Cell Biology Laboratory, Korea Research Institute of Bioscience and Biotechnology, Taejon, South Korea
Correspondence: Jong-Seok Lim, Ph.D., Cell Biology Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusung P.O. Box 115, Taejon 305-600, South Korea. E-mail: jslim{at}kribb4680.kribb.re.kr
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: CD40 signaling • dexamethasone • apoptosis
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Glucocorticoids (GC) are potent anti-inflammatory and immunosuppressive
agents that act on a variety of immune cells, including T cells,
monocytes/macrophages, osteoclasts, and DCs [13
14
15
16
]. GC
have also been reported to play physiological regulatory roles, such as
in the induction of apoptosis [17
, 18
],
involvement in thymic selection [19
], and in the
regulation of the Th1/Th2 balance [20
]. So far, effects
of GC on antigen-presenting cells (APC) have been studied mainly on
monocytes and macrophages where GC have the ability to down-regulate
the production of a large number of cytokines, including IL-1, IL-6,
IL-8, IL-12, tumor necrosis factor 
(TNF-), and
granulocyte-macrophage colony-stimulating factor (GM-CSF)
[21
22
23
]. Although monocytes and macrophages can present
Ag to Th cells, DC are regarded as professional APC during the onset of
the immune response, since they have the particular ability to activate
naive Th lymphocytes.
The effects of GC on DC death have been poorly described. Recently, one study showed that GC are potent inhibitors of production of the bioactive IL-12p70 heterodimer by human DC [24 ]. On the other hand, GC did not affect the level of expression of costimulatory molecules by DC or the ability of DC to take up Ag and stimulate the proliferation of CD4+ Th cells in vitro. Based on these findings, the conclusion was formed that suppression of T cell-mediated inflammation by GC not only relies on a direct effect on T cells, but also on various effects on DC.
In this study, we investigated whether GC might evoke apoptosis in human DC derived from monocytes. By determining the annexin V-positive cells and mitochondrial membrane potential of DC as the sign of early apoptosis, we could clearly demonstrate that GC used in physiological concentrations are strong inducers of apoptosis in human DC. The GC-induced apoptosis of DC was independent of caspase activation because it was not inhibited by the broad caspase inhibitor, Z-VAD-fmk. Furthermore, agonistic CD40 antibody completely inhibited dexamethasone (Dex)-induced cell death, suggesting that the signals generated after cross-linking of CD40 receptor may modulate at an early stage the GC-mediated death signal. Our findings thus suggest that in addition to the inhibition of DC maturation, the well-known anti-inflammatory and immunosuppressive effects of GC are mediated, at least in part, by the direct induction of DC apoptosis. In addition, our results indicate that CD40 activation may play a role in the immune responses under apoptotic stimuli.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Monocyte preparation and generation of DC from peripheral blood
mononuclear cells (PBMC)
For DC generation a method used by Sallusto et al.
[3
] was slightly modified. Briefly, PBMC from healthy
donors (Red Cross Blood Center, Taejon, South Korea) were isolated by
density centrifugation on Histopaque 1077 (Sigma), and then washed
twice with RPMI 1640 medium without serum. After the lysis of
erythrocytes, the populations were resuspended in RPMI 1640 medium
supplemented with L-glutamine (2 mM), penicillin (100
U/mL), streptomycin (100 µg/mL), HEPES (10 mM), and 10%
heat-activated FBS. The plastic-adherent cells (2 h, 37°C) were
cultured overnight, and nonadherent cells were removed. The cells were
cultured in RPMI containing 200 ng/mL of rhGM-CSF and 10 ng/mL of
rhIL-4. DCs were usually harvested after a culture of 6–7 days,
counted, and used for assay. The plastic-adherent monocytes that show a
high expression of CD14 and negative for CD1a were prepared from the
overnight culture.
DC maturation
DC (3 x 105) were treated with IL-1ß
(Endogen, Woburn, MA), TNF- (Strathmann Biotech, Hannover, Germany),
lipopolysaccharide (LPS), or purified agonistic CD40 antibody for
48 h and compared with untreated DC for their CD83 expression by
flow cytometric analysis with FACSCalibur (Becton Dickinson, San Jose,
CA). FITC- and PE-conjugated murine IgG1 mAb of unrelated specificity
was always used as a control. For determining the effect of Dex on DC
maturation, Dex was co-treated with cytokines, LPS, or CD40 antibody
for 24 h, respectively, and CD83 expression on the DC surface was
determined.
Induction of apoptosis and treatment with caspase inhibitor
DC were incubated in RPMI 1640 medium supplemented with 10%
heat-inactivated FBS, rhGM-CSF, and rhIL-4 for 24–72 h in the absence
or presence of Dex. For the comparison, Jurkat cells were incubated for
6 h in the absence or presence of stimulating human Fas antibody,
CH-11 (Upstate Biotechnology, Lake Placid, NY). Inhibition by caspase
inhibitor was performed with Z-VAD-fmk (Enyzme Systems, Livermore, CA),
which was simultaneously added with Dex or stimulating human Fas
antibody.
Flow cytometric analysis for apoptosis
For the analysis of mitochondrial membrane potential and
cell-surface exposure of phosphatidylserine, primary monocytes, DC
(3 x 105), Jurkat, or U937 cells (5 x
105) were incubated with 100 nM MitoTracker for 15 min at
37°C and then washed twice with PBS. The cells were incubated with
1.5 µg/mL FITC-conjugated annexin V in 10 mM HEPES/NaOH (pH 7.4), 140
mM NaCl, 2.5 mM CaCl2 for 15 min at room temperature. The
proportion of apoptotic cells in the prepared cells was determined by
flow cytometric analysis using FACSCalibur. Data were analyzed using
CellQuest software (Becton Dickinson).
Detection of mRNA expression of apoptosis-related genes by RNase
protection assay (RPA)
Total RNA was prepared by RNAzolTM B. The specific
mRNAs for anti- or pro-apoptotic genes were detected by multi-probe
template set, hAPO-2c (RiboQuant; PharMingen, San Diego, CA), which
contains templates for bcl-w, bcl-x, bfl-1, bad, bik, bak, bax, bcl-2,
and mcl-1. In brief, antisense RNA probes were generated from DNA
templates using T7 DNA-dependent RNA polymerase in the presence of
[-32P]UTP (Amersham Pharmacia Biotech, Uppsala,
Sweden). Labeled probes were hybridized with total RNA (15 µg)
overnight at 56°C. Unhybridized RNA was digested with RNase according
to PharMingen’s supplied procedures. RNase-protected probes were
resolved on denaturing 5% polyacrylamide gels. The gels were dried and
exposed to film (CP-BU, AGFA) at -70°C for 1 day.
Detection of Bcl-xL and Bcl-2 mRNA
Relative levels of Bcl-xL and Bcl-2 mRNA were
measured in DC using a reverse transcriptase-polymerase chain reaction
(RT-PCR) method. Total RNA from Dex- or Dex plus CD40 Ab-treated DC was
isolated using RNAzol B (Tel-Test, Friendswood, TX) according to the
manufacturer’s instructions. First-strand cDNA was synthesized using
ProSTARTM First-Strand RT-PCR Kit (Stratagene, La Jolla,
CA). The reaction mixture in a total volume of 20 µL was heated at
95°C for 5 min before adding 2 U of Taq DNA polymerase (Qiagen,
Valencia, CA), followed by 28 cycles (Bcl-xL) or 35 cycles
(Bcl-2) of 94°C (1 min), 55°C (1 min), 72°C (1 min), and a final
extension step at 72°C for 10 min. The primer sequences were:
Bcl-xL; sense 5’-TTGGACAATGGACTGGTTGA-3’, antisense
5’-GGTAGAGTGGATGGTCAGTG-3’, Bcl-2; sense
5’-CACCTGTGGTCCACCTGAC-3’, antisense
5’-ACAGTTCCACAAAGGCATCC-3’. RT-PCR primers for ß-actin were
purchased from Clontech Labs (Palo Alto, CA). PCR products were
analyzed by electrophoresis on a 1.5% agarose gel and visualized by
staining ethidium bromide.
Statistical analysis
Results were expressed as the means ± SD. The
comparison between variables was analyzed using the Student’s unpaired
t test for comparisons of percentages of cell death.
Statistical significance was considered if P was <
0.05.
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Figure 1. Dex mediates cell death of DC in a dose- and time-dependent manner. DC
differentiated from peripheral blood monocytes of healthy donors
in vitro for 1 week were seeded at 3 x
105/mL in RPMI plus 10% FBS containing GM-CSF and IL-4.
(A) Dex was added in several concentrations as indicated. Percentages
of cell death from triplicate cultures after 24, 48, or 72 h were
measured by annexin V binding using flow cytometry. The experiment is
representative of two experiments using DC from different donors. (B)
Apoptotic cells 48 h after treatment with 10-7 M
Dex were determined by double-staining the cells with 100 nM
MitoTracker to detect mitochondrial damage and with 1.5 µg/mL annexin
V-FITC to detect phosphatidylserine. DC underwent significant cell
death showing decrease of mitochondrial membrane potential.
