| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Hospital for Special Surgery, Cornell University Medical Center, New York, NY 10021; and
The Rockefeller University, New York, NY 10021
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and/or TNF-
or by blocking the DC
survival signals TNF-related activation-induced cytokine and CD40L.
However, apoptosis could be induced with C2-ceramide, suggesting that
signals proximal to the generation of ceramide might mediate resistance
to Fas. Analysis of protein expression of several anti-apoptotic
mediators revealed that expression of the intracellular inhibitor of
apoptosis Fas-associated death domain-like IL-1-converting
enzyme-inhibitory protein was significantly higher in Fas-resistant DCs
than in Fas-sensitive macrophages, suggesting a possible role for
Fas-associated death domain-like IL-1-converting enzyme-inhibitory
protein in DC resistance to Fas-mediated apoptosis. Our results
demonstrate that murine DCs differ significantly from other APC
populations in susceptibility to Fas-mediated apoptosis during cognate
presentation of Ag. Because DCs are most notable for initiation of an
immune response, resistance to apoptosis may contribute to this
function. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) (4, 5) through the Fas pathway. The interaction is Ag specific, MHC
restricted, and may play an important role in limiting Ag presentation
to Th1 CD4+ T cells (1, 4).
Dendritic cells (DCs) are the most potent of the known APCs
(6). They are professional APCs specialized in Ag capture,
migration to secondary lymphoid organs, and T cell priming
(6). Compared with M and B cells, DCs have the unique
capacity to trigger primary T cell responses. This has been attributed
to their possession of a prominent intracellular class II compartment
(7), the ability of DCs to synthesize high levels of class
II molecules (6), their constitutively high levels of
cell-surface adhesion and costimulatory molecule expression (8, 9), and expression of a unique chemokine specific for
CD45RA+ naive T cells (10).
Recently, DCs have also been shown to be superior APCs for triggering
proliferative responses and IFN- production by mature T cells
through secretion of IL-12 (11, 12, 13).
To determine whether DCs are negatively regulated by Th1 CD4+ T cells in the same way as other APCs, we studied the expression and function of Fas in murine DCs. Because several studies have demonstrated the heterogeneity of murine DCs depending on their developmental origin and maturation state (14, 15), we examined both bone marrow (BM)-derived and splenic DCs. Our study reveals that although DCs expressed Fas upon maturation, they were not susceptible to Fas-mediated cell death. These results reveal an important difference in immunoregulation of different types of APCs during cognate interaction with T cells and suggest that resistance to Fas-mediated apoptosis may contribute to the potency of DCs as professional APCs.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
MRL/MpJ-Faslpr (MRL/lpr), MRL/MpJ+ (MRL/+), C3H/HeSnJ (C3H), C57BL/6, and BALB/c mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and subsequently bred at the Hospital for Special Surgery. The mutant strain, CBA/K1Jms/lprcg/lprcg (CBA/lprcg) and the wild type, CBA/K1Jms (CBA/+) were kindly provided by Akio Matsuzawa (Institute of Medical Science, Tokyo, Japan) (16). MRL, C3H, and CBA are all H-2k strains, BALB/c is H-2d, and C57B/6 is H-2b.
Cell lines and clones
A.E7, a CD4+ Th1 T cell clone (17), was kindly provided by Dr. R. H. Schwartz (National Institutes of Health, Bethesda, MD). A.E7 is specific for the carboxyl-terminal fragment of pigeon cytochrome c (peptide 81–104) presented by I-Ek (18). A.E7 was maintained as previously described (19) using moth DASp (kindly provided by Marc Jenkins, University of Minnesota, MN), a peptide comprising moth residues 86–90 spliced to pigeon cytochrome c residues 94–104. This peptide has been shown to induce more vigorous proliferation of A.E7 than pigeon cytochrome c peptide 81–104; that is the A.E7 response to DASp is heteroclitic (18). The surface IgG+ B cell lymphoma line, A20, was maintained as previously described (20).
Dendritic and M cell cultures
BM suspensions were prepared from 6- to 8-wk-old mice as
previously described (21). Briefly, cells were treated
with mAbs and complement to deplete cells bearing MHC class II, CD4,
CD8, and B220 markers. Approximately 1 x
106 cells were suspended in 1 ml of culture
medium (RPMI 1640 medium/5% FCS/50 mm 2-ME, gentamicin; 20 µg/ml) in
the presence of 1000 U/ml recombinant mouse GM-CSF (1 x
107 U/mg; Kirin Brewery, Maebashi, Gumma, Japan).
At days 2 and 4, most of the nonadherent cells, representing developing
granulocytes, were removed and replaced with fresh medium. Clusters of
proliferating DCs were removed from the underlying stroma and adherent
M on day 8. The percentage of mature DCs was determined by
cell-surface Ag staining for CD86 and MHC class II and lack of staining
for F4/80 and RB6 as previously described (21). In some
experiments, DCs were sort-purified (see below).
Two populations of splenic DCs were prepared. Day 0 DCs were isolated from single-cell suspensions of spleens that were prepared by a mild collagenase D digestion at 37°C for 60 min. Positive selection of splenic DCs was performed within 2 h by magnetic cell sorting with the Midi-MACS (Miltenyi Biotech, Camberley, Surrey, U.K.), using N418 MicroBeads to select DCs. Positively selected cells were passed over the selection column twice to increase their purity. Purity of the cells, assessed by staining with FITC-conjugated CD11c, was >85%. DCs allowed to mature overnight were prepared by incubating splenocytes from collagenase-digested spleen overnight in complete medium at 2 x 106 cells/ml. Nonadherent cells were collected the following day, and dead cells were removed by Ficoll-Hypaque centrifugation. Positive selection of DCs was performed as described above. Overnight-matured DCs isolated using this method were >90% viable.
