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,,
*
Department of Surgery, Division of Biologic Therapeutics and Surgical Oncology,
Department of Pathology, and
University of Pittsburgh Cancer Institute, Pittsburgh, PA 15261; and
University of Pittsburgh Mass Spectrometry Facility, University of Pittsburgh Center for Biotechnology and Bioengineering, Pittsburgh, PA 15219
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, failed to do so.
The protective activity of
12-O-tetradecanoylphorbol-13-acetate is abrogated by
pretreatment with phosphoinositide 3-kinase (PI3K) inhibitor, LY294002.
Therefore, down-regulation of PI3K is the major facet of tumor-induced
DC apoptosis. Tumor SN, N-oleoylethanolamine, or PDMP
suppressed Akt, NF-
B, and bcl-xL in DC, suggesting that
the accumulation of ceramide impedes PI3K-mediated survival signals.
Taken together, ceramide mediates tumor-induced DC apoptosis by
down-regulation of the PI3K pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To achieve a better T cell response against tumors, the presence of functionally active APC or DC in T cell areas may be required. Additionally, the longevity of DC in vivo also contributes to obtaining a favorable T cell response (6), suggesting that DC primed toward apoptosis are unable to support T cell reactions. The increase of apoptosis in DC coincubated with tumor cells and the paucity of DC in implanted tumors have been reported both in human and mouse systems (7, 8). These findings postulated the existence of tumor-derived apoptotic factors; however, such factors have yet to be characterized or identified. Although the pretreatment of DC with IL-12 or CD40 ligand (7) or the transduction of bcl-xL (8) is efficacious in rescuing DC from tumor-induced apoptosis to some extent, the underlying mechanisms involved in increasing DC susceptibility to apoptosis remain unsolved.
DC apoptosis is a physiological phenomenon involved both in the development of DC from precursors and in the elimination of end-stage mature cells. The susceptibility of DC to apoptosis is regulated in the course of their development (9). The signal transduction pathway of DC apoptosis or survival must be fully explored to modulate DC survival.
Cumulative reports have focused on the importance of ceramides as bioeffector molecules involved in cellular stress responses as well as in programmed cell death (10). Intracellular ceramide interacts with several signaling pathways to transduce signals and determine cell fate (10). Nevertheless, the relevance of ceramide for apoptosis is still controversial (11), which is mainly due to the differences in the procedures of ceramide quantification, types of cells used, or the stimuli used to induce apoptosis. Currently, little is known about the implication of ceramide in DC biology, except for the positive impact on DC maturation as assessed by the decrease of phagocytic activity (12).
In this study, we investigated the mechanisms of tumor-induced DC
dysfunction or apoptosis. We found that several tumor supernatants (SN)
increased ceramide and induced apoptosis in bone marrow (BM)-derived
DC. We demonstrate that ceramide mediates tumor-induced DC apoptosis
through the down-regulation of phosphoinositide 3-kinase (PI3K) and its
downstream signals, Akt, NF-B, or bcl-xL. DC
survival factors, including LPS or TNF-, failed to prevent
tumor-induced DC apoptosis. However, the reduction of ceramide was
effective for protecting DC from apoptosis and promoting DC survival in
the presence of tumor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three- to 5-wk-old female C57BL/6 mice (H-2Kb, I-Ab) and BALB/c mice (H-2Kd, I-Ad) were purchased from The Jackson Laboratory (Bar Harbor, ME).
Reagents
Murine GM-CSF and IL-4 were provided by Schering-Plough (Kenilworth, NJ). Cell-permeable inhibitors specific for caspase-3 (N-acetyl-Asp-Glu-Val-Asp-CHO, Ac-DEVD-CHO), were obtained from BD PharMingen (San Diego, CA) and caspase-1 inhibitor, Z-Val-Ala-Asp (OMe)-fluorometylketone (Z-VAD-fmk), from Alexis Biochemicals (San Diego, CA), respectively. Staphylococcus aureus Cowan I (SAC), N-acetyl-D-erythro-sphingosine (C2 ceramide), fumonisin B1 (FB1), L-cycloserine, and 12-O-tetradecanoylphorbol-13-acetate (TPA) were obtained from Sigma (St. Louis, MO). N-dihydroacetyl-D-erythro-sphingosine (C2 dihydroceramide), glucosylceramide synthase inhibitor, D-L-threo 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), and protein kinase C (PKC) inhibitor, bisindoylmaleiamide I were purchased from Calbiochem (La Jolla, CA). Inhibitors of extracellular signal-related kinase (ERK) (PD98059) and p38 stress-activated protein kinase (SAPK) (SB20358) were obtained from Alexis Biochemicals. PI3K inhibitor (LY294002) and acid ceramidase inhibitor (N-oleoylethanolamine, NOE) were obtained from Biomol (Plymouth Meeting, PA).
Cell lines and culture SN
Cell lines syngeneic to C57BL/6 were used to collect culture SN; B16, BL6 (melanoma), MC38 (adenocarcinoma), MCA205, MCA102, and MCA207 (fibrosarcoma). Allogeneic cell lines, SCC-VII (squamous cell carcinoma) and TBJ (neuroblastoma), were also used. As controls for these tumor cell lines, we used the mouse melanocyte cell line (Melan-A) and the mouse fibroblast cell line (L cells). Melan-A was provided from Dr. E. Gorelik (University of Pittsburgh, Pittsburgh, PA) and L cells were purchased from American Type Culture Collection (Manassas, VA), respectively. All cells were maintained in complete medium composed of RPMI 1640 (Life Science Technologies, Gaithersburg, MD) supplemented with 10% FBS, 2 mM L-glutamine, 1% nonessential amino acids, and 100 U/ml penicillin and streptomycin. To prepare the culture SN, 1–2 x 106 cells were seeded in 20 ml of complete medium on 165-cm2 flasks and cultured for 3 or 4 days. Before reaching confluence, SN were collected and centrifuged at 1500 x g for 5 min to remove residual dead cells. All SN were stored at -80°C until use.
Generation of BM DC
BM DC were generated according to the method described previously (7), with some modification. In brief, BM cells were depleted of CD4-, CD8-, or B220-positive cells with the treatment of anti-mouse CD4-, CD8-, and B220 Abs (recovered from hybridoma cell lines RL174, TIB145, and TIB146, respectively) and rabbit complement. Nonadherent cells were cultured in complete medium containing 1000 U/ml each GM-CSF and IL-4 at 37°C, 5% CO2. On day 7 of culture, CD11c-positive cells were separated using anti-mouse CD11c Ab-bound magnetic beads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. The cells were given 20–50% (v/v) cellular SN and were cultured thereafter.
Flow cytometric analysis
The expression of surface molecules on DC was analyzed by FACS (BD Immunocytometry Systems, San Jose, CA). For the staining of MHC class-I (H-2Kb), MHC class-II (I-Ab), CD11c, CD40, CD80, and CD86, FITC- or PE-labeled rat mAbs were used. FITC- or PE-labeled rat IgG was substituted for specific Abs to obtain negative controls. All Abs were purchased from BD PharMingen.
MLR
Allogeneic T cells were separated from BALB/c mice by nylon wool column procedures. The percentage of CD3-positive cells after the separation was >85%, as assessed by FACS. After irradiation of DC, they were suspended in complete medium and seeded at 1 x 102 –2 x 104/well on 96-well flat-bottom culture plates. The responder T cells were mixed with DC at 1 x 105/well and cultured for 5 days at 37°C, 5% CO2. During the last 16–18 h of incubation, 1 µCi/well of [3H]thymidine (New England Nuclear Life Science, Boston, MA) was added. Assays were performed in triplicate. On day 5, the cells were harvested and [3H]thymidine incorporated into T cells was measured by a beta counter.
IL-12 ELISA
After day 7 DC had been cultured with or without tumor SN for 3 days, 1 x 106 of DC were stimulated with a 200–400/1 dilution of SAC (v/v) for 48 h. IL-12 p70 was assayed by an OptEIA ELISA kit (BD PharMingen). The threshold of the assay was 15 pg/ml.
Apoptosis assay
Cardiolipin located on inner mitochondria membranes was stained with 10-nonylacridine orange (NAO; Molecular Probes, Eugene, OR) (13). After the samples had been adjusted to 1 x 106/500 µl, NAO was added at 0.2 µg/ml and incubated for 15 min at 37°C. To evaluate mitochondrial membrane potential, 1 x 106/500 µl of cells were stained with 10 µM of potential sensitive fluorescent dye, rhodamine 123 (Rh123; Calbiochem) for 15 min at 37°C (14). Cells were then washed and subjected to FACS analysis.
The DNA content in samples was analyzed by staining with propidium iodide (PI; Sigma) (14). After DC had been fixed with 50% ethanol for 30 min at 4°C, the samples were treated with RNase A (Sigma) at 0.1 mg/ml for 30 min at 37°C. Then, PI was added to samples at 0.1 mg/ml and incubated for 15 min at room temperature. The samples were subjected to FACS for cell cycle analysis. The population of hypoploidic DNA content (sub G0/G1 fraction) is determined to be apoptotic cells.
To protect DC from apoptosis, we treated day 7 DC with cytokines, caspase inhibitors, or some reagents before the addition of tumor SN. The apoptosis assay was performed 24–72 h following treatment.
Caspase-3 assay
Caspase-3 activity in cells was measured by a FluorAce apopain assay kit (Bio-Rad, Hercules, CA). The assay is based on the detection of fluorogenic substrates specifically cleaved by caspase-3. After 2 x 106 of day 7 DC had been cultured in the presence or absence of SN, the lysates were prepared by freeze-thaw. Caspase-3 activity was measured according to the manufacturer’s instructions.
Analysis of ceramide by mass spectrometry
Cellular lipids were extracted from at least 2 x 105 DC as reported previously (13). Extracts were dissolved in chloroform/methanol (1:2, v/v) before analysis by mass spectrometry. Lipids were analyzed by direct infusion into a Quattro II triple quadrupole mass spectrometer (Micromass, Manchester, U.K.) as reported previously (13). Mass spectra were obtained by scanning the range of 400–950 mass to charge ratio (m/z) every 1.6 s and summing individual spectra. The levels of ceramide were expressed as the sum of the total ion currents (TIC) per cell for each of the ceramide species. In some samples, a similar analysis was performed under positive mode. For the analysis of sphingoid bases, the spectra were obtained by scanning 250–480 m/z every 0.7 s under positive mode. The comparison of ceramide levels was performed among the series of samples on which MS analysis had been performed on the same day under the same settings.
EMSA
Nuclear extracts or cytoplasmic protein were obtained from DC according to the methods described previously (15), with some modification. The cells were suspended in lysis buffer containing 10 mM Tris-HCl (pH 7.5), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF, 0.1 mM Na3VO4, and 0.1% Triton X-100. Then the samples were incubated on ice for 20 min and were centrifuged at 7500 x g for 10 min at 4°C. The pellets were suspended in extraction buffer containing 20 mM Tris-HCl (pH 7.5), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 0.1% Triton X-100, 25% glycerol, 0.5 mM EDTA, 0.5 mM PMSF, and 0.1 mM Na3VO4. After a 30-min incubation on ice, they were centrifuged at 14,000 x g for 20 min at 4°C. The SN were obtained and used as nuclear extracts. The protein concentration was determined by a protein assay kit (Bio-Rad).
Two micrograms of extracts was incubated for 30 min in 20 µl of
reaction buffer containing 10 mM Tris-HCl (pH 7.5), 1 mM
MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, and
4% glycerol with [32P] end-labeled,
double-stranded oligonucleotide probe specific for B site (Promega,
Madison, WI) and 2.0 µg of poly(dI-dC) (Amersham Pharmacia Biotech,
Piscataway, NJ). In some experiments, unlabeled NF-B or mutant
NF-B oligonucleotide (Santa Cruz Biotechnology, Santa Cruz,
CA) was added to the samples at 500-fold excess before the addition of
labeled probe. The complexes were resolved on 5% polyacrylamide gels
in Tris-HCl (pH 8.0)-borate-EDTA buffer. Dried gels were placed with
Kodak (Rochester, NY) OMAT x-ray film for 12–48 h at -70°C.
Western blotting
To isolate the whole cell lysates for immunoblotting, the cell pellets were suspended in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.6 mM PMSF, 0.1% SDS, and 1% Triton X-100. The samples were incubated at 4°C for 30 min and then centrifuged at 14,000 x g for 30 min. The SN were collected, and total protein was quantified. Ten to 100 µg of lysate was separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane blocked with 5% nonfat dry milk. Immunostaining was performed with rabbit polyclonal primary Abs specific for Bcl-xL/S (Santa Cruz Biotechnology) or Akt (Cell Signaling Technology, Beverly, CA) followed by incubation with goat polyclonal anti-rabbit IgG Ab conjugated to HRP (Santa Cruz Biotechnology). Immunoreactive bands were visualized using ECL reagents (New England Nuclear Life Science).
Akt kinase assay
Activity of Akt kinase was measured by an Akt kinase assay kit
(Cell Signaling Technology). Briefly, after DC had been cultured with
or without SN for 3–4 days, whole cell lysates were collected and Akt
was immunoprecipitated from >200 µg of lysate with immobilized mouse
monoclonal anti-Akt Ab (1G1, IgG2a). Kinase reactions were
performed at 30°C for 30 min with precipitated Akt and 1 µg of
GSK-3 fusion protein as substrates in the presence of 200 µM ATP. The
proteins were separated by 10% SDS-PAGE and transferred onto
polyvinylidene difluoride membranes. Phosphorylated GSK-3 was reacted
with rabbit polyclonal anti-phospho-GSK-3
(Ser21/9) Ab and HRP-conjugated anti-rabbit IgG Ab, and
was visualized by LumiGro reagents (Cell Signaling Technology).
Statistical analysis
The Mann-Whitney U test was used to compare the values where appropriate. Values of p < 0.05 were considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After the separation of cells on day 7, >90% of the cells exhibited characteristic DC morphology. After culturing in the presence or absence of tumor SN for an additional 3 days, no significant difference in expression was seen for CD11c, CD40, CD80, CD86, H-2Kb, and I-Ab Ags among the groups (data not shown).
When tumor SN were added to day 7 DC and cultured thereafter with
GM-CSF and IL-4, the viability of B16SN-treated cells decreased more
rapidly than those in the other groups (data not shown). To test
whether tumor SN induced DC apoptosis, we stained cells with Rh123,
NAO, or PI (13, 14). Among the cell lines tested, B16SN
exhibited the most potent proapoptotic activity on DC in these
different assays after 72 h of incubation with SN (Fig. 1
, A and B).
Fibrosarcoma cell lines MCA207 and MCA102 also showed a similar
activity (Fig. 1B). The SN collected from the mouse
melanocyte cell line (Melan-A) and the fibroblast cell line (L cell)
did not induce DC apoptosis (Fig. 1B), indicating that tumor
SN-induced DC apoptosis is not related to the origin of tumor cells.
For the remaining studies, we used the B16SN to examine the mechanisms
of tumor-induced DC apoptosis. When we minimized the DC-B16SN
incubation time, we determined that a 6-h exposure was sufficient to
induce apoptosis after 48 h (data not shown). This indicated that
tumor-induced DC apoptosis was executed by certain apoptotic signals,
and not simply by the lack of nutrients.
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To compare the ability of DC to stimulate T cell proliferation, an
allogeneic MLR assay was performed. Day 10 DC treated with B16SN
exhibited a suppressed T cell response as compared with other SN (Fig. 2A). In addition, with the SAC
stimulation, the production of IL-12 p70 from B16SN-treated day 10 DC
was significantly lower than the other SN (Fig. 2B). These
results suggested that the B16SN had an overall suppressive effect on T
cell stimulatory activity of DC.
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We analyzed lipid profiles of DC by negative and positive
electrospray ionization mass spectrometry (ESI-MS). In the negative ion
mode, the prominent peaks were found at m/z 572, 682, and
684 (Fig. 3A). The other peaks
at m/z 794, 796, and 885 were identified as
phosphatidylcholine (PC)-chloride (Cl-) adducts
(m/z 794, 796) and phosphatidylinositol (PIn, m/z
885), respectively. In DC treated with B16SN, the relative amount of
the 572, 682, and 684 mass ions increased as compared with controls
(Fig. 3), whereas PC and PIn did not significantly change regardless of
the treatments (Fig. 3). Similar results were obtained with DC treated
with MCA207 or MCA102SN (data not shown).
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C). The accumulation of C16 or C24 ceramides correlated
well with the degree of DC apoptosis induced by various tumor SN
(correlation coefficient of TIC and percentage of apoptosis, 0.906).
These results indicated that C16 and/or C24 ceramide accumulation was
closely related with tumor-induced DC apoptosis. To exclude the
possibility that the increase of ceramide is a result of apoptosis, we
examined lipid profiles from day 9 DC that were deprived of GM-CSF and
IL-4 for 2 days. Under these conditions, apoptosis occurred in >40%
of the DC. However, neither C16 nor C24 ceramide increased as compared
with controls (data not shown), suggesting that ceramide is not
generated as a result of apoptosis. Ceramide mediates tumor-induced DC apoptosis
To examine whether ceramide induces DC apoptosis, we added C2
ceramide or its analog C2 dihydroceramide to day 7 DC and examined
apoptosis after 24 h. C2 ceramide, but not C2 dihydroceramide,
induced apoptosis in DC (Fig. 4A).
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B) in a
dose-dependent fashion (data not shown). Such treatments led to DC
apoptosis (Fig. 4C), thus demonstrating that the increase of
endogenous ceramide induces DC apoptosis. When these inhibitors were
used in the presence of B16SN, additive effects were observed in
enhancing ceramide levels and DC apoptosis with enhanced kinetics as
early as 24 h after treatment (Fig. 4, B and
C). Such additive effects were also observed with MCA207 SN
(data not shown). Thus, some tumor SN increase endogenous ceramide
levels in DC, which then contribute directly to the induction of
apoptosis.
Ceramide can be generated by either SM breakdown induced by acid or
neutral sphingomyelinase (SMase) and/or by de novo ceramide synthesis
(10). To block acid or neutral SMase, we used
chlorpromazine (18) or glutathione (19), but
failed to prevent tumor-induced DC apoptosis (data not shown). However,
when DC were treated with L-cycloserine, an inhibitor of serine
palmitoyltransferase (20), ceramide levels and the degree
of apoptosis were reduced (Fig. 5, A and B). The ability of
L-cycloserine to prevent tumor-induced DC
apoptosis was abrogated by the addition of C2 ceramide (Fig. 5B). These results indicate that the increase of ceramide
that is involved in tumor-induced apoptosis is generated via the de
novo synthesis pathway. FB1, an inhibitor of ceramide synthase
(21), also reduced ceramide mass (Fig. 5A);
however, it did not prevent DC apoptosis (Fig. 5B). It has
been reported that the inhibition of ceramide synthase increases
sphingoid bases (sphinganine) that are growth inhibitory and cytotoxic
to some cells (22). In B16SN-treated DC, the sphinganine
level was higher than that in control DC (TIC per cell of C16
sphinganine, 0.45 vs 0.26). FB1 (50 µM) increased sphinganine (TIC,
10.0) and apoptosis (45%) in DC in the presence of B16SN (Fig. 5B). However, the addition of 100 µM
L-cycloserine to FB1-treated culture reduced both
sphinganine and apoptosis to baseline levels (TIC, 0.29; apoptosis
22%) in B16SN-treated DC. These results demonstrate that the
accumulation of sphinganine is critically involved in the failure of
FB1 to prevent DC apoptosis, despite the reduction of C16 and C24
ceramides.
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To test whether a caspase-dependent pathway is involved in DC
apoptosis, caspase-3 activity was measured. Caspase-3 activity was
higher in B16SN-treated DC than in untreated cells (Fig. 6A). On day 7, DC were
pretreated with 25–50 µM caspase-3 inhibitor Ac-DEVD-CHO or
caspase-1 inhibitor Z-VAD-fmk before the addition of tumor SN. Although
the increase of caspase-3 activity in B16SN-treated DC was suppressed
by these inhibitors (data not shown), neither of these rescued DC from
B16 or MCA207 SN-induced apoptosis (Fig. 6B). It is well
known that agonistic anti-Fas Ab (clone CH-11) induces apoptosis in
Jurkat cells in a caspase-dependent fashion. To confirm that the
concentration of these caspase inhibitors is sufficient to block
caspase-dependent apoptosis, we treated Jurkat cells with 200 ng/ml
anti-Fas Ab (CH-11; Upstate Biotechnology, Lake Placid, NY) in the
presence or absence of these inhibitors. After 24 h of incubation,
75% of Jurkat cells were apoptotic by NAO staining in the absence of
the inhibitors. Both Z-VAD-fmk (25 µM) and Ac-DEVD-CHO (25 µM)
strongly inhibited anti-Fas-induced Jurkat cell apoptosis
(apoptotic cells, 7 and 28%, respectively). These results indicate
that the inhibitors were sufficient to prevent caspase-dependent
apoptosis. Therefore, tumor SN-induced DC apoptosis is mainly executed
by a caspase-independent mechanism(s).
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Several molecules are known to improve DC survival, such as
LPS (23, 24), TNF- (25), IL-12
(7), TNF-related activation-induced cytokine
(6), and CD40 ligand (25). We used
10–100 ng/ml LPS, 10–50 ng/ml IL-12, or 10–50 ng/ml recombinant
TNF- to rescue DC from tumor-induced apoptosis. We stimulated DC by
cross-linking CD40 with hamster anti-mouse CD40 mAb and subsequent
anti-hamster IgG Ab (26). However, these reagents
failed to improve DC survival in the presence of tumor SN (data not
shown). These reagents had no significant effect on NOE- or
PDMP-induced DC apoptosis, suggesting that ceramide-mediated DC
apoptosis is resistant to these survival factors (data not shown).
Phorbol ester, TPA, is a potent activator of serine/threonine kinases,
such as PKC, ERK, or PI3K, and a known antagonizer of some types of
ceramide-mediated cell death (27). We added 10 ng/ml TPA
to DC 15 min before the addition of B16 or MCA207 SN. Our results
indicated that TPA protected DC from SN-induced apoptosis (Fig. 7A) and delayed the decrease
of viable DC for up to 6 days (Fig. 7B). Furthermore, TPA
decreased DC apoptosis caused by NOE, but did not in PDMP-treated DC
(Fig. 7A). TPA suppressed ceramide increases induced by
tumor SN or NOE, but not by PDMP (Fig. 7C). These results
demonstrate that TPA is efficacious for the blocking of tumor-induced
or ceramide-mediated DC apoptosis. Such protection by TPA correlated
with the reduction of ceramide levels, suggesting that the inhibition
of ceramide accumulation is one of the mechanisms of preventing DC from
tumor-induced apoptosis.
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A). Alternatively, treatment
with the ERK inhibitor (PD98059) or the p38 SAPK inhibitor (SB23085)
also led to DC apoptosis, but to a much lower level than LY294002 (Fig. 8A). The PKC inhibitor, bisindoylmaleiamide-I had no effect
on DC apoptosis (Fig. 8A). When 10 ng/ml TPA was used before
these inhibitor treatments, DC apoptosis induced by LY294002 or PD98059
was reduced, but that induced by SB23085 was enhanced (Fig. 8A). These data indicate that the PI3K pathway is critically
involved in DC survival, and ERK and p38 SAPK pathways are at least
partially involved. In addition, TPA is able to activate PI3K and ERK
but not p38 SAPK in DC. To examine which pathway was responsible
for TPA-induced protection against tumor-induced DC apoptosis, we
pretreated DC with kinase inhibitors before the addition of TPA and
tumor SN. The protective effect of TPA was abolished with LY294002,
suggesting that activation of PI3K is one of the mechanisms TPA uses in
preventing DC apoptosis (Fig. 8B). Conversely, the
inhibition of PI3K is a major facet of tumor-induced DC apoptosis.
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B, and
bcl-xL in DC
One of the downstream targets of PI3K is Akt/protein kinase B
(28). We examined Akt kinase activity in DC treated with
or without tumor SN. Akt activity decreased in DC incubated with B16SN,
whereas the expression of Akt did not differ (Fig. 9). A downstream Akt target is NF-B,
an essential transcription factor that up-regulates multiple survival
genes in many types of cells (29). EMSA revealed that
NF-B activation was suppressed in B16SN-treated DC (Fig. 10A). The binding of the
probe to the samples was inhibited with consensus but not with mutant
NF-B oligonucleotide, indicating that the binding is specific for
the B sequence (Fig. 10A). In Western blot analysis, the
expression of nuclear NF-B p50 and Rel-B in B16SN-treated DC was
lower than those in untreated DC (data not shown), also confirming the
suppression of NF-B activity. The Bcl-2 family of
genes plays pivotal roles in protecting a variety of cells from
ceramide-mediated apoptosis (30). The expression of
bcl-xL in B16SN-treated DC was reduced compared
with controls (Fig. 10B), whereas bcl-2 expression did not
differ between the two conditions (data not shown).
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B activation, or the expression of
bcl-xL in DC was suppressed by NOE or PDMP (Figs. 9 and 10), suggesting that intracellular ceramide down-regulates Akt,
NF-B, and bcl-xL. The treatment of DC with
LY294002 also impaired these molecules (Figs. 9 and 10), confirming
that Akt, NF-B, and bcl-xL are downstream of
PI3K. The pretreatment of DC with TPA or L-cycloserine
before the addition of B16SN restored NF-B and
bcl-xL levels (Fig. 10), which indicated that
these molecules are up-regulated by the reduction of ceramide and/or
the activation of PI3K. Therefore, these results demonstrate that
ceramide accumulates in DC exposed to some tumor SN, which subsequently
down-regulates PI3K and its downstream Akt kinase,
bcl-xL, and NF-B. All of these changes reduce
the survival potential of DC and subsequently enhance the
susceptibility of DC to apoptosis. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We demonstrated the involvement of ceramide in DC apoptosis through several lines of evidence. First, ceramide accumulated in DC incubated with tumor SN, which resulted in DC apoptosis and correlated well with the degree of apoptosis. Second, the accumulation of endogenous ceramide by treatment with inhibitors of ceramide metabolism induced DC apoptosis. Third, these inhibitors enhanced ceramide mass and accelerated apoptosis in the presence of tumor SN. Finally, the blocking of ceramide synthesis with L-cycloserine prevented DC from tumor-induced apoptosis.
We used ESI-MS to analyze each of the ceramide species in DC (13). ESI-MS of lipids offers several advantages over existing techniques: 1) total lipid profiles are able to be analyzed without any prior purification or chemical derivatization, 2) collision-induced dissociation of the lipids allows direct confirmation of their structure, and 3) changes in structure are able to be assessed directly at the molecular level (13). Using this approach, we previously reported that C16 ceramide is up-regulated during the later phases of apoptosis induced by ionizing radiation or Fas ligation in multiple cell types (13). In this study, we identified C16, C24:1, and C24:0 ceramides as the predominant species that increased in tumor-induced apoptotic DC as determined by the TIC of each of the ceramide species. Like previous studies including our own (31, 32), we used synthetic ceramide as internal standards and confirmed the proportional relationship between the TIC of each of the ceramide species and their ratio to standards (data not shown). In contrast with C16 or C24 ceramide, PC levels did not change regardless of the treatments. Thus, the comparison of ceramide levels by TIC as performed in this study reflects an accurate assessment of ceramide levels.
It has been reported that ceramide changes not only quantitatively but also qualitatively in some stress responses (33). In apoptotic DC, increases in C24:1 and C24:0 ceramides were more distinctive than C16 ceramide, which was opposite to that seen in Jurkat T cells (13). Such differences may be explained in part by the different patterns of lipid profiles based on cell type (31). However, it should be noted that each of the ceramide species may have different biological activities. The proapoptotic activity of C16 and C24 ceramide was evidenced in prior studies in addition to our own (13, 34). On the contrary, some metabolites of ceramide may signal proliferative and anti-apoptotic responses in some cells. Sphingosine-1-phosphate, which is generated from ceramide by the action of ceramidase and sphingosine kinase, has proliferative activity and antagonizes ceramide-induced apoptosis (35). Thus, mass spectrometric analyses of lipid profiles are necessary to identify each species involved at the molecular level.
Many researchers use cell-permeable short chain (C2 or C6) ceramides to mimic the influences of ceramide on cells in vitro (10). We confirmed that C2 ceramide induced DC apoptosis in a dose-dependent manner. However, it is still unclear whether exogenous ceramide analogs accurately mimic the effects of intracellular ceramide (36). Some studies showed that exogenous ceramide acts through different sites of action from endogenous ceramide in terms of caspase activation and apoptosis (36, 37). Thus, the data obtained with such ceramide analogs need to be evaluated carefully. A balance between synthesis and metabolism of ceramide through multiple enzymes determines intracellular ceramide levels. As a substitute for short chain ceramide, we used inhibitors of acid ceramidase (NOE) or glucosylceramide synthase (PDMP) to elevate endogenous ceramide levels in DC. In some types of cells, these inhibitors cause ceramide accumulation by the inhibition of ceramide metabolism and then induce apoptosis (16, 17). With concomitant incubation with tumor SN, these inhibitors accelerated apoptosis and ceramide accumulation in DC as compared with those with SN alone. Therefore, the pathways involving these enzymes are activated in DC to metabolize accumulated ceramide in the presence of tumor SN. In contrast, tumor-derived factors may have an inhibitory activity on these pathways.
The direct downstream targets of ceramide may be different according to cell type, magnitude of ceramide generation, and/or its localization. Ceramide is synthesized mainly by the SMase pathway and the de novo synthesis pathway (10). The enzymes involved in these pathways reside in different intracellular compartments, causing different localizations of ceramide (10, 36). Thus, it is conceivable that cells may respond differently to some forms of stress by generating ceramide from different sources (38, 39). From the sequential analysis of SM mass and the inability of SMase inhibitors to prevent DC death, the involvement of this pathway in DC apoptosis may be unlikely.
Ceramide is also synthesized from sphingosine or sphinganine through the action of serine palmitoyltransferase and ceramide synthase (10, 36). Recently, additional studies implicated that ceramide generated via the de novo pathway has a signaling function in apoptosis (40, 41). Perry et al. (40) reported that this ceramide pathway plays a regulatory role in mediating membrane-related apoptotic events, which were independent of the caspase activation cascade. Most of the signals transduced by the de novo pathway are unknown; however, Raf-1/ERK has been recently reported as its direct downstream target (41). In this study, L-cycloserine was effective in preventing tumor-induced DC apoptosis, but FB1 was not. Several explanations may be possible for such differences in activity. First, FB1 is a mycotoxin and possesses proapoptotic activity in some cell types (42). It has been reported that blocking ceramide synthase with FB1 results in the increase of sphinganine, which is cytotoxic to some cells (22). In our study, sphinganine levels, as well as apoptosis, increased in DC with the FB1 treatment in the presence of B16SN. The combination of L-cycloserine and FB1 reduced sphinganine levels and apoptosis, demonstrating the involvement of sphinganine in FB1-induced enhancement of B16SN-induced DC apoptosis. FB1 exhibited a toxic effect on DC at concentrations as low as 50 µM, whereas L-cycloserine was tolerated up to 200 µM (data not shown). Second, serine palmitoyltransferase is the initial and rate-limiting enzyme in the pathway and governs the production of ceramide (10, 40). L-cycloserine prevents C16, C24, and sphinganine increases in DC, thus resulting in the protection of DC from tumor-induced apoptosis. Based on these data, the putative tumor-derived factor is considered to stimulate the de novo pathway in DC, thus leading to increases in endogenous ceramide levels.
Our study shows that tumor-induced DC apoptosis occurs via a caspase-independent pathway, or at least is not critical for DC apoptosis. One of the mechanisms of caspase-independent apoptosis is the disruption of mitochondria (43). Ceramide has direct effects on isolated mitochondria and its functions, resulting in membrane permeability transition and generation of reactive oxygen species, which are followed by loss of membrane potential (10). In the present study, the involvement of reactive oxygen species as an effector in DC apoptosis is unlikely, because the scavenging agent or mitochondrial respiratory chain inhibitor (44) did not prevent tumor-induced DC apoptosis (data not shown).
Tumor-induced or ceramide-mediated DC apoptosis was not fully reversed
by other DC survival factors, such as LPS, IL-12, TNF-, or agonistic
CD40 Ab. These results suggested that tumor-induced DC apoptosis is
executed by unique and multifactorial mechanisms. The inability of
TNF- or CD40 Ab to rescue DC from tumor- or ceramide-induced
apoptosis may be related to their potential to increase endogenous
ceramide levels in DC (12). We found that phorbol ester,
TPA, is the most effective in protecting DC from tumor-induced
apoptosis. The protective effect of TPA in our system was mostly
attributed to its ability to activate PI3K to reduce intracellular
ceramide mass. The importance of PI3K or ERK pathways for survival or
protection from apoptosis has been demonstrated in many types of cell,
including human or murine DC (23, 24). Downstream of PI3K,
Akt executes anti-apoptotic effects by way of several mechanisms,
such as phosphorylation of Bc1-2/Bc1-xL-associated death
promoter, activation of NF-B, up-regulation of bcl-2 or
bcl-xL, or maintaining the integrity of
mitochondria (28). Whereas some cytokines or growth
factors are known to activate PI3K, stress-induced or exogenous
ceramide has been reported to suppress the PI3K/Akt pathway (45, 46). Also in this study, we confirmed that an endogenous
ceramide increase is able to suppress Akt activity by treating DC with
NOE. In DC pretreated with TPA or L-cycloserine, apoptosis
was attenuated and the expression of NF-B and
bcl-xL were restored, suggesting the importance
of these molecules for DC survival in the presence of tumor SN. Thus,
it is conceivable that the ceramide increase induces DC apoptosis by
the inhibition of the PI3K/Akt and/or ERK-dependent survival pathways,
not by the enhancement of caspase-dependent apoptotic pathways. In our
hands, bcl-2 expression did not differ in the presence of tumor SN.
Different roles or sites of action have been implicated between bcl-2
and bcl-xL in ceramide-mediated apoptosis
(47).
We found that several human tumor cell lines induced maturation and subsequent apoptosis in human monocyte-derived DC within 24–72 h of incubation (data not shown). Kiertscher et al. (48) reported similar findings when tumor SN was added to DC from the beginning of culture. In our study, ceramide accumulated before the occurrence of apoptosis in human DC incubated with tumor SN (data not shown). These results imply the possibility of ceramide playing an essential role in tumor-induced DC apoptosis in both murine and human systems.
The question remains as to the nature of the tumor-derived proapoptotic
factor. It is well known that TNF- or Fas ligand (FasL) is an
inducer of apoptosis in various types of cells, accompanied by the
elevation of ceramide. The possibility that TNF- is responsible for
the observed effects is low, because the addition of rTNF- did not
lead to DC apoptosis and anti-TNF- Ab did not restore
tumor-induced apoptosis (data not shown). Murine BM-derived DC are
reported to express both Fas and FasL. BM DC generated from Fas
knockout (lpr) mice were also susceptible to tumor
SN-induced apoptosis, suggesting that Fas-FasL interaction is not
required (data not shown). Biochemical analyses with B16SN are
currently underway to identify tumor-derived factors.
In summary, our study indicates that some tumor cells produce
proapoptotic factors that act on BM DC. Tumor-induced DC apoptosis is
mediated by ceramide, which down-regulates PI3K, accompanied by the
suppression of NF-B and bcl-xL. TPA protects
DC from apoptosis by activating PI3K and/or enhancing ceramide
metabolism. It has been demonstrated that the longevity of DC in vivo
is necessary to prime and maintain effective anti-tumor immune
responses, which may be attained by protecting DC from apoptosis.
Transduction of anti-apoptotic genes into DC is one approach to
meet these needs (8). Recently, metabolizing endogenous
ceramide by the transduction of acid ceramidase genes or direct
administration of sphingosine-1-phosphate has been shown to attenuate
TNF- or radiation-induced apoptosis (16, 49).
Therefore, the modulation of ceramide synthesis or
ceramide-metabolizing pathways needs to be considered as potential
therapeutic manipulations to prevent DC apoptosis in the clinical
setting.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andrew A. Amoscato, Department of Surgery, University of Pittsburgh, Center for Biotechnology and Bioengineering, 300 Technology Drive, Suite 206, Pittsburgh, PA 15219. E-mail address: amoscatoaa{at}msx.upmc.edu
3 Abbreviations used in this paper: DC, dendritic cell; Ac-DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp-CHO; BM, bone marrow; C2 ceramide, N-acetyl-D-erythro-sphingosine; C2 dihydroceramide, N-dihydroacetyl-D-erythro-sphingosine; ERK, extracellular signal-related kinase; ESI-MS, electrospray ionization mass spectrometry; FB1, fumonisin B1; m/z, mass to charge ratio; NAO, 10-nonylacridine orange; NOE, N-oleoylethanolamine; PC, phosphatidylcholine; PDMP, D-L-threo 1-phenyl-2-decanoylamino-3-morpholino-1-propanol; PI, propidium iodide; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; Rh123, rhodamine 123; SAC, Staphylococcus aureus Cowan I; SAPK, stress-activated protein kinase; SM sphingomyelin; SMase, sphingomyelinase; SN, supernatant; TIC, total ion current; TPA, 12-O-tetradecanoylphorbol-13-acetate; Z-VAD-fmk, Z-Val-Ala-Asp (Ome)-fluorometylketone; PIn, phosphatidylinositol; FasL, Fas ligand.
Received for publication March 20, 2001. Accepted for publication August 1, 2001.
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, p50-p50
homodimers; 
, p50-p65 or p50-RelB, p50-cRel heterodimers.
B, Ten micrograms of cell lysates collected from DC was
separated on 10% polyacrylamide gel. Bcl-xL expression was
examined by Western blot.