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Prepublished online as a Blood First Edition Paper on September 5, 2002; DOI 10.1182/blood-2002-06-1769.
IMMUNOBIOLOGY
From the Thomas E. Starzl Transplantation Institute and
Department of Surgery, Department of Dermatology and University of
Pittsburgh Cancer Institute and Department of Physiology and Center for
Biological Imaging, University of Pittsburgh Medical Center, PA.
Under steady-state conditions, internalization of self-antigens
embodied in apoptotic cells by dendritic cells (DCs) resident in
peripheral tissue followed by DC migration and presentation of
self-peptides to T cells in secondary lymphoid organs are key steps for
induction and maintenance of peripheral T-cell tolerance. We show here
that, besides this traffic of apoptotic cells mediated by peripheral
tissue-resident DCs, splenic marginal zone DCs rapidly ingest
circulating apoptotic leukocytes, process apoptotic cell-derived peptides into major histocompatibility complex class II (MHC-II) molecules, and acquire CD8 Continual transport of self-antigen
(self-Ag) by migrating dendritic cells (DCs) to secondary
lymphoid organs is a crucial step for induction/maintenance of
peripheral tolerance.1 Apoptotic cells resulting from
cellular turnover in peripheral tissues are an important source of
self-Ag. Immature DCs endocytose apoptotic cells in vitro and
DC-bearing apoptotic cells have been detected in vaginal epithelium and
intestinal lamina propria in rodents.2-6 The observation
by Huang et al6 that intestinal DC-bearing apoptotic
epithelial cell fragments traffic to mesenteric lymph nodes under
steady-state conditions suggests that endocytosis of apoptotic cells by
DCs in peripheral tissues followed by transport and presentation of
self-peptides to naive T lymphocytes in secondary lymphoid organs may
be involved in T-cell peripheral tolerance. There are possible reasons
to explain why, under steady-state conditions, endocytosis and
processing of apoptotic cells by antigen-presenting cells (APCs) does
not disrupt self-tolerance: (1) interaction with apoptotic cells does
not induce DC maturation,7-9 and (2) ingestion of
apoptotic cells by macrophages decreases secretion of proinflammatory
factors and induces release of the anti-inflammatory interleukin 10 (IL-10) and transforming growth factor Although there is extensive information about the receptors used by
macrophages during phagocytosis of apoptotic cells, little is known
about the mechanisms used by DCs. The integrins,
Mice
Reagents
DCs The method for isolation of spleen DCs was modified from that described by Inaba et al.18,19 Briefly, B10 spleens were flushed with 100U/mL collagenase (type IV, Sigma), teased apart with fine forceps, and digested with 400U/mL collagenase (30 minutes at 37°C). After digestion, cells were diluted in ice-cold Ca++-free 0.01 M EDTA (ethylenediaminetetraacetic acid) Hanks balanced salt solution (HBSS). Splenic DC-enriched suspensions (20%-30% DC purity) were obtained by gradient centrifugation (1800 rpm, 20 minutes at 4°C) over 16% (wt/vol) metrizamide (Sigma). Thereafter, DCs were washed with ice-cold Ca++-free 0.01 M EDTA HBSS, then labeled with bead-conjugated anti-CD11c monoclonal antibody (mAb; clone N418, Miltenyi Biotec, Auburn, CA), and immunobead sorted by positive selection using VS+ separation columns (Miltenyi Biotec). At the end of the procedure, the DC purity was 92% or greater assessed by CD11c and IAhi expression by 2-color flow cytometry.Bone marrow (BM) DCs were generated as described.20
Briefly, BM cells were removed from femurs and tibias of B10 mice and depleted of erythrocytes by hypotonic lysis. Erythroid precursors, T
and B lymphocytes, natural killer (NK) cells, granulocytes, and
IA+ cells were removed by complement depletion
using a cocktail of mAbs (anti-TER-119, anti-CD3 Phagocytosis of apoptotic cells BALB/c splenocytes were used as a source of apoptotic cells. Apoptosis was induced by UVB irradiation (ULTRA-LUM, Claremont, CA, lamp model UVB16). After culture in RPMI 1640 (or PBS) the percentage of splenocytes undergoing early apoptosis (annexin V+, propidium iodide-negative [PI ] cells) increased from
25% (1 hour after UV irradiation) to 85% (3 hours after UV
irradiation) with less than 10% of cells in late apoptosis (annexin
V+ PI+ cells). To avoid accumulation of cells
in late apoptosis for in vitro studies, apoptotic splenocytes were
mixed with DCs no longer than 1 hour after UV irradiation. For in vivo
analysis, apoptotic cells were injected 3 hours following UV
irradiation. For most of the in vitro studies, BALB/c splenocytes were
labeled (red) with PKH26 before UV irradiation and then mixed with
PKH67-GL-labeled (green), immunobead-sorted (B10) splenic or BM DCs.
