CpG oligodeoxynucleotides induce IL-8 expression in CD34+ cells via mitogen-activated protein kinase-dependent and NF-{kappa}B-independent pathways

Jung Mogg Kim1, Nam In Kim1, Yu-Kyoung Oh2, Young-Jeon Kim3, Jeehee Youn4 and Myung-Ju Ahn5

1 Department of Microbiology and Institute of Biomedical Science, Hanyang University College of Medicine, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791, Korea
2 School of Life Sciences and Biotechnology, Korea University, Korea
3 Department of Science, Joongbu University, Choongnam, Korea
4 Department of Anatomy and Cell Biology and 5 Department of Internal Medicine, Hanyang University College of Medicine, Seoul, Korea

Correspondence to: J. M. Kim; E-mail: jungmogg{at}hanyang.ac.kr


    Abstract
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 Abstract
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 Methods
 Results
 Discussion
 References
 
To elucidate the role of Toll-like receptor 9 (TLR9) activation along with the intracellular signaling pathways triggered by CpG DNA in CD34+ cells, we investigated whether synthetic oligodeoxynucleotides (ODNs), containing unmethylated CpG motifs, could induce IL-8 expression in CD34+ cells through mitogen-activated protein kinase (MAPK) or nuclear factor-{kappa}B (NF-{kappa}B) pathway. We demonstrated evidence for the first time that CD34+ cells constitutively expressed TLR9. Exposure of the cells to CpG ODN resulted in a time- and dose-dependent increase of IL-8 expression, and activation of phosphorylated ERK1/2 and phosphorylated p38. In addition, CpG ODN stimulated AP-1, but not NF-{kappa}B, signals. Moreover, inhibitors of MAPK (U0126 and SB203580) significantly reduced the IL-8 production, while the inhibition of NF-{kappa}B (pyrrolidinedithiocarbamate and retrovirus containing dominant-negative I{kappa}B{alpha} plasmid) did not affect the IL-8 expression increased by CpG ODN. Moreover, co-stimulation with LPS and CpG synergistically up-regulates IL-8 in CD34+ cells. These results suggest that CpG DNA, acting on TLR9, activates CD34+ cells to express IL-8 through MAPK-dependent and NF-{kappa}B-independent pathways.

Keywords: AP-1, ERK1/2, p38, Toll-like receptor 9


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Primitive hematopoietic stem cells (HSCs) are pluripotent cells with the capacity to give rise to all lineages of blood cells. During commitment, progenitor cells are composed mainly of cells with the potential for differentiation into one or two lineages. The HSCs express CD34 molecules in their surfaces. The numbers of CD34+ stem cells are significantly higher in umbilical cord blood than in adult peripheral blood (1). Especially, the numbers of colony-forming unit-granulocyte–macrophages are much higher in umbilical cord blood than in peripheral blood (2). Therefore, umbilical cord blood has been proposed as an ideal alternative to bone marrow and peripheral blood for HSC transplantation.

We recently demonstrated that Escherichia coli provokes the expression of several pro-inflammatory cytokines, including IL-8, in CD34+ cells (3). This suggests that CD34+ cells can recognize the molecular patterns associated with pathogens and subsequently initiate the transcription of inflammatory genes. These molecular patterns may be specifically conserved components of microbes, such as LPS from Gram-negative bacteria, CpG DNA and flagellin (46). Among these specifically conserved components of microbes, unmethylated 5'-CpG-3' dinucleotides with certain flanking bases have been suggested to be responsible for the immunogenicity of bacterial DNA (7). Synthetic oligodeoxynucleotides (ODNs), containing this CpG motif, have been shown to have immunostimulatory effects similar to those of bacterial DNA (7). However, little information is available on the biological effect of bacterial CpG DNA on the CD34+ cells.

