PGE2 confers survivin-dependent apoptosis

0 downloads 0 Views 388KB Size Report
Sherven Sharma, Karen Reckamp, Mariam Dohadwala, and Steven M. Dubinett1. UCLA Lung Cancer Research Program of the Jonsson Comprehensive ...
PGE2 confers survivin-dependent apoptosis resistance in human monocyte-derived dendritic cells Felicita Baratelli, Kostyantyn Krysan, Nathalie Heuze´-Vourc’h, Li Zhu, Brian Escuadro, Sherven Sharma, Karen Reckamp, Mariam Dohadwala, and Steven M. Dubinett1 UCLA Lung Cancer Research Program of the Jonsson Comprehensive Cancer Center and the Division of Pulmonary and Critical Care Medicine, Department of Medicine, Geffen School of Medicine at University of California Los Angeles; and Molecular Gene Medicine Laboratory, Veteran’s Affairs Greater Los Angeles Healthcare System, California

Abstract: Control of apoptosis is fundamental for dendritic cell (DC) homeostasis. Numerous factors maintain DC viability throughout their lifespan, including inhibitor of apoptosis proteins. Among them, survivin is overexpressed in many human malignancies, but its physiological function in normal cells has not been fully delineated. Prostaglandin E2 (PGE2), also overproduced in several malignancies, has shown to induce proapoptotic and antiapoptotic effects in different cell types, including immune cells. In DC, PGE2 predominantly affects maturation and modulates immune functions. Here, we show that exposure of monocyte-derived DC to PGE2 (10ⴚ5 M) for 72 h significantly increased DC survivin mRNA and protein expression. In contrast, DC, matured with lipopolysaccharide or tumor necrosis factor ␣, did not reveal survivin induction in response to PGE2. Following exposure to apoptotic stimuli, DC treated with PGE2 exhibited an overall increased viability compared with control DC, and this effect was correlated inversely with caspase-3 activation. Moreover, PGE2-treated, survivin-deficient DC demonstrated reduced viability in response to apoptotic stimuli. Further analysis indicated that PGE2 induced DC survivin expression in an E prostanoid (EP)2/EP4 receptor and phosphatidylinositol-3 kinase-dependent manner. These findings suggest that PGE2-dependent regulation of survivin is important in modulating apoptosis resistance in human DC. J. Leukoc. Biol. 78: 555–564; 2005. Key Words: lipid mediators 䡠 immune cells 䡠 antiapoptotic proteins

INTRODUCTION Apoptosis is crucial for the normal development and homeostasis of the immune system. This process can be initiated by a variety of signals leading to the elimination of unwanted cells during differentiation or following completion of their effector function. Survivin, a member of the inhibitor of apoptosis (IAP) gene family, has been implicated in preservation of cell viability as well as control of mitosis in proliferating cells [1, 2]. 0741-5400/05/0078-555 © Society for Leukocyte Biology

Amply expressed in fetal tissues and overexpressed in cancer, survivin has also been described in endothelial cells and normal hematopoietic cells, including CD34⫹ cells, in which it regulates cell-cycle entry and proliferation [3–11]. Survivin expression has also been reported in activated T and B cells, neutrophils, and dendritic cells (DC) [12–15]. Unlike other members of the IAP family, survivin suppresses apoptosis induced by a broad spectrum of stimuli. However, the inhibitory mechanisms underlying apoptosis suppression have not been elucidated completely. Previous reports indicate that survivin inhibits caspase-3 and -7 directly [16, 17], and an inverse correlation between survivin and active caspase-3 occurs in CD34⫹ cells [9 –11] or in tumor cells [18] exposed to apoptotic stimuli. In contrast, recent data provide evidence for an essential interaction of survivin with a second mitochondrial activator of caspase, Smac-DIABLO, in the inhibition of taxolinduced apoptosis, demonstrating the inability of survivin to bind to caspase in this system [19]. DC, the most efficient antigen-presenting cells, are uniquely able to prime effector T cells and control immune responses [20, 21]. DC function involves highly regulated processes, from antigen acquisition in peripheral tissues to migration toward lymphoid organs and antigen presentation to T cells. Through the different phases of their life cycle, DC fate is influenced by a multiplicity of environmental factors that interplay with immunomodulators and affect cell homeostasis and viability. During inflammation or at the tumor site, prostaglandins (PGs) have been identified as key regulators of immune responses and may modulate immune cell survival [22]. PGE2 has been noted to cause differential effects in a cell typespecific and maturation phase-dependent manner [23–30]. PGE2, produced by macrophages and thymic stromal cells, has been shown to inhibit apoptosis in a variety of cells including monocytes [23–25]. PGE2, however, has also been shown to mediate apoptosis in T cells, thymocytes, and B cell lymphomas [26 –28]. In contrast, in nonsmall cell lung cancer

1 Correspondence: Lung Cancer Research Program, Division of Pulmonary and Critical Care Medicine, UCLA Geffen School of Medicine, 37-131 CHS, 10833 Le Conte Ave., Room 37-131 CHS, Los Angeles, CA 90095. E-mail: [email protected] Received October 7, 2004; revised February 24, 2005; accepted April 21, 2005; doi: 10.1189/jlb.1004569.

Journal of Leukocyte Biology Volume 78, August 2005 555

(NSCLC) cells, PGE2 overproduction has been associated with apoptosis resistance and increased survivin expression [18]. Our recent report implicated PGE2-dependent modulation of apoptosis protection in NSCLC via ubiquitination-dependent survivin stabilization [18]. Here, we investigate the effect of PGE2 on survivin expression in human DC. This is the first report documenting that PGE2 up-regulates survivin and leads to heightened apoptosis resistance in human DC.

antigen (HLA)-DR (BD Biosciences PharMingen). The cells were also stained with the corresponding FITC- or PE-conjugated, isotype-matched control antibody (BD Biosciences PharMingen). Viability of the cells was measured using 7-amino-actinomycin-D (7-AAD; Calbiochem). A live gate was set by forwardand side-scatter and 7-AAD staining. Ten thousand cells with high forwardscatter and high side-scatter were counted. Events were acquired by fluorescein-activated cell sorter using a Life Science Research (LSR) instrument (Becton Dickinson, Biosciences Immunocytometry System, San Jose, CA) and CELLQuest software in the University of California, Los Angeles, Jonsson Comprehensive Cancer Center Flow Cytometry Core Facility. Data analysis was performed by CELLQuest software.

MATERIALS AND METHODS

Survivin enzyme-linked immunosorbent assay (ELISA)

Generation of human monocyte-derived DC DC were derived from peripheral blood mononuclear cells (PBMC), as described previously, with minor modifications [29, 30]. Briefly, PBMC were generated from a leukocyte-enriched buffy coat from healthy donors (leucopack). Each leucopack was obtained with Institutional Review Board approval, and all donors signed informed consent. After centrifugation with FicollPaqueTM Plus (Amersham Bioscienses, Piscataway, NJ), the light-density fraction from the 42.5–50% interface was recovered and used to obtain monocytes using CD14⫹ Microbeads (Miltenyi Biotec, Auburn, CA), according to the manufacturer’s instructions. Alternatively, the mononuclear cells were resuspended in RPMI 1640 (Cellgro, Mediatech, Herndon, VA) supplemented with 20 mM HEPES buffer (Cellgro, Mediatech), 100 units/ml penicillinstreptomycin, and 2 mM glutamine (Invitrogen, Grand Island, NY) and allowed to adhere to tissue-culture flasks. After 2 h at 37°C, nonadherent cells were removed. CD14⫹ monocytes or plastic-adherent mononuclear cells were cultured for 8 –11 days in complete RPMI medium supplemented with 10% human serum AB (Gemini Bio-Products, Woodland, CA), 75 ng/ml recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF; Peprotech, Rocky Hill, NY; specific activity ⱖ1⫻107 units/mg), and 75 ng/ml rh interleukin (IL)-4 (Peprotech; specific activity ⱖ5⫻106 units/mg). Cytokines were replaced every 2 days. To induce maturation, lipopolysaccharide (LPS; 5 ␮g/ml, Sigma Chemical Co., St. Louis, MO) or tumor necrosis factor ␣ (TNF-␣; 50 ng/ml, Peprotech) was added to DC on day 6 of culture for 48 h.

DC culture conditions PGE2-treated DC were generated by addition of 16,16-dimethyl-PGE2 (dmPGE2; Cayman Chemical, Ann Harbor, MI) for 72 h on day 5 of culture or on day 8 for LPS- and TNF-␣-matured DC. dmPGE2 is a synthetic analog of PGE2 with a prolonged half-life in vivo and acts as an E prostanoid (EP) receptor agonist. dmPGE2 (PGE2) was used in all the experiments because of its greater stability in vitro. In stimulation experiments, to analyze the effects of EP2/EP4 receptor agonists and antagonists on day 5 of culture, DC were plated in six-well plates at 106/3 ml complete 10% AB medium. Cells were stimulated for 72 h with PGE2 (1.5⫻10⫺5 M, corresponding to 5 ␮g/ml), pertussis toxin (50 ng/ml, Calbiochem, La Jolla, CA), pertussis toxin and PGE2, cholera toxin (1 ␮g/ml, Sigma Chemical Co.), forskolin (20 ␮M, Biomol Research Laboratories, Plymouth Meeting, PA), butaprost (1 ␮M, Cayman Chemical), and sulprostone (1 ␮M, Cayman Chemical). To study the involvement of phosphatidylinositol-3 kinase (PI-3K) and mitogen-activated protein kinase (MAPK) pathways, we used the specific inhibitors [2-(4-morpholinyl)8-phenyl-(4H)-1-benzopyran-4-one] LY294002 and 2⬘-amino-3⬘-methoxyflavone PD98059 (both from Calbiochem), respectively. Briefly, on days 5–7 of culture, DC were incubated with LY294002 (1–5 ␮M) or PD98059 (40 ␮M) or the appropriate concentration of vehicle (dimethyl sulfoxide) for 1 h at 37°C. Cells were washed off the inhibitors and cultured in complete medium with or without PGE2 (1.5⫻10⫺5 M), as described above.

Immunophenotypic analysis of DC by flow cytometry The DC phenotype was analyzed on day 8 of culture. Briefly, cultured cells were stained directly with the following monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE) fluorochromes: CD3, CD11c, CD14, CD40, CD80, CD86 (all from BD Biosciences PharMingen, San Diego, CA), CD83 (Coulter Immunology, Hialeah, FL), and human leukocyte

556

Journal of Leukocyte Biology Volume 78, August 2005

DC cultured under the conditions described above were analyzed for survivin protein expression by Duo Set威 IC ELISA Development System (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. For total protein extracts, (107/ml) cultured DC were solubilized in extraction buffer [6 M urea, 1 mM EDTA, 0.005% Tween 20, 0.5% Triton X-100, 100 ␮M phenylmethylsulfonyl fluoride (PMSF), and 1⫻ CompleteTM protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) in phosphate-buffered saline (PBS), pH 7.2–7.4]. For the ELISA, a 96-well microplate (Nalge Nunc International, Denmark) was coated overnight at room temperature with 4 ␮g/ml rabbit anti-human survivin antibody and blocked for 1 h with blocking buffer (1% bovine serum albumin, 5% sucrose, 0.05% sodium azide in PBS). Standard survivin or DC extracts were incubated for 2 h prior to addition of a biotinylated rabbit anti-human survivin followed by 20 min incubation with a streptavidin-horseradish peroxidase (HRP)-conjugated antibody. For detection, the plate was incubated with a substrate solution (H2O2 and 3,3⬘,5,5⬘tetramethylbenzidine). The reaction was stopped by adding 1 M sulfuric acid (Fisher Scientific, Pittsburgh, PA), and the optical density was read at 450 nm in a Benchmark microplate reader (Bio-Rad Laboratories, Hercules, CA). The sensitivity limit of the ELISA assay was 280 pg/ml.

Induction of apoptosis Apoptosis was induced by the combination of serum starvation and ultraviolet (UV)C irradiation or by staurosporine (Sigma Chemical Co.). Briefly, monocytederived DC cultured in GM-CSF and IL-4 for 5 days were plated at 2 ⫻ 106/6 ml into six-well plates in the presence or absence of serum. Exogenous PGE2 (1.5⫻10⫺5 M) was added to serum-containing or serum-free DC cultures for 72 h. On day 8, only serum-deprived DC with or without PGE2 were exposed for 9 min to a 9000-␮W/cm2 UV source [CL-1000 UV cross-linker UVP, peak emission 280 nm (1 J/cm2 UVC)] and incubated overnight at 37°C, 5% CO2, along with the control cultures. In other experiments, apoptosis was induced by 1 h incubation with staurosporine (1 ␮M).

Annexin-V staining Assessment of apoptosis was accomplished by measuring the translocation of membrane phosphatidylserine using the Annexin-V FITC kit (Biosource International, Camarillo, CA), according to the manufacturer’s instructions. Annexin-V⫹/propidium iodide⫹ cells, which are comprised of early and late apoptotic cells, were analyzed by flow cytometry using a Becton Dickinson LSR instrument and CellQuest威 software (Becton Dickinson). The ratio of apoptotic cells was obtained by dividing the percentage of apoptotic cells detected upon stimulation with serum starvation and UVC irradiation by the percentage of apoptotic cells detected without apoptotic stimulation.