Apoptotic splenocytes and DCs were incubated in round-bottom
polypropylene tubes at 5:1 or 2:1 ratios for 2 hours in the absence of
serum or in the presence of different concentrations of normal or
heat-inactivated (56°C, 45 minutes) B10 mouse serum. Thereafter, the
cells were washed with ice-cold 0.1% sodium azide/PBS and fixed with
2% (vol/vol) paraformaldehyde. The percentage of green DCs with
internalized/attached red apoptotic cells (double-positive cells) was
analyzed by flow cytometry. For blocking experiments, DCs were
preincubated (30 minutes at 4°C) with 10 or 25 µg/mL of each of the
following azide-free/low-endotoxin mAbs (all from BD Pharmingen):
anti-CD11b (clone M1/70), anti-CD11c (HL3), anti-CD18 (clone GAME-46),
anti-CD14 (clone rmC5-3), anti-CD21/CD35 (clone 7G6), anti-CD35
(clone-8C12), anti-CD51 (clone H9.2B8), anti-CD61 (clone2G9.G2), or
IgG control.
Immunofluorescence staining of tissue sections Spleen blocks were embedded in Tissue-Tek OCT (Miles Laboratories, Elkhart, IN), snap frozen in isopentane/liquid nitrogen, and stored at 80°C. Cryostat sections (8 µm) and cytospins
(Shandon cytocentrifuge; 230g) were fixed in 96% ethanol
(10 minutes), blocked with 10% normal goat serum, and incubated
overnight (4°C) with each of the following biotin-mAbs: anti-CD11c,
anti-CD11b, anti-CD8 , anti-H2Dd (all from BD
Pharmingen), anti-MOMA 1 (Bachem, King of Prussia, PA), anti-F4/80
(Bachem), or anti-ER-TR9 (Bachem). As a second step, slides were
incubated with 1:3000 Cy3-streptavidin (Jackson Immunoresearch
Lab, West Grove, PA), for 30 minutes at room temperature. Cell nuclei
were stained with DAPI (4,6 diamidino-2-phenylindole; Molecular
Probes, Eugene, OR). For some experiments, cytospins were stained with
FITC-TUNEL (In Situ Cell Death Detection Kit, FITC, Roche,
Indianapolis, IN) following by Cy3 anti-CD11c or Cy3
anti-H2Dd. Slides were fixed in 2% paraformaldehyde,
mounted in glycerol/PBS, and examined with a Zeiss Axiovert 135 microscope equipped with appropriate filters and a cooled CCD camera
(Photometrics CH250, Tucson, AZ). Signals from different fluorochromes
were acquired independently, and montages edited using the Adobe
Photoshop software program (Adobe Systems, Mountain View, CA).
Confocal and 2-photon confocal microscopy CD11c+ immunobead-sorted splenic DCs from mice injected with PKH67-apoptotic cells that had been attached to poly-L-lysine-treated slides were fixed with 2% paraformaldehyde, labeled with Cy3-CD11c. They were imaged with a Leica TCS-NT confocal microscope (Leica Microsystems, Deerfield, IL) with filters for detection of Cy3 and FITC at 1024 × 1024 resolution with a section interval of 0.8 µm. Two-photon microscopy was used to analyze the intrasplenic localization of PKH67-BALB/c apoptotic cells in B10 recipients. Then, 3-mm3 spleen fragments were fixed with 2% paraformaldehyde for 2 hours. Single x-y-axis images were acquired using a multiphoton laser scanning confocal microscope comprising a titanium-sapphire ultrafast tunable laser (Coherent Mira Model 900-F), Olympus Fluoview confocal scanning electronics, an Olympus IX70 inverted system microscope, and custom built input-power attenuation and external photomultiplier detection systems. Single-plane image acquisition used 2-photon excitation at 870 nm with an Olympus 20 ×, UApo 0.7NA water immersion objective.RPA The analysis of cytokine mRNAs transcribed by DCs was performed by RNAse protection assay (RPA) as described.20 RNA was isolated using a total RNA Isolation Kit (BD Pharmingen) from 5 × 106 BM DCs (per experimental variable) purified by negative selection by immunomagnetic sorting. The RPA was performed using the RiboQuant Multi-Probe RPA System (BD Pharmingen). Two multiprobe customized template kits containing cDNAs encoding mouse IL-1, IL-1 , IL-1ra, IL-4, IL-6, IL-10, IL-12p35, IL-12p40,
interferon (IFN-), IFN-, IFN- , TNF-, TGF1, GM-CSF,
MIF, and the housekeeping genes L32 and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) were used as templates for the T7
polymerase-directed synthesis of -32P]-UTP-labeled
antisense RNA probes. Hybridization (16 hours at 56°C) of 5 µg of
each target mRNA with the antisense RNA probe sets was followed by
RNAse and proteinase K treatment, phenol-chloroform extraction, and
ammonium acetate precipitation of protected RNA duplexes. In each RPA,
the corresponding antisense RNA probe set (3 × 103 cpm)
was included as molecular weight standard, and to examine probe
set integrity. Mouse RNA (positive control) and RNA degradation controls were included. Yeast tRNA served as negative control. Samples
were electrophoresed on acrylamide-urea sequencing gels. Dried gels
were exposed on Fujifilm x-ray film, at 80°C. Quantification of
bands was performed by densitometry (Personal densitometers 1;
Molecular Dynamics, Sunnyvale, CA). The signals from specific mRNAs
were normalized to signals from housekeeping genes (L32 and
GADPH) run on each lane to adjust for loading differences.
