Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, K. D.
Right arrow Articles by Lim, J.-S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, K. D.
Right arrow Articles by Lim, J.-S.
(Journal of Leukocyte Biology. 2001;69:426-434.)
© 2001 by Society for Leukocyte Biology

Inhibition of glucocorticoid-mediated, caspase-independent dendritic cell death by CD40 activation

Kwang Dong Kim, Yong-Kyung Choe, In Seong Choe and Jong-Seok Lim

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucocorticoids (GC) are potent anti-inflammatory and immunosuppressive agents that act on a variety of immune cells, including T cells, monocytes/macrophages, osteoclasts, and dendritic cells (DC). However, the mechanism(s) by which GC exert anti-inflammatory effects is still largely unknown. It is already well known that GC treatment inhibits DC maturation and interleukin (IL)-12 production by DC. In this study, we investigated the apoptosis induction of DC by a synthetic GC, dexamethasone (Dex). The stimulation with Dex resulted in DC apoptosis in a dose- and time-dependent manner as it was measured by determining annexin V-positive cells and mitochondrial potential. In contrast, monocytes that are precursor cells of DC are resistant to Dex-mediated apoptosis. The Dex-induced apoptosis of DC was independent of caspase activation because it was not inhibited by the broad caspase inhibitor, Z-VAD-fmk. It is interesting that agonistic CD40 antibody completely inhibited Dex-induced cell death, whereas other inflammatory stimuli did not show the same effect, suggesting that CD40 signaling may selectively modulate GC-mediated DC apoptosis. Taken together, our findings revealed an important role of GC and CD40 signaling in the regulation of immune responses in which DC play a key role in the inflammatory process of various immunomediated diseases.

Key Words: CD40 signaling • dexamethasone • apoptosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) are the most potent antigen-presenting cells found in lymphoid organs that have the unique ability to prime naive T lymphocytes. The understanding of the origins, trafficking, and function of DC has dramatically increased in recent years, and is still emerging [1 ]. On activation, immature DC undergo maturation and migrate to the lymph nodes where they encounter naive T cells [2 ]. DC maturation is accompanied by the occurrence of CD83 antigen on the cell surface and the diminished expression of the mannose receptor (MR) and antigen uptake capacities [3 , 4 ]. In contrast, the knowledge of the fate of DC after their activation of neighboring T cells is relatively lacking and there is little information about the negative regulation on newly activated DC. Recently, some studies have demonstrated that DC derived from monocytes, bone marrow (BM), or CD34+ progenitor cells expressed Fas upon maturation, but they were resistant to Fas-mediated apoptosis [5 6 7 ]. It was postulated in their studies that a high level of FLIP or up-regulation of bcl-2 protein expression in DC is involved in the resistance to Fas-mediated death. A cytokine such as interleukin (IL)-10 may have inhibitory effects on DC functions including cell differentiation, expression of costimulatory molecules, and IL-12 production [8 , 9 ]. However, there has been little evidence to indicate induction of DC death by the treatment of negative regulatory cytokines except for IL-10 inducing apoptotic death of mature Langerhans cells (mLC) [10 ]. In addition, widely used immunomodulators like steroids may block DC maturation [11 , 12 ].

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 {alpha} (TNF-{alpha}), 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents for cell culture, cytokines, and antibodies
All cultures were performed in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; GIBCO, Grand Island, NY). Growth factors used in the primary cultures of DC precursors were recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; provided by LG Biotech, Iksan, South Korea) and rhIL-4 (R & D Systems, Minneapolis, MN). Flow cytometric analysis for determining apoptosis of DC was performed using annexin V-fluorescein isothiocyanate (FITC) (PharMingen, San Diego, CA) and MitoTracker Orange CMTMRos (Molecular Probes, Eugene, OR). The following mAbs were used: phycoerythrin (PE)-conjugated CD83 (Immunotech, Marseille, France), 3C10 directed against CD14 (TIB-228), and the affinity-purified form from ascites prepared with G28.5 hybridoma directed against CD40 (HB-9110, ATCC, Rockville, MD). Control Abs included mouse IgG1, FITC/PE-coupled IgG1 (PharMingen), and FITC-coupled goat F(ab’)2 anti-mouse IgG (Biosource International, Camarillo, CA) as isotype controls and a secondary reagent, respectively. Dexamethasone and mifepristone (RU486) were purchased from Sigma.