Peritoneal exudate M (PEMs) were isolated as previously described
(4). Briefly, mice were injected with 2 ml of
thioglycollate (TG) broth (Difco, Detroit, MI) followed by peritoneal
lavage 3–4 days later. M were isolated from peritoneal exudate
cells by adherence to plastic and cultured at 37°C in complete RPMI
1640 medium in a humidified incubator with 5%
CO2. In certain experiments, M or DCs were
cultured in the presence of 100 U/ml IFN- (Life Technologies,
Gaithersburg, MD) or 10 ng/ml TNF- (Genzyme, Cambridge, MA). After
1–2 days, the adherent PEMs or BM-derived M were removed by gentle
lavage with PBS containing 2.5 µM EDTA.
Monoclonal Abs, flow cytometry analysis, and recombinant proteins
Abs reactive against murine cell-surface markers, F4/80, B220
(RA3-3A1/6.1), Fc- (2.4G2), CD4 (GK1.5), CD8 (3.155), and
anti-I-A (TIB 120) were purchased from the American Type Culture
Collection (Manassas, VA) and used as either supernatants or grown as
ascites in pristane-treated mice as described (22).
PE-conjugated anti-mouse FasL Ab (MFL3), PE-conjugated Jo-2 Ab,
FITC-conjugated rat anti-mouse B7.2 (CD86), and FITC-conjugated rat
anti-mouse CD11c were purchased from PharMingen (San Diego, CA).
Abs to the granulocyte-specific marker RB6, to CD40L (MR1), and to a
neutralizing anti-TNF- Ab were gifts from Dr. R. M.
Steinman (The Rockefeller University, New York, NY), Dr. R. Noelle
(Dartmouth Medical School, Hanover, NH), and Dr. F. Finkelman
(University of Cincinnati, OH), respectively. TNF-related
activation-induced cytokine (TRANCE) receptor (TRANCE-R)-Fc, a
recombinant protein of the extracellular domain of TRANCE-R fused to
the constant region of human IgG1 (23), and human
CD8-TRANCE, a recombinant molecule of the extracellular domain of
murine TRANCE fused to human CD8 (24), were kind gifts
of Dr. Y. Choi (The Rockefeller University). Soluble FasL protein was
prepared as previously described (25). The following
secondary Abs were purchased from Jackson ImmunoResearch (West Grove,
PA): FITC and B-PE-conjugated streptavidin, FITC-conjugated mouse
F(ab')2 anti-rat IgG (Fc fragment-specific),
and FITC-conjugated anti-human IgG (Fc fragment-specific). Before
staining M or DCs, Fc- receptors were blocked with the mAb,
2.4G2. For single-color analysis, 2 x 104
cells were incubated for 30 min at 4°C with either directly
conjugated Ab or the primary Ab followed by a second 30-min incubation
with FITC- or PE-conjugated secondary Abs. Analysis was performed on a
FACScan instrument (Becton Dickinson, Mountain View, CA). Debris and
dead cells were excluded from the analysis by propidium iodide (PI)
staining and appropriate gating.
Sorting of DCs
Day 8 BMDCs from C3H mice were washed in PBS 0.1% BSA and resuspended at a concentration of 2 x 106/ml. Saturating concentrations of F4/80, RB6, and B220 mAbs were added and the cells were incubated for 45 min at 4°C. The cells were washed twice in PBS and the secondary Ab, FITC-conjugated anti-rat IgG, was added under the same conditions. The stained cells were washed and resuspended at 1 x 107 cells/ml. DCs were sorted based on forward scatter, side scatter, and lack of FITC staining using a FACScan Plus (Becton Dickinson Immunocytometry Systems, San Jose, CA) cell sorter. The cells obtained were >95% pure as judged by B7.2high staining (26).
Lipid analogs and evaluation of apoptosis
C2-ceramide (N-acetylsphingosine) and C2-dihydroceramide (N-acetyldihydrosphingosine) were obtained from Biomol (Plymouth Meeting, PA), and stock solutions were prepared in DMSO and 100% ethanol, respectively. The final concentrations of DMSO and ethanol in the incubations (0.2 and 0.1%, respectively) did not induce apoptosis. Apoptosis was quantified by PI uptake. In brief, PI was added to cells at a final concentration of 50 µg/ml in PBS in the presence of 10 µg RNase and allowed to equilibrate for >2 h in the dark. The percentage of PI-positive cells was then assessed by flow cytometry (FACScan; Becton Dickinson). The percentage of DNA in the subdiploid region reflects the proportion of apoptotic cells (27). Doublets and cell clumps were excluded using a double discrimination FACS module.
Cytotoxicity and cell viability assays
Peritoneal exudate cells were adhered to plastic dishes,
cultured for 1–2 days in the presence of IFN-, and gently
resuspended as described above. BMDCs and adherent BM-derived M were
harvested on day 8 of culture with GM-CSF. Splenic DCs were isolated
from spleen and used immediately after isolation (Day 0 DCs) or were
isolated and used after an overnight incubation of splenocytes
(overnight-matured DCs) as described above. M and DC preparations
were preincubated with optimal concentrations of the DASp peptide
(19, 28). Then, 1–2 x 106
PEMs, BM-derived M, or DC targets were labeled with 100 µCi
51Cr (New England Nuclear, Boston, MA) for 1
h in 37°C, washed, and then used as targets in 4-h cytotoxicity
assays with effector cells. Triplicate wells (5 x
103 target cells per well in 96-well microtiter
plates) were set up to assay spontaneous release (no effector cells),
experimental release (with 2.5–20 x 104
effector T cells), and total release (determined by lysing target cells
with 1% Triton X-100). After incubation for 4 h at 37°C, the
cells were pelleted by centrifugation, and 50 µl of supernatant was
collected and counted in a Microbeta Trilux Counter (Wallac,
Gaithersburg, MD). Mixed target experiments were performed as above
except that the target population was made up of 5 x
103 51Cr-labeled DCs mixed with 5 x
103 unlabeled M per well. Effector A.E7 cells
were used at 5–20 x 104 cells per well to
maintain E:T ratios as indicated.
Cell viability was assessed by a fluorometric assay using the dye Alamar blue (Alamar Bio-Sciences, Sacramento, CA) as described previously (20). Alamar blue was added at 10% final concentration for 4 h at 37°C. Fluorescence was read on a Cytofluor 2350 plate reader (Millipore, Bedford, MA) with excitation at 530 and emission at 590 nm. The results were expressed as: 100 - (fluorescence intensity of test sample/fluorescence intensity of control).
Proliferation assays
The ability of the DCs and M to stimulate A.E7 proliferation
was assessed by thymidine incorporation. A total of 2 x
104 to 1 x 105 A.E7
cells were cultured with decreasing numbers of irradiated DCs or M
(3000 rad to prevent any possible proliferation of the APCs) in 0.2 ml
of RPMI 1640:Eagle Hank’s amino acid complete medium in 96-well
round-bottom tissue culture plates. The cells were either incubated in
the absence or presence of 0.2 µM DASp for 3 days or in some
experiments APCs were pulsed with Ag for 2 h after which the cells
were washed free of Ag before the proliferation assay. Then, 1 µCi of
[3H]thymidine (New England Nuclear) was added
to each well for the final 18 h of culture. Cells were harvested
onto filter mats using a Tomtec Mach III cell harvester (Orange,
CT), and thymidine incorporation was determined on the
scintillation counter. In some experiments, a stimulation index was
calculated as a ratio of the
[3H]thymidine incorporation of A.E7 cells
stimulated in the presence of DCs and 0.2 µM DASp Ag divided by the
[3H]thymidine incorporation of A.E7 cells
stimulated in the presence of DCs without Ag.
Western blot analysis of FLIP, Bcl-2, Bcl-xL, and caspase-1
Peritoneal M, splenic DCs, BMDCs, and BM-derived M were
cultured in RPMI 1640 in the presence or absence of IFN- for 24
h. The cells were lysed, and 50 µg of protein from each sample was
resolved on a 12% SDS-PAGE gel and transferred to Immobilon-P
membranes (Millipore). The blots were blocked in milk, probed with
-Bcl-2 (3F11; PharMingen), -Bcl-xL (S-18; Santa Cruz
Biotechnology, Santa Cruz, CA), -caspase-1 (p10; Santa Cruz
Biotechnology) or -Fas-associated death domain-like IL-1-converting
enzyme (FLICE)-inhibitory protein (FLIP)L
(Upstate Biotechnologies, Lake Placid, NY) Abs and detected with the
appropriate HRP-conjugated secondary Abs and enhanced chemiluminesence
substrate (Amersham, Arlingon Heights, IL). To correct for variations
in protein loading, immunoblots were probed with anti-ribosomal P
Ab (29) followed by HRP-conjugated sheep anti-human
IgG (Amersham) and developed as above.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Two-color flow cytometry analyses revealed that 90% of Day 8
GM-CSF-cultured BM cells were Fas+ and that 60%
of cells were CD86high (all of which was
Fas+) (Fig. 1
).
Similar results were obtained with BMDCs derived from BALB/c, C3H, or
MRL/+ mice. The Fas+ CD86-
cells (Fig. 1) were granulocytes as determined by staining by the
granulocyte-specific marker, RB6 (data not shown). Incubation of BMDCs
for an additional 24 h with either IFN- or TNF- did not
alter Fas expression (data not shown).
|
A shows that
anti-Fas induced little to no death of DCs. In contrast, Jo-2
killed 68% of the Fas-sensitive B cell lymphoma, A20, as previously
described (20). Because priming peritoneal M with the
cytokines TNF- and IFN- sensitized the cells to Fas-mediated
death (4), BMDCs were cultured with IFN- (100 U/ml) or
TNF- (10 ng/ml) for an additional 24 h before addition of the
anti-Fas mAb. Preincubation of BMDCs with these cytokines did not
influence susceptibility to Fas-mediated apoptosis. Extending the
incubation period with Jo-2 to 48 and 72 h, increasing the
concentration of the Jo-2 Ab up to 7.5 µg/ml or extending the
preincubation period of BMDCs with IFN- and TNF- to 48 h as
well as including cycloheximide during the incubations, did not
increase susceptibility of BMDCs to Fas-mediated cell death by Jo-2
relative to control hamster IgG (data not shown). To determine whether
authentic FasL would induce cell death, BMDCs were incubated with a
soluble FasL protein (25). BMDCs remained resistant to
cell death whereas the Fas-sensitive A20 cell line was fully
susceptible to the FasL fusion protein as expected (data not
shown).
|
We previously observed that T cell-mediated apoptosis of M was
more efficient than that induced by the agonist Ab (4). To
examine whether additional signals via adhesion molecules or
coreceptors were required for efficient apoptosis of BMDCs, we used
A.E7, a CD4+ Th1 cell line known to express FasL
following activation (32).
