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Originally published online as doi:10.1189/JLB.1008639 on June 18, 2009

Published online before print June 18, 2009
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(Journal of Leukocyte Biology. 2009;86:691-699.)
© 2009 Society for Leukocyte Biology

Synergistic production of interleukin-23 by dendritic cells derived from cord blood in response to costimulation with LPS and IL-12

Mi Seon Jang*, Young Min Son*, Gi Rak Kim*, Yeo Jin Lee*, Woon Kyu Lee§, Seok Ho Cha{dagger}, Seung Hyun Han{ddagger} and Cheol-Heui Yun*,1

* Protein Engineering and Comparative Immunology, Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, and
{ddagger} Department of Oral Microbiology and Immunology, BK21 Program, and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea; and
{dagger} Department of Pharmacology and Toxicology and
§ Center for Advanced Medical Education by BK21 Project, College of Medicine, Inha University, Incheon, Republic of Korea

1. Correspondence: Protein Engineering and Comparative Immunology, Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea. E-mail: cyun{at}snu.ac.kr

ABSTRACT

This study was performed to provide insight for the optimization and regulation of immune homeostasis, which should be taken into account in the development of cell therapy using DCs and/or cytokine. Human CBDCs costimulated with LPS and IL-12 were examined for cytokine expression compared with ABDCs. Our results showed that costimulation with IL-12 and LPS in CBDCs resulted in increased expression of IL-23. Concomitantly, the phosphorylation of ERKs and p38 MAPK was increased, suggesting that these kinases are important signaling components for IL-23 induction in CBDC costimulated with LPS and IL-12. Furthermore, production of IL-23 in CBDC costimulated with LPS and IL-12 caused CD4+CD45RO+ memory cells to increase IFN-{gamma} production. Taken together, CBDCs, costimulated with LPS and IL-12, show a synergistic increase in IL-23 production via enhanced phosphorylation of ERK1/2 and p38 MAPK and consequently, an induction of IFN-{gamma} production in the memory cells.

Key Words: human • antigen-presenting cells • cytokines

Introduction

Umbilical CB is a rich source of hematopoietic progenitors and contains numerous leukocytes characterized uniquely to participate in immune responses [1 ]. As host defense and hematopoiesis are developmentally immature in the neonatal when compared with those of adult, CB has been investigated in transplantation and has become important for therapeutic purposes [2 , 3 ]. CB is superior to AB for allogeneic stem cell therapy, as it has reduced expression levels of MHC class II molecules and Th1 cell-polarizing APC activity [4 , 5 ]. Indeed, DCs derived from CB have been used in clinical trials to optimize a disease-specific immunity [6 , 7 ]. DCs are one of the most potent APCs that phagocytose foreign antigens and display antigenic peptides to T lymphocytes, thereby contributing to adaptive immune responses. After capturing foreign antigens, immature DCs migrate from peripheral tissue to lymphoid organs and begin to mature. During the maturation process, DCs up-regulate the expression of costimulatory and MHC molecules, as well as the receptors for proinflammatory cytokines and chemokines, and they down-regulate their endocytotic ability [8 , 9 ]. DCs are known to display a number of receptors, such as TLRs, nucleotide oligomerization domain-like receptors, and retinoic acid-inducible gene 1-like receptors, on the cell surface or in the cytoplasm, and these receptors recognize not only cytokines and chemokines but also foreign antigens [8 , 10 , 11 ]. LPS, a major component of Gram-negative bacterial cell walls, is recognized by TLR4 with LPS-binding protein, CD14, and myeloid-differentiation protein 2 and induces various cytokines in vitro and in vivo, resulting in local inflammation and sometimes, septic shock when the response is overwhelming [12 13 14 ].

IL-12 and IL-23 are typically produced by APCs and are composed of a specific polypeptide, where IL-12p35 for IL-12 and IL-23p19 for IL-23 are linked to IL-12/IL-23p40 by disulfide bonding to form biologically functional proteins [15 , 16 ]. Although IL-12p35 and IL-23p19 require IL-12p40 to be secreted, monomers or homodimers of IL-12p40 can form and antagonize IL-12 activity [17 ]. IL-12 plays a pivotal role in promoting differentiation of naïve CD4+ T cells into Th1 effector cells and in stimulating the NK cells and CD8+ T cells to produce IFN-{gamma} [18 ]. Given the structural similarities between IL-12 and IL-23, it is interesting that IL-23 is essential for the pathogenesis of autoimmune diseases such as autoimmune encephalomyelitis, collagen-induced arthritis, and inflammatory bowel disease, and IL-23 plays a crucial role in promoting early inflammatory responses [18 19 20 21 ].