Western blot analysis For survivin protein, Western blot was performed on lysate from DC and PGE2-treated DC harvested on day 8 of culture. For active caspase-3, cell lysate was obtained from DC, PGE2-treated DC, and serum-depleted DC, with or without PGE2 4 h after treatment with UVC or staurosporine (1 ␮M), as described previously. Briefly, DC and PGE2-treated DC (107/ml) were lysed in PBS containing 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM sodium chloride, 1 mM EDTA, 1 mM PMSF, and 1⫻ Complete™ protease inhibitor cocktail (Roche Diagnostics). The protein concentration in the cell lysate was determined using a bicinchoninic acid assay (Pierce, Rockford, IL). Protein-normalized aliquots of cell lysate were electrophoresed on a 12%

http://www.jleukbio.org

sodium dodecyl sulfate-polyacrylamide gel and transferred onto nitrocellulose membranes. Survivin was immunodetected with 1:1000 rabbit anti-human survivin polyclonal antibody (Novus Biologicals, Littleton, CO) and with 1:15,000 HRP-conjugated anti-rabbit immunoglobulin (Ig). Active caspase-3 was detected with 1:5000 rabbit anti-human caspase-3 polyclonal antibody (BD Biosciences PharMingen) followed by incubation with 1:15,000 HRP anti-rabbit Ig (Santa Cruz Biotechnologies, CA). The anti-caspase-3 antibody used recognizes the 32-kDa unprocessed procaspase-3 and the 17-kDa subunit of cleaved caspase-3. Immunoblots were developed using an enhanced chemiluminescence detection system (Supersignal West Pico Chemiluminescence, Pierce) followed by autoradiography. Equal protein loading was confirmed by immunodetecting the membranes with antiactin antibody (Santa Cruz Biotechnologies). A549 tumor cell lysate was used as a positive control to evaluate survivin protein expression. Lysate from untreated and campthothecin-treated Jurkat cell lines (BD Biosciences PharMingen) was used as negative and positive control, respectively, for detection of caspase-3. Protein quantification was verified by computerized densitometric analysis using Scion Image software (Version 1.62c, Frederick, MD).

Inhibition of survivin by small interfereing (siRNA) To inhibit survivin expression, on day 5 of culture, DC were transfected with siRNA survivin or siRNA glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the control (both from Ambion Inc., Austin, TX) using the TransMessenger transfection reagent kit (Qiagen, Valencia, CA) according to the manufacturer’s instruction. The concentration of survivin siRNA and of the transfection reagents used in our experiments was in accord with those previously established by our published data [31] and as recommended by the manufacturer’s protocol. Briefly, 2 ⫻ 106 DC were transfected with siRNA survivin (1.6 ␮g) or siRNA GAPDH (1.6 ␮g) for 3 h. Transfected DC were washed thoroughly and cultured with PGE2 (1.5⫻10⫺5 M) for 72 h in the presence or absence of serum. On day 8, only serum-deprived, PGE2-treated DC were exposed to UVC. All transfected DC were incubated for 14 –16 h prior to detection of apoptosis as described previously. To test siRNA transfection efficiency, survivin expression was analyzed in the cell lysate 24 – 48 h after gene silencing by specific ELISA, as described previously.

IL-12p70 and IL-10 ELISA To induce IL-12 production, (105) DC and PGE2-treated DC were primed with interferon-␥ (IFN-␥; 50 ng/ml, Peprotech) for 2 h and then stimulated with LPS (1 ␮g/ml) for 48 h. To induce IL-10 production, (105) DC and PGE2-treated DC were stimulated with LPS (1 ␮g/ml) for 48 h. IL-12 p70 and IL-10 concentrations were measured in the supernatant of unstimulated or stimulated DC by specific ELISA as described previously [32]. For detection of IL-12p70, antibodies and standards were purchased from R&D Systems. For detection of IL-10, antibodies and standards were obtained from BD Biosciences PharMingen. The sensitivity limit of the ELISA for IL-12 p70 and IL-10 was ⬍1 pg/ml.

Real-time, quantitative polymerase chain reaction (PCR) analysis To analyze the effect of PGE2 in the regulation of survivin mRNA expression, total RNA was extracted from 2 ⫻ 106 DC and PGE2-treated DC using the RNeasy mini kit (Qiagen), and cDNA was prepared using a standard kit from Invitrogen, according to the manufacturer’s instructions. Survivin mRNA levels were quantified by real-time reverse transcriptase-PCR using the SYBR Green quantitative PCR kit from Finnzymes (Espoo, Finland) in an iCycler (Bio-Rad) following the manufacturer’s protocol. Amplification was carried out in a total volume of 20 ␮l for 40 cycles of 15 s at 95°C, 1 min at 55.6°C. Survivin mRNA levels were determined after normalization of RNA concentration with ␤-actin, and values were expressed as fold-increased expression above DC control. All samples were run in triplicate. Primers were designed to span intron-exon junctions to minimize amplification of genomic DNA. Primers were synthesized by Integrated DNA Technologies (Coralville, IA). Survivin primers were as follows: forward, 5⬘-GGCCCAGTGTTTCTTCTGCTT-3⬘; reverse, 5⬘-TTGACAGAAAGGAAAGCGCAAC-3⬘ [32]. ␤-actin primers were forward, 5⬘-GATGAGATTGGCATGGCTTT-3⬘; reverse: 5⬘-CACCTTCACCGTTCCAGTTT-3⬘.

Statistics The unpaired two-tailed Student's t-test was used to compare differences in DC and PGE2-treated DC groups; a P value of less than or equal to 0.05 was considered statistically significant.

RESULTS Generation of DC and PGE2-treated DC To generate human DC in vitro, we followed an established protocol using purified CD14⫹ monocytes or plastic adherent monocytes cultured in GM-CSF and IL-4 for 8 –11 days as described previously [29, 30]. The resulting DC population exhibited morphologic and phenotypic features consistent with DC at an intermediate stage of maturation: lack of the lineagespecific markers CD3 and CD14 (data not shown); high levels of CD11c, CD86, and HLA-DR; expression of CD40 and CD80; and some expression of CD83 (Fig. 1A). To study the impact of PGE2 in regulation of DC survivin expression, we treated day 5 DC with exogenous PGE2 for 72 h using concentrations (10⫺5 M), which we speculated to be relevant in vivo [33], and proven effective in vitro based on our previous research [34] and preliminary experiments. The phenotype of PGE2-tretaed DC remained unchanged with the exception of a reduced mean fluorescence intensity of CD11c (307/629) and HLA-DR (219/267) and a slight up-regulation of CD83 (Fig. 1B). PGE2, alone or in combination with multiple cytokines and growth factors, has been shown to affect DC biology and immune function profoundly [35–37]. In particular, PGE2 suppresses IL-12 but promotes IL-10 production, favoring a T helper cell type 2-immune response [35–37]. Under our experimental conditions, we found that PGE2, used at 10⫺5 M concentration, not only reduced production of IL-12 p70 (Fig. 1C) but also secretion of IL-10 (Fig. 1D).

Exogenous PGE2 induces survivin expression in DC There is accumulating evidence that PGE2 is capable of modulating apoptosis in different cell types exhibiting proapoptotic and antiapoptotic functions, depending on the maturation stage and tissue localization of target cells [23–28]. PGE2 overproduction increases cell survival and reduces tumor cell apoptosis [18, 38 – 40]. Moreover, PGE2 has been demonstrated to cause apoptosis resistance in NSCLC cells by up-regulation of survivin expression [18]. Therefore, we sought to investigate the effect of PGE2 in DC by analyzing the antiapoptotic protein survivin. Lysates from DC and PGE2-treated DC cultured for 8 days in GM-CSF and IL-4 were used to detect survivin protein levels (Fig. 2) by ELISA (Fig. 2A) or Western blot (Fig. 2B). Both methods showed that survivin protein was expressed in DC as described previously [15]. It is interesting that when DC where incubated with PGE2 for 72 h, we observed a significant increase in survivin (Fig. 2, A and B). Treatment of DC with escalating doses of PGE2 for 72 h resulted in maximum survivin expression at 1.5 ⫻ 10⫺5 M, reaching a plateau at 3 ⫻ 10⫺5 M (Fig. 2C). All subsequent experiments were thus performed with 1.5 ⫻ 10⫺5 M PGE2. In a time-course study, in which DC were cultured with PGE2 for 72 h, and survivin was Baratelli et al. PGE2-dependent modulation of DC survivin

557

Fig. 1. DC and PGE2-treated DC surface phenotype. Magnetic-purified CD14⫹ monocytes were differentiated into DC in 10% AB medium supplemented with GM-CSF and IL-4, with or without PGE2. On day 8 of culture, cells were labeled directly with fluorochrome-conjugated antibodies as described in Materials and Methods. Histograms show surface expression in DC (A) and PGE2-treated DC (PGE2-DC) (B). Bold line indicates surfacemarker staining and dotted line, isotype control. Numbers indicate the percentage of positive cells. PGE2-treated DC secrete low levels of IL-12p70 and IL-10 (105) DC and PGE2-DC were plated into a 24-well plate in 1 ml culture medium in the presence or absence of the appropriate stimulation for IL-12p70 (IFN-␥ and LPS) or IL-10 (LPS). After 48 h induction, supernatants from stimulated and nonstimulated DC (DC and PGE2-DC) were harvested, and IL-12p70 (C) and IL-10 (D) production was determined by specific ELISA. Values refer to cytokine concentration expressed in nanograms per million cells 48 h after stimulation and represent the arithmetic mean ⫾ SD of three separate experiments. *, Statistically significant difference compared with control DC.

analyzed over a time period of 96 h, we observed maximal PGE2 survivin up-regulation at 96 h (Fig. 2D). Next, we analyzed survivin mRNA expression in DC cultured with or without PGE2 by real-time PCR. Our results show that PGE2treated DC significantly expressed higher levels of survivin mRNA with an average 2.7-fold increase compared with control DC (Fig. 2E).

PGE2-mediated survivin is not induced in LPSor TNF-␣-matured DC Factors modulating DC survival act differentially throughout the DC life-span, and various studies have reported susceptibility or resistance to apoptosis, depending on the maturation state and apoptotic stimuli [41– 45]. With regard to survivin expression in DC exposed to maturation factors, we found that LPS- and TNF-␣-stimulated DC analyzed on day 8 of culture expressed survivin at levels similar to unstimulated, control DC (Fig. 3A). However, when these matured DC were subsequently exposed to exogenous PGE2 for 72 h, they appeared to be refractory to PGE2-mediated induction of survivin, suggesting a differential effect of PGE2 related to their maturational stage (Fig. 3B). When the maturation factors and PGE2 were added at the same time, the levels of survivin expression did not change significantly (data not shown). 558

Journal of Leukocyte Biology Volume 78, August 2005

PGE2 induction of DC survivin confers protection to apoptosis and is associated with decreased active caspase-3 Survivin is known to suppress apoptosis caused by a variety of stimuli such as TNF-␣/Fas ligand, Bax, caspases, and chemotherapeutic drugs [1, 2, 5, 9]. We speculated that PGE2dependent survivin induction would affect DC viability by conferring protection to apoptosis. Therefore, we induced apoptosis in DC and PGE2-treated DC by a combination of serum starvation and UVC irradiation. Total apoptotic cells were detected by annexin-V/phosphatidylinositol (PI) staining by flow cytometry after 14 –16 h incubation following induction of apoptosis (Fig. 4). It is interesting that PGE2-treated DC showed increased viability compared with control DC, suggesting that PGE2 may reduce DC susceptibility to apoptotic stimuli (Fig. 4A). Next, to determine the specific effect of survivin in PGE2-mediated apoptosis resistance, we inhibited survivin expression in PGE2-treated DC by siRNA. Efficiency of gene silencing was assessed by ELISA in the cell lysate (Fig. 4B). We found that suppression of survivin increased cell death in PGE2-treated DC after exposure to apoptosis (Fig. 4C, upper right) compared with PGE2-treated DC transfected with the siRNA control GAPDH (Fig. 4D, lower right), confirming http://www.jleukbio.org

Fig. 2. PGE2 induces survivin expression in DC. DC were cultured in GM-CSF and IL-4 for 8 days with or without PGE2 added on day 5 for 72 h. Survivin protein expression was measured by ELISA (A) and Western blotting (B). (A) Results are expressed as means (⫾SD) of six independent experiments, each performed in duplicate. *, Statistically significant difference compared with control DC. (B) One representative experiment of three is shown. PGE2 induces DC survivin in a dose- and time-dependent manner. (C) DC on day 5 were treated with dmPGE2 at 0.6, 1.5, and 3 ⫻ 10⫺5 M. Survivin was measured by ELISA on day 8. (D) In a time-course experiment, PGE2 was added for 72 h to DC on day 5. Survivin was measured by ELISA from days 6 through 9. (C and D) Expressed as mean (⫾SD) of one representative experiment of three performed in duplicate. (E) PGE2 up-regulates survivin mRNA in DC. DC cultured with or without PGE2, were analyzed for survivin gene expression by real-time PCR. Data were normalized by ␤-actin gene expression, and values are represented as fold-increased expression above the control sample (untreated DC). Data are expressed as means (⫾SD) of three independent experiments performed in triplicate. *, Statistically significant difference compared with control DC.

the regulatory effect of PGE2 in DC survivin expression and maintenance of viability. Caspase-3 is a key protease activated during the early stages of apoptosis and like other members of the caspase family, is synthesized as an active proenzyme. Controversial findings in diverse cell systems and experimental conditions have linked survivin-mediated inhibition of apoptosis to its specific binding to caspase-3 [16 –18]. Moreover, an inverse correlation between survivin and active caspase-3 has been demonstrated in CD34⫹ cells or tumor cells exposed to different apoptotic stimuli [9, 10, 18]. Therefore, we analyzed whether PGE2induced DC survivin was similarly correlated with reduced activation of caspase-3. DC and PGE2-treated DC, in serum and serum-free condition, were analyzed for active caspase-3 after 4 h exposure to UVC radiation, as described previously. As shown in Figure 5, DC and PGE2-treated DC expressed cleaved caspase-3 at baseline. However, upon serum depletion and UVC radiation, active caspase-3 expression was decreased only in PGE2-treated DC (Fig. 5). A similar pattern of caspase-3

activation was observed in DC and PGE2-treated DC following exposure to staurosporine, consistent with the hypothesis of PGE2-induced resistance to DC apoptosis (Fig. 5). These findings are in correlation with the pattern of cell viability obtained by annexin-V/PI staining represented in Figure 4A.

EP2/EP4 receptor agonists induce survivin in DC Four distinct receptors, designated EP1– 4, are known to mediate PGE2 function [46]. Previous studies have demonstrated the expression of EP2 and EP4 receptor mRNA in DC and PGE2-treated DC [47], and recently, we have found that human monocyte-DC predominantly express the EP2 receptor [30]. Based on these findings, we investigated the functional implication of EP2/EP4 signaling in mediating DC survivin induction. As EP2 and EP4 receptors stimulate cyclic adenosine monophosphate (cAMP) production, we incubated DC with selective agonists or with chemicals able to activate the G␣s subunit of G proteins, thus mimicking Gs-coupled receptor signaling. As shown in Figure 6, the selective EP2 receptor Baratelli et al. PGE2-dependent modulation of DC survivin

559

Fig. 3. LPS- and TNF-␣-matured DC express survivin at levels similar to control DC. DC were cultured for 8 days with or without PGE2 as described previously. LPS or TNF-␣ were added on day 6 for 48 h. Survivin protein expression was analyzed by ELISA on day 8 (A). LPS- and TNF-␣-matured DC are refractory to PGE2-survivin up-regulation. LPS- and TNF-␣ matured DC were generated as described above. PGE2 was added to the mature DC for 72 h, and survivin was analyzed by ELISA on day 11 (B). Results are expressed as means (⫾SD) of three independent experiments, each performed in duplicate. *, Statistically significant difference compared with control. ns, Not significant.

agonist butaprost could induce a significant increase in survivin expression in DC. Similarly, cholera toxin, which activates the G␣s subunit of G proteins, significantly up-regulated survivin expression in DC and forskolin, and a pharmacologi-

cal activator of adenylate cyclase showed a similar trend of survivin up-regulation. Moreover, the substantial contribution of EP2/EP4 receptors was corroborated by sulprostone, a nonselective EP3 ⬎⬎ EP1 receptor agonist, as it did not replicate

Fig. 4. PGE2 increases viability of immature DC. (A) DC were cultured for 9 days with or without PGE2 as described previously. Apoptosis was induced in DC and PGE2-DC by serum depletion followed by UVC irradiation as described in Materials and Methods. The percentage of apoptotic cells was quantified by annexin-V/PI staining on day 9 in untreated and apoptotic-treated DC and PGE2-DC. Results represent the ratio of apoptotic cells and are expressed as means (⫾SD) of three separate experiments. *, Statistical significance compared with control value. Survivin gene expression was silenced by survivin siRNA transfection. (B) Day 5 DC were transfected with siRNA survivin or siRNA GAPDH as described in Materials and Methods. Survivin protein expression was analyzed in nontransfected DC (DC control), DC transfected with siRNA survivin, or siRNA GAPDH, 48 h after transfection. Data are expressed as means (⫾SD) of one representative experiment of two performed in duplicate. Inhibition of survivin expression decreases viability of DC treated with exogenous PGE2. (C and D) Day 5 DC were transfected with siRNA survivin (C) or siRNA GAPDH (D) followed by 72 h incubation with exogenous PGE2. Apoptosis was induced by serum depletion and UVC irradiation (right panels), as described previously. Cell viability was assessed by annexin-V/PI staining on day 9. The density plots show annexin-V and PI-stained cells. Numbers in the quadrants indicate the percentage of positive cells. One representative result of two separate experiments is shown.

560

Journal of Leukocyte Biology Volume 78, August 2005

http://www.jleukbio.org

Fig. 5. PGE2-induced survivin is inversely correlated with active caspase-3 in DC exposed to apoptotic stimuli. Active caspase-3 (17 kDa) was measured by Western blot analysis in DC and PGE2-treated DC 4 h after induction of apoptosis as described in Materials and Methods. (A) Representative experiments of two. (B) The densitometric analysis of the Western blot in A.

PGE2-mediated induction of survivin. Finally, pertussis toxin, which acts by suppressing Gi-coupled receptors such as EP3, did not significantly inhibit the PGE2-mediated increase in survivin.

Inhibition of PI-3K but not MAPK kinase (MEK) pathway blocks PGE2-induced DC survivin As PI-3K/Akt and MEK pathways are important in many cell types in the regulation of cell survival, proliferation,

and differentiation [48], we evaluated the participation of these pathways in mediating PGE2-induced survivin in DC. Survivin expression was analyzed following pretreatment of DC with the inhibitors LY294002 and PD98059, which suppress the PI-3K and the MEK pathways, respectively. As shown in Figure 7, treatment of DC with LY294002 (Fig. 7A) but not with PD98059 (Fig. 7B) could effectively block PGE2-induced up-regulation of survivin. Induction of apoptosis followed by analysis of cell viability by annexin-V and PI staining was not performed in parallel to the experiments shown in Figures 6 and 7, as the reagents used to stimulate the DC could generate nonspecific, proapoptotic effects, which could interfere with the outcome and the interpretation of the results.

DISCUSSION

Fig. 6. EP2/EP4 receptor agonists replicate the PGE2-dependent induction of survivin in DC. DC were cultured under the stimulatory conditions indicated. Survivin protein expression was detected by ELISA as described previously. Data are expressed as means (⫾SD) of one representative experiment of three performed in triplicate. *, Statistically significant difference compared with control DC. **P ⫽ 0.003. ns, Not significant.

Although survivin has been studied extensively in tumor cells, less is known regarding its role in nontransformed cells. Fukuda et al. [9 –11] have demonstrated that survivin is not a cancerspecific protein and plays a role in the proliferation and survival of normal hematopoietic cells. Accumulating evidence has now indicated that survivin is expressed in other cell systems including neutrophils [14], activated lymphocytes, and DC [15]. Here, we report that monocyte-derived DC treated with exogenous PGE2 up-regulated survivin expression significantly. The eicosanoids may function in the regulation of immune cell homeostasis. In particular, PGE2 has been shown to exert diverse effects in a variety of immune cells [23–30]. The current study focuses on the impact of PGE2 in DC survivin expression. Although PGE2 induced survivin in the absence of maturation signals, PGE2, added at the same time (from day 5 for 72 h) with LPS or TNF-␣ (data not shown) or following DC exposure to these maturation agents (from day 5 for 48 h), did not affect DC survivin Baratelli et al. PGE2-dependent modulation of DC survivin

561

Fig. 7 . PI-3K but not MAPK pathway mediates PGE2-induced DC survivin. DC were stimulated with LY294002 (A) or with PD98059 (B), with or without PGE2, as described in Materials and Methods. Survivin protein expression was detected by ELISA, as described previously. Results are expressed as means (⫾SD) of three independent experiments each performed in duplicate. *, Statistically significant difference compared with control DC.

expression significantly. These findings suggest that PGE2 regulates survivin in a maturation-dependent manner. It is possible that alteration of in vitro culture conditions could modify these outcomes. For example, Schmidt et al. [15] reported that adherent monocytes cultured with GM-CSF and IL-4 in the presence of TNF-␣ (10 ng/ml), added from day 1 to day 7, express higher levels of survivin compared with the same cells cultured in the absence of TNF-␣. Our findings show that PGE2 pretreatment heightened the threshold of apoptosis of serum-deprived, immature DC exposed to UVC. Moreover, when survivin was suppressed by specific inhibition of its gene expression via siRNA, these PGE2-treated DC no longer demonstrated enhanced resistance to apoptotic stimuli. These observations suggest that under stress- or apoptosis-inducing stimuli, a PGE2-rich environment may increase the viability of DC at an immature stage of differentiation. The actual outcome of this effect and its significance in the immune response are complex, especially when the multiple biologic effects of PGE2 are considered [22, 35–37]. For example, PGE2 could promote the survival of immature DC, thus favoring antigen capture by immature DC that have heightened capacity for endocytosis [20]. The fact that PGE2 may have this impact could be important in the modulation of the immune response. Each developmental DC stage is associated with specific phenotypic and biologic features, which enhance or limit the immune response [20, 49]. Thus, a prolonged half-life of immature DC could, in certain circumstances, augment immune responses because of the increase of a population of DC with high endocytic capacity. In contrast, in some settings, such as the tumor environment, apoptosis resistance of immature DC could interfere with effective cell-mediated immunity [50, 51]. PGE2 has multiple effects on DC biology and function including migration [30, 47] and modulation of cytokine production [35, 37]. In the present study, PGE2 not only contributed to survivin up-regulation but also modulated DC phenotype and cytokine secretion. Activated PGE2-treated DC secreted significantly less IL-12p70 than did control DC. IL-10 production was also markedly reduced. Our findings, in agreement with previous reports [30, 36, 37], suggest that PGE2 may facilitate survival of DC that have immunosuppressive capacities. Thus, in circumstances in which it is produced at heightened levels, such as those described in several malignancies [38 – 40], 562

Journal of Leukocyte Biology Volume 78, August 2005

PGE2 may contribute to the tumor-induced immunosuppression [50, 52]. Therefore, the impact of modulatory factors, such as PGE2, which can affect immature DC survival, could have important immunological repercussions. The activation of distinct apoptotic processes controls DC development and homeostasis. Maturation facilitates DC survival and thus has an antiapoptotic function [42, 53]; however, it has also been shown that developmentally and functionally, end-stage DC undergo apoptotic cell death [41]. In addition, functionally distinct apoptotic schedules may be associated with different phases of DC development. Lundqvist et al. [53], for example, demonstrated that mature DC cultured under serum-free condition were protected from Fas/CD95-mediated apoptosis. In another report, monocyte-derived DC, although expressing the CD95 molecule on their surface, did not undergo apoptosis on CD95 ligation unless treated with cycloheximide, which sensitized DC to apoptosis by preventing synthesis of antiapoptotic proteins [45]. In agreement with these studies, we were not able to induce apoptosis by Fas-Fas L interaction, although our cells expressed Fas, thus arguing against the involvement of the extrinsic pathway (data not shown). Although survivin expression is correlated to apoptosis resistance in cancer cells, the cellular and molecular mechanisms underlying this association are not elucidated completely. As a member of the IAP family, survivin is characterized by the presence of a baculovirus IAP repeat domain, in which certain reports have been demonstrated to be critical for the interaction with caspases and to prevent their activation [1, 2, 16, 17]. Our results show that DC and PGE2-treated DC expressed similar baseline levels of cleaved caspase-3, indicating ongoing apoptosis in the absence of exogenous apoptotic stimuli. However, when we induced apoptosis by serum depletion and UVC or by staurosporine, only PGE2-treated DC exhibited a decrease of active caspase-3 compared with control DC, suggesting an inverse correlation between survivin and caspase-3 activation, as described previously [9 –11, 18]. Previous studies have reported that PGE2 and cAMP analogs induce antiapoptotic effects in T cell receptor-mediated, activation-induced cell death of peripheral T lymphocytes, T cell hybridomas, circulating human monocytes, and an immature CD4⫹ CD8⫹ double-positive T cell line [23–26]. Herein, we http://www.jleukbio.org

studied the implication of the EP receptors EP2 and EP4, which are expressed in human DC and signal through adenylate cyclase-mediated mechanisms [30, 46, 47]. It has been shown that PGE2-containing stimuli modulate DC surface EP receptor expression, thus influencing the response of DC to exogenous PGE2 [30, 47]. Indeed, we observed that forskolin, a physiological cAMP modulator, cholera toxin, a Gs-coupled protein agonist, and butaprost, a selective EP2 agonist, replicated the PGE2-mediated induction of DC survivin, consistent with the involvement of EP2 and/or EP4 receptors. Previous studies have implicated specific signal-transduction events in DC homeostasis and viability. Particularly, the PI-3K/Akt and MAPK pathways have been highlighted in LPS-dependent survival of monocyte-derived DC [42]. Ardeshna et al. [42], in fact, showed that LPS activated PI-3K/ Akt and MAPK p42/p44 pathways, and the specific inhibitors LY294002 and PD98059 suppressed phosphorylation of these pathways in human DC. However, in this report, inhibition of PI-3K for 48 h also led to a decreased viability of LPSstimulated DC. In our current study, only the PI-3K/Akt pathway appeared to mediate PGE2-induced survivin, as preincubation of DC with LY294002 suppressed survivin expression with no effect on DC viability. Similarly, studies in normal CD34⫹ cells have implicated the PI-3K/Akt pathway, as LY294002 blocked growth factor-induced up-regulation of survivin in these cells [11]. The PI-3K/Akt pathway has also been associated with cytokine regulation of survivin expression in acute myeloid leukemia cells, where survivin down-regulation was accompanied by cell-cycle arrest [54]. Finally, the PI-3K/ Akt pathway has been linked to resistance to apoptosis in hematopoietic cells by regulating the expression and function of several Bcl-2 family members [10, 53]. The ubiquitin-proteasome pathway has been implicated in post-translational regulation of survivin [55]. We recently found that PGE2 caused reduced ubiquitination of survivin in NSCLC, thus inducing protein stabilization and thereby promoting resistance to apoptosis [18]. However, in DC, PGE2-dependent induction of survivin did not involve changes in survivin ubiquitination, suggesting a different regulatory pathway (data not shown). Analysis of survivin expression by real-time PCR demonstrated that PGE2 upregulated survivin mRNA expression levels in DC. Therefore, in nontransformed cells, transcriptional or post-transcriptional mechanisms may be operative in the regulation of survivin expression. Regulation of apoptosis is crucial for the control of the immune response. Any imbalance in favor of apoptosis inhibitors can lead to a prolonged half-life of effector cells. Conversely, this same effect can be detrimental if the cell exhibits altered function. It is well established that PGE2 production is enhanced through up-regulation of cyclooxygenase-2 in several pathologic conditions, including various types of cancer [38 – 40]. As immune effector cells, DC are therefore exposed to PGE2 present in the microenvironment. Thus, the observation that PGE2 affects DC viability through survivin up-regulation and apoptosis protection has diverse and important implications in the immune response.

ACKNOWLEDGMENTS This study was supported by the UCLA Lung Cancer SPORE P50 CA 90388, National Institutes of Health (NIH) R01 CA85686, the Medical Research Funds from the Department of Veteran Affairs, the Research Enhancement Award Program in Cancer Gene Medicine, and the Tobacco-Related Disease Research Program of the University of California. We thank Lauren Winter and Sandra Tran for technical assistance. We thank the Flow Cytometry Core Facility of UCLA Jonsson Comprehensive Cancer Center and AIDS Research, which are supported by NIH Awards CA-16042 and AI-28697, the Jonsson Cancer Center, the UCLA AIDS Institute, and the UCLA Geffen School of Medicine.