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-{alpha} (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 [{alpha}-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GC induces DC death
DC generated by culturing human blood monocytes for 7 days with GM-CSF and IL-4 exhibited characteristic DC cell morphology and typically expressed high levels of cell adhesion and costimulatory molecules [25 ]. They did not express monocyte surface marker, CD14, and had immature phenotypes of DC defined not only by a weak CD83 antigen expression, but also by a high capacity of Ag uptake. We initially examined apoptosis induction of monocyte-derived DC from peripheral blood of healthy individuals through the use of synthetic GC, Dex. Cell death was detected by determining the annexin V-positive cells. As shown in Figure 1A , Dex treatment specifically induced DC apoptosis in a dose- and time-dependent manner. When cells were double-stained with annexin V and the DNA dye propidium iodide (PI), we observed that Dex increased annexin V-positive cells, whereas the cells were partially PI negative, confirming again the process of apoptotic cell death in DC (data not shown). The specific death did not result from the minimal contamination of lymphocytes residing in the DC culture, because DC having high forward scatter (FSC) and side scatter (SSC) values could be easily gated on FACS analysis. The significant increase of apoptosis was obtained after the treatment with 0.1–1 µM Dex for 48 h when compared with DC cultured in the absence of it. Furthermore, when mitochondrial membrane potential, whose decrease is also thought to be an early event in apoptosis in DC, was determined with MitoTracker Orange, Dex treatment indeed induced a change of membrane potential (Fig. 1B) . Collectively, these data suggest that Dex is able to induce DC apoptosis mostly due to mitochondrial damage.



View larger version (54K):
[in this window]
[in a new window]
  		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.

 
Inhibition of Dex-induced DC death by blocking the GC receptor with mifepristone
To determine whether Dex evokes GC receptor-mediated DC death, DC were incubated with GC receptor blocker, mifepristone (RU486), in the presence of Dex (10-7 M). We observed a substantially decreased rate of apoptosis in DC that were co-treated with Dex and mifepristone (48.9 vs. 24.6%; Dex vs. Dex plus 10-6 M mifepristone, P<0.05; Fig. 2 ), indicating that Dex specifically induces cell death via GC receptor on DC. However, the inhibition effect was reduced by a treatment with 10-5 M mifepristone, showing its cellular toxicity in high concentration.



View larger version (29K):
[in this window]
[in a new window]
  		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).

 
Differential susceptibility of monocytes and monocyte-derived DC to GC-mediated cell death
To determine whether Dex-induced cell death is cell type-specific, not due to the monocyte death by Dex, we treated monocytes and monocyte-derived DC with Dex for 48 h and examined cell death by performing dual staining for surface phosphatidylserine and mitochondrial membrane potential. DC were sensitive to Dex treatment when judged from the loss of plasma membrane integrity and decrease of mitochondrial membrane potential, whereas monocytes expressing CD14 antigen were relatively resistant to it (Fig. 3 ). The staining with anti-CD14 antibody revealed that ~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 this window]
[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.