Moreover, IL-23 has a unique role in stimulating effector and memory T cells to induce the production of IL-17, which appears to be involved in the regulation of T cell effector function [22 23 24 25 ]. For the biological effect of IL-12, the IL-12R chains IL-12Rβ1 and -β2 are coexpressed predominantly on activated T cells and NK cells, but they are also found at low levels on human monocytes, macrophages, and DCs, where they activate the JAK/STAT pathway [26 27 28 29 ]. Although the activity of IL-12 has been studied intensively in T cells and NK cells, the immunobiology of DCs in response to IL-12 is largely unknown. Furthermore, little is known about the autocrine or paracrine loop of the IL-12 signaling pathway in human CB-derived DCs, which could be used in cell therapy. Therefore, in the present study, we compared the IL-23 production between CBDCs and ABDCs in response to costimulation with LPS and IL-12.

MATERIALS AND METHODS

Reagents and antibodies
rhIL-12 and LPS from Escherichia coli (026:B6) were purchased from the R&D Systems (Minneapolis, MN, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. For inhibition experiments, U0126, SB203580, and LY294002 were obtained from Calbiochem (San Diego, CA, USA), and SP600125 was purchased from Sigma-Aldrich. Inhibitors were resuspended in DMSO and then used to pretreat cells 1 h before stimulation in each experiment. Antibodies specific for p-ERK1/2 (Thr202/Tyr204), p-p38 MAPKs (Thr180/Tyr182), p-stress-activated protein kinase/JNK (Thr183/Tyr185), p-I{kappa}B{alpha} (Ser32), I{kappa}B{alpha}, and STAT4 were obtained from Cell Signaling (Danvers, MA, USA), and ERK1/2 (Chemicon, Temecula, CA, USA), p-STAT4 (BD Biosciences, San Diego, CA, USA), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for Western blot analysis.

Preparation of human blood monocyte-derived DCs
All experiments using human CB were performed under the approval of the Institutional Review Board of the Seoul National University (Republic of Korea; IRB No. 0705/001-002, 0806/001-002). CBMCs were obtained by density gradient centrifugation using Ficoll-Paque PlusTM (Amersham Healthcare, Buckinghamshire, UK). Then, monocytes were isolated from the CBMC using the IMagTM anti-human CD14 antibody, a magnetic bead-based positive selection kit (BD Biosciences). This procedure routinely yields over 90% pure CD14-positive cells, as determined by flow cytometry (data not shown). To generate immature DCs, CD14-positive cells were treated with 500 U/ml rhIL-4 (R&D Systems) and 800 U/ml rhGM-CSF (R&D Systems) at 1 x 106 cells/ml in RPMI 1640 (Invitrogen, Grand Island, NY, USA), supplemented with heat-inactivated 10% FBS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 ug/ml streptomycin (all from Invitrogen), and 0.05 mM 2-ME (Sigma-Aldrich) in a six-well tissue-culture plate (Costar Corp., Cambridge, MA, USA) for 5 days at 37°C under 5% CO2 with a media change every 2 days.

ELISA to detect IL-23, IL-12p40, TNF-{alpha}, IP-10, and IL-1β
To measure cytokine production, CBDCs were cultured in the presence of different stimuli in a 96-well culture plate (Becton Dickinson, Lincoln Park, NJ, USA) at 37°C under 5% CO2. The culture media were collected at the indicated times after stimulation, and the production of IL-23 (eBiosceience, San Diego, CA, USA), IL-12/IL-23p40, IL-12p70, IP-10, TNF-{alpha}, and IL-1β (R&D Systems) was measured by ELISA, according to the manufacturer’s instruction. Colorization was terminated by the addition of 50 µl sulfuric acid (2 N), and the A450–570 was measured in an ELISA microplate reader (Spectramax 340PC, Molecular Devices, Sunnyvale, CA, USA).