Rosetting and polystyrene bead assays Erythocytes bearing IgM and mouse C3bi were prepared as described.21,22 Briefly, sheep erythrocytes (Colorado Serum, Denver, CO) were washed 3 times in veronal-buffered saline (VBS, Sigma), and a 5% vol/vol erythrocyte suspension incubated with a 1:100 subagglutinating dilution of antisheep red blood cell IgM mAb (Cedarlane). The sensitized erythrocytes (EAs) were washed in VBS and a 5% EA suspension incubated with one tenth mouse (B10) serum at 37°C for 30 minutes. Under these conditions, most of the bound C3 generated is primarily in the C3bi form.23 In preliminary experiments (not shown), 90% to 95% of immature BM DCs rosetted C3biEAs (and did not bind EAs), a phenomenon blocked by anti-CD11b (M1-70) mAb. Based on the fact that spleen and BM DCs do not express CR for C1q, C3b, C3d, C3dg, or C4b (ie, C1qR,24 CR1, and CR2) and that the I domain of CD11b (blocked by M1/70 mAb) is the main C3bi receptor expressed by DCs, these results confirmed the presence of murine C3bi on C3biEAs (no commercial mAb against mouse C3bi is available). DCs were incubated with C3biEAs or EAs (erythrocyte/DC ratio = 25:1) for 16 hours at 37°C in culture medium with 2% wt/vol bovine serum albumin (BSA fraction V; protease free, Roche) and 0.5 mg/mL soybean trypsin inhibitor (Sigma). Polystyrene beads with bound anti-rat IgG (Dynabeads M-450; Dynal, Lake Success, NY) were coated with 10 µg/mL anti-CD11b (M1/70), anti-CD18 (GAME-46), anti-CD11a (leukocyte function associated antigen 1 [LFA-1]), or irrelevant IgG in PBS. DCs were incubated with mAb-coated beads (bead/DC ratio = 8:1) first for 3 hours at 4°C and then for 13 hours at 37°C.Statistical analysis Results are expressed as mean ± SD. Comparisons between different means were performed by ANOVA, followed by the Student Newman Keuls test. Comparison between 2 means was performed by Student t test. A P < .05 was considered significant.
Preferential uptake of apoptotic cells by spleen MZ DCs The kinetics of phagocytosis of apoptotic cells by splenic DCs was analyzed in vivo. As early as 1 hour after intravenous injection of 2 × 107 PKH67 apoptotic (BALB/c) splenocytes in allogeneic (B10) recipients, 10% to 15% of splenic CD11c+ DCs internalized apoptotic cells as determined by flow cytometry, with maximum levels of endocytosis 24 hours after apoptotic cell injection (Figure 1A). After 1 hour or 3 hours of injection, CD8 DCs were the main DC subset that had
internalized apoptotic cells. Whereas the percentage of
CD8+ DCs with apoptotic cell inclusions increased after
24 hours, a proportional decrease in the number of CD8
DCs with apoptotic cells was observed (Figure 1B). This effect may be
ascribed to either up-regulation of CD8 by CD8 DCs,
or to transfer of PKH67-apoptotic cells from CD8 to
CD8+ DCs. The capacity of spleen DCs to process and
present allopeptides contained in the apoptotic splenocytes was
examined with Y-Ae mAb. This recognizes the IAb
(B10)-IE52-68 peptide (IE from BALB/c)
complex.25 Y-Ae positivity was detected in splenic DCs
from animals intravenously injected with BALB/c apoptotic splenocytes
at 24 hours (35% ± 10% of splenic DCs) and at 48 hours
(62% ± 15% of splenic DCs) after injection (4 independent
experiments) and not in DCs from B10 mice injected with
IE (B10) apoptotic leukocytes (Figure 1C).