 
Activation of caspase is not essential for DC death by glucocorticoid
Caspases that are abundantly expressed in lymphoid cells have been found to play a critical role in mediating apoptosis [26 ]. To investigate the effect of caspase inhibition on Dex-mediated DC death, DC were treated with Dex in the absence or presence of generic pharmacological caspase inhibitor Z-VAD-fmk. We found that treatment of 10 µM Z-VAD-fmk did not prevent Dex-induced cell death in 48 h (Fig. 4 ). Dex-induced DC death was not influenced by the presence of Z-VAD-fmk at concentrations up to 100 µM (data not shown). In a similar experiment using caspase-3 inhibitor, DEVD-cho, again no inhibition of cell death was observed (data not shown). In contrast, when Fas-mediated apoptosis was measured with Jurkat cells for the comparison, it was almost completely inhibited by the caspase inhibitors (Fig. 4) . Accordingly, the caspase inhibitors including Z-VAD-fmk, which significantly inhibited Fas-mediated mitochondrial transmembrane potential loss and membrane phosphatidyl-serine externalization, had no preventive effect on Dex-induced changes in DC.



View larger version (52K):
[in this window]
[in a new window]
  		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.

 
CD40-mediated signal blocks GC-induced DC death
Because the CD40 signal rescues DC from spontaneous cell death and renders them stimulus for survival through activation, experiments were set up to examine whether CD40 cross-linking could alter Dex-induced apoptosis of DC. Indeed, the addition of 1 µg purified anti-CD40 mAb (G28.5) into the DC culture almost completely prevented the DC apoptosis induced by Dex, whereas isotype-matched control antibody did not change the cell death by Dex (Fig. 5 A and B ). In contrast, cell death remained unchanged during antibody treatment without Dex (see below). Therefore, these data suggest that CD40 ligation could counteract Dex-mediated DC death. In addition, it was important to exclude the possibility that cytokines were modulating DC death. However, the presence of cytokines (GM-CSF plus IL-4) in the DC culture did not significantly change the Dex-induced DC death, excluding the cytokine effect on DC susceptibility to Dex-induced cell death (Fig. 5A) .



View larger version (36K):
[in this window]
[in a new window]
  		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).

 
There have been several reports that the inflammatory stimuli including CD40 ligation can induce DC maturation and/or activation [27 28 29 ]. Therefore, we then investigated whether the inflammatory signals involved in DC maturation could prevent DC cell death to the same degree as CD40 activation. To confirm the effect of different maturation signals on DC development, DC derived from three healthy individuals were treated with TNF-{alpha}, 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-{alpha}, 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-{alpha} 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-{alpha} (200 ng/mL) or IL-1ß (50 ng/mL) were employed (Dex + TNF-{alpha}, 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-{alpha}. 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 this window]
[in a new window]
  		Figure 6. Differential effect of DC maturation signals on Dex-mediated DC death. (A) Inflammatory signals including TNF-{alpha}, 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-{alpha}, IL-1ß, and LPS, but partially by CD40 ligation. DC were cultured with TNF-{alpha} (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).

 
Expression of cell death inhibitor genes in DC
Using the RNase protection assay, we compared the mRNA expression of apoptosis-related genes by DC cultured in the absence or presence of Dex and Dex plus CD40 antibody for 12 h (Fig. 7 ). Bcl-x mRNA was constitutively expressed in DC, but only low levels of Bcl-2 and Bfl-1 mRNAs were detected. Dex moderately down-regulated antiapoptotic gene expressions, whereas the stimulation with CD40 antibody significantly enhanced mRNA expression of these genes. In contrast, the significant levels of pro-apoptotic genes including Bad, Bak, Bax, were detected in DC whether stimulated or not with Dex or Dex plus CD40 antibody. When we determined the mRNA expression pattern of Bcl-xL and Bcl-2 by RT-PCR using RNA sources from different donors, expressions of Bcl-xL and Bcl-2 in DC were decreased up to an undetectable level of transcripts at 6 and 12 h after Dex treatment, respectively (Fig. 8 ). Simultaneous addition of CD40 antibody to the DC culture not only provided protection against early down-regulation of Bcl-xL and Bcl-2 gene expression by Dex, but also induced the overexpression of them at 12 h after the stimulation with CD40 antibody compared with that observed in controls. These data collectively indicate that Dex might induce DC death by the down-regulation of anti-apoptotic gene expression and CD40 activation could counteract Dex-mediated DC death by up-regulating anti-apoptotic gene expression.