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Figure 2. GC receptor blockade with mifepristone decreases Dex-induced DC death.
DC were cultured for 48 h in the presence of Dex (10-7
M). In addition, they were stimulated with 1 or 10 µM mifepristone.
Apoptosis was measured by annexin V-binding using flow cytometry. Data
represent means ± SD of three independent
experiments. **P < 0.05
(compared with Dex without mifepristone).
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90% of
the cells used for the FACS analysis were CD14+ (data not
shown). Consistent with the relative resistance of primary monocytes to
Dex, the myelomonocytic cell line, U937, remained highly resistant to
cell death induced by Dex (Fig. 3)
, indicating that Dex may
differentially induce cell death in monocytes and DC.
 View larger version (49K): [in a new window] |
Figure 3. Dex induces cell death in DC but not primary monocytes or U937 cells.
Monocytes were enriched by a 90-min adherence to culture plates
followed by overnight culture. Monocyte-derived DC were obtained by 1
week of culture in DC medium. DC, monocytes, and U937 cells were
cultured for 48 h in medium alone or in the presence of
10-7 M Dex, respectively. To determine apoptosis
percentage, cells were stained with 100 nM MitoTracker to detect
mitochondrial damage and with 1.5 µg/mL annexin V-FITC to detect
phosphatidylserine. Data are from one of two experiments with similar
results.
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Figure 4. Caspase inhibitor Z-VAD-fmk does not prevent Dex-mediated cell death in
DC while suppressing Fas-mediated Jurkat cell death. DC were incubated
with 10-7 M Dex in the absence or presence of 10 µM
Z-VAD-fmk for 48 h. Jurkat cells were exposed to 100 ng/mL
anti-Fas mAb (CH-11) for 6 h. After cell harvest, percentages of
apoptotic cells was determined by double-staining the cells with 100 nM
MitoTracker to detect mitochondrial damage and with 1.5 µg/mL annexin
V-FITC to detect phosphatidylserine. Cell death of neither was affected
by the treatment with 10 µM Z-VAD-fmk alone (data not shown). This is
representative of two separate experiments with similar results using
DC from different donors.
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Figure 5. The presence of IL-4 and GM-CSF in culture does not affect DC death
induced by Dex, and CD40 cross-linking prevents Dex-mediated DC death.
(A) DC derived from blood monocytes after 1 week of culture were
further incubated in culture medium without (a, c) or with cytokines
(b, d) for 48 h. Dex (10-7 M) plus control mouse IgG1
mAb (b, c) or Dex plus anti-CD40 mAb (1 µg/mL) (d) was added at the
beginning of the culture. (B) Inhibition of Dex-mediated cell death by
a co-treatment of stimulating anti-CD40 mAb. DC were incubated with Dex
in the absence or presence of anti-CD40 mAb (1 µg/mL) for 48 h.
The mean ± SD of 10 independent experiments (total
n = 16) performed with DC from different donors are
shown. **P < 0.001 (compared
with medium control or Dex plus anti-CD40-treated group).
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, IL-1ß, LPS, and CD40
antibody, respectively. As shown in Figure 6A
, these different signals all were found to induce DC maturation
with respect to the up-regulation of CD83 expression. These
inflammatory stimuli alone were not able to increase DC death when
compared to that of untreated DC (Fig. 6B)
. When the same amount of
TNF-, IL-1ß, or LPS was added to DC cultures containing GC, CD83
expression was completely inhibited (Fig. 6C)
. However, CD40 ligation
was able to partly up-regulate CD83 expression even in the presence of
Dex. In addition, CD40 ligation but to a lesser extent TNF- was able
to inhibit Dex-induced DC death that was not affected by a treatment of
IL-1ß or LPS (Fig. 6B)
. Similar data were obtained when high amounts
of TNF- (200 ng/mL) or IL-1ß (50 ng/mL) were employed (Dex +
TNF-, 27.7 ± 0.7%; Dex + IL-1ß, 55.0 ± 0.6%;
Dex, 52.2 ± 3.1%, mean ± SD, n = 3). DC death after GC treatment was reduced to the level of
spontaneous death by the stimulation with CD40 antibody. These findings
indicate that GC can induce DC apoptosis even in the presence of
maturation signals such as LPS and IL-1ß, but partially in the
presence of TNF-. We conclude therefore that the stimulus by CD40
ligation among various inflammatory signals may provide the signal not
only for the induction of DC maturation, but also for the inhibition of
GC-induced DC death.
 View larger version (29K): [in a new window] |
Figure 6. Differential effect of DC maturation signals on Dex-mediated DC death.
(A) Inflammatory signals including TNF-, IL-1ß, LPS, and CD40
ligation are biologically active and induce DC maturation when
determined by an increase of CD83 expression in DC. DC obtained from
three different individuals were cultured in the presence of each
stimulus for 48 h, and CD83 expression was determined by flow
cytometric analysis. (B) CD40 ligation inhibits Dex-mediated DC death,
whereas IL-1ß and LPS are ineffective at inhibiting DC apoptosis.
Cells were incubated with each stimulus at the same concentration as
shown in panel A, in the absence or presence of 10-7 M Dex
for 48 h and cell death was measured by annexin V-binding using
flow cytometry. The mean ± SD of three independent
experiments performed with DC from different donors are shown.
**P < 0.05 (compared with
medium control). (C) Dex completely inhibits DC maturation by TNF-,
IL-1ß, and LPS, but partially by CD40 ligation. DC were cultured with
TNF- (250 ng/mL), IL-1ß (50 ng/mL), LPS (500 ng/mL), or CD40 mAb
(1 µg/mL) in the absence (dashed line) or presence (solid line) of
10-7 M Dex for 24 h, and CD83 expression was
determined by flow cytometric analysis. The negative control was
stained with mouse FITC/PE-coupled IgG1 (dotted line).
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Figure 7. Detection of apoptosis-related gene mRNA using RPA. Detection of mRNA
was done as described in the procedures supplied by PharMingen. (A)
Lane 1, mRNA of RNase unprotected probes; lane 2, untreated DC; lane 3,
Dex-treated DC; lane 4, Dex plus anti-CD40 antibody-treated DC. (B)
Scanning of the autoradiograms of the multi-probe template sets carried
out with Fujifilm BAS-1500 image analyzer. The values were obtained
using the TINA 2.0 program. Fold increases represent the ratio of each
band from treated DC to that from untreated DC after background values
of L32 mRNA were subtracted. Selected data from one of three
independent experiments are shown.
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Figure 8. Determination of Bcl-xL and Bcl-2 expression level in DC
with the use of RT-PCR. RT-PCR was performed on total cellular RNA
prepared from control DC, DC cultured with Dex, or Dex plus CD40
antibody (1 µg/mL) for 6, 12, and 24 h, respectively. cDNA
derived from control DC, Dex-, or Dex plus anti-CD40 mAb-treated DC was
amplified with specific primers for Bcl-xL or Bcl-2. The
expression of control ß-actin is shown in the bottom panel.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies with murine and human DC generated in vitro have
shown that corticosteroids down-regulate the ability of DC to stimulate
T cells [11
], and in vitro cultured human DC
showed a reduced IL-12p70 production after treatment with
corticosteroid [24
]. Furthermore, corticosteroids had a
unique and profound inhibitory effect on the generation and function of
DC [30
]. However, the effects of GC on DC death have
been poorly described. Only one recent study showed that after in
vivo corticosteroid treatment DC in rat tracheal mucosa rapidly
decreased due to their apoptosis [31
]. It has been shown
that stimulation with GC resulted in monocyte apoptosis, and continuous
treatments of monocytes with IL-1ß could almost completely prevent
GC-induced monocyte death [32
]. They also reported that
GC-induced monocyte apoptosis was diminished by simultaneous treatment
with caspase inhibitor. In contrast, we found that GC induced
caspase-independent DC death, as defined by the loss of plasma membrane
integrity and decrease of mitochondrial membrane potential, whereas
monocytes were relatively resistant to GC treatment. Although it is
unclear whether this divergence reflects the different method used for
the monocyte preparation, a possible explanation may be the low GC
concentration used in our study. In fact, they observed significant
cell death in CD14-positive monocytes at 10-6 M, but not at
10-8 M. Caspase inhibitors, even at a concentration of 100
µM showing partial toxicity after 48 h, did not provide any
protection against GC-induced cell death, excluding the possibility
that they were ineffective because they were present intracellularly at
inadequate concentrations (Fig. 4
and not shown). With respect to this
observation, it is highly likely that a recently described
mitochondrial apoptosis-inducing factor (AIF) may be more related to
the caspase-independent cell death of DC [33
,
34
]. They suggested that the apoptotic activity of AIF is
maintained in the presence of caspase inhibitor, Z-VAD-fmk. Currently,
we are examining whether Dex-treatment can induce apoptosis through AIF
release into cytosol and its translocation into nucleus of these cells.
DC death was GC-specific because GC-induced cell death was
significantly prevented by a co-treatment with GC receptor blocker,
mifepristone. We further demonstrated that when co-treated with
microbial product, LPS, or with proinflammatory cytokines such as
IL-1ß or TNF-, GC-induced DC death was hardly or only partially
affected. Therefore, we conclude that there is a difference in
susceptibility to GC treatment between monocytes and DC, indicating
phenotypical alterations during monocyte differentiation into DC.
Similarly, a recent work showed that different DC populations and
macrophage were differentially susceptible to HLA-DR-mediated cell
death [35
]. It has also been shown that treatment with
GC results in lower numbers of splenic DC, accompanied by an increase
in splenic macrophages [11
].
In our work TNF-, IL-1ß, LPS, or CD40 ligation induced the
membrane expression of CD83, a marker of mature DC, whereas except for
CD40 ligation, inflammatory signal-induced DC maturation was completely
blocked by the presence of GC. Similar to these observations, these
signals were not effective for inhibiting GC-induced DC death. Of note,
TNF- had a partial inhibitory effect on GC-induced cell death,
although the presence of GC blocked DC maturation by TNF-,
indicating the possibility that DC maturation and survival are
regulated by different signaling pathways, as previously described
[36
]. However, CD40-mediated signal provided almost
complete protection against GC-induced DC death.
Although the nature of the CD40 signaling pathway in DC has not been elucidated, CD40 activation may elicit its protective effect against steroid-induced apoptosis in DC through a variety of mechanisms. Early studies showed that CD40 ligation not only activates B cells, but also enhances the survival of monocytes and DC [10 , 37 ]. In particular, it is suggested that survival of DC is mediated through engagement of extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway [36 ]. Although much remains to be done to connect this pathway into the inhibition of GC-mediated cell death by several inflammatory stimuli, our results indicate that GC may differentially influence CD40-dependent and other stimuli-dependent pathways of DC survival.
Despite the enormous strides made in our understanding of regulated cell death, the mechanism(s) by which GC cause apoptosis is still largely unknown. Our data indicate that the caspase-independent DC death induced by GC treatment may be mediated in these cells by alterations in mitochondrial function. A number of different stimuli are able to induce caspase-independent cell death of lymphocytes [38 39 40 ], DC [35 ], and tumor cells [41 ]. Mitochondria are suggested to play a pivotal role as both initiators and targets in a cell death pathway [42 ]. Perturbations of mitochondria allow the release of cytochrome c [43 ], which upon binding to apoptotic protease-activating factor-1 (Apaf-1) and in the presence of dATP leads to the activation of a caspase cascade [44 ]. These events are responsible for many of the biochemical changes characteristic of apoptosis. Anti-apoptotic members of Bcl-2 family stabilize the mitochondrial membrane barrier function and inhibit apoptosis. Bcl-xL has been shown to interact with caspase 9 and Apaf1, resulting in the inhibition of caspase activation [45 , 46 ]. Increased levels of Bcl-xL induced by PKB kinase activity may form inhibitory complexes with caspase 9, Apaf1, and cytochrome c after apoptotic insult, delaying caspase activation and slowing the progression of cell death in T cells. PKB-mediated regulation of Bcl-xL may also antagonize the apoptotic effects of Fas. In fact, Bcl-xL has been associated with inhibition of Fas-induced apoptosis in T cells and B cells [37 , 47 ]. A recent work showed that expression of antiapoptotic gene such as Bcl-2 might be regulated by the inflammatory cytokine milieu [48 ]. In this study, we demonstrated a novel correlation in DC between CD40 activation and expression of anti-apoptotic genes such as Bcl-xL and Bcl-2, and showed that CD40 activation promotes DC survival in the presence of GC through enhanced Bcl-xL and Bcl-2 expression.
In conclusion, we demonstrate that GC used in physiological concentrations are strong inducers of apoptosis in human DC by determining the annexin V-positive cells and mitochondrial membrane potential of DC as the sign of early apoptosis. The GC-induced apoptosis of DC was independent of caspase activation because it was not inhibited by a broad caspase inhibitor, Z-VAD-fmk. Furthermore, agonistic CD40 antibody completely inhibited Dex-induced cell death, suggesting that CD40 activation may selectively modulate GC-mediated DC apoptosis. Therefore, our findings suggest that in addition to the inhibition of DC maturation and activation, the well-known anti-inflammatory and immunosuppressive effects of GC are mediated, at least in part, by the direct induction of DC apoptosis. In addition, the results presented herein further expand the role of CD40 in inflammation in that the enhancement of DC longevity through CD40 signaling may augment and perpetuate inflammatory responses during corticosteroid treatment.
Received September 18, 2000; revised October 26, 2000; accepted October 27, 2000.
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