Three-dimensional analysis by 2-photon confocal microscopy,
followed by 2-color immunofluorescence, showed that circulating apoptotic cells accumulated initially (first 24 hours) at the periphery
of splenic follicles, mostly within CD11c+ DCs of the MZ
(Figure 2A-B,D-E). By 48 to 72 hours, DCs
with PKH67 inclusions mobilized to the T-cell area of the follicle center (Figure 2C,F). The preferential entrapment of circulating apoptotic cells into the MZ was confirmed by labeling
MOMA-1+ metallophillic macrophages (inner limit of MZ) and
red pulp F4/80hi macrophages (outer limit).
MOMA-1+ metallophillic macrophages, ER-TR9+ MZ
macrophages, and F4/80hi red pulp macrophages also
internalized apoptotic cells (Figure 2G-I). Analysis of
immunobead-sorted splenic DCs from B10 mice injected intravenously with
BALB/c PKH67-apoptotic cells by confocal microscopy demonstrated PKH67
fragments within the cytoplasm of CD11c+ DCs (Figure
3A-B). Coexpression of donor (BALB/c) H2
molecules (H2Dd) and PKH67 or TUNEL in CD11c+
splenic DCs confirmed that donor apoptotic cells (H2Dd+,
TUNEL+) were indeed taken up by MZ DCs (Figure 3C-E). This
observation excluded the possibility that PKH67 could have been
transferred passively between PKH67-apoptotic cells and DCs. Cytospin
preparations of DCs isolated 1 hour after injection of apoptotic cells
confirmed that the apoptotic cells were initially internalized by
CD8
Uptake of apoptotic cells by CD8 DCs exhibited a higher level of phagocytosis of
apoptotic cells than CD8+ DCs may have been due to more
efficient endocytosis or to a more appropriate anatomic localization of
the former cells. The proximity of CD8 DCs to the
marginal sinus, through which circulating cells traffic, may favor the
interaction of circulating apoptotic cells with CD8
DCs rather than with CD8+ DCs, which are more numerous
in T-cell areas.26-28 In vitro experiments showed that
18% to 50% (33% ± 5%) of freshly isolated PKH67-splenic (B10) DCs phagocytosed PKH26-apoptotic (BALB/c) splenocytes within 2 hours, as determined by the presence of double-positive DCs by flow
cytometry (Figure 4A). Phagocytosis
assays were performed in the presence of 10% normal B10 mouse serum.
The fact that uptake of apoptotic cells by splenic DCs decreased in the
presence of 2 mM EDTA, 10 µm cytochalasin D, or at 4°C, confirmed
that the assay detected internalization of apoptotic cells rather than binding of apoptotic cells to DCs (Figure 4A). To test the phagocytic capacity of CD8+ DCs, unlabeled splenic (B10) DCs were
mixed with (BALB/c) PKH26 apoptotic cells (2 hours, 37°C), fixed in
paraformaldehyde, and labeled with Cychrome-anti-CD8 and
FITC-anti-IAb or FITC-anti-H2Dd
(anti-H2Dd mAb cannot penetrate the DC membrane, and thus
labels free or partially internalized BALB/c apoptotic cells). With
this approach, it was possible to distinguish: (1) free apoptotic cells
(PKH26+, H-2Dd+, FSClow,
SSClow); (2) apoptotic cells attached to or partially
endocytosed by B10 DC (PKH26+, H-2Dd+,
IAb+, FSChi, SSCint), and (3)
apoptotic cells completely internalized by B10 DC (PKH26+,
H-2Dd, IAb+, FSChi,
SSCint; Figure 4B-E). Although most of the apoptotic cells
associated with B10 DCs were completely internalized
(PKH26+, IAb+, and H2Dd), the
majority of the apoptotic cells associated with CD8+ DCs
remained outside the DC (PKH26+, H2Dd+; Figure
4B,C and 4D,E, respectively). This result demonstrates that the
entrapment of circulating apoptotic splenocytes in vivo is initially
performed mainly by CD8 DCs that acquire CD8
expression during their migration to the follicular T-cell area
(Figures 1 and 2A-F).