H-2k-positive BMDCs obtained from CBA mice were
pulsed with cytochrome c peptide for 12 h and examined
for their ability to be killed by the Ag-specific, MHC-restricted A.E7
cells in 4-h 51Cr release assays. Peptide-pulsed,
IFN--primed peritoneal M from CBA mice were included for
comparison. Whereas the primed M were susceptible to Th1-mediated
cell death in a dose-dependent manner (Fig. 2
B) as
previously described (4), BMDCs were relatively resistant
to cell death. As expected, both M and BMDCs obtained from a
Fas-deficient CBA/lprcg mouse were
resistant to cell death. Incubation of BMDCs for an additional 24
h with either TNF- or IFN- before exposure to Th1 cells did not
induce susceptibility to cell death (data not shown). Similar findings
were observed in BMDCs that were isolated on a cell sorter to a purity
of >95% (Fig. 3). Taken together with
the resistance to anti-Fas-mediated apoptosis shown above,
these findings demonstrate that BMDCs are relatively Fas resistant in
vitro.
|
in a mixed target experiment. 51Cr-labeled DCs
were mixed with equal numbers of unlabeled M so that each population
was present at equal concentrations as described in Materials and
Methods. As a positive control, a portion of the M were
51Cr-labeled and used in a cytotoxicity assay
performed in parallel to demonstrate adequate activation of the A.E7
cells. As shown in Fig. 2C, the
51Cr-labeled M stimulated the Th1 cells to
kill in a dose-dependent manner as expected. In contrast, the
51Cr-labeled DCs remained resistant to
Fas-mediated cell death despite the presence of M in the target
population. These data suggest that DC resistance to Fas-mediated cell
death is not a result of failure to adequately activate the Th1 cells
as DCs remained resistant despite being in the presence of Th1 cells
stimulated by M APC known to stimulate FasL function in A.E7 cells.
In addition, M and DCs caused equivalent up-regulation of FasL
surface expression on A.E7 cells after a 4-h incubation with Ag (data
not shown). To further examine the activation potential of DCs as
compared with M, proliferation assays were performed. Fixed numbers
of T cells were incubated with decreasing numbers of Ag-pulsed
BMDCs in the presence or absence of DASp Ag. T cell
proliferation was evaluated by [3H]thymidine
incorporation. The DCs were efficient stimulators of A.E7 proliferation
(see Fig. 5C) further suggesting that failure to stimulate
the A.E7 cells is an unlikely explanation for lack of killing of BMDCs.
Taken together, these data, along with DC resistance to Ab-mediated
cell death, suggest that failure to undergo Fas-mediated apoptosis with
Th1 cells is a result of resistance to Fas-mediated cell death by DCs
and not a failure to adequately activate the A.E7 cells. However, BMDCs
were not totally resistant to apoptosis, because C2 ceramide, but not
the inactive precursor of ceramide, dihydroceramide, readily induced
apoptosis of these cells (Fig. 4).
|
|
are susceptible to Fas-mediated cell death
The DCs used in these experiments were generated from progenitors
by inducing differentiation with GM-CSF, a cytokine known to inhibit
polymorphonuclear neutrophil apoptosis (33). To determine
whether GM-CSF inhibited apoptosis of all myeloid-derived cells, we
examined T cell-mediated apoptosis of BM-derived M incubated with
GM-CSF under the identical conditions used for DCs. As shown in Fig. 3B, BM-derived M cultured in the presence of GM-CSF and
peritoneal M were equally susceptible to Fas-mediated cell death by
A.E7 cells, while DCs from the same culture were resistant. Additional
experiments revealed that peritoneal M cultured in the absence or
presence of GM-CSF were equally susceptible to T cell-mediated
apoptosis (data not shown). These findings indicate that GM-CSF does
not block apoptosis of all BM-derived hemopoietic cells, although we
cannot exclude the possibility that BMDCs are more susceptible to the
anti-apoptotic effect of GM-CSF.
Splenic DCs up-regulate Fas upon maturation in vitro but remain resistant to Fas-mediated cell death
To determine whether the observations reported above were
generalizable, Fas expression and function were analyzed on splenic
DCs. As shown in Fig. 5A,
freshly isolated Day 0 DCs were 86% pure as defined by high-level
expression of CD11c (N418), a characteristic of murine spleen DCs
(34). As has been previously reported, a significant
portion of the spleen DCs were CD8-positive (35)
although considerable variability was noted between different
preparations (range, 8–35%). Staining of DCs for Fas expression
revealed that there was little or no Fas expression on the freshly
isolated splenic DCs (Fig. 5A), most likely accounting for
their resistance to A.E7-mediated cytotoxicity (data not shown). When
splenic DCs were allowed to mature overnight, 88% of the cells
expressed high levels of CD11c, and these cells became uniformly
Fas-positive (data not shown). As previously reported
(14), spontaneous cell death of Day 0 splenic DCs was
observed after overnight maturation in vitro (60–80%). This high rate
of spontaneous cell death was not Fas or TNF- dependent as
lpr-derived DCs showed similar levels of cell death, and the addition
of a neutralizing anti-TNF- Ab (10 µg/ml) also failed to
inhibit the spontaneous death in either the lpr or wild-type DCs (not
shown). As previously reported, GM-CSF enhanced the survival of splenic
DCs (14).
As the rate of spontaneous cell death of purified DCs allowed to mature
overnight was too high to allow accurate assessment of susceptibility
to FasL-mediated death by the A.E7 cells, we devised an alternative
method to isolate mature spleen DCs overnight. Whole spleen was
collagenase treated, and splenocytes were prepared. This splenocyte
preparation was cultured overnight, and the DCs were isolated with
CD11c beads as described in Materials and Methods the
following day. Viability of the CD11c-positive population allowed to
mature overnight in the milieu of surrounding splenocytes was >90%,
and, as shown in Fig. 5A, these cells expressed high levels
of Fas. This population of mature viable DCs (termed overnight-matured
spleen DCs (O/N SPDCs) in Fig. 5) was resistant to Fas-mediated
apoptosis by the Th1 cells despite high expression of Fas on their
surface (Fig. 5B). To determine whether these findings could
be explained by failure to present Ag, the DC populations were
irradiated and compared with irradiated BMDC and peritoneal M
populations in proliferation assays with A.E7 cells. As shown in Fig. 5C, all the APC populations displayed potent Ag-presenting
function with M yielding lower proliferation counts as compared with
DCs as previously reported (36). These data generated on
splenic DCs isolated in the absence of GM-CSF suggest that resistance
to Fas-mediated apoptosis is shared by different populations of
DCs.
TRANCE and CD40L are not responsible for resistance of BMDC to Th1-mediated cell death
TRANCE (37) or receptor activator of NF-
B
(38), a novel ligand of the TNF family, is expressed on T
cells and, like CD40L (39), promotes the survival of
mature DCs (24). To examine the role of TRANCE and CD40L
in inhibiting Th1-mediated cell death of BMDCs, as well as their
potential role in Th1-mediated death of M, we first examined
expression of these two molecules on A.E7 cells during the 4-h
incubation period with APCs and Ag. As shown in Fig. 6A, flow cytometric analysis
of the A.E7 cells clearly demonstrated up-regulation of both ligands
during the 4-h 51Cr release assay with either
M or DC APCs. We next investigated whether blocking TRANCE and/or
CD40L function using a TRANCE-R-Fc fusion protein or MR1 anti-CD40L
Ab, respectively, would render the DCs susceptible to A.E7-mediated
apoptosis or would alter the death of M targets to Th1-mediated cell
death. As shown in Fig. 6B, inclusion of TRANCE-R-Fc or MR1
neither enhanced death of BMDCs by A.E7 cells nor altered the death of
the M as compared with isotype controls. Both the MR1 Ab and
TRANCE-R-Fc were functional inhibitors at the concentrations used (Fig. 6, C and D) as previously reported (23, 40). These data reveal that the resistance of BMDCs to
FasL-expressing Th1 cells cannot be overcome by blocking the functions
of TRANCE and CD40L.
|
To further examine the mechanism of DC resistance to Fas-mediated
apoptosis, the expression of several inhibitors of apoptosis were
examined in DC and M populations from the same mice. FLIP interacts
with Fas-associated death domain and FLICE (caspase 8) and potently
inhibits apoptosis induced by both Fas and TNF receptor ligation
(41, 42, 43). To investigate whether FLIP plays a role in DC
resistance to Fas-mediated apoptosis, we compared the levels of FLIP
expression in the M and DC populations by Western blot analysis. As
shown in Fig. 7A, expression
of FLIPL was significantly higher in
Fas-resistant DCs than in Fas-sensitive M, providing a possible
explanation for the relative resistance of DCs compared with M. In
contrast, the levels of Bcl-2 and Bcl-xL did not correlate with
susceptibility or resistance to Fas-mediated cell death (Fig. 7B). Similarly expression of caspase 1 (44, 45)
was equivalent in the different APC populations (not shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
that promote their terminal differentiation, initiate
a T cell response (26). In the current study, we show
that, unlike B cells (1, 46) and M (4, 5),
DCs are highly resistant to Fas-mediated killing. Resistance of DCs to
Fas-mediated apoptosis was not explained by trivial factors such as in
vitro addition of cytokines. GM-CSF is known to be anti-apoptotic
(33), probably through activation of the src kinase, lyn
(47), and to enhance the survival of
splenic DCs after overnight culture (14). However, GM-CSF
did not inhibit Fas-mediated cell death of BM M, and splenic DCs
that were not exposed to GM-CSF were also resistant to T cell-mediated
cell apoptosis. Similarly, although we have previously shown that
Fas-mediated apoptosis of M APCs was more efficient when induced by
CD4+Th1 T cells compared with anti-Fas Ab,
and that sensitivity of the targets was enhanced by cytokines such as
IFN- or TNF- (4), none of these factors could
account for DC resistance in the current study.