Western blot analysis
DCs treated with different stimuli were washed with cold PBS and lysed in cold RIPA lysis buffer containing 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris, pH 7.4, a protease inhibitor cocktail (Roche, Mannheim, Germany), 2 mM NaF, 0.1 mM sodium orthovanadate, and 2 mM glycerol phosphate. After centrifugation at 28,000 g for 5 min at 4°C, the insoluble material was removed, and the protein concentration was measured by Bradford Assay (Bio-Rad Laboratories, Hercules, CA, USA) with BSA as a standard. Lysates (20 µg) were separated by SDS-PAGE and transferred to a polyvinylidene difluoride microporous membrane (Amersham Bioscience, Piscataway, NJ, USA), which was blocked with 3% BSA in TBST (0.1 M Tris, 0.9% NaCl, and 0.1% Tween 20) for 90 min at room temperature before being probed with the primary antibodies described previously. The primary antibodies were detected using HRP-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) or HRP-conjugated mouse anti-rabbit IgG (Chemicon) and visualized by the ECL system (GE Healthcare, Buckinghamshire, UK).

RNA extraction and RT-PCR analysis
Cells (5x105) were collected in 1 ml TRI Reagent (Invitrogen), and RNA was extracted using chloroform and isopropyl alcohol. Samples were kept frozen at –80ºC until use. Total RNA was quantitated by A260 measurements (NanoDrop® ND-1000 spectrophotometer, Amersham Bioscience). RNA was reverse-transcribed into cDNA using Moloney murine leukemia virus RT together with oligo dT (Invitrogen). Then, 1 µl each RT reaction mixture was used for PCR. Sense and antisense oligonucleotide primers were designed for RT-PCR using DNA sequence information obtained from the Genome Database (National Center for Biotechnology Information). Primers were synthesized by Bioneer (Korea), and the sequences are as follows: 5'-gaggcctgtttaccattgga-3', 5'-cggttcttcaagggaggatt-3' for IL-12p35; 5'-ggaccagagcagtgaggtctt-3', 5'-ctccttgttgtcccctctga-3' for IL-12/IL-23p40; 5'-gctgtaatgctgctgttgct-3', 5'-gaaggctcccctgtgaaaat-3' for IL-23p19; 5'-catatccggatgcagactca-3', 5'-gccaacttggacaccttgat-3' for IL-12R β1; 5'-gctgtgtctgcagcaaattc-3', 5'-tccccttttcctctgtgatg-3' for IL-12R β2; and 5'-tcaccatcttccaggagcga-3', 5'-agtgatggcatggactgtgg-3' for GAPDH. PCR was performed in a thermal cycler (Bio-Rad Laboratories) under the following conditions: predenaturation at 95°C for 4 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min; an additional elongation step for 5 min at 72°C was performed. The PCR products were separated on 1.2% agarose gel containing ethidium bromide. The intensity of expression was analyzed using a densitometer with Multi Gauge software (Fujifilm, Tokyo, Japan).

Flow cytometric analysis
Immature DCs or DCs stimulated with E. coli LPS and/or rhIL-12 for 1–9 h were stained with IL-12Rβ1-PE and IL-12Rβ2-PE (BD Biosciences). DCs were plated in 24-well plates at a density of 5 x 105 cells/ml in RPMI complete media. The DCs were stimulated with LPS and/or IL-12 for 24 h. DCs were then harvested and stained with CD80-PE, CD83-FITC, CD86-allophycocyanin, CD40-PE, CD11c-allophycocyanin, or CCR7-FITC (BD Biosciences). After staining for 20 min at 4°C, DCs were washed three times, and differences in the expression of cell-surface molecules were detected using a FACSCalibur with CellQuest (BD Biosciences). All flow cytometric data were analyzed by FlowJo software (Tree Star, San Carlos, CA, USA).

For intracellular staining of IFN-{gamma}, primed DCs were cocultured with PBMCs and stimulated with the T cell activation/expansion kit (Miltenyi Biotec, Auburn CA, USA) in the presence of GolgiPlugTM (BD Biosciences). After 10 h of stimulation, cell-surface molecules were detected by staining with CD4-FITC and CD45RO-allophycocyanin (BD Biosciences). After fixation and permeabilization, cells were washed twice with BD Perm/WashTM buffer and then stained with PE-labeled anti-IFN-{gamma} (BD Biosciences).

IFN-{gamma} and IL-17 secretion in T cells costimulated with primed DCs
DCs (2x105) were costimulated with LPS (100 ng/ml) and IL-12 (100 ng/ml) in 96-well plates for 5 h. After stimulation, LPS and IL-12 were removed from the media, and the DCs were cultured for another 19 h. PBMCs were cocultured with DCs pretreated with LPS and IL-12 in the presence of the T cell activation/expansion kit (Miltenyi Biotec) for 48 h. To ensure that the IFN-{gamma} and IL-17 expression was induced specifically by IL-23, anti-human IL-23p19 antibody (R&D Systems) was used for the neutralization of IL-23 secreted from DCs. The supernatants from PBMCs were evaluated for IFN-{gamma} and IL-17 (R&D Systems) production by ELISA.

Statistical analysis
The mean value ± SD was determined for each treatment group in a given experiment, and all experiments were performed at least three times. Treatment groups were compared with the appropriate control group, and statistical significance was measured using a two-tailed paired t-test. Differences were considered significant when P < 0.05.

RESULTS

Synergistic increase of IL-23 production in DCs costimulated with LPS and IL-12
A critical function of DCs is the secretion of inflammatory cytokines in response to pathogen-associated molecular patterns. IL-12 is one of the major inflammatory cytokines that primarily causes T cells to promote an adaptive immune response. There have also been reports that DCs derived from human peripheral blood monocytes express IL-12Rs that are involved in the tyrosine phosphorylation of several intracellular proteins in response to IL-12 [26 ].

To examine the effects of IL-12 on the capability of DCs to produce cytokines in an autocrine and/or paracrine manner, we stimulated immature CBDCs with LPS, alone or together with IL-12. We found the synergistic expression of IL-12p40 and IL-23 in CBDCs costimulated with LPS and IL-12. It should be noted that the rIL-12 used in the present study was not detected by the IL-12p40 and IL-23 ELISA (Fig. 1 ). It is to note that LPS or IL-12 alone induced low, if there were any, levels of IL-12p70 (data not shown). Other inflammatory cytokines, IL-6, IL-10, and TNF-{alpha}, and chemokines were not affected by the addition of IL-12 (Fig. 1) .


Figure 1
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  		Figure 1. Production of cytokines in DCs costimulated with LPS and IL-12. Monocyte-derived immature CBDCs were treated with IL-12 (100 ng/ml) or LPS (100 ng/ml), alone or together with IL-12 and LPS in a 96-well culture plate for 24 h. After stimulation, the culture supernatant was collected and analyzed for the expression of TNF-{alpha}, IL-6, IL-10, IP-10, IL-12p40, and IL-23 by ELISA. To ensure that the minimal detection of rIL-12 was used in the present study, cell-culture media treated with rIL-12 for 24 h in the absence of DCs were used for the analysis of IL-12p40 and IL-23 by ELISA. Values are mean ± SD of three replicates. Cont, Negative control. *, A significant difference at P < 0.05 compared with control.

Kinetics of IL-23 production
We next checked the kinetics of IL-23 production in CBDCs stimulated with LPS and IL-12. Although there is a difference in the absolute concentration of IL-23 among individuals, production of IL-23 was increased synergistically in a dose-dependent manner (Fig. 2A ). Moreover, the synergistic expression of IL-23 was constitutively maintained in CBDCs stimulated with LPS at 100 ng/ml (data not shown) or 250 ng/ml (Fig. 2A) . Induction of IL-23 in CBDCs costimulated with LPS and IL-12 was also increased in a time-dependent manner (Fig. 2B) . The secretion of IL-23 was obvious at 12 h after costimulation and reached a maximum at 18 h (Fig. 2B) , and the production of the IL-12/IL-23p40 subunit increased gradually in a time-dependent manner until 24 h (data not shown). This result suggests that the production of IL-23 may be influenced by transcriptional levels of IL-23p19, although expression of IL-12p40 was also high.


Figure 2
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  		Figure 2. Kinetics of IL-23 production in DCs in response to costimulation with LPS and IL-12. CBDCs were costimulated with (A) the indicated concentration of LPS and IL-12 for 24 h or (B) LPS (100 ng/ml) and IL-12 (100 ng/ml) for the indicated times. Then, the cell culture supernatants were analyzed for IL-23 by ELISA. Values are mean ± SD of triplicates. *, A significant difference at P < 0.05 compared with control.

Preferential induction of IL-23p19 in CBDCs
To confirm the synergistic expression of IL-23 from CBDCs in response to IL-12 and LPS, mRNA levels of IL-23p19, IL-12p35, and IL-12p40 were analyzed by RT-PCR. As shown in Figure 3 A and B , mRNA expression of IL-23p19 in CBDCs costimulated with LPS and IL-12 increased in a dose-dependent manner. Although the expression of IL-12p35 was also increased, it was relatively low compared with the expression of IL-12p19. Based on these results, we suggest that although LPS alone can sufficiently induce the expression of IL-12p40 in CBDCs, expression of IL-23p19 is critical for the production of the IL-23 heterodimer. This expectation is supported by the fact that IL-23 production is determined mainly by the expression of the IL-23p19 subunit, rather than by IL-12/IL-23p40 [30 ]. Furthermore, we detected the expression of IL-12Rs on immature DCs (Fig. 4B ), and the expression of IL-12Rs increased in CBDCs stimulated with LPS, demonstrating positive regulation of IL-12 signaling (Fig. 4A) .


Figure 3
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  		Figure 3. Preferential increase of IL-23p19 expression in CBDCs costimulated with LPS and IL-12. (A) Immature CBDCs were treated with LPS and/or IL-12 for 8 h. Total RNA was isolated, and mRNA levels of IL-12p35, IL-12/IL-23p40, IL-23p19, and GAPDH were analyzed by RT-PCR. (B) Intensities of subunit IL-23p19, IL-12p40, and IL-12p35 bands were normalized to GAPDH. The results are a representative of three independent experiments.


Figure 4
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  		Figure 4. Expression of IL-12Rs on CBDCs. (A) Immature DCs were stimulated with LPS alone or LPS and IL-12 (100 ng/ml) for 6 h, and mRNA levels of IL-12Rβ1, IL-12Rβ2, and GAPDH were analyzed. (B) The expression of IL-12Rβ1 and IL-12Rβ2 on immature CBDCs was detected using a FACSCalibur, and flow cytometric data were analyzed by FlowJo software. The results are a representative of three independent experiments.

Pattern of IL-23 production in CBDCs compared with ABDCs
It has been reported that DCs from newborn express a high level of IL-23p19 mRNA after LPS stimulation in comparison with ABDCs [31 ]. Thus, we analyzed the induction of IL-23p19 in ABDCs. It was interesting that unlike in CBDCs, mRNA expression of IL-23p19 and IL-12p40 in ABDCs was not increased synergistically (Fig. 5 A and B ), and there was no significant change in the expression of IL-23 in ABDCs after stimulation with LPS and/or IL-12 (Fig. 5C) . These data support the idea that IL-12 has no effect on the production of IL-23 in ABDCs at the mRNA or protein level.


Figure 5
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  		Figure 5. IL-12 and IL-23 expression in ABDCs. (A) Monocyte-derived ABDCs were costimulated with LPS and/or IL-12 for 8 h. Total RNA was analyzed by RT-PCR to determine the mRNA levels of IL-23p19, IL-12/IL-23p40, IL-12p35, and GAPDH. (B) The intensities of the IL-23p19 and IL-12p35 bands were normalized to GAPDH. (C) ABDCs were costimulated with the indicated concentrations of LPS and IL-12 for 24 h. Cell supernatants were collected and used for measurement of IL-23 by ELISA. The result is a representative of three independent experiments.

ERK1/2 activation-dependent production of IL-23
It is well known that the cellular effects of IL-12 are mainly a result of the activation of STAT4 [29 , 32 ]. NF-{kappa}B is an important transcription factor that induces the gene expression of cytokines and costimulatory molecules in cells upon LPS stimulation [33 ]. The activation of NF-{kappa}B is achieved by phosphorylation and degradation of I{kappa}B. To gain insight into the mechanism for IL-23 production, we analyzed phosphorylation of STAT4 and I{kappa}B{alpha} in the cell lysates of CBDCs stimulated with LPS and/or IL-12. I{kappa}B{alpha} was phosphorylated and degraded only upon LPS stimulation (Fig. 6A ), and phosphorylation of STAT4 was induced by IL-12 but not by LPS (Fig. 6B) .


Figure 6
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  		Figure 6. Synergistic expression of IL-23 is dependent on ERK and p38 MAPKs. Immature CBDCs were stimulated with LPS (100 ng/ml) and/or IL-12 (100 ng/ml) for the indicated time-points. After stimulation, DC lysates were prepared using RIPA buffer for Western blotting. For the assay, specific antibodies to (A) p-I{kappa}B{alpha}, I{kappa}B{alpha}, and β-actin, (B) p-STAT4 and STAT4, and (C) p-JNK/JNK, p-ERK/ERK, or p-p38 MAPK/p38 MAPK were used. (D) Immature CBDCs were pretreated with 10 µM U0126 (ERK inhibitor), SB203580 (p38 MAPK inhibitor), or SP600125 (JNK inhibitor) for 1 h prior to stimulation with LPS (100 ng/ml) only or LPS plus IL-12 (100 ng/ml). NT, Inhibitor-nontreated control. After 24 h stimulation, the cell-culture supernatants were collected and analyzed for the production of IL-23 and IL-12p40 by ELISA. Results are from three independent experiments. Costimulation control of LPS and IL-12 has a significant difference compared with LPS-treated control (P<0.05). Values are mean ± SD of triplicates. *, A significant difference at P < 0.05 compared with inhibitor-nontreated control.

The MAPKs participate in cellular responses to different extracellular stimuli, such as growth factors or stresses, and also play an important role in cytokine production [34 ]. So, we decided to verify whether MAPKs are involved in IL-23 production in cells stimulated by LPS and IL-12. As shown in Figure 6C , ERK1/2 and p38 MAPK appear to be important signaling components of IL-23 expression. To confirm the functional relevance of enhanced p-ERK1/2 in response to LPS and IL-12, CBDCs were pretreated for 1 h with an ERK inhibitor (U0126), a p38 MAPK inhibitor (SB203580), or JNK inhibitor (SP600125) before stimulation with LPS and IL-12. Consistent with the aforementioned finding, although ERK and p38 inhibitor decreased the IL-23 production in CBDCs treated with LPS only, the production of IL-23 was reduced markedly by ERK and p38 MAPK inhibitors in CBDCs costimulated with LPS and IL-12 (Fig. 6D) . However, other inhibitors of JNK or PI3K did not show any effect on IL-23 production (data not shown). Based on these results, the activation of ERK1/2 and p38 MAPK appears to play an important role in IL-23 production in CBDCs costimulated with LPS and IL-12.

Bioactive IL-23-induced IFN-{gamma} expression
It has been suggested that IL-23 induction of IFN-{gamma} production is important for the formation of memory T cells in humans [35 ]. In addition, it has been reported that IL-23 promotes induction of IFN-{gamma} and proliferation primarily in memory T cells rather than naïve T cells [35 ]. Therefore, we wanted to determine whether the IL-23 produced in CBDCs, costimulated with LPS and IL-12, had a biological function. We hypothesized that IFN-{gamma} production in the memory T cells should be increased by the IL-23 produced by CBDCs. For this study, we choose the time-point for media exchange based on the induction of IL-23 shown in Figure 2B . We primed CBDCs with LPS and IL-12 for 5 h, stimulants were withdrawn, and the cells were incubated further for another 19 h in a fresh complete media. We treated primed CBDCs with or without IL-23p19 neutralizing antibody prior to coculture of CBDCs and PBMCs and then examined IFN-{gamma}-producing cells with a memory phenotype. As expected, the number of memory cells (CD4+CD45RO+) producing IFN-{gamma} was increased (Fig. 7 A and B ) significantly (P<0.05). To confirm that the production of IFN-{gamma} was influenced by IL-23 and not IL-12, we added IL-23p19 neutralizing antibody for 1 h before the coculture of DCs and PBMCs. As a result, we found a significant decrease in the number of memory cells producing IFN-{gamma}. This result was confirmed by analyzing IFN-{gamma} production in the culture supernatants using ELISA (Fig. 7 C). These results provide evidence that production of IFN-{gamma} in memory T cells is induced by IL-23. Moreover, IL-23 production by CBDCs can induce the production of IL-17 (Fig. 7D) . Recently, it has been reported that IL-1β in culture supernatants of activated DCs plays an important role in IL-23-mediated IL-17 production by CD4+ T cells [36 ]. Therefore, we examined the IL-1β production in CBDCs costimulated with LPS and/or IL-12 to identify the impact of IL-1β on the expression of IL-17. As a result, there are no differences in the expression of CBDCs stimulated with LPS and IL-12 compared with that of LPS alone (Fig. 7E) .


Figure 7
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  		Figure 7. Impact of CBDCs stimulated with LPS and IL-12 on T cells with memory phenotype. (A–D) Primed DCs derived from CB were cocultured with PBMCs stimulated with a T cell activation/expansion kit. (A) IFN-{gamma}-expressing memory T cells were detected by the intracellular staining method described in Materials and Methods. (B) Changes of IFN-{gamma}-expressing memory T cells are shown as relative geometric mean. The cell culture supernatants were analyzed subsequently by ELISA for the expression of (C) IFN-{gamma} and (D) IL-17 following stimulation for 48 h. (E) Monocyte-derived immature CBDCs were treated with LPS and/or IL-12 for 24 h. After stimulation, the culture supernatant was collected and analyzed for the expression of IL-1β by ELISA. Values are mean ± SD of triplicates. *, A significant difference at P < 0.05 compared with control.

DISCUSSION

In the present study, we have shown that production of IL-23 in CBDCs costimulated with LPS and IL-12 is increased synergistically in an IL-12 dose-dependent manner. The IL-23 produced in CBDCs promoted IFN-{gamma} production in memory T cells. It is generally believed that T cells and NK cells are the major responders to IL-12. However, previous reports have shown that IL-12 can influence APCs via autocrine regulation [26 , 37 , 38 ]. Our results reveal that in CBDCs, IL-12, in conjunction with LPS, can increase the production of IL-23 synergistically through an increase in the transcriptional and protein levels of IL-23p19. Although heterodimeric cytokine IL-12 shares the IL-12/IL-23p40 subunit with IL-23, only newly synthesized IL-23 had such an effect. It is important to note that production of IL-6, IL-10, TNF-{alpha}, and IP-10 was not affected significantly by IL-12. Furthermore, costimulation with LPS and IL-12 did not influence the expression of the cell-surface molecules CD80, CD83, CD86, CCR7, or CD40 when compared with those molecules induced by LPS only.

It is well known that the signaling pathway triggered by IL-12 is associated with phosphorylation of Jak2/Tyk2 and consequent activation of STAT4 leading to the production of IFN-{gamma} in T cells and NK cells [39 ]. Interestingly, IL-12R is also expressed in human DCs but at levels much lower than in T cells and NK cells. In DCs, IL-12R plays a role in the early events of activation in autocrine and/or paracrine pathways [26 , 38 , 40 ]. We confirmed the increased expression of transcriptional IL-12Rβ1 and IL-12Rβ2 after LPS stimulation in ABDCs (data not shown) and CBDCs. However there was no difference in the expression of IL-12Rs on the surface of CBDCs stimulated with LPS and/or IL-12 for 1–9 h (data not shown). Furthermore, our data suggest that LPS plays a dominant role in the maturation of DCs, which is not affected by the addition of IL-12 (data not shown).

One of the major differences between CBDCs and ABDCs is the capacity to produce IL-12 and IL-23 in response to LPS alone or LPS together with IL-12. It has been reported that IL-12 production in CBDCs is impaired, as the transcription of IL-12p35 is strongly inhibited by defected nucleosome remodeling [41 ]. Therefore, the production of IL-23, heterodimer of IL-12/IL-23p40 and IL-23p19 by CBDCs, is preferentially increased. These findings support our data further, showing that mRNA expression of IL-23p19 in CBDCs, costimulated with LPS and IL-12, is dominant and increases in an IL-12 dose-dependent manner. The transcriptional expression of IL-23p19 was higher than that of IL-12p35 in CBDCs, and this was reversed in ABDCs under the same conditions. Furthermore, we could not detect any induction of IL-23p19 in ABDCs treated with IL-12. It is probable that such a difference resulted from a difference in stage or lineage and/or a distinct microenvironmental milieu between peripheral blood and CB during the stimulation. It has been suggested that in CBDCs, IL-23 has a supplementary effect on the IL-12 deficiency in the neonatal immune response.

The IL-12p35 and IL-23p19 subunits appear to be expressed poorly and require IL-12/IL-23p40 as a partner to form an active disulfide-linked heterodimer [15 ]. Our results are also concordant with reports that IL-23 production depends primarily on expression of IL-12p19 rather than IL-12/IL-23p40. Also, it makes sense that the synergistic increase of IL-23p19 expression is indispensable for the synergistic production of IL-23. Similar to results from our study, it has shown that down- or up-regulation of IL-23p19 expression results in the production of IL-23, regardless of IL-12p40 expression.

The above results led us to investigate further the signaling pathway for IL-23 production in CBDCs. Upon cotreatment with IL-12 and LPS, CBDCs initiate the intracellular signaling cascade by recruiting several adaptor proteins and activating transcription factors. It is well known that LPS activates NF-{kappa}B via MyD88-dependent and Toll/IL-1R domain-containing adaptor-inducing IFN-β-dependent pathways to induce the expression of target cytokines [42 ]. Moreover, it has been reported that IL-12 binds IL-12Rs and activates Jak2/Tyk2 and STAT4 transcription factors [43 , 44 ]. Therefore, we investigated the activation of NF-{kappa}B and STAT4 in CBDCs stimulated with LPS and/or IL-12. NF-{kappa}B activation induced by LPS alone was confirmed by the detection of an increased level of p-I{kappa}B in CBDCs, as the activation of NF-{kappa}B is initiated by I{kappa}B kinase-induced degradation of I{kappa}B proteins. However, there was no difference in the levels of p-STAT4 in CBDCs when stimulated with LPS alone. Ursula Grohmann et al. [33 ] reported that IL-12 acts directly on DCs to promote NF-{kappa}B but not STAT4 expression. On the contrary, we could detect increased phosphorylation of STAT4 but not I{kappa}B activation in CBDCs after IL-12 stimulation alone. Although activation of NF-{kappa}B and STAT4 was observed in CBDCs costimulated with LPS and IL-12, no significant change was found when compared with a single treatment with LPS or IL-12.

In the present study, in CBDCs stimulated with IL-12 alone, we observed the phosphorylation of ERK1/2 and p38 MAPK. Although LPS can induce all three MAPKs, our data show that the phosphorylation of ERK1/2 and p38 MAPK in CBDCs, costimulated with LPS and IL-12, was higher than in cells stimulated with LPS alone. These results demonstrate that the IL-12-mediated phosphorylation of ERK1/2 and p38 MAPK appears to be important for the induction of IL-23 expression in CBDCs. However, LPS-mediated JNK and I{kappa}B, together with IL-12-induced STAT4 phosphorylation, were not affected by further stimulation with IL-12.

There have been several reports about how the biological impact of IL-23 varies according to the immunological environment. Recent reports showed the effect of IL-23 on the induction of IL-17 expression in Th17 cells, a new, additional subset of CD4+ Th cells [45 , 46 ], and induction of IFN-{gamma} production in memory T cells [35 ]. In the present study, we have found a marginal increase of IFN-{gamma}-producing memory cells, which we consider important and of biological significance as a result of the relatively small percentage of basal memory cells. We also observed increased expression of IL-17, although the level was minimal. It is probable that IL-23 together with other cytokines such as IL-1β act synergistically to induce IL-17. Indeed, our concept was supported by the fact that IL-1β contained in supernatants of activated DCs plays an important role in IL-23-mediated IL-17 production by human CD4+ T cells [36 ]. Furthermore, we have found no significant changes by other cytokines including IL-6, IL-10, and TNF-{alpha} in CBDCs costimulated with LPS and IL-12. IL-17 is known to be involved in many inflammatory diseases, such as rheumatoid arthritis, asthma, systemic lupus erythematosus, and allograft rejection [21 , 47 , 48 ]. IL-23 also plays an important role in promoting cellular immunity against bacterial infection and metastatic activity [49 , 50 ]. It has been suggested that the role of IL-23 as an immunological regulator might be to balance between Th1 and Th17 [51 , 52 ].

Our observations suggest that IL-23 produced by CBDCs could play a critical role in newborns during encounters with foreign antigens. Furthermore, it has been suggested that IL-23 can compensate for the relatively small amount of IL-12 produced in CBDCs [31 , 53 ]. However, the production of IL-23 in CBDCs treated with LPS and IL-12 could induce a different profile of cytokine expression dependent on the microenvironmental milieu. Although CB-derived cell therapies together with various cytokine treatment trials are under intensive investigation recently, we need to proceed cautiously for the induction of unexpected and/or possibly unwanted cytokine production. In conclusion, our study provides insight into the synergistic induction of IL-23 secretion and activation of IFN-{gamma}-producing memory cells in CBDCs exposed to LPS and IL-12.

ACKNOWLEDGMENTS

This work was supported by a grant from the Biogreen 21 Program (20080401034054), Rural Development Agency, and GRCMVP for Technology Development Program of Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea. We greatly appreciate the Seoul Metropolitan Public Cord Blood Bank (ALLCORD) and the 21C Frontier Microbial Genomics and Application Center Program, respectively, for helpful supply of cord blood.

FOOTNOTES

Abbreviations: A450–570=absorbance at 450–570 nm, AB=adult blood, ABDC=AB-derived DC, APC=antigen presenting cell, CB=cord blood, CBDC=CB-derived DC, CBMC=CB mononuclear cell, DC=dendritic cell, h=human, IP-10=IFN-inducible protein 10, p=phospho, RIPA=radioimmunoprecipitation assay, TLR=Toll-like receptor

Received October 17, 2008; revised April 27, 2009; accepted April 28, 2009.

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