Unlike LPS, which is recognized by Toll-like receptor 4 (TLR4), CpG DNA is recognized by TLR9 (8). The signaling of these two receptors, however, shares similar downstream pathways, including activation of mitogen-activated protein kinases (MAPKs) and I{kappa}B kinase complex (9, 10). Several studies have shown that CpG DNA has strong stimulatory effects on lymphocytes (7, 1114). These stimulatory effects include triggering B cell proliferation, resistance to apoptosis, release of IL-6 and IL-12, NK cell secretion of IFN-{gamma}, increased lytic activity and monocyte/macrophage secretion of IFN-{alpha}/ß, IL-6, IL-12, granulocyte–monocyte colony-stimulating factor, chemokines and tumor necrosis factor (TNF)-{alpha}. Since there is no report regarding TLR9 and CpG DNA-induced signaling in CD34+ cells, we investigated the role of TLR9 activation along with the intracellular signaling pathways triggered by CpG DNA in CD34+ cells. In the present study, we provide evidence for the first time that CD34+ cells constitutively express TLR9 mRNA. Exposure of the cells to CpG ODN increased the expression of IL-8 through MAPK-dependent and nuclear factor-{kappa}B (NF-{kappa}B)-independent pathways. We further show that LPS and CpG ODN have a synergistic effect on the induction of IL-8 response in CD34+ cells.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Reagents
Phosphorothioate-modified CpG ODNs were obtained from GeneTech (Daejon, Korea) [CpG-A ODN 2216, 5'-ggGGGA CGATCGTCgggggG-3' (lower case letters, phosphorothioate linkage; capital letters, phosphodiester linkage 3' of the base and bold, CpG dinucleotides)] (15). No endotoxin could be detected in the CpG ODN preparation (<0.03 EU ml–1, Limulus amebocyte lysate assay; BioWhittaker, Walkersville, MD, USA). The CpG ODN was re-suspended in Tris-ethylene diaminetetraacetic acid (TE) buffer and diluted in PBS (15). Escherichia coli plasmid DNA (pEGFP-N1; Clontech, Palo Alto, CA, USA) was purified by endotoxin-free plasmid kit (Qiagen, Germany). U0126, which inhibits MAPK and ERK kinase (MEK) 1/2 that phosphorylates extracellular signal-regulated kinase (ERK) 1/2, and SB203580, which specifically inhibits p38, were purchased from Calbiochem (San Diego, CA, USA). Pyrrolidinedithiocarbamate (PDTC), which inhibits NF-{kappa}B, and chloroquine were purchased from Sigma Chemical Company (St Louis, MO, USA).

Isolation of CD34+ cells from umbilical cord blood
Normal umbilical cord blood scheduled for discarding after delivery was obtained with a maternal consent. Low-density mononuclear cells were isolated on Ficoll-Paque (1.077 g ml–1) (Amersham Pharmacia Biotech, Piscataway, NJ, USA), washed twice with PBS containing 2 mM EDTA and centrifuged for 10 min at 200 x g at 20°C. The cell pellet was re-suspended in a final volume of 300 µl of the buffer per 108 total cells. CD34+ cells were incubated with a monoclonal anti-human CD34+ antibody (QBEND/10), followed by positive selection by immunomagnetic beads and MACS separators according to the manufacturer's recommendations (Miltenyi Biotech, Auburn, CA, USA) (3). The purity of CD34+ preparations routinely exceeded ~98%, as determined by FACS analysis (Becton Dickinson, San Jose, CA, USA). The CD34+ cells (1 x 105 ml–1) were seeded in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) in 35-mm dishes at 37°C in a humidified atmosphere of 5% CO2.

Reverse transcription–PCR analysis and ELISA
CD34+ cells were incubated with CpG ODN for indicated times, after which total cellular RNA was extracted from the cells using Trizol reagent (GIBCO BRL, Gaithersburg, MD, USA). For TLR9, the PCR cycle consisted of denaturation (94°C for 1 min), annealing (55°C for 1 min) and an elongation step (72°C for 1.5 min) for 38 cycles, followed by an additional extension step (72°C for 10 min). The primer sequences of TLR9 were as follows: 5'-GCATCTTCTTCCGCTCACTC-3' (sense) and 5'-AGCCACGAAGCTGAAGTTGTG-3' (antisense) (16). PCR amplification using these oligonucleotide primers yielded PCR products with a length of 598 bp. The positive control for TLR9 mRNA expression was used in the RNA extracted from RPMI 8226 human B cell line [American Type Tissue Collection (ATCC) CCL-155] and the negative control was from Jurkat human T cell line (ATCC TIB-152). These cell lines were used previously as positive and negative controls for the TLR mRNA expression by Takeshita et al. (17). Quantitative reverse transcription (RT)–PCR using internal standards was used to quantify IL-8 and ß-actin mRNA levels, as described previously (3). Synthetic standard RNA was kindly provided by Kagnoff of the University of California, San Diego. PCR amplification consisted of 35 cycles of 1-min denaturation at 95°C, 2.5-min annealing and extension at either 60 (IL-8) or 72°C (ß-actin). RT and PCR amplification of the internal RNA standards, using IL-8 and ß-actin primers, yielded 401-bp fragment and 520-bp fragment, respectively. IL-8 and ß-actin primers were used to amplify 289-bp and 661-bp fragment from cellular RNA, respectively.