Recently, two members of the TNF family have been shown to promote DC
survival. CD40L up-regulates MHC and costimulatory molecule expression
in DCs, induces the expression of a variety of cytokines (e.g., IL-12),
and increases DC survival (48). TRANCE (receptor activator
of NF-B ligand) is a DC-restricted survival factor that mediates T
cell-DC communication (24, 38) and inhibits apoptosis of
mouse BMDCs and human monocyte-derived DCs. Although we demonstrated
up-regulation of both TRANCE and CD40L on A.E7 cells following
incubation with DCs and Ag, resistance to FasL-mediated cell death
could not be overcome by inhibiting the function of TRANCE and CD40L
either alone or in combination. This suggests that a mechanism
independent of TRANCE and CD40L leads to the resistance of DCs to
FasL-mediated apoptosis during the early stages of an immune response
and indicates that resistance to Fas-mediated apoptosis is a third
independent factor contributing to DC survival.
We considered several other explanations for Fas-mediated resistance of
DCs. Whereas resistance of freshly isolated splenic DCs could possibly
be due to low Fas expression, BMDCs that had matured for 8 days or
splenic DCs that matured overnight had high Fas expression and were
very efficient at Ag presentation. Therefore, Fas resistance must be
explained by downstream anti-apoptotic pathways as has been seen in
a number of other cell types (2, 49). Because BMDCs were
susceptible to ceramide-mediated apoptosis, the block in Fas signaling
most likely occurs at, or proximal to, caspase 3 (50).
FLIP, a homologue of FLICE, interferes with the most proximal step of
Fas signal transduction (41). Significantly higher levels
of FLIP protein expression were detected in DC compared with M
obtained from the same mice. These data are consistent with the idea
that high levels of FLIP expression accounts for resistance of DC to
Fas-mediated cell death. Bcl-2 and Bcl-xL inhibit Fas-mediated
apoptosis of some cell types (51) and promote DC survival
in certain circumstances (24, 48). Whereas BMDCs and M
both expressed Bcl-2 and Bcl-xL, expression did not correlate with
susceptibility to Fas-mediated apoptosis. IFN- renders M
susceptible to Fas-mediated apoptosis (4); however, this
cytokine did not effect expression of Bcl-2 or Bcl-xL in BM M or
caspase 1 in BMDCs or M.
The unique finding of this report is that murine DCs from different
sources and at different levels of maturation are resistant to
FasL-bearing Th1 cells during a cognate T cell-APC interaction. In the
afferent limb of an immune response, where DCs activate very small
numbers of Ag-reactive T cells, resistance to apoptosis would provide
DCs with a superior ability to initiate an immune response. In
addition, during an encounter with activated Th1 cells expressing FasL,
resistance to Fas-mediated apoptosis would prolong the DC:T cell
interaction and contribute to the potency of DC APC function. This
would be consistent with recent reports demonstrating the superiority
of DCs over B cells and M in stimulating proliferation and IFN-
production by Th1 cells (12, 13).
Because CD4+ killing of the cognate APCs has been described as a mechanism of down-modulating an immune response, our finding raises the question of how a cognate interaction between a DC and a T cell is terminated. In vivo studies have shown that labeled DCs disappear from lymph nodes following interaction with T cells (52), although it was not known whether the DCs migrated out of the lymph node or were killed by the responding T cells. DeSmedt et al. reported that LPS induced the activation and disappearance of DCs in the spleen and that DC activation by inflammatory cytokines may lead to elimination of the DC and termination of a cognate interaction with T cells (53). Because a similar loss of DCs was observed in wild-type and FasL mutant mice, the findings in this study are entirely consistent with the in vitro studies reported here and indicate that the Fas pathway is unlikely to be involved in DC elimination. Recently, the ability of DCs to acquire and cross-present Ag from apoptotic cells has been demonstrated (54, 55). Therefore, the regulation of cell death in DCs is critical for understanding their proposed role in cross-priming and cross-tolerance (54, 55).
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Dalit Ashany, Hospital for Special Surgery, Cornell University Medical Center, Research Building, Room 324, 535 East 70th Street, New York, NY 10021. E-mail address:
3 Abbreviations used in this paper: FasL, Fas ligand; M, macrophage; DC, dendritic cell; BM, bone marrow; PEM, peritoneal exudate M; TG, thioglycollate; TRANCE, TNF-related activation-inducedd cytokine; TRANCE-R, TRANCE receptor; PI, propidium iodide; BMDC, bone marrow dendritic cell; FLICE, Fas-associated death domain-like IL-1-converting enzyme; FLIP, FLICE-inhibitory protein; O/N SPDC, overnight-matured spleen DC.
Received for publication January 7, 1999. Accepted for publication September 7, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production by T helper 1 cells. Eur. J. Immunol. 26:659.[Medline]
T cells by TNF-. J. Immunol. 160:5221. induces ice gene expression and enhances cellular susceptibility to apoptosis in the U937 leukemia cell line. Biochem. Biophys. Res. Commun. 229:21.[Medline]
modulates a p53-independent apoptotic pathway and apoptosis-related gene expression. J. Biol. Chem. 272:16351.This article has been cited by other articles:
|
|
 |
|
  M. Paulsen, B. Mathew, J. Qian, M. Lettau, D. Kabelitz, and O. Janssen FasL cross-linking inhibits activation of human peripheral T cells Int. Immunol., May 1, 2009; 21(5): 587 - 598. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Mohamood, M. L. Guler, Z. Xiao, D. Zheng, A. Hess, Y. Wang, H. Yagita, J. P. Schneck, and A. R. A. Hamad Protection from Autoimmune Diabetes and T-Cell Lymphoproliferation Induced by FasL Mutation Are Differentially Regulated and Can Be Uncoupled Pharmacologically Am. J. Pathol., July 1, 2007; 171(1): 97 - 106. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Izawa, N. Ishimaru, K. Moriyama, M. Kohashi, R. Arakaki, and Y. Hayashi Crosstalk between RANKL and Fas signaling in dendritic cells controls immune tolerance Blood, July 1, 2007; 110(1): 242 - 250. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Pradhan, J. Genebriera, W. L. Denning, K. Felix, C. A. Elmets, and L. Timares CD4 T Cell-Induced, Bid-Dependent Apoptosis of Cutaneous Dendritic Cells Regulates T Cell Expansion and Immune Responses J. Immunol., November 1, 2006; 177(9): 5956 - 5967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kawasaki, W. J. Hubbard, M. A. Choudhry, M. G. Schwacha, K. I. Bland, and I. H. Chaudry Trauma-Hemorrhage Induces Depressed Splenic Dendritic Cell Functions in Mice J. Immunol., October 1, 2006; 177(7): 4514 - 4520. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. N. Mueller, C. M. Jones, A. T. Stock, M. Suter, W. R. Heath, and F. R. Carbone CD4+ T Cells Can Protect APC from CTL-Mediated Elimination. J. Immunol., June 15, 2006; 176(12): 7379 - 7384. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Shi, J. Wolfe, J. Q. Russell, K. Fortner, N. Hardin, J. Anguita, and R. C. Budd Fas Ligand Deficiency Impairs Host Inflammatory Response against Infection with the Spirochete Borrelia burgdorferi Infect. Immun., February 1, 2006; 74(2): 1156 - 1160. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Franchi, F. Malisan, B. Tomassini, and R. Testi Ceramide catabolism critically controls survival of human dendritic cells J. Leukoc. Biol., January 1, 2006; 79(1): 166 - 172. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-G. de Goer de Herve, D. Durali, T.-A. Tran, G. Maigne, F. Simonetta, P. Leclerc, J.-F. Delfraissy, and Y. Taoufik Differential effect of agonistic anti-CD40 on human mature and immature dendritic cells: the Janus face of anti-CD40 Blood, October 15, 2005; 106(8): 2806 - 2814. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Guo, M. Zhang, H. Tang, and X. Cao Fas signal links innate and adaptive immunity by promoting dendritic-cell secretion of CC and CXC chemokines Blood, September 15, 2005; 106(6): 2033 - 2041. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Baratelli, K. Krysan, N. Heuze-Vourc'h, L. Zhu, B. Escuadro, S. Sharma, K. Reckamp, M. Dohadwala, and S. M. Dubinett PGE2 confers survivin-dependent apoptosis resistance in human monocyte-derived dendritic cells J. Leukoc. Biol., August 1, 2005; 78(2): 555 - 564. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Craft, K. W. Bruhn, B. D. Nguyen, R. Prins, J. W. Lin, L. M. Liau, and J. F. Miller The TLR7 Agonist Imiquimod Enhances the Anti-Melanoma Effects of a Recombinant Listeria monocytogenes Vaccine J. Immunol., August 1, 2005; 175(3): 1983 - 1990. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Dohrman, T. Kataoka, S. Cuenin, J. Q. Russell, J. Tschopp, and R. C. Budd Cellular FLIP (Long Form) Regulates CD8+ T Cell Activation through Caspase-8-Dependent NF-{kappa}B Activation J. Immunol., May 1, 2005; 174(9): 5270 - 5278. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Vassiliou, V. Sharma, H. Jing, F. Sheibanie, and D. Ganea Prostaglandin E2 Promotes the Survival of Bone Marrow-Derived Dendritic Cells J. Immunol., December 1, 2004; 173(11): 6955 - 6964. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. C. Alaniz, S. Sandall, E. K. Thomas, and C. B. Wilson Increased Dendritic Cell Numbers Impair Protective Immunity to Intracellular Bacteria Despite Augmenting Antigen-Specific CD8+ T Lymphocyte Responses J. Immunol., March 15, 2004; 172(6): 3725 - 3735. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Gorbachev and R. L. Fairchild CD4+ T Cells Regulate CD8+ T Cell-Mediated Cutaneous Immune Responses by Restricting Effector T Cell Development through a Fas Ligand-Dependent Mechanism J. Immunol., February 15, 2004; 172(4): 2286 - 2295. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Guo, M. Zhang, H. An, W. Chen, S. Liu, J. Guo, Y. Yu, and X. Cao Fas ligation induces IL-1{beta}-dependent maturation and IL-1{beta}-independent survival of dendritic cells: different roles of ERK and NF-{kappa}B signaling pathways Blood, December 15, 2003; 102(13): 4441 - 4447. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Colino and C. M. Snapper Two Distinct Mechanisms For Induction of Dendritic Cell Apoptosis in Response to Intact Streptococcus pneumoniae J. Immunol., September 1, 2003; 171(5): 2354 - 2365. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. T. Semnani, A. Y. Liu, H. Sabzevari, J. Kubofcik, J. Zhou, J. K. Gilden, and T. B. Nutman Brugia malayi Microfilariae Induce Cell Death in Human Dendritic Cells, Inhibit Their Ability to Make IL-12 and IL-10, and Reduce Their Capacity to Activate CD4+ T Cells J. Immunol., August 15, 2003; 171(4): 1950 - 1960. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hoves, S. W. Krause, D. Halbritter, H.-G. Zhang, J. D. Mountz, J. Scholmerich, and M. Fleck Mature But Not Immature Fas Ligand (CD95L)-Transduced Human Monocyte-Derived Dendritic Cells Are Protected from Fas-Mediated Apoptosis and Can Be Used as Killer APC J. Immunol., June 1, 2003; 170(11): 5406 - 5413. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. TURECI, H. BIAN, F. O. NESTLE, L. RADDRIZZANI, J. A. ROSINSKI, A. TASSIS, H. HILTON, M. WALSTEAD, U. SAHIN, and J. HAMMER Cascades of transcriptional induction during dendritic cell maturation revealed by genome-wide expression analysis FASEB J, May 1, 2003; 17(8): 836 - 847. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Buonocore, F. Paulart, A. Le Moine, M. Braun, I. Salmon, S. Van Meirvenne, K. Thielemans, M. Goldman, and V. Flamand Dendritic cells overexpressing CD95 (Fas) ligand elicit vigorous allospecific T-cell responses in vivo Blood, February 15, 2003; 101(4): 1469 - 1476. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Dreher, M. Kok, C. Obregon, S. G. Kiama, P. Gehr, and L. P. Nicod Salmonella virulence factor SipB induces activation and release of IL-18 in human dendritic cells J. Leukoc. Biol., October 1, 2002; 72(4): 743 - 751. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Tada, J. O-Wang, Y. Takiguchi, K. Tatsumi, T. Kuriyama, S. Okada, T. Tokuhisa, S. Sakiyama, and M. Tagawa Cutting Edge: A Novel Role for Fas Ligand in Facilitating Antigen Acquisition by Dendritic Cells J. Immunol., September 1, 2002; 169(5): 2241 - 2245. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Medema, D. H. Schuurhuis, D. Rea, J. van Tongeren, J. de Jong, S. A. Bres, S. Laban, R. E.M. Toes, M. Toebes, T. N.M. Schumacher, et al. Expression of the Serpin Serine Protease Inhibitor 6 Protects Dendritic Cells from Cytotoxic T Lymphocyte-Induced Apoptosis: Differential Modulation by T Helper Type 1 and Type 2 Cells J. Exp. Med., September 3, 2001; 194(5): 657 - 668. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Micheau, S. Lens, O. Gaide, K. Alevizopoulos, and J. Tschopp NF-{kappa}B Signals Induce the Expression of c-FLIP Mol. Cell. Biol., August 15, 2001; 21(16): 5299 - 5305. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Fields, C. L. Sokol, A. Eaton-Bassiri, S.-j. Seo, M. P. Madaio, and J. Erikson Fas/Fas Ligand Deficiency Results in Altered Localization of Anti-Double-Stranded DNA B Cells and Dendritic Cells J. Immunol., August 15, 2001; 167(4): 2370 - 2378. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Kalled, A. H. Cutler, and L. C. Burkly Apoptosis and Altered Dendritic Cell Homeostasis in Lupus Nephritis Are Limited by Anti-CD154 Treatment J. Immunol., August 1, 2001; 167(3): 1740 - 1747. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Nicolo, B. Tomassini, M. R. Rippo, and R. Testi UVB-induced apoptosis of human dendritic cells: contribution by caspase-dependent and caspase-independent pathways Blood, March 15, 2001; 97(6): 1803 - 1808. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. D. Kim, Y.-K. Choe, I. S. Choe, and J.-S. Lim Inhibition of glucocorticoid-mediated, caspase-independent dendritic cell death by CD40 activation J. Leukoc. Biol., March 1, 2001; 69(3): 426 - 434. [Abstract] [Full Text]
|
|
|
|
|
|
B. A. Bladergroen, M. C. M. Strik, N. Bovenschen, O. van Berkum, G. L. Scheffer, C. J. L. M. Meijer, C. E. Hack, and J. A. Kummer The Granzyme B Inhibitor, Protease Inhibitor 9, Is Mainly Expressed by Dendritic Cells and at Immune-Privileged Sites J. Immunol., March 1, 2001; 166(5): 3218 - 3225. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Rescigno, V. Piguet, B. Valzasina, S. Lens, R. Zubler, L. French, V. Kindler, J. Tschopp, and P. Ricciardi-Castagnoli FAS Engagement Induces the Maturation of Dendritic Cells (Dcs), the Release of Interleukin (Il)-1{beta}, and the Production of Interferon {gamma} in the Absence of IL-12 during Dc-T Cell Cognate Interaction: A New Role for FAS Ligand in Inflammatory Responses J. Exp. Med., December 4, 2000; 192(11): 1661 - 1668. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. O. Funk, H. Walczak, C. Voigtlander, S. Berchtold, T. Baumeister, P. Rauch, S. Rossner, A. Steinkasserer, G. Schuler, and M. B. Lutz Cutting Edge: Resistance to Apoptosis and Continuous Proliferation of Dendritic Cells Deficient for TNF Receptor-1 J. Immunol., November 1, 2000; 165(9): 4792 - 4796. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Willems, Z. Amraoui, N. Vanderheyde, V. Verhasselt, E. Aksoy, C. Scaffidi, M. E. Peter, P. H. Krammer, and M. Goldman Expression of c-FLIPL and resistance to CD95-mediated apoptosis of monocyte-derived dendritic cells: inhibition by bisindolylmaleimide Blood, June 1, 2000; 95(11): 3478 - 3482. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