Increased uptake of apoptotic cells by splenic DCs in the presence of normal serum The role of heat-labile serum factors in the internalization of apoptotic cells by splenic DCs was analyzed in vitro in the absence of serum, or in the presence of normal (noncomplement inactivated) or heat-inactivated (56°C, 45 minutes) mouse serum. In the presence of 10% normal (B10) mouse serum, PKH67-splenic (B10) DCs exhibited an increase in their ability to phagocytose PKH26-apoptotic (BALB/c) splenocytes of at least 3-fold compared with their activity in the absence of serum. This effect reached a plateau at 12.5% serum concentration and after 2 hours of incubation (Figure 4F-G). Depletion of heat-labile serum protein activity decreased the number of DCs that phagocytosed apoptotic cells 2-fold (Figure 4G), an effect that was dependent on time and serum concentration. Similar results were obtained when syngeneic (B10) splenocytes or thymocytes were used as sources of apoptotic cells (data not shown). Reduced uptake of apoptotic cells in the presence of heat-inactivated serum suggests that complement factors (ie, C1q, C3bi that are known to enhance the process) may be involved in the adhesion/internalization of apoptotic cells by splenic DCs.15,16,29 The fact that similar results were obtained with BM-derived immature CD11c+ CD86 (B10) DCs, which did not require
purification by positive selection with anti-CD11c mAb (clone N418),
demonstrated that purification with N418 mAb did not interfere with
C3bi recognition by DCs (Figure 4H), as shown
previously.30
Role of CRs in internalization of apoptotic cells by splenic DCs Because heat-labile serum factors optimized the uptake of apoptotic cells by DCs and because C3bi on the surface of apoptotic cells facilitates their phagocytosis by macrophages,15,16 we assayed the role of the C3bi receptors CD11b/CD18 and CD11c/CD18 on uptake of apoptotic cells by splenic DCs. PHK67-splenic (B10) DCs were mixed with PHK26-apoptotic (BALB/c) splenocytes in the presence of mAbs (10-25 µg/mL) specific for CRs. Apoptotic cells were preincubated (60-90 minutes) with normal (B10) mouse serum, washed, and then used for phagocytosis assays in the presence of 10% complement-inactivated mouse (B10) serum. This approach prevented DC death from antibody-dependent complement activation. Inhibition of up to 40% of apoptotic cell uptake by splenic DCs was observed following blocking of the and chains of CD11b/CD18 (CR3) with mAbs M1/70 and GAME-46,
respectively (Figure 5A). A less
pronounced inhibition of apoptotic cell uptake was detected after
blocking CD11c with mAb HL3. Inhibition was not detected with either
irrelevant IgG, mAbs directed against CRs not expressed by murine DCs
(anti-CD21/35 or anti-CD21), or anti-CD14 mAb (involved in uptake of
apoptotic cells by macrophages but not expressed by DCs; Figure 5A). As
positive controls, blocking of the v (CD51) and
3 (CD61) integrins with specific mAb also inhibited the
uptake of apoptotic cells by DCs.
The role of complement factors in the uptake of apoptotic cells by splenic DCs was analyzed in vivo in (B10) mice made hypocomplementemic with CVF before intravenous injection of 2 × 107 PKH67-apoptotic (BALB/c) splenocytes. CVF is a C3-like polypeptide that functions as a C3 convertase resistant to inactivation by factors H and I, a fact that, in mice, results in a severe inactivation of C3 (and secondary C5) without effect on factors like C1q that precede C3 in the complement cascade.31 After 12 hours, the percentage of splenic DCs that internalized circulating apoptotic cells was reduced significantly (P < .001) in hypocomplementemic mice compared with non-CVF-treated controls (Figure 5B-C). Thus complement factors derived from C3 or later in the complement cascade are involved in the uptake of circulating apoptotic cells by splenic MZ DCs in vivo. Interaction with apoptotic cells regulates the pattern of DC cytokines The effect that apoptotic cells exert on cytokine gene transcription and secretion by DCs was analyzed by RPA and enzyme-linked immunosorbent assay (ELISA), respectively. Due to the high numbers of DCs required to perform semiquantitative mRNA analysis by RPA, we used immature BM-derived (B10) DCs negatively selected by immunomagnetic sorting (purity of CD11c+ CD86
DC < 95%). These BM DCs exhibit similar characteristics to MZ DCs
(CD8 CD11b+ CD11c+
CD86/lo F4/80dim ER-TR9
MOMA-1 and take up apoptotic cells with similar
efficiency; Figure 4G-H). BM DCs were cocultured with BALB/c apoptotic
splenocytes (1:4 ratio) for 4 or 16 hours and the level of cytokine
mRNAs assessed by RPA. Interaction/ingestion of apoptotic cells reduced
the levels of IL-1, IL-1, IL-6, IL-12p35, IL-12p40, and TNF-
mRNAs (Figure 6A-B). A proportional
decrease in cytokine mRNA copies was also detected in DCs cocultured
with apoptotic splenocytes followed by stimulation with 200 ng/mL
lipopolysaccharide (LPS; Figure 6A-B). Ingestion of apoptotic
cells exerted minimal or no effect on the levels of IL-1ra, MIF, and
TGF-1 mRNAs. The absence of detectable mRNA in extracts from
equivalent numbers of apoptotic cells used in the phagocytosis assays
demonstrated that the changes in cytokine mRNA levels were not caused
by mRNA from apoptotic cells (Figure 6A). Similar results were obtained
by ELISA for the amounts of cytokines secreted by DCs in the culture
supernatant. Levels of TNF-, IL-1, IL-1, and IL-6 decreased
significantly following incubation of DCs with apoptotic cells, even on
stimulation with LPS (200 ng/mL; Figure 6C). Incubation with
apoptotic cells blocked the stimulatory effect of LPS on IL-12p70
secretion and the inhibition that LPS exerted on TGF-1 secretion
(Figure 6C).
Interaction of C3bi with CD11b/CD18 mimics the effects of apoptotic cells on DC cytokines Binding of C3bi on the apoptotic cell surface to CD11b/CD18 on DCs may be involved in recognition/internalization of apoptotic cells and may also instruct DCs to regulate cytokine production, as occurs in macrophages.32,33 To analyze the effect of C3bi on DC cytokines, immunobead-sorted immature BM DCs were mixed with C3biEA (prepared with mouse serum) or EAs in rosetting assays, or with medium alone. The levels of TNF-, IL-6, IL-1, and IL-1 decreased significantly when DCs were incubated with C3biEA, even in
the presence of LPS (200 ng/mL; Figure
7). Similarly, incubation with C3biEA
decreased the levels of IL-12p70 secreted by BM DCs in response to LPS
stimulation. C3biEA interaction did not modify the levels of TGF-1
secreted by DCs. To establish whether the effect of C3bi on DC
cytokines was mediated via CD11b/CD18, immature BM DCs were incubated
with anti-CD11b mAb (M1/70)-coated 4.5-µm polystyrene beads in the
presence of 200 ng/mL LPS. BM DCs incubated with anti-CD11b (M1/70)
beads decreased significantly their secretion of TNF-, IL-6,
IL-1, IL1-, and IL-12p70 compared with DCs incubated with
irrelevant IgG-coated beads (Figure 8). A
similar reduction (with the exception of IL-6) was induced by anti-CD18
mAb (GAME-46)-coated beads. As control, incubation with anti-CD11a mAb
(LFA-1)-coated beads did not affect cytokine levels.
Models of peripheral tolerance have demonstrated that migratory BM-derived APCs, which constitutively transport and process peripheral tissue-specific self-Ag to lymph nodes, are able to silence Ag-specific T-cell-receptor transgenic CD4+ or CD8+ T cells.34,35 Interestingly, migratory DCs internalize and transport apoptotic intestinal epithelial cells to T-cell areas of mesenteric lymph nodes.6 These and other observations suggest that, in the absence of inflammation, tissue-resident DCs take up apoptotic cells, process the self-Ag contained within them and, without receiving a maturation or danger signal, migrate constitutively via lymphatic vessels to lymph nodes.18,19 Once in the T-cell area, DCs maintain peripheral tolerance by induction of self-Ag-specific T-cell anergy/deletion or generation of T-regulatory cells.1,36-40 In the present study, we demonstrate that besides the constitutive
traffic of apoptotic cell fragments within DCs from the periphery to
lymph nodes via lymphatic vessels, circulating apoptotic cells may also
be internalized by spleen-resident MZ DCs. This alternative and
efficient mechanism may account for the billions of apoptotic
leukocytes that are removed from the circulation every day without
breaching self-tolerance. MZ DCs are in a strategic localization to
capture blood-borne particulate antigen, including circulating
apoptotic leukocytes.41 We show here that MZ DCs rapidly
internalize circulating allogeneic PKH-67+ apoptotic
leukocytes. Several facts confirmed that the fragments found within MZ
DCs were apoptotic cells. Most of the PKH-67+ inclusions
stained specifically for the donor allogeneic antigen H2Dd and TUNEL, and some of the TUNEL+
inclusions coexpressed H2Dd. Our results also reveal that
the uptake of circulating apoptotic cells is initially performed mainly
by CD8 Our results support the recent demonstration that splenic
CD8 It is likely that the consequences of apoptotic cell clearance for
tolerance versus immunity depend on the interaction of apoptotic cells
with DC surface receptors, on the fact that apoptotic cells are rapidly
internalized preventing the release of intracellular proteases that
activate APCs, and on the DC microenvironment where the endocytosis
takes place.44-47 The cell membrane of apoptotic cells
undergoes molecular changes that allow its recognition by receptors
expressed by phagocytes.44-46 Among them, CD36
(thrombospondin receptor) and the integrins
We show here that after DCs internalized apoptotic cells they produced
significantly lower levels of IL-1 In conclusion, our results demonstrate that, under steady-state
conditions, DCs of the splenic MZ rapidly take up apoptotic leukocytes
from the circulation. Together with other ligands, C3bi deposition
optimizes apoptotic cell uptake and instructs DCs both to reduce the
synthesis of proinflammatory and Th1-driving cytokines, and to maintain
high levels of the immunoregulatory factor TGF-
We thank Bridget L. Colvin, Susan Specht, and Jan Urso for technical assistance. We thank the Schering-Plough Research Institute for the gifts of cytokines and Dr Charles A Janeway (Yale University) for the Y-Ae mAb.
Submitted June 14, 2002; accepted August 23, 2002.
Prepublished online as Blood First Edition Paper, September 5, 2002; DOI 10.1182/blood-2002-06-1769.
Supported by grants from National Institutes of Health: R21 HL69725 (A.E.M.); R01 AI43916 and P01 CA73743 (L.D.F.); R01 DK49745 and R01 AI41011 (A.W.T.); and the Dermatology Foundation (A.T.L.).
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: Adrian E. Morelli or Angus W. Thomson, E1504 Biomedical Science Tower, 200 Lothrop St, Pittsburgh, PA 15213; e-mail: morelli{at}imap.pitt.edu or thomsonaw{at}msx.upmc.edu.
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  M. L. Salem, A. A. AL-Khami, S. A. EL-Naggar, C. M. Diaz-Montero, Y. Chen, and D. J. Cole Cyclophosphamide Induces Dynamic Alterations in the Host Microenvironments Resulting in a Flt3 Ligand-Dependent Expansion of Dendritic Cells J. Immunol., February 15, 2010; 184(4): 1737 - 1747. [Abstract] [Full Text] [PDF]
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E. M. Behrens, Y. Ning, N. Muvarak, P. W. Zoltick, A. W. Flake, and S. Gallucci Apoptotic Cell-Mediated Immunoregulation of Dendritic Cells Does Not Require iC3b Opsonization J. Immunol., September 1, 2008; 181(5): 3018 - 3026. [Abstract] [Full Text] [PDF]
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M.-L. Santiago-Raber, S. Kikuchi, P. Borel, S. Uematsu, S. Akira, B. L. Kotzin, and S. Izui Evidence for Genes in Addition to Tlr7 in the Yaa Translocation Linked with Acceleration of Systemic Lupus Erythematosus J. Immunol., July 15, 2008; 181(2): 1556 - 1562. [Abstract] [Full Text] [PDF]
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K. Kalies, P. Konig, Y.-M. Zhang, M. Deierling, J. Barthelmann, C. Stamm, and J. Westermann Nonoverlapping Expression of IL10, IL12p40, and IFN{gamma} mRNA in the Marginal Zone and T Cell Zone of the Spleen after Antigenic Stimulation J. Immunol., April 15, 2008; 180(8): 5457 - 5465. [Abstract] [Full Text] [PDF]
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F. Wermeling, Y. Chen, T. Pikkarainen, A. Scheynius, O. Winqvist, S. Izui, J. V. Ravetch, K. Tryggvason, and M. C.I. Karlsson Class A scavenger receptors regulate tolerance against apoptotic cells, and autoantibodies against these receptors are predictive of systemic lupus J. Exp. Med., October 1, 2007; 204(10): 2259 - 2265. [Abstract] [Full Text] [PDF]
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E. M. Behrens, U. Sriram, D. K. Shivers, M. Gallucci, Z. Ma, T. H. Finkel, and S. Gallucci Complement Receptor 3 Ligation of Dendritic Cells Suppresses Their Stimulatory Capacity J. Immunol., May 15, 2007; 178(10): 6268 - 6279. [Abstract] [Full Text] [PDF]
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A. Maeda, A. Schwarz, K. Kernebeck, N. Gross, Y. Aragane, D. Peritt, and T. Schwarz Intravenous Infusion of Syngeneic Apoptotic Cells by Photopheresis Induces Antigen-Specific Regulatory T Cells J. Immunol., May 15, 2005; 174(10): 5968 - 5976. [Abstract] [Full Text] [PDF]
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J. Dalgaard, K. J. Beckstrom, F. L. Jahnsen, and J. E. Brinchmann Differential capability for phagocytosis of apoptotic and necrotic leukemia cells by human peripheral blood dendritic cell subsets J. Leukoc. Biol., May 1, 2005; 77(5): 689 - 698. [Abstract] [Full Text] [PDF]
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T. T. Brandhorst, M. Wuthrich, B. Finkel-Jimenez, T. Warner, and B. S. Klein Exploiting Type 3 Complement Receptor for TNF-{alpha} Suppression, Immune Evasion, and Progressive Pulmonary Fungal Infection J. Immunol., December 15, 2004; 173(12): 7444 - 7453. [Abstract] [Full Text] [PDF]
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M.-K. Chang, C. J. Binder, Y. I. Miller, G. Subbanagounder, G. J. Silverman, J. A. Berliner, and J. L. Witztum Apoptotic Cells with Oxidation-specific Epitopes Are Immunogenic and Proinflammatory J. Exp. Med., December 6, 2004; 200(11): 1359 - 1370. [Abstract] [Full Text] [PDF]
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A. E. Morelli, A. T. Larregina, W. J. Shufesky, M. L. G. Sullivan, D. B. Stolz, G. D. Papworth, A. F. Zahorchak, A. J. Logar, Z. Wang, S. C. Watkins, et al. Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells Blood, November 15, 2004; 104(10): 3257 - 3266. [Abstract] [Full Text] [PDF]
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M. Epardaud, M. Bonneau, F. Payot, C. Cordier, J. Megret, C. Howard, and I. Schwartz-Cornil Enrichment for a CD26hi SIRP- subset in lymph dendritic cells from the upper aero-digestive tract J. Leukoc. Biol., September 1, 2004; 76(3): 553 - 561. [Abstract] [Full Text] [PDF]
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X. Chen, K. Doffek, S. L. Sugg, and J. Shilyansky Phosphatidylserine Regulates the Maturation of Human Dendritic Cells J. Immunol., September 1, 2004; 173(5): 2985 - 2994. [Abstract] [Full Text] [PDF]
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A. J. Nauta, G. Castellano, W. Xu, A. M. Woltman, M. C. Borrias, M. R. Daha, C. van Kooten, and A. Roos Opsonization with C1q and Mannose-Binding Lectin Targets Apoptotic Cells to Dendritic Cells J. Immunol., September 1, 2004; 173(5): 3044 - 3050. [Abstract] [Full Text] [PDF]
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M. D. Rosenblum, E. Olasz, J. E. Woodliff, B. D. Johnson, M. C. Konkol, K. A. Gerber, R. J. Orentas, G. Sandford, and R. L. Truitt CD200 is a novel p53-target gene involved in apoptosis-associated immune tolerance Blood, April 1, 2004; 103(7): 2691 - 2698. [Abstract] [Full Text] [PDF]
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K. Tada, M. Tanaka, R. Hanayama, K. Miwa, A. Shinohara, A. Iwamatsu, and S. Nagata Tethering of Apoptotic Cells to Phagocytes through Binding of CD47 to Src Homology 2 Domain-Bearing Protein Tyrosine Phosphatase Substrate-1 J. Immunol., December 1, 2003; 171(11): 5718 - 5726. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
, anti-B220,
anti-NK-1.1, anti-Gr1, and anti-IAb; BD
Pharmingen) followed by incubation (45 minutes, 37°C) with low-toxicity rabbit complement (Cedarlane, Hornby, ON, Canada). Remaining BM cells were cultured in RPMI 1640 (Life Technologies, Grand
Island, NY) in 75-cm2 flasks (5 × 106
cells/flask) with 10% vol/vol heat-inactivated fetal calf serum (FCS;
Life Technologies), glutamine, nonessential amino acids, sodium
pyruvate, HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 2-mercaptoethanol (2-ME), and
penicillin/streptomycin, supplemented with mrGM-CSF (1000 U/mL) and
mrIL-4 (1000 U/mL). Culture medium and exogenous cytokines were
renewed every other day.
.01; **P 