Before measurement of IL-8, the supernatants of culture media were filtered through a 0.22-µm filter to remove any contaminants. The levels of human IL-8 were determined by Quantikine immunoassay kit (R&D Systems, Minneapolis, MN, USA). Each sample was tested in triplicate.

Electrophoretic mobility shift assays
Cells were harvested, and nuclear extracts were prepared as described (18). The concentrations of proteins in the extracts were determined by the Bradford assay (Bio-Rad, Hercules, CA, USA). Electrophoretic mobility shift assays (EMSAs) were performed according to the protocol of the manufacturer (Promega, Madison, WI, USA). In brief, nuclear extracts were incubated for 30 min at room temperature with {gamma}32P-labeled oligonucleotide probe corresponding to an AP-1-binding site or an NF-{kappa}B-binding site. After incubation, bound and free DNAs were resolved on 5% native polyacrylamide gels, as described previously (18). For competition assays, nuclear extracts were pre-incubated with AP-1 oligomer (5'-CGC TTG ATG ACT CAG CCG GAA-3'), AP-1 oligomer mutant (5'-CGC TTG ATG ACT TGG CCG GAA-3') or NF-{kappa}B oligomer (5'-AGT TGA GGG GAC TTT CCC AGG C-3') for 1 h at 4°C.

Recombinant retrovirus and retrovirus infection
Dominant-negative I{kappa}B{alpha} (S32A, S36A) (19) was amplified with sense (5'-aaccATGGCATACCCATACGACGTCCCAGACTACGCTttccaggcggccgagcgcccccaggag-3') and anti-sense (5'-aaaaGGATCCtcataacgtcagacgctggcct-3') primers using high fidelity Taq polymerase (GIBCO BRL). The capital letters represent nucleotides encoding a haemagglutinin tag. The PCR products were digested with Nco-1 and BamH1 restriction enzymes. These enzymes were incubated with the buffer [150 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2, 1 mM dithiothreitol (pH 7.9) and 100 µg ml–1 BSA] at 37°C for 2 h. The digested PCR products were cloned into the corresponding sites in MFG retroviral vector by replacing the GFP sequence of MFG.GFP.IRES.puro. The retroviral plasmids obtained were introduced into 293 gpg retrovirus packaging cell line (20) by transient transfection. Briefly, the plasmid DNA (4 µg) was mixed with lipofectamine (GIBCO BRL) in optiMEM (GIBCO BRL) at room temperature for 30 min. The mixture was overlaid to the 293 gpg retrovirus packaging cell line (3 x 106 cells per 60-mm culture dish) and incubated for 8 h at 37°C. Subsequently, DMEM supplemented with 10% FBS and puromycin (2 µg ml–1, Sigma) were added. At 24 and 48 h later, the culture media were changed to new media. After 72 h, the supernatants were harvested and used for retroviral infection. The virus titers, measured in NIH3T3 cell line by puromycin-resistant colony formation, were between 105 and 5 x 105 ml–1 (retrovirus-I{kappa}B{alpha}-AA). The infection and selection of target cells with puromycin were performed as described previously (18).

Immunoblots
Cells were washed with ice-cold PBS and lysed in 0.5 ml per well lysis buffer [150 mM NaCl, 20 mM Tris (pH 7.5), 0.1% Triton X-100, 1 mM phenylmethylsulphonylfluoride and 10 µg ml–1 aprotonin] as described previously (3). Protein concentrations in the lysates were determined by the Bradford method (Bio-Rad). A total of 5–15 µg protein per lane was size-fractionated on a denaturing, non-reducing 6% polyacrylamide minigel (Mini-PROTEIN II; Bio-Rad) and electrophoretically transferred to a nitrocellulose membrane (0.1-µm pore size). Specific proteins were identified with polyclonal antibodies for pan-ERK1/2 (p44/p42) (catalog no. 9102), phospho-ERK1/2 (catalog no. 9101), pan-p38 (catalog no. 9212) and phospho-p38 (catalog no. 9212) (all from Cell Signaling Technology, Inc., Beverly, MA, USA). The immunoreactive proteins were visualized using goat anti-rabbit secondary antibodies conjugated to HRP (Transduction Laboratories, Lexington, KY, USA), which was followed by enhanced chemiluminescence (ECL system; Amersham Life Science, Buckinghamshire, UK) and exposure to X-ray film (XAR5; Eastman Kodak Company, Rochester, NY, USA).

Statistical analysis
Data are presented as mean ± SD for quantitative RT–PCR and mean ± SEM for ELISA. Wilcoxon's rank sum test was used for statistical analysis. A level of statistically significant difference was accepted at P < 0.05.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of TLR9 mRNA in CD34+ cells
RT–PCR analysis of RNA from CD34+ cells revealed constitutive expression of TLR9 transcripts, and stimulation of CD34+ cells with CpG ODN did not change the TLR9 mRNA expression (Fig. 1). In this experiment, RPMI 8226 human B cell line showed TLR9 mRNA expression as a positive control, whereas Jurkat human T cell line did not express TLR9 as a negative control. Subsequent nucleotide sequence analyses of the RT–PCR product from CD34+ cells revealed 100% identity with the published human TLR9 cDNA sequence, confirming that the primers used in this study amplified the correct sequences.



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Fig. 1. TLR9 mRNA expression in CD34+ cells, revealed by RT–PCR analysis. The cells (1 x 106) in the 6-well tissue culture plate were treated with CpG ODN (10 µM) for the indicated periods. The expression of mRNA for TLR9 and ß-actin was assessed by RT–PCR using specific primers. (+) and (–) represent positive and negative controls, respectively. Shown in the figure are the representative data of three separate experiments.

 
CpG DNA induces IL-8 expression in human CD34+ cells
After demonstrating constitutive expression of TLR9 in CD34+ cells, we then examined whether CpG DNA could induce immune response in these cells. As shown in Fig. 2, CpG ODN-induced increases of IL-8 mRNA in CD34+ cells were dose dependent and followed a characteristic time course. However, the ß-actin mRNA levels in stimulated and unstimulated cells remained relatively constant throughout the same period (Fig. 2B; ~6 x 106 transcripts per microgram RNA). To determine whether increased IL-8 mRNA levels were accompanied by increased secretion of IL-8 protein, we measured the amount of IL-8 proteins in culture supernatants. The increase of IL-8 mRNA expression was associated with an increase of IL-8 secretion 24 h post-stimulation (control, 0.8 ± 0.3 ng ml–1; CpG, 9.8 ± 1.1 ng ml–1; mean ± SEM, n = 5).



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Fig. 2. CpG ODN-induced increases of IL-8 expression in CD34+ cells. (A) After treatment of CD34+ cells with various doses of CpG ODN, expression of IL-8 mRNA was analyzed by quantitative RT–PCR. (B) IL-8 mRNA expression in CD34+ cells at different times following treatment with CpG ODN (10 µM). Data are presented as numbers of mRNA transcripts per microgram of total RNA (mean ± SD, n = 5).

 
We next determined whether E. coli plasmid DNA might be effective in inducing IL-8 expression. Thus, CD34+ cells were treated with plasmid DNA (20 µg ml–1), and IL-8 expression was examined 12 h later by quantitative RT–PCR. Escherichia coli plasmid DNA also up-regulated IL-8 mRNA expression in CD34+ cells (control, 3.6 x 105; E. coli DNA, 5.7 x 107; mean numbers of IL-8 mRNA transcripts per microgram RNA, n = 5). In this experiment, LPS levels in E. coli plasmid DNA was <0.03 EU ml–1.

Chloroquine blocked CpG ODN-induced up-regulation of IL-8 in CD34+ cells
Internalization and endosomal maturation have been shown to be required for CpG DNA to activate TLR9 signaling in immune cells (21, 22). As shown in Fig. 3, chloroquine, which effectively blocks endosomal maturation (21), significantly inhibited the CpG ODN-induced increase of IL-8 production in CD34+ cells, indicating similar ODN-mediated signaling between CD34+ cells and immune cells.



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Fig. 3. Activation of CD34+ cells by CpG ODN is dependent on endosomal maturation. CD34+ cells were treated with chloroquine (10 or 50 µg ml–1) for 30 min, followed by CpG ODN treatment (10 µM) for 24 h. IL-8 production was determined by ELISA analysis (mean ± SEM, n = 5; *P < 0.05 compared with control, **P < 0.05 compared with group treated with CpG ODN only, ***P < 0.01 compared with group treated with CpG ODN only).

 
CpG DNA activates AP-1 signals in CD34+ cells
AP-1 activation is known to induce the expression of IL-8 (23). Therefore, to determine whether CpG DNA could activate AP-1 signals in CD34+ cells, AP-1–DNA binding studies were performed by EMSA using nuclear extracts after stimulation of CD34+ cells with CpG ODN. As shown in Fig. 4(A), stimulation of these cells with CpG ODN increased the binding activity. The specificity of AP-1 signals was confirmed by competition assay. The addition of AP-1 oligomer to nuclear extracts after stimulation of CD34+ cells with CpG ODN for 2 h was shown to suppress the AP-1 signal, however, the addition of AP-1 mutant oligomer or NF-{kappa}B oligomer did not change the signals (Fig. 4B). In contrast to AP-1 signals, stimulation of the cells with CpG ODN did not activate NF-{kappa}B activity (Fig. 4C). Consistent with this, degradation of I{kappa}B{alpha} was not observed in CpG ODN-stimulated CD34+ cells, as determined by immunoblot analysis.



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Fig. 4. CpG ODN activates AP-1 signals in CD34+ cells. CD34+ cells were stimulated with CpG ODN (10 µM) for the indicated periods of time. (A) AP-1 activity was assessed by EMSA. (B) Competition assay for AP-1 was performed using each oligomer to the nuclear extracts of CD34+ cells. (C) NF-{kappa}B DNA-binding activity was also assessed by EMSA at the indicated times. Immunoblots for concurrent I{kappa}B{alpha} and actin under the same condition are provided beneath each EMSA time point. (+) represents a positive control, where CD34+ cells were treated with TNF-{alpha} (20 ng ml–1) for 3 h. (–) represents negative control. These results are representatives of three independent experiments.

 
Effect of signaling inhibitors on CpG ODN-induced IL-8 expression in CD34+ cells
CpG ODN has been shown to stimulate several signaling pathways, including ERK1/2 and NF-{kappa}B in macrophages and other immune cells (2426). As shown in Fig. 5(A), treatment of CD34+ cells with CpG ODN induced activation of phosphorylated ERK1/2 and p38. Next, we determined the pharmacological sensitivities of these pathways that might have contributed to the expression of IL-8 in CD34+ cells after CpG ODN exposure. Figure 5(B) shows that MEK1/2 inhibitor U0126 and p38 inhibitor SB203580 significantly inhibited the CpG ODN-induced increase of IL-8 production. The ERK inhibitor (U0126) or p38 inhibitor (SB203580) prevented CpG DNA-mediated phosphorylation of ERK1/2 or p38 in CD34+ cells (Fig. 5C). However, NF-{kappa}B inhibitor PDTC had no effect on the CpG ODN-stimulated IL-8 expression. To confirm that the inhibition of NF-{kappa}B activity had no effect on the CpG ODN-stimulated IL-8 expression, transfection with retrovirus-I{kappa}B{alpha}-AA was performed. Our recent study showed that activation of NF-{kappa}B signals was completely inhibited when the CD34+ cells were transfected with retrovirus-I{kappa}B{alpha}-AA (3). In the present study, IL-8 production in response to CpG ODN was not attenuated in NF-{kappa}B-suppressed CD34+ cells (control, 0.6 ± 0.3 ng ml–1; CpG, 10.1 ± 1.5 ng ml–1; CpG + retrovirus-I{kappa}B{alpha}-AA, 10.2 ± 0.8 ng ml–1; mean ± SEM, n = 3). These results suggest that CpG ODN up-regulates IL-8 in CD34+ cells through the activation of MAPK signaling pathways.



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Fig. 5. CpG ODN activates MAPK in CD34+ cells. (A) After CD34+ cells were stimulated with CpG ODN (10 µM) for the indicated times, cell lysates were prepared and subjected to immunoblot analysis for ERK1/2 and p38. (B) CD34+ cells were treated with U0126 (50 µM), SB203580 (10 µM) or PDTC (100 µM) for 30 min, followed by CpG ODN treatment (10 µg ml–1). ELISA analysis of IL-8 production 24 h, following CpG ODN treatment (mean ± SEM, n = 5; *P < 0.05 compared with group treated with CpG ODN only). (C) Phosphorylation of ERK1/2 and p38 was detected in CD34+ cells 30 min after stimulation with CpG ODN (10 µg ml–1). Pre-treatment with the U0126 (50 µM) for 30 min prevented phosphorylation of ERK1/2 activity, whereas pre-treatment with SB203580 (10 µM) blocked phosphorylation of p38.

 
Co-stimulation with LPS and CpG synergistically up-regulates IL-8 in CD34+ cells
The above studies clearly showed that CpG ODN was effective in inducing IL-8 response in CD34+ cells. Because CD34+ cells are likely to be concomitantly exposed to both LPS and bacterial DNA during bacterial infection, we then examined whether LPS and CpG have a synergistic effect in inducing IL-8 expression in CD34+ cells. As shown in Fig. 6, there was a little increase in the levels of IL-8 when the cells were treated with low concentrations of either LPS (100 ng ml–1) or CpG ODN (1 µM). However, combination of both led to a dramatic enhancement in IL-8 response. These results suggest that co-stimulation of CD34+ cells with CpG ODN and LPS induces synergistic up-regulation of immune responses.



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Fig. 6. Co-stimulation with CpG ODN and LPS induces synergistic up-regulation of IL-8 expression in CD34+ cells. After CD34+ cells were stimulated with CpG ODN alone (1 µM), LPS alone (100 ng ml–1) or combination of both for 24 h, amounts of IL-8 in the culture supernatants were examined by ELISA. Data shown are mean ± SEM of five independent experiments. *P < 0.05 compared with a group treated with CpG ODN only or a group treated with LPS only.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The innate immune system is important in host defense against invading pathogens such as bacteria and viruses. TLRs, a family of receptors, are involved in innate immune response and function by recognizing specific conserved components of microbes (27). Recently, TLR9 was found to be critical in the recognition of unmethylated CpG motifs in microbial DNA. Its expression is a prerequisite for the responsiveness to CpG DNA and has been shown to be expressed in many types of immune cells (17). In the present study, we showed for the first time that CD34+ cells express TLR9 and that CpG DNA triggers an IL-8 response in CD34+ cells, as demonstrated by an increase in IL-8 mRNA expression and protein secretion.

The availability of blood cells is sustained by pluripotent hematopoietic progenitors. These HSCs express CD34 surface molecules and are pluripotent with the capacity to differentiate into erythrocytes, granulocytes, monocytes, megakaryocytes and lymphocytes (28). Considering that human HSCs are relatively resistant to infection by bacteria, compared with viral infection (29, 30), our study supports a possible role for CpG DNA-mediated CD34+ immune response in the host defense system.

CpG ODN activation has been shown to be dependent on internalization and endosomal maturation in macrophage and other immune cells (7, 21, 22). A similar observation was also made in CD34+ cells in the present study. Pre-treatment with chloroquine, an inhibitor of vesicular acidification and endosomal maturation, significantly inhibited the CpG ODN-induced up-regulation of IL-8 expression.

The molecular mechanism by which CpG DNA activates CD34+ cells has not been fully elucidated. In the present study, treatment with CpG DNA showed activation of AP-1 signals in CD34+ cells. AP-1 activation induced by CpG stimulation began 15 min after CpG stimulation, reached the maximal activity at 60 min, and then decreased. However, DNA-binding activity of AP-1 at 3 h after stimulation was still at high level compared with unstimulated control. This may be due to the difference of turnover rates between MAPK and AP-1.

In the present study, it is of interest to note that the stimulation with CpG ODN did not activate NF-{kappa}B signals in CD34+ cells. In our recent study, infection of CD34+ cells with live E. coli activated NF-{kappa}B (3). This difference might have been due to the fact that CpG DNA constitutes only a small part of E. coli, suggesting that live E. coli may involve a variety of signaling pathways. Although CD34+ cells are homogeneous in CD34 expression, there is a possibility that CD34+ cells may be still heterogeneous. Therefore, target cells for CpG DNA may be minor population. This might be one of the causes why CpG DNA-induced NF-{kappa}B activation was undetectable.

Our study also showed that MAPK, such as ERK1/2 and p38, of CD34+ cells was activated following treatment with CpG ODN. Furthermore, the CpG ODN-induced IL-8 expression was inhibited by ERK inhibitor (U0126) and p38 inhibitor (SB203580), but not by NF-{kappa}B inhibitor (PDTC and retrovirus-I{kappa}B{alpha}-AA), suggesting that CpG DNA up-regulates IL-8 expression via MAPK-dependent and NF-{kappa}B-independent signaling pathways. The addition of an ERK inhibitor (U0126) or a p38 inhibitor (SB203580) showed ~60% inhibition of IL-8 production in CD34+ cells stimulated with CpG DNA. The partial suppression may be due to other pathways which can activate AP-1 signals.

Simultaneous exposure to different antigenic components of bacteria, such as LPS and lipoprotein or CpG DNA plus LPS, has been shown to synergistically act to induce cytokine production in cultured immune cells and in intact animals (31, 32). Recently, a study demonstrated that co-stimulation of TLR4 and TLR2 or TLR9 induced synergistic release of the Th1 cytokines, IFN-{gamma} and TNF-{alpha} (33). In the present study, we also showed that co-stimulation of CD34+ cells with CpG DNA and LPS led to a synergistic increase of IL-8 expression. Although different TLRs are known to induce distinct cellular and systemic responses to infection, TLR4 and TLR9 induce similar pro-inflammatory responses (31). Considering LPS-mediated TLR4 activation in many cells (34), it is quite possible that the increase of IL-8 expression was mediated by TLR4 in LPS-stimulated CD34+ cells, indicating a cooperation between TLR9 and TLR4 in stimulation of immune response in the cells.

Results obtained from the present study may have important implications in host defense and gene therapy strategies. In particular, a new role of CpG DNA and CD34+ cells in systemic bacterial infection is possible. To date, LPS has been the major focus in studying the Gram-negative sepsis. The fact that CpG DNA was also active in inducing pro-inflammatory response in CD34+ cells may suggest its involvement in the development and/or progression of sepsis. This is particularly highlighted by the fact that LPS and CpG have a synergistic effect in inducing IL-8 response in CD34+ cells. The results also have important implications in CD34+ gene transfer. The fact that expression plasmid had a direct CpG-mediated biological effect on CD34+ cells raises not only a safety concern, but also suggests caution in interpreting experimental data. Furthermore, attention should be paid to distinguish the therapeutic gene-mediated biological effect from the CpG-mediated non-specific effect. Strategies are currently being developed to modify the plasmid expression vectors to decrease CpG immunostimulatory activity.

In conclusion, we have demonstrated here that CD34+ cells constitutively express TLR9 mRNA. Exposure to CpG DNA induced an IL-8 response in CD34+ cells via MAPK-dependent signaling pathways. This study suggests a possible role of CpG DNA-mediated CD34+ immune response in the host defense system.


    Acknowledgements
 
The experiments comply with the current laws of our country in which the experiments were performed. We thank Martin F. Kagnoff for gifts of standard RNAs, Hee-Young Chung for retrovirus containing I{kappa}B{alpha} superrepressor and Joo Hyoung Lee, Jin Young Lee, Hye-Sook Lee and Han-Jin Lee for their excellent technical help. This work was supported by National Research Laboratory program (M10400000019-04J0000-01910) and Korea Science and Engineering Foundation (R01-2002-000-00024-0).


    Abbreviations
 
ATCC   American Type Tissue Collection
EMSA   electrophoretic mobility shift assay
ERK   extracellular signal-regulated kinase
FBS   fetal bovine serum
HSC   hematopoietic stem cell
MAPK   mitogen-activated protein kinase
MEK   MAPK and ERK kinase
NF-{kappa}B   nuclear factor-{kappa}B
ODN   oligodeoxynucleotide
PