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Medical Research Council Center for Inflammation Research, University of Edinburgh, Edinburgh, Scotland, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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but have a diminished ability to produce IL-12 in response to
external stimuli, a property that corresponds to a failure to
up-regulate CD86. By single-cell analysis we demonstrate that these
inhibitory effects are restricted to those DCs that have engulfed
apoptotic cells, with bystander DCs remaining unaffected. These changes
were independent of the production of anti-inflammatory cytokines
TGF-
1 and IL-10 and corresponded with a diminished capacity to
stimulate naive T cells. Thus, the ingestion of apoptotic cells is not
an immunologically null event but is capable of modulating DC
maturation. These results have important implications for our
understanding of the role of clearance of dying cells by DCs not only
in the normal resolution of inflammation but also in control of
subsequent immune responses to apoptotic cell-derived
Ags. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immature DCs acquire Ag by many pathways including uptake of soluble Ag
or protein complexes by endocytosis and macropinocytosis and ingestion
of entire cells by phagocytosis. Ingestion of certain necrotic cells is
capable of inducing DC maturation, while ingesting apoptotic cells
fails to activate DCs, appearing to be an immunologically null event
(4, 5). However, such DCs are capable of responding to
strong external stimuli, such as monocyte-conditioned medium or
IFN-
, to mature and present Ag derived from the ingested apoptotic
cells to T cells (6, 7, 8, 9). Apoptotic cells are a
preferential source of many autoantigens (10), often found
localized to apoptotic blebs, and the ability of DCs to present such
Ags unchecked might initiate autoimmunity. In support of this,
perturbations in apoptotic cell death and clearance of these cells have
been shown to contribute to the induction of autoimmunity (11, 12).
However, a growing body of evidence implicates DCs that ingest dying
cells in maintaining self-tolerance, by constantly sampling peripheral
self Ags and presenting them in a tolerogenic way to the adaptive
immune system. Thus, a dichotomy exists in responses of DCs that may be
either "friend or foe" (13). The ability of a DC to
deliver "signal 2," either as costimulation or IL-12, singly or in
combination, appears key in determining subsequent immune responses and
is likely to be tightly controlled. Interestingly, the ingestion of
apoptotic cells by macrophages generates an active
anti-inflammatory response through the production of TGF-1 and
other anti-inflammatory molecules and down-regulates subsequent
release of proinflammatory cytokines (14, 15, 16). Because of
the close lineage relationship of macrophages and myeloid DCs, we
postulated that apoptotic cell ingestion by DCs might similarly
modulate their effector functions.
In this paper we confirm that immature murine bone marrow-derived DCs
ingest apoptotic cells and, after phagocytosis, become functionally
distinct. We demonstrate that endotoxin-induced production of IL-12,
but not TNF-, is selectively diminished in DCs that have ingested
apoptotic cells. In addition, endotoxin-driven up-regulation of the
costimulatory molecule CD86 is inhibited in those DCs that had
phagocytosed apoptotic cells, but not in neighboring DCs. We show the
functional consequences of these changes by demonstrating that these
DCs have a reduced capacity to stimulate T cell proliferation. Thus,
phagocytosis of apoptotic cells affects subsequent maturation of DCs in
a manner analogous to the anti-inflammatory effects in macrophages,
generating mature, CD86low cells that produce
less IL-12. These data confirm that the apoptotic cell is not
immunologically null, but, by inhibiting DC activation, may contribute
to down-regulation of the response to apoptotic cell-derived self Ag
and maintenance of self tolerance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice were purchased from B & K Universal (Hull, U.K.) and were used at 8 wk for bone marrow-derived DC and macrophage preparation. T cells were isolated from the DO11.10 transgenic mice expressing TCR specific for the chicken OVA peptide, OVA323–339, in association with I-Ad. The mice were typed for the presence of the transgene using Abs against CD4 (BD PharMingen, San Diego, CA) and biotin peak 2 (KJ1-26; Scottish Antibody Production Unit, Lanarkshire, U.K.) and streptavidin-PE (BD PharMingen) on PBL.
DC and macrophage culture
Murine cells were cultured in RPMI 1640 supplemented with 2 mM
L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin,
5 x 10-5 M 2-ME, and 15% heat-inactivated
FBS unless otherwise stated. DC complete medium also contained 10–15%
conditioned supernatant from a hybridoma (gift from Prof. D. Gray,
Edinburgh, U.K.) expressing rGM-CSF, resulting in a final GM-CSF
concentration of 20–30 ng/ml. This hybridoma also produces IL-10, at a
final concentration of 
2 ng/ml, but no TNF- or IL-12. All culture
reagents were obtained from Life Technologies (Grand Island, NY) unless
otherwise stated.
DCs were cultured as described previously (17). Briefly,
femurs from BALB/c mice were removed, dipped in 70% ethanol for
10 s, and then placed in DC complete medium. Bone marrow was
flushed from femurs, and 10 ml of a single-cell suspension of bone
marrow cells at 2 x 105/ml was plated in
non-tissue culture grade petri dishes. On day 3 an additional 10 ml of
fresh medium was added to the cultures. On day 6 half the medium was
removed, and the cells were pelleted, resuspended in fresh medium, and
added back to the petri dishes. On day 7 nonadherent cells were
removed, leaving strongly adherent macrophages on the plate. These
cells were pelleted, resuspended at 2 x 105
cells/ml, and replated before use. On day 7 these were a heterogeneous
population, 65–80% of the cells having surface phenotype and
morphology of immature DCs (Fig. 1
A), with granulocytes being
the main contaminant. Maturation was initiated on day 7 with 0.1–1
µg/ml LPS (Escherichia coli serotype 026:B6;
Sigma-Aldrich, St. Louis, MO) or 0.02% (w/v) Staphylococcus
aureus (Cowan strain) (SAC; Calbiochem, La Jolla, CA)
(3), and cells were assessed on day 8.
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Generation of apoptotic cells
Neutrophils were extracted from peripheral blood of healthy volunteers as described previously (18). Briefly, blood was separated using dextran sedimentation and a Percoll gradient. This yielded highly pure human neutrophils (>90%), which were allowed to undergo constitutive apoptosis by aging overnight in Iscove’s medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% autologous serum. After this period the cells were 40–80% apoptotic by cytospin morphology. This method of generating apoptotic bodies was preferred because there was no significant necrosis (<1%) by trypan blue exclusion, as confirmed by annexin-propidium iodide staining and flow cytometry. Apoptotic murine thymocytes were also generated for use in some experiments by treating single-cell suspensions of thymocytes with dexamethasone for 4–6 h. This method yielded apoptotic cells, but these preparations often contained contaminating postapoptotic cells and other nonapoptotic thymic cells. For most experiments cells were stained using a green cell tracker dye (Molecular Probes, Eugene, OR) before overnight culture.
DC-apoptotic cell coculture
Fluorescently labeled apoptotic cells were cocultured with day 7 DCs at a ratio of 2–5:1, apoptotic cells:DC. Interaction of DCs with apoptotic cells was assessed by removing cells after 2 h and staining with allophycocyanin-CD11c for FACS analysis. All FACS analyses were conducted on a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA). Maximum interaction was seen at ratios of 5:1 apoptotic cells/DCs, but the large numbers of uningested apoptotic cells reduced the efficiency of cell sorting, so ratios of 2:1 were used in most experiments. For fluorescent microscopy DCs were grown on chamber well slides, allowed to interact with red fluorescent apoptotic cells, then fixed with 4% paraformaldehyde. Slides were stained in PBS with 0.5% BSA and 0.2% sodium azide with I-Ad-FITC (BD PharMingen) in the presence of 10% normal mouse serum. Slides were mounted and examined under x63 oil lens using an inverted microscope (Zeiss, New York, NY), and images were captured using Open Lab software (Improvision, Coventry, U.K.) and CoolSnap digital camera (Media Cybernetics, Silver Spring, MD).
T cell proliferation assays
CD4 T cells were isolated from spleens of DO11.10 transgenic mice. In brief, spleens were made into a single-cell suspension by passing through a 53-µm pore size filter in PBS, and debris and red cells were removed by density gradient sedimentation through Lympholyte-M (Cedarlane Laboratories, Ontario, Canada) according to the manufacturer’s instructions. Cells were then isolated using L3T4 (CD4) microbeads and the MACS purification system (Miltenyi Biotec, Auburn, CA). Day 7 DCs were cocultured with apoptotic cells for 4 h, stimulated with LPS overnight, and then pulsed with 5 µg/ml OVA peptide (OVA323–339; Albachem Laboratories, Edinburgh University, Edinburgh, U.K.) for 2 h. They were then washed thoroughly, and cells were sorted into DCs containing apoptotic cells and those not, using a FACSVantage cell sorter (BD Biosciences). Contaminating apoptotic cells could be excluded from the sort by their smaller size and bright fluorescence. OVA-TCR-transgenic T cells (3 x 105) were cocultured in 24-well tissue culture dishes with varying doses of DCs for 5 days in a final volume of 2 ml. Proliferation was assessed by removing triplicate 100-µl samples pulsed with 1 µCi/well [3H]thymidine (Sigma-Aldrich) for 16 h. Cells were harvested, and thymidine incorporation was measured using a scintillation counter. Interactions were performed in duplicate.
FACS analysis of costimulatory molecules
DCs were cocultured with apoptotic cells for 4 h before overnight stimulation with LPS. Nonadherent cells were then removed from plates and resuspended in PBS with 0.5% BSA and 0.2% sodium azide. Blocking was performed using 10% mouse serum for 15 min, then cells were stained with relevant Abs at 4°C in the dark for 30 min. Cells were then washed and resuspended in 200 µl of FACS wash and analyzed using FACSCalibur and FlowJo software. The following Abs were used (all from BD PharMingen unless otherwise stated): FITC-I-Ad/I-Ed, PE-CD40, PE-CD86, PE-CD54, PE-CD11c, allophycocyanin-CD11c, and FITC-F4/80 (Serotec, Oxford, U.K.). All samples were compared with appropriate isotype controls. The geometric mean fluorescence of cells positive to isotype control was used in analysis unless stated otherwise.
Cytokine detection
For intracellular cytokine staining, cells were cocultured with
apoptotic cells for 4 h and stimulated with LPS for 5 h in
the presence of GolgiPlug (BD PharMingen) according to the
manufacturer’s instructions. Cells were harvested and stained for cell
surface markers as described above. Cells were then fixed using 4%
paraformaldehyde and permeabilized with 2% saponin in PBS with 0.5%
BSA, 0.2% sodium azide, and 10% mouse serum while staining with
allophycocyanin-TNF-, IL-10, and IL-12p40/p70 (BD PharMingen). IL-10
could not be reliably detected above background by intracellular
staining and so was also measured in the supernatant after 24–48 h of
interaction with apoptotic cells and LPS using a Quantakine ELISA kit
(R&D Systems, Minneapolis, MN). Interactions were performed in
duplicate wells, and triplicate readings of each supernatant were made.
Soluble forms of IL-10 and TGF-R were obtained from R&D Systems and
used at the recommended concentrations (1.25 and 0.5 µg/ml,
respectively).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Day 7 murine myeloid DCs cultured as described above were immature
by cell surface phenotype (CD11c+MHC class
II+CD40-CD80lowCD86low)
and could be matured (as evidenced by up-regulation of costimulatory
molecules and MHC class II) over 24 h by the addition of LPS in a
dose-dependent manner (Fig. 1
A). Similar results were seen
by stimulation with SAC (data not shown). Apoptotic cells stained with
a fluorescent cell-tracker dye were cultured with DCs at a ratio of
5:1, and interactions with DCs were quantified by flow cytometry. In a
typical experiment, 2 h after coincubation 50% of
CD11c+ cells had interacted with apoptotic cells.
Such interaction was exhibited by <5% of DCs at 4°C, demonstrating
that the interaction assay used predominantly detected phagocytosis
rather than binding (Fig. 1B). Ingestion was further
confirmed by fluorescence microscopy (Fig. 1C). Similar
rates of phagocytosis were seen when dexamethasone-treated murine
thymocytes were used as an alternative source of apoptotic cells (data
not shown). Thus, immature murine DCs exhibited a capacity for
phagocytosis of apoptotic cells similar in magnitude to that reported
in studies of immature human DC (7, 8).
Ingestion of apoptotic cells specifically inhibits the ability of DCs to up-regulate CD86
To ascertain whether ingestion of apoptotic cells alters DC
phenotype, cell surface expression of activation markers was studied by
flow cytometry after phagocytosis. No significant difference in cell
surface expression of the costimulatory molecules CD40, CD80, and CD86
was seen between immature DCs that had ingested apoptotic cells
(ac+) and those that had not
(ac-) either immediately (2 h, data not shown)
or 24 h (Fig. 2) after phagocytosis,
confirming that ingestion of apoptotic cells did not activate DCs.
However, on DC maturation with LPS a marked difference in surface
expression of CD86 was detected between ac+ DCs
and ac- DCs. Immature DCs were predominantly
CD86low, with a small population of
CD86high cells. Upon maturation driven by LPS the
proportion of CD86high cells increased in a
dose-dependent manner (Fig. 1A). However, fewer
ac+ DCs became CD86high
compared with ac- DCs; in a typical experiment
at 0.1 µg/ml LPS only 13.2% of ac+ DCs became
CD86high vs 42% ac- DCs,
and this difference was maintained at the highest LPS dose of 0.5
µg/ml, with only 24% of ac+ DCs becoming
CD86high compared with 46% of
ac- DCs (Fig. 2). Similar results were seen when
DCs were matured with SAC (data not shown). Mature DCs were
heterogeneous for CD54 with distinct populations of
CD54low and CD54high cells,
becoming most apparent at the highest dose of LPS (0.5 µg/ml).
Despite the general increase in fluorescent intensity of the
ac+ DCs after phagocytosis, it is apparent that
the percentage of CD54high cells was lower in
ac+ than ac- populations
(Fig. 2). CD40 expression was unaffected by apoptotic cells, with
maturation in response to 0.5 µg/ml generating a single
CD40+ population (Fig. 2), and no statistically
significant difference was seen in MHC class II or CD80 expression
(data not shown). Hence, the failure to up-regulate CD86 in the
ac+ DCs was not due to general unresponsiveness
to LPS but appeared to affect a subset of costimulatory molecules.
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, A and
B), the presence of apoptotic cells resulted in
significantly lower CD86 fluorescence for the whole DC population
compared with the control. No such difference would have been
detectable had the capacity to ingest apoptotic cells merely marked a
subpopulation of DCs destined not to up-regulate CD86 in response to
LPS. This and the observation that no significant difference in
costimulatory molecule expression was detected between the
ac+ DCs and ac- DC
population immediately after ingestion (data not shown) support the
fact that phagocytosis had not preferentially occurred in a
subpopulation destined to be CD86low. Also, this
inhibitory effect was not merely a result of particle ingestion, as
immature DCs, when cocultured with latex beads and oxidized lipid,
showed remarkably high levels (>85%) of phagocytosis and endocytosis,
respectively, and exhibited no defect in LPS-driven up-regulation of
CD86 expression, exhibiting, instead, apparently enhanced expression
(Fig. 3C). Taken together these data support the hypothesis
that apoptotic cell ingestion alters the subsequent response of DCs to
maturation stimuli.
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Cytokines produced by DCs are especially important in determining
subsequent T cell responses. We therefore examined the effect of
ingestion of apoptotic cells on cytokine production by DCs by combining
the fluorescent phagocytosis assay and intracellular cytokine staining
of cells, allowing us to study the production of cytokines by
individual DCs (Fig. 4). The autocrine
response to TNF- produced after LPS stimulation is an important
factor in terminal maturation and activation of DCs as well as
recruitment and activation of neighboring effector cells.
Interestingly, virtually all the DCs containing apoptotic cells
expressed TNF- after stimulation with LPS for 5 h,
demonstrating their functional viability and continuing responsiveness
to LPS stimulation. A small population of the
ac- DCs failed to produce TNF- and probably
represented a population of fully matured or "exhausted" DCs
(19) (Fig. 4). IL-12 is produced predominately by DCs and
orchestrates both the innate and adaptive immune responses. DCs express
a functional IL-12R, ligation of which by bioactive IL-12p70 augments
LPS maturation. In contrast to TNF-, ac+ DCs
failed to express IL-12 even when stimulated with 0.5 µg/ml LPS (Fig. 4). Similar results were seen when DCs were stimulated with SAC (data
not shown).
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1
IL-10 is an important anti-inflammatory cytokine associated
with induction of tolerance, resolution of inflammation, inhibition of
production of proinflammatory cytokines, and DC maturation
(20). IL-10 has been shown to inhibit DC maturation,
acting in both a paracrine and an autocrine manner (21).
Interaction of apoptotic cells with monocytes (22), but
not macrophages (14), has been shown to induce the
production of IL-10. To investigate whether changes in IL-10 expression
by DCs ingesting apoptotic cells might contribute to the different
phenotype, intracellular IL-10 production and release into the
supernatant were studied. IL-10 was detectable in DC culture
supernatant but was unaffected by interaction with apoptotic cells or
the addition of LPS (Fig. 5A).
Intracellular IL-10 was difficult to detect reliably over background
staining, and no differences in levels of IL-10 staining between
ac+ and ac- DCs were
detectable (data not shown). Furthermore, blockade of functional IL-10
by soluble IL-10R did not differentially affect costimulatory molecule
expression in the two DC subpopulations (data not shown). Finally, LPS
activation of DCs derived from bone marrow of IL-10-deficient mice was
also inhibited by the ingestion of apoptotic cells. Interestingly,
these DCs demonstrated a heightened responsiveness to LPS, confirming
an autocrine feedback role for IL-10 in DC maturation (Fig. 5B).
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-1 is another important inhibitory cytokine implicated in
anti-inflammatory effects of apoptotic cells. TGF- is found in
apoptotic cells, preferentially localized to the mitochondria
(23), as well as being secreted by macrophages ingesting
apoptotic cells (14, 15, 16). Although TGF-1 could be found
in our culture supernatants, levels of serum contamination made
determining its origin difficult (data not shown). However, when a
soluble TGF-R was used to neutralize active TGF-1 released by DCs
they were still inhibited after ingesting apoptotic cells (Fig. 5C). In contrast, soluble TGF-R was capable of blocking
inhibition of TNF- production by macrophages that had ingested
apoptotic cells in a parallel system (Fig. 5D). Taken
together these data mitigate against a role for autocrine IL-10 or
TGF-1 in inhibiting the DC response to LPS while confirming the
previously reported role for TGF- in the inhibition of macrophages
that have ingested apoptotic cells. Ingestion of apoptotic cells generates DCs with diminished capacity to sustain Ag-dependent unprimed T cell proliferation despite LPS maturation
To determine whether these cytokine and surface CD86 differences
reflected a distinct functional phenotype of DCs, we chose to examine
the capacity of ac- DCs and
ac+ DCs to sustain Ag-dependent naive T cell
proliferation, a process critically dependent on IL-12 production and
expression of costimulatory molecules. The use of unprimed T cells from
DO11.10 TCR-transgenic mice allowed us directly to compare T cell
proliferation in response to mature ac+
DCs vs ac- DCs, pulsed in both cases with
OVA323–339 peptide after LPS maturation.
Interestingly, ac+ DCs retained the ability to
sustain naive T cell proliferation but were only 30% as effective
as stimulators compared with ac- DCs or DCs
matured without apoptotic cells when cultured at a ratio of 10:1, T
cells:DCs (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bone marrow-derived DCs and macrophages arise from common myeloid
precursors and share many characteristics while maintaining subtle
differences in responses and effector functions. Ingestion of apoptotic
cells stimulates macrophages to adopt an anti-inflammatory
phenotype, inhibiting LPS-induced release of TNF- and up-regulating
release of TGF-1 and other anti-inflammatory mediators
(24, 25). Furthermore, previous reports and our
unpublished data emphasize that this phenotypic change in macrophages
ingesting apoptotic cells is spread to surrounding cells through the
paracrine action of cytokine release triggered by the
ingestion of apoptotic cells. This is in contrast to the response
of DCs ingesting apoptotic cells, which, in this current study, did
not affect the ability of neighboring DCs that had not ingested
apoptotic cells to mature or stimulate T cells. This would implicate a
direct and cell-specific effect of apoptotic cell ingestion on DC
expression of CD86 and cytokine production, rather than a paracrine
effect of secreted anti-inflammatory cytokines. Our data lend
weight to the possibility that the different recognition mechanisms
employed for ingestion of apoptotic cells by monocyte-derived
phagocytes might determine the different responses seen between
macrophages and DCs (7, 26), although we have not set out
to define the receptors for apoptotic cells implicated in the
inhibition of DC maturation in this study. The preferential inhibition
of IL-12 has been demonstrated by ligation of a large number of
phagocytic receptors used by macrophages, including some that are also
expressed on DCs (27, 28). The CD36/integrin
v5/thrombospondin
complex has been implicated as the major receptor for apoptotic cells
in DC phagocytosis, and recently binding of both malaria-infected
erythrocytes (29) or apoptotic cells (30) to
this complex has been shown to inhibit human DC maturation. Although
these studies did not address whether apoptotic cell internalization
must occur, our data imply that a direct interaction is necessary for
these effects to be seen.
Apoptotic cells are poorly immunogenic and, unless they overload normal
clearance mechanisms (probably becoming secondarily necrotic) or are
associated with danger signals, rarely incite an immune response
(31). Furthermore, UV irradiation, characterized by
widespread apoptosis, is associated with generalized immunological
hyporesponsiveness, demonstrating a potential immunosuppressive effect
of apoptotic cells on the adaptive immune system. In an interesting
recent report, injection of apoptotic cells was able
to promote bone marrow engraftment even across MHC barriers in a
species-independent manner (32). Although the exact
mechanisms of immunosuppression in such systems are not fully
understood, a bone marrow-derived cell, likely to be the DC, has been
implicated. Exactly how this occurs is controversial, but an
increasingly accepted view is that the immature DC, with low levels of
costimulatory molecule expression, would fail to deliver signal 2 and
induce anergy or deletion of an interacting T cell. In support of this,
repeated immunization with immature DCs does appear to induce Treg/Tr1
cells. In contrast, mature DCs, which are able to secrete a potent
stimulatory cytokine, IL-12, and express high levels of costimulatory
molecule expression, induce strong adaptive immunity. IL-12 has been
shown to have a myriad of functions, including modulating Th1 vs Th2
switching, activation of NK cells, and production of IFN-.
Furthermore, the autocrine effects of IL-12 have been shown to augment
DC responses to exogenous stimuli, underscoring the importance of this
cytokine in DC effector functions and the subsequent adaptive immune
response. Although failure of ac+ DCs to produce
high levels of IL-12 might contribute to some of the subsequent
phenotypic changes, including diminished T cell stimulation, we have no
direct evidence of whether this is indeed the case and is the subject
of ongoing investigation. Nevertheless, failure to produce IL-12 after
apoptotic cell ingestion appears to correlate closely with these
changes.
The importance of ingestion of apoptotic cells by DCs is underscored by circumstantial evidence implicating such DCs in maintaining tolerance. For example, a specific population of rat lymph DCs, characterized by being OX41-CD4-, has been described. These cells represent a major population found in the lymph draining the intestinal epithelium and have blunt pseudopodia and coarse granular inclusions, identified as being derived from apoptotic intestinal epithelial cells. Functionally, these OX41-CD4- rat lymph DCs also demonstrate an impaired ability to stimulate T cells in vitro and have been implicated in the ability of the gut to handle large amounts of foreign Ags in a tolerogenic fashion (33, 34, 35). Interestingly, our in vitro cultured ac+ DCs also share some of these physical characteristics with the OX41-CD4- rat lymph DCs (our personal observations). While comparisons between in vivo/ex vivo studies of rat DCs and our in vitro work on murine DCs need to be made with caution, taken together these data lend strong support to the concept that ingestion of apoptotic cells by DCs modulates their function.
In conclusion, it is essential for DCs to mature before they can activate naive T cells, and our data and two recent studies (4, 5) confirm that ingestion of apoptotic cells alone did not provide sufficient maturation stimulus. However, some necrotic cells or virally infected apoptotic cells are effective stimulators of DC maturation. Therefore, DCs ingesting apoptotic cells must be exposed to additional agents, such as necrotic cells, monocyte-conditioned medium, or viral products, before they become capable of stimulating T cells. Many of these agents will be present in inflamed sites alongside apoptotic cells in vivo, and the potential for DCs both to acquire apoptotic cell-derived self-Ags and receive maturation signals is high. However autoimmunity is uncommon, and the response of the DC is likely to be tightly regulated. We suggest that ingestion of apoptotic cells is not immunologically null, but is capable of regulating DC maturation, providing a counterbalance for inflammatory stimuli. A failure to see these inhibitory effects of apoptotic cells in other studies may reflect the percentage of DCs ingesting apoptotic cells and the strength of the maturation stimulus used. In the future, defining whether apoptotic cells themselves are sufficient to alter DCs effector functions in vivo will be of great interest. Further investigating this process will increase our understanding of the mechanisms controlling peripheral self-tolerance while giving us new insights into strategies for Ag delivery that might generate tolerance rather than immunity. In contrast, understanding how apoptotic tumor cells or pathogens might also use this phenomenon for immune evasion will increase our understanding of tumor immunology and infectious disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lynda M. Stuart, Medical Research Council Center for Inflammation Research, University of Edinburgh, Teviot Place, Edinburgh, Scotland, U.K. EH8 9AG. E-mail address: lynda.stuart{at}ed.ac.uk
3 Abbreviations used in this paper: DC, dendritic cell; ac, apoptotic cell; SAC, Staphylococcus aureus (Cowan strain).
Received for publication September 6, 2001. Accepted for publication December 12, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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v5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J. Exp. Med. 188:1359., PGE2, and PAF. J. Clin. Invest. 101:890.[Medline]
v3). J. Immunol. 161:6250. released by apoptotic T cells contributes to an immunosuppressive milieu. Immunity 14:715.[Medline]
v5 integrin recruits the CrkII-Dock180-rac1 complex for phagocytosis of apoptotic cells. Nat. Cell Biol. 2:899.[Medline]
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P. Hubert, N. Jacobs, J.-H. Caberg, J. Boniver, and P. Delvenne The cross-talk between dendritic and regulatory T cells: good or evil? J. Leukoc. Biol., October 1, 2007; 82(4): 781 - 794. [Abstract] [Full Text] [PDF]
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M. Gray, K. Miles, D. Salter, D. Gray, and J. Savill Apoptotic cells protect mice from autoimmune inflammation by the induction of regulatory B cells PNAS, August 28, 2007; 104(35): 14080 - 14085. [Abstract] [Full Text] [PDF]
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U. Johansson, L. Walther-Jallow, A. Smed-Sorensen, and A.-L. Spetz Triggering of Dendritic Cell Responses after Exposure to Activated, but Not Resting, Apoptotic PBMCs J. Immunol., August 1, 2007; 179(3): 1711 - 1720. [Abstract] [Full Text] [PDF]
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 L.-P. Erwig and P. M. Henson Immunological Consequences of Apoptotic Cell Phagocytosis Am. J. Pathol., July 1, 2007; 171(1): 2 - 8. [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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P. Sen, M. A. Wallet, Z. Yi, Y. Huang, M. Henderson, C. E. Mathews, H. S. Earp, G. Matsushima, A. S. Baldwin Jr, and R. M. Tisch Apoptotic cells induce Mer tyrosine kinase-dependent blockade of NF-{kappa}B activation in dendritic cells Blood, January 15, 2007; 109(2): 653 - 660. [Abstract] [Full Text] [PDF]
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 Y. S. Kim, D. H. Choi, M. L. Block, S. Lorenzl, L. Yang, Y. J. Kim, S. Sugama, B. P. Cho, O. Hwang, S. E. Browne, et al. A pivotal role of matrix metalloproteinase-3 activity in dopaminergic neuronal degeneration via microglial activation FASEB J, January 1, 2007; 21(1): 179 - 187. [Abstract] [Full Text] [PDF]
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A. Krispin, Y. Bledi, M. Atallah, U. Trahtemberg, I. Verbovetski, E. Nahari, O. Zelig, M. Linial, and D. Mevorach Apoptotic cell thrombospondin-1 and heparin-binding domain lead to dendritic-cell phagocytic and tolerizing states Blood, November 15, 2006; 108(10): 3580 - 3589. [Abstract] [Full Text] [PDF]
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 T.-C. Tzeng, J.-L. Suen, and B.-L. Chiang Dendritic cells pulsed with apoptotic cells activate self-reactive T-cells of lupus mice both in vitro and in vivo Rheumatology, October 1, 2006; 45(10): 1230 - 1237. [Abstract] [Full Text] [PDF]
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M. Lucas, L. M. Stuart, A. Zhang, K. Hodivala-Dilke, M. Febbraio, R. Silverstein, J. Savill, and A. Lacy-Hulbert Requirements for Apoptotic Cell Contact in Regulation of Macrophage Responses J. Immunol., September 15, 2006; 177(6): 4047 - 4054. [Abstract] [Full Text] [PDF]
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M. Skoberne, S. Somersan, W. Almodovar, T. Truong, K. Petrova, P. M. Henson, and N. Bhardwaj The apoptotic-cell receptor CR3, but not {alpha}vbeta5, is a regulator of human dendritic-cell immunostimulatory function Blood, August 1, 2006; 108(3): 947 - 955. [Abstract] [Full Text] [PDF]
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D. A. Fraser, S. S. Bohlson, N. Jasinskiene, N. Rawal, G. Palmarini, S. Ruiz, R. Rochford, and A. J. Tenner C1q and MBL, components of the innate immune system, influence monocyte cytokine expression J. Leukoc. Biol., July 1, 2006; 80(1): 107 - 116. [Abstract] [Full Text] [PDF]
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 M. A Wallet, P. Sen, and R. Tisch Immunoregulation of Dendritic Cells Clin. Med. Res., August 1, 2005; 3(3): 166 - 175. [Abstract] [Full Text] [PDF]
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E. Yu. Nikitina, S. A. Desai, X. Zhao, W. Song, A. Z. Luo, R. D. Gangula, K. M. Slawin, and D. M. Spencer Versatile Prostate Cancer Treatment with Inducible Caspase and Interleukin-12 Cancer Res., May 15, 2005; 65(10): 4309 - 4319. [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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L. M. Stuart, K. Takahashi, L. Shi, J. Savill, and R. A. B. Ezekowitz Mannose-Binding Lectin-Deficient Mice Display Defective Apoptotic Cell Clearance but No Autoimmune Phenotype J. Immunol., March 15, 2005; 174(6): 3220 - 3226. [Abstract] [Full Text] [PDF]
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P. R. Hoffmann, J. A. Kench, A. Vondracek, E. Kruk, D. L. Daleke, M. Jordan, P. Marrack, P. M. Henson, and V. A. Fadok Interaction between Phosphatidylserine and the Phosphatidylserine Receptor Inhibits Immune Responses In Vivo J. Immunol., February 1, 2005; 174(3): 1393 - 1404. [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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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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W.-K. Ip and Y.-L. Lau Distinct Maturation of, but Not Migration between, Human Monocyte-Derived Dendritic Cells upon Ingestion of Apoptotic Cells of Early or Late Phases J. Immunol., July 1, 2004; 173(1): 189 - 196. [Abstract] [Full Text] [PDF]
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 F. Ebstein, C. Sapede, P.-J. Royer, M. Marcq, C. Ligeza-Poisson, I. Barbieux, L. Cellerin, G. Dabouis, and M. Gregoire Cytotoxic T Cell Responses against Mesothelioma by Apoptotic Cell-pulsed Dendritic Cells Am. J. Respir. Crit. Care Med., June 15, 2004; 169(12): 1322 - 1330. [Abstract] [Full Text] [PDF]
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 H.-J. Anders, B. Banas, and D. Schlondorff Signaling Danger: Toll-Like Receptors and their Potential Roles in Kidney Disease J. Am. Soc. Nephrol., April 1, 2004; 15(4): 854 - 867. [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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V. S. Zimmermann, A. Bondanza, A. Monno, P. Rovere-Querini, A. Corti, and A. A. Manfredi TNF-{alpha} Coupled to Membrane of Apoptotic Cells Favors the Cross-Priming to Melanoma Antigens J. Immunol., February 15, 2004; 172(4): 2643 - 2650. [Abstract] [Full Text] [PDF]
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J. Colino and C. M. Snapper Opposing Signals from Pathogen-Associated Molecular Patterns and IL-10 Are Critical for Optimal Dendritic Cell Induction of In Vivo Humoral Immunity to Streptococcus pneumoniae J. Immunol., October 1, 2003; 171(7): 3508 - 3519. [Abstract] [Full Text] [PDF]
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M. Lucas, L. M. Stuart, J. Savill, and A. Lacy-Hulbert Apoptotic Cells and Innate Immune Stimuli Combine to Regulate Macrophage Cytokine Secretion J. Immunol., September 1, 2003; 171(5): 2610 - 2615. [Abstract] [Full Text] [PDF]
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A. E. Morelli, A. T. Larregina, W. J. Shufesky, A. F. Zahorchak, A. J. Logar, G. D. Papworth, Z. Wang, S. C. Watkins, L. D. Falo Jr, and A. W. Thomson Internalization of circulating apoptotic cells by splenic marginal zone dendritic cells: dependence on complement receptors and effect on cytokine production Blood, January 15, 2003; 101(2): 611 - 620. [Abstract] [Full Text] [PDF]
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 K. G. Brinker, H. Garner, and J. R. Wright Surfactant protein A modulates the differentiation of murine bone marrow-derived dendritic cells Am J Physiol Lung Cell Mol Physiol, January 1, 2003; 284(1): L232 - L241. [Abstract] [Full Text] [PDF]
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 V. L. M. Esnault Apoptosis: the central actor in the three hits that trigger anti-neutrophil cytoplasmic antibody-related systemic vasculitis Nephrol. Dial. Transplant., October 1, 2002; 17(10): 1725 - 1728. [Full Text] [PDF]
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A. D. Chernysheva, K. A. Kirou, and M. K. Crow T Cell Proliferation Induced by Autologous Non-T Cells Is a Response to Apoptotic Cells Processed by Dendritic Cells J. Immunol., August 1, 2002; 169(3): 1241 - 1250. [Abstract] [Full Text] [PDF]
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) or
medium alone (
) is shown, demonstrating that apoptotic cells can
suppress both LPS- and SAC-driven DC maturation. Apoptotic cells were
excluded from FACS analysis on the basis of small size and bright green
fluorescence. The mean fluorescence ± SD for triplicate
measurements from one culture is shown. Similar results were seen in
three (SAC) or five (LPS) independent experiments. *,
p < 0.05 (by ANOVA). C, DCs were
cultured with latex beads (
); compared with
incubation with no particles (
