levin for
assistance with figures; Drs. S. Silletti and D. Cheresh for providing us with the 3 and
5 primer pairs; F. Isdell for assistance with the FACSort®; T. de Lange for use of the microscope; and S. Turley for assistance with the immunofluorescence.
Supported by National Institutes of Health (NIH) grants HL-42540 and EY-10967 (to S.F.A. Pearce and R.L. Silverstein); NIH MSTP grant GM-07793 (to M.L. Albert); NIH grant AI-39516, and grants from the SLE foundation (to N. Bhardwaj).
Abbreviations used in this paper Ann V, Annexin V; DC, dendritic cell; MCM, monocyte-conditioned medium; PI, propidium iodide; PS, phosphatidylserine.
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