REFERENCES 1. Li, F., Ambrosini, G., Chu, E. Y., Plescia, J., Tognin, S., Marchisio, P. C., Altieri, D. C. (1998) Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396, 580 –584. 2. Conway, E. M., Pollefeyt, S., Cornelissen, J., DeBaere, I., Steiner-Mosonyi, M., Ong, K., Baens, M., Collen, D., Schuh, A. C. (2000) Three differentially expressed survivin cDNA variants encode proteins with distinct antiapoptotic functions. Blood 95, 1435–1442. 3. Carter, B. Z., Milella, M., Altieri, D. C., Andreeff, M. (2001) Cytokineregulated expression of survivin in myeloid leukemia. Blood 97, 2784 – 2790. 4. Mahotka, C., Krieg, T., Krieg, A., Wenzel, M., Suschek, C. V., Heydthausen, M., Gabbert, H. E., Gerharz, C. D. (2002) Distinct in vivo expression patterns of survivin splice variants in renal cell carcinomas. Int. J. Cancer 100, 30 –36. 5. Shankar, S. L., Mani, S., O’Guin, K. N., Kandimalla, E. R., Agrawal, S., Shafit-Zagardo, B. (2001) Survivin inhibition induces human neural tumor cell death through caspase-independent and -dependent pathways. J. Neurochem. 79, 426 – 436. 6. Papapetropoulos, A., Fulton, D., Mahboubi, K., Kalb, R. G., O’Connor, D. S., Li, F., Altieri, D. C., Sessa, W. C. (2000) Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway. J. Biol. Chem. 275, 9102–9105. 7. O’Connor, D. S., Schechner, J. S., Adida, C., Mesri, M., Rothermel, A. L., Li, F., Nath, A. K., Pober, J. S., Altieri, D. C. (2000) Control of apoptosis during angiogenesis by survivin expression in endothelial cells. Am. J. Pathol. 156, 393–398. 8. Blanc-Brude, O. P., Yu, J., Simosa, H., Conte, M. S., Sessa, W. C., Altieri, D. C. (2002) Inhibitor of apoptosis protein survivin regulates vascular injury. Nat. Med. 8, 987–994. 9. Fukuda, S., Pelus, L. M. (2001) Regulation of the inhibitor-of-apoptosis family member survivin in normal cord blood and bone marrow CD34(⫹) cells by hematopoietic growth factors: implication of survivin expression in normal hematopoiesis. Blood 98, 2091–2100. 10. Fukuda, S., Pelus, L. M. (2002) Elevation of survivin levels by hematopoietic growth factors occurs in quiescent CD34⫹ hematopoietic stem and progenitor cells before cell cycle entry. Cell Cycle 1, 322–326. 11. Fukuda, S., Foster, R. G., Porter, S. B., Pelus, L. M. (2002) The antiapoptosis protein survivin is associated with cell cycle entry of normal cord blood CD34(⫹) cells and modulates cell cycle and proliferation of mouse hematopoietic progenitor cells. Blood 100, 2463–2471. 12. Kornacker, M., Verneris, M. R., Kornacker, B., Scheffold, C., Negrin, R. S. (2001) Survivin expression correlates with apoptosis resistance after lymphocyte activation and is found preferentially in memory T cells. Immunol. Lett. 76, 169 –173. 13. Xing, Z., Conway, E. M., Kang, C., Winoto, A. (2004) Essential role of survivin, an inhibitor of apoptosis protein, in T cell development, maturation, and homeostasis. J. Exp. Med. 199, 69 – 80. 14. Altznauer, F., Martinelli, S., Yousefi, S., Thurig, C., Schmid, I., Conway, E. M., Schoni, M. H., Vogt, P., Mueller, C., Fey, M. F., ZangemeisterWittke, U., Simon, H. U. (2004) Inflammation-associated cell cycleindependent block of apoptosis by survivin in terminally differentiated neutrophils. J. Exp. Med. 199, 1343–1354. 15. Schmidt, S. M., Schag, K., Muller, M. R., Weck, M. M., Appel, S., Kanz, L., Grunebach, F., Brossart, P. (2003) Survivin is a shared tumor-associ-

Baratelli et al. PGE2-dependent modulation of DC survivin

563

16.

17.

18.

19. 20. 21. 22. 23. 24. 25. 26.

27. 28. 29.

30.

31. 32.

33.

34.

35.

ated antigen expressed in a broad variety of malignancies and recognized by specific cytotoxic T cells. Blood 102, 571–576. Tamm, I., Wang, Y., Sausville, E., Scudiero, D. A., Vigna, N., Oltersdorf, T., Reed, J. C. (1998) IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res. 58, 5315–5320. Shin, S., Sung, B. J., Cho, Y. S., Kim, H. J., Ha, N. C., Hwang, J. I., Chung, C. W., Jung, Y. K., Oh, B. H. (2001) An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and -7. Biochemistry 40, 1117–1123. Krysan, K., Merchant, F. H., Zhu, L., Dohadwala, M., Luo, J., Lin, Y., Heuze-Vourc’h, N., Pold, M., Seligson, D., Chia, D., Goodglick, L., Wang, H., Strieter, R., Sharma, S., Dubinett, S. (2004) COX-2-dependent stabilization of survivin in non-small cell lung cancer. FASEB J. 18, 206 –208. Song, Z., Yao, X., Wu, M. (2003) Direct interaction between survivin and Smac/DIABLO is essential for the anti-apoptotic activity of survivin during taxol-induced apoptosis. J. Biol. Chem. 278, 23130 –23140. Banchereau, J., Steinman, R. M. (1998) Dendritic cells and the control of immunity. Nature 392, 245–252. 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. Harris, S., Padilla, J., Koumas, L., Ray, D., Phipps, R. (2002) Prostaglandins as modulators of immunity. Trends Immunol. 23, 144 –150. Goetzl, E. J., An, S., Zeng, L. (1995) Specific suppression by prostaglandin E2 of activation-induced apoptosis of human CD4⫹ CD8⫹ T lymphoblasts. J. Immunol. 154, 1041–1047. Porter, B. O., Malek, T. R. (1999) Prostaglandin E2 inhibits T cell activation-induced apoptosis and Fas-mediated cellular cytotoxicity by blockade of Fas-ligand induction. Eur. J. Immunol. 29, 2360 –2365. Heidenreich, S., Otte, B., Lang, D., Schmidt, M. (1996) Infection by Candida albicans inhibits apoptosis of human monocytes and monocytic U937 cells. J. Leukoc. Biol. 60, 737–743. Pica, F., Franzese, O., D’Onofrio, C., Bonmassar, E., Favalli, C., Garaci, E. (1996) Prostaglandin E2 induces apoptosis in resting immature and mature human lymphocytes: a c-Myc-dependent and Bcl-2-independentassociated pathway. J. Pharmacol. Exp. Ther. 277, 1793–1800. Suzuki, K., Tadakuma, T., Kizaki, H. (1991) Modulation of thymocyte apoptosis by isoproterenol and prostaglandin E2. Cell. Immunol. 134, 235–240. Brown, D. M., Phipps, R. P. (1996) Bcl-2 expression inhibits prostaglandin E2-mediated apoptosis in B cell lymphomas. J. Immunol. 157, 1359 –1370. Riedl, K., Baratelli, F., Batra, R. K., Yang, S. C., Luo, J., Escuadro, B., Figlin, R., Strieter, R., Sharma, S., Dubinett, S. (2003) Overexpression of CCL-21/secondary lymphoid tissue chemokine in human dendritic cells augments chemotactic activities for lymphocytes and antigen presenting cells. Mol. Cancer 2, 35. Baratelli, F., Heuze-Vourch, N., Krysan, K., Dohadwala, M., Riedl, K., Sharma, S., Dubinett, S. M. (2004) PGE2-dependent enhancement of TIMP-1 production limits dendritic cell migration through extracellular matrix. J. Immunol. 173, 5458 –5466. Krysan, K., Dalwadi, H., Sharma, S., Pold, M., Dubinett, S. M. (2004) Cyclooxygenase 2-dependent expression of survivin is critical for apoptosis resistance in non-small cell lung cancer. Cancer Res. 64, 6359 – 6362. Tyagi, A. K., Agarwal, C., Singh, R. P., Shroyer, K. R., Glode, L. M., Agarwal, R. (2003) Silibinin down-regulates survivin protein and mRNA expression and causes caspases activation and apoptosis in human bladder transitional-cell papilloma RT4 cells. Biochem. Biophys. Res. Commun. 312, 1178 –1184. Altorki, N. K., Keresztes, R. S., Port, J. L., Libby, D. M., Korst, R. J., Flieder, D. B., Ferrara, C. A., Yankelevitz, D. F., Subbaramaiah, K., Pasmantier, M. W., Dannenberg, A. J. (2003) Celecoxib, a selective cyclo-oxygenase-2 inhibitor, enhances the response to preoperative paclitaxel and carboplatin in early-stage non-small-cell lung cancer. J. Clin. Oncol. 21, 2645–2650. Stolina, M., Sharma, S., Lin, Y., Dohadwala, M., Gardner, B., Luo, J., Zhu, L., Kronenberg, M., Miller, P. W., Portanova, J., Lee, J. C., Dubinett, S. (2000) Specific inhibition of cyclooxygenase 2 restores antitumor reactivity by altering the balance of IL-10 and IL-12 synthesis. J. Immunol. 164, 361–370. Whittaker, D. S., Bahjat, K. S., Moldawer, L. L., Clare-Salzler, M. J. (2000) Autoregulation of human monocyte-derived dendritic cell maturation and IL-12 production by cyclooxygenase-2-mediated prostanoid production. J. Immunol. 165, 4298 – 4304.

564

Journal of Leukocyte Biology Volume 78, August 2005

36. Steinbrink, K., Paragnik, L., Jonuleit, H., Tuting, T., Knop, J., Enk, A. H. (2000) Induction of dendritic cell maturation and modulation of dendritic cell-induced immune responses by prostaglandins. Arch. Dermatol. Res. 292, 437– 445. 37. Kalinski, P., Hilkens, C. M., Snijders, A., Snijdewint, F. G., Kapsenberg, M. L. (1997) IL-12-deficient dendritic cells, generated in the presence of prostaglandin E2, promote type 2 cytokine production in maturing human naive T helper cells. J. Immunol. 159, 28 –35. 38. Dohadwala, M., Batra, R. K., Luo, J., Lin, Y., Krysan, K., Pold, M., Sharma, S., Dubinett, S. (2002) Autocrine/paracrine prostaglandin E2 production by non-small cell lung cancer cells regulates matrix-metalloproteinase-2 and CD44 in cyclooxygenase-2 dependent invasion. J. Biol. Chem. 277, 50828 –50833. 39. Huang, M., Stolina, M., Sharma, S., Mao, J., Zhu, L., Miller, P., Wollman, J., Herschman, H., Dubinett, S. (1998) Non-small cell lung cancer cyclooxygenase-2-dependent regulation of cytokine balance in lymphocytes and macrophages: up-regulation of interleukin 10 and down-regulation of interleukin 12 production. Cancer Res. 58, 1208 –1216. 40. Sheng, H., Shao, J., Washington, M. K., DuBois, R. N. (2001) Prostaglandin E2 increases growth and motility of colorectal carcinoma cells. J. Biol. Chem. 276, 18075–18081. 41. 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 longterm cultures. J. Exp. Med. 185, 317–328. 42. Ardeshna, K. M., Pizzey, A. R., Devereux, S., Khwaja, A. (2000) The PI3 kinase, p38 SAP kinase, and NF-␬B signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells. Blood 96, 1039 –1046. 43. 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. 44. Leverkus, M., Walczak, H., McLellan, A., Fries, H. W., Terbeck, G., Brocker, E. B., Kampgen, E. (2000) Maturation of dendritic cells leads to up-regulation of cellular FLICE-inhibitory protein and concomitant downregulation of death ligand-mediated apoptosis. Blood 96, 2628 –2631. 45. 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 monocytederived dendritic cells: inhibition by bisindolylmaleimide. Blood 95, 3478 –3482. 46. Breyer, R. M., Bagdassarian, C. K., Myers, S. A., Breyer, M. D. (2001) Prostanoid receptors: subtypes and signaling. Annu. Rev. Pharmacol. Toxicol. 41, 661– 690. 47. Scandella, E., Men, Y., Gillessen, S., Forster, R., Groettrup, M. (2002) Prostaglandin E2 is a key factor for CCR7 surface expression and migration of monocyte-derived dendritic cells. Blood 100, 1354 –1361. 48. Martindale, J. L., Holbrook, N. J. (2002) Cellular response to oxidative stress: signaling for suicide and survival. J. Cell. Physiol. 192, 1–15. 49. Lutz, M. B., Schuler, G. (2002) Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity? Trends Immunol. 23, 445– 449. 50. Sharma, S., Stolina, M., Yang, S. C., Baratelli, F., Lin, J. F., Atianzar, K., Luo, J., Zhu, L., Lin, Y., Huang, M., Dohadwala, M., Batra, R. K., Dubinett, S. M. (2003) Tumor cyclooxygenase 2 -dependent suppression of DC function. Clin. Cancer Res. 9, 961–968. 51. Yang, L., Carbone, D. P. (2004) Tumor-host immune interactions and dendritic cell dysfunction. Adv. Cancer Res. 92, 13–27. 52. Yang, L., Yamagata, N., Yadav, T., Brandon, S., Courtneyt, R. L., Morrow, J. D., Shyr, Y., Boothby, M., Joyce, S., Carbone, D. P., Breyer, R. M. (2003) Cancer-associated immunodefiency and dendritic cell abnormalities mediated by the prostaglandin EP2 receptor. J. Clin. Invest. 111, 727–735. 53. Lundqvist, A., Nagata, T., Kiessling, R., Pisa, P. (2002) Mature dendritic cells are protected from Fas/CD95-mediated apoptosis by upregulation of Bcl-X(L). Cancer Immunol. Immunother. 51, 139 –144. 54. Milella, M., Kornblau, S. M., Estrov, Z., Carter, B. Z., Lapillonne, H., Harris, D., Konopleva, M., Zhao, S., Estey, E., Andreeff, M. (2001) Therapeutic targeting of the MEK/MAPK signal transduction module in acute myeloid leukemia. J. Clin. Invest. 108, 851– 859. 55. Zhao, J., Tenev, T., Martins, L. M., Downward, J., Lemoine, N. R. (2000) The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner. J. Cell Sci. 113, 4363– 4371.

http://www.jleukbio.org