View larger version (36K):
[in this window]
[in a new window]
  		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.

 


View larger version (33K):
[in this window]
[in a new window]
  		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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we were able to demonstrate that GC induce apoptosis in human DC in a time- and dose-dependent manner. GC-induced DC death was not attenuated by inhibition of caspases, which was enough to block Fas-induced apoptosis in Jurkat cells, indicating that GC are able to induce apoptosis of DC, independent of caspase activation. To our knowledge, this study is the first to show that treatment with corticosteroids in vitro induces the cell death in DC generated from monocytes in the presence of GM-CSF and IL-4.

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-{alpha}, 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-{alpha}, 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-{alpha} had a partial inhibitory effect on GC-induced cell death, although the presence of GC blocked DC maturation by TNF-{alpha}, 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.


   ACKNOWLEDGEMENTS
 
This work was supported in part by Good Health R & D Project (HMP-98-B-1-0002) from the Ministry of Health and Welfare of South Korea.

Received September 18, 2000; revised October 26, 2000; accepted October 27, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y. J., Pulendran, B., Palucka, K. (2000) Immunobiology of dendritic cells Annu. Rev. Immunol. 18,767-811[Medline]
  2. Steinman, R. M., Swanson, J. (1995) The endocytic activity of dendritic cells J. Exp. Med. 182,283-288[Free Full Text]
  3. Sallusto, F., Lanzavecchia, A. (1994) Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha J. Exp. Med. 179,1109-1118[Abstract/Free Full Text]
  4. Sallusto, F., Cella, M., Danieli, C., Lanzavecchia, A. (1995) Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products J. Exp. Med. 182,389-400[Abstract/Free Full Text]
  5. Bjorck, P., Banchereau, J., Flores-Romo, L. (1997) CD40 ligation counteracts Fas-induced apoptosis of human dendritic cells Int. Immunol. 9,365-372[Abstract/Free Full Text]
  6. Ashany, D., Savir, A., Bhardwaj, N., Elkon, K. B. (1999) Dendritic cells are resistant to apoptosis through the Fas (CD95/APO-1) pathway J. Immunol. 163,5303-5311[Abstract/Free Full Text]
  7. Willems, F., Amraoui, Z., Vanderheyde, N., Verhasselt, V., Aksoy, E., Scaffidi, C., Peter, M. E., Krammer, P. H., Goldman, M. (2000) Expression of c-FLIP(L) and resistance to CD95-mediated apoptosis of monocyte-derived dendritic cells: inhibition by bisindolylmaleimide Blood 95,3478-3482[Abstract/Free Full Text]
  8. Koch, F., Stanzl, U., Jennewein, P., Janke, K., Heufler, C., Kampgen, E., Romani, N., Schuler, G. (1996) High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10 J. Exp. Med. 184,741-746[Abstract/Free Full Text]
  9. Steinbrink, K., Wolfl, M., Jonuleit, H., Knop, J., Enk, A. H. (1997) Induction of tolerance by IL-10-treated dendritic cells J. Immunol. 159,4772-4780[Abstract]
  10. Ludewig, B., Graf, D., Gelderblom, H., Becker, Y., Kroczek, R., Pauli, G. (1995) Spontaneous apoptosis of dendritic cells is efficiently inhibited by TRAF (CD40-ligand) and TNF-{alpha}, but strongly enhanced by interleukin-10 Eur. J. Immunol. 25,1943-1950[Medline]
  11. Moser, M., De Smedt, T., Sornasse, T., Tielemans, F., Chentoufi, A. A., Muraille, E., Van Mechelen, M., Urbain, J., Leo, O. (1995) Glucocorticoids down-regulate dendritic cell function in vitro and in vivo Eur. J. Immunol. 25,2818-2824[Medline]
  12. Sacedon, R., Vicente, A., Varas, A., Jimenez, E., Munoz, J. J., Zapata, A. G. (1999) Glucocorticoid-mediated regulation of thymic dendritic cell function Int. Immunol. 11,1217-1224[Abstract/Free Full Text]
  13. Zacharchuk, C. M., Mercep, M., Chakraborti, P. K., Simons, S. S., Jr, Ashwell, J. D. (1990) Programmed T lymphocyte death. Cell activation- and steroid-induced pathways are mutually antagonistic J. Immunol. 145,4037-4045[Abstract]
  14. Blotta, M. H., DeKruyff, R. H., Umetsu, D. T. (1997) Corticosteroids inhibit IL-12 production in human monocytes and enhance their capacity to induce IL-4 synthesis in CD4+ lymphocytes J. Immunol. 158,5589-5595[Abstract]
  15. Weinstein, R. S., Jilka, R. L., Parfitt, A. M., Manolagas, S. C. (1998) Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone J. Clin. Invest. 102,274-282[Medline]
  16. Piemonti, L., Monti, P., Allavena, P., Sironi, M., Soldini, L., Leone, B. E., Socci, C., Di Carlo, V. (1999) Glucocorticoids affect human dendritic cell differentiation and maturation J. Immunol. 162,6473-6481[Abstract/Free Full Text]
  17. Gruber, J., Sgonc, R., Hu, Y. H., Beug, H., Wick, G. (1994) Thymocyte apoptosis induced by elevated endogenous corticosterone levels Eur. J. Immunol. 24,1115-1121[Medline]
  18. Brunetti, M., Martelli, N., Colasante, A., Piantelli, M., Musiani, P., Aiello, F. B. (1995) Spontaneous and glucocorticoid-induced apoptosis in human mature T lymphocytes Blood 86,4199-4205[Abstract/Free Full Text]
  19. Iwata, M., Hanaoka, S., Sato, K. (1991) Rescue of thymocytes and T cell hybridomas from glucocorticoid-induced apoptosis by stimulation via the T cell receptor/CD3 complex: a possible in vitro model for positive selection of the T cell repertoire Eur. J. Immunol. 21,643-648[Medline]
  20. Ramierz, F., Fowell, D. J., Puklavec, M., Simmonds, S., Mason, D. (1996) Glucocorticoids promote a TH2 cytokine response by CD4+ T cells in vitro J. Immunol. 156,2406-2412[Abstract]
  21. Joyce, D. A., Steer, J. H., Abraham, L. J. (1997) Glucocorticoid modulation of human monocyte/macrophage function: control of TNF-alpha secretion Inflamm. Res. 46,447-451[Medline]
  22. Kunicka, J. E., Talle, M. A., Denhardt, G. H., Brown, M., Prince, L. A., Goldstein, G. (1993) Immunosuppression by glucocorticoids: inhibition of production of multiple lymphokines by in vivo administration of dexamethasone Cell. Immunol. 149,39-49[Medline]
  23. Almawi, W. Y., Beyhum, H. N., Rahme, A. A., Rieder, M. J. (1996) Regulation of cytokine and cytokine receptor expression by glucocorticoids J. Leukoc. Biol. 60,563-572[Abstract]
  24. Vieira, P. L., Kalinski, P., Wierenga, E. A., Kapsenberg, M. L., de Jong, E. C. (1998) Glucocorticoids inhibit bioactive IL-12p70 production by in vitro-generated human dendritic cells without affecting their T cell stimulatory potential J. Immunol. 161,5245-5251[Abstract/Free Full Text]
  25. Kim, K. D., Lee, H. G., Kim, J. K., Park, S. N., Choe, I. S., Choe, Y. K., Kim, S. J., Lee, E., Lim, J. S. (1999) Enhanced antigen-presenting activity and tumour necrosis factor-alpha-independent activation of dendritic cells following treatment with Mycobacterium bovis bacillus Calmette-Guerin Immunology 97,626-633[Medline]
  26. Nicholson, D. W., Thornberry, N. A. (1997) Caspases: killer proteases Trends Biochem. Sci. 22,299-306[Medline]
  27. Winzler, C., Rovere, P., Rescigno, M., Granucci, F., Penna, G., Adorini, L., Zimmermann, V. S., Davoust, J., Ricciardi-Castagnoli, P. (1997) Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures J. Exp. Med. 185,317-328[Abstract/Free Full Text]
  28. De Smedt, T., Pajak, B., Muraille, E., Lespagnard, L., Heinen, E., De Baetselier, P., Urbain, J., Leo, O., Moser, M. (1996) Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo J. Exp. Med. 184,1413-1424[Abstract/Free Full Text]
  29. Cella, M., Engering, A., Pinet, V., Pieters, J., Lanzavecchia, A. (1997) Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells Nature 388,782-787[Medline]
  30. Woltman, A. M., de Fijter, J. W., Kamerling, S. W., Paul, L. C., Daha, M. R., van Kooten, C. (2000) The effect of calcineurin inhibitors and corticosteroids on the differentiation of human dendritic cells Eur. J. Immunol. 30,1807-1812[Medline]
  31. Brokaw, J. J., White, G. W., Baluk, P., Anderson, G. P., Umemoto, E. Y., McDonald, D. M. (1998) Glucocorticoid-induced apoptosis of dendritic cells in the rat tracheal mucosa Am. J. Respir. Cell Mol. Biol. 19,598-605[Abstract/Free Full Text]
  32. Schmidt, M., Pauels, H. G., Lugering, N., Lugering, A., Domschke, W., Kucharzik, T. (1999) Glucocorticoids induce apoptosis in human monocytes: potential role of IL-1 beta J. Immunol. 163,3484-3490[Abstract/Free Full Text]
  33. Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I., Snow, B. E., Brothers, G. M., Mangion, J., Jacotot, E., Costantini, P., Loeffler, M., Larochette, N., Goodlett, D. R., Aebersold, R., Siderovski, D. P., Penninger, J. M., Kroemer, G. (1999) Molecular characterization of mitochondrial apoptosis-inducing factor Nature 397,441-446[Medline]
  34. Daugas, E., Susin, S. A., Zamzami, N., Ferri, K. F., Irinopoulou, T., Larochette, N., Prevost, M. C., Leber, B., Andrews, D., Penninger, J., Kroemer, G. (2000) Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis FASEB J 14,729-739[Abstract/Free Full Text]
  35. Bertho, N., Drenou, B., Laupeze, B., Berre, C. L., Amiot, L., Grosset, J. M., Fardel, O., Charron, D., Mooney, N., Fauchet, R. (2000) HLA-DR-mediated apoptosis susceptibility discriminates differentiation stages of dendritic/monocytic APC J. Immunol. 164,2379-2385[Abstract/Free Full Text]
  36. Rescigno, M., Martino, M., Sutherland, C. L., Gold, M. R., Ricciardi-Castagnoli, P. (1998) Dendritic cell survival and maturation are regulated by different signaling pathways J. Exp. Med. 188,2175-2180[Abstract/Free Full Text]
  37. Lee, H. H., Dadgostar, H., Cheng, Q., Shu, J., Cheng, G. (1999) NF-kappaB-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes Proc. Natl. Acad .Sci. USA 96,9136-9141[Abstract/Free Full Text]
  38. Drenou, B., Blancheteau, V., Burgess, D. H., Fauchet, R., Charron, D. J., Mooney, N. A. (1999) A caspase-independent pathway of MHC class II antigen-mediated apoptosis of human B lymphocytes J. Immunol. 163,4115-4124[Abstract/Free Full Text]
  39. Deas, O., Dumont, C., MacFarlane, M., Rouleau, M., Hebib, C., Harper, F., Hirsch, F., Charpentier, B., Cohen, G. M., Senik, A. (1998) Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes J. Immunol. 161,3375-3383[Abstract/Free Full Text]
  40. Skov, S., Klausen, P., Claesson, M. H. (1997) Ligation of major histocompatability complex (MHC) class I molecules on human T cells induces cell death through PI-3 kinase-induced c-Jun NH2-terminal kinase activity: a novel apoptotic pathway distinct from Fas-induced apoptosis J. Cell Biol. 139,1523-1531[Abstract/Free Full Text]
  41. Sarin, A., Williams, M. S., Alexander-Miller, M. A., Berzofsky, J. A., Zacharchuk, C. M., Henkart, P. A. (1997) Target cell lysis by CTL granule exocytosis is independent of ICE/Ced-3 family proteases Immunity 6,209-215[Medline]
  42. Green, D. R., Reed, J. C. (1998) Mitochondria and apoptosis Science 281,1309-1312[Abstract/Free Full Text]
  43. Kluck, R. M., Bossy-Wetzel, E., Green, D. R., Newmeyer, D. D. (1997) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis Science 275,1132-1136[Abstract/Free Full Text]
  44. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., Wang, X. (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade Cell 91,479-489[Medline]
  45. Hu, Y., Benedict, M. A., Wu, D., Inohara, N., Nunez, G. (1998) Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation Proc. Natl. Acad. Sci. USA 95,4386-4391[Abstract/Free Full Text]
  46. Pan, G., O’Rourke, K., Dixit, V. M. (1998) Caspase-9, Bcl-XL, and Apaf-1 form a ternary complex J. Biol. Chem. 273,5841-5845[Abstract/Free Full Text]
  47. Boise, L. H., Thompson, C. B. (1997) Bcl-x(L) can inhibit apoptosis in cells that have undergone Fas-induced protease activation Proc. Natl. Acad. Sci. USA 94,3759-3764[Abstract/Free Full Text]
  48. Wakisaka, S., Suzuki, N., Takeba, Y., Shimoyama, Y., Nagafuchi, H., Takeno, M., Saito, N., Yokoe, T., Kaneko, A., Asai, T., Sakane, T. (1998) Modulation by proinflammatory cytokines of Fas/Fas ligand-mediated apoptotic cell death of synovial cells in patients with rheumatoid arthritis (RA) Clin. Exp. Immunol. 114,119-128[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
L. Arruvito, S. Giulianelli, A. C. Flores, N. Paladino, M. Barboza, C. Lanari, and L. Fainboim
NK Cells Expressing a Progesterone Receptor Are Susceptible to Progesterone-Induced Apoptosis
J. Immunol., April 15, 2008; 180(8): 5746 - 5753.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
M. Dauer, K. Schad, J. Junkmann, C. Bauer, J. Herten, R. Kiefl, M. Schnurr, S. Endres, and A. Eigler
IFN-{alpha} promotes definitive maturation of dendritic cells generated by short-term culture of monocytes with GM-CSF and IL-4
J. Leukoc. Biol., August 1, 2006; 80(2): 278 - 286.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
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]

Home page
 Int ImmunolHome page
H. Igarashi, K. L. Medina, T. Yokota, M. I. D. Rossi, N. Sakaguchi, P. C. Comp, and P. W. Kincade
Early lymphoid progenitors in mouse and man are highly sensitive to glucocorticoids
Int. Immunol., May 1, 2005; 17(5): 501 - 511.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
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]

Home page
 J. Leukoc. Biol.Home page
S. M. L. Tamma and R. F. Coico
IgD-receptor (IgD-R) cross-linking partially protects murine T cells from dexamethasone-induced apoptosis
J. Leukoc. Biol., June 1, 2003; 73(6): 764 - 770.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
A. M. Woltman and C. van Kooten
Functional modulation of dendritic cells to suppress adaptive immune responses
J. Leukoc. Biol., April 1, 2003; 73(4): 428 - 441.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, K. D.
Right arrow Articles by Lim, J.-S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, K. D.
Right arrow Articles by Lim, J.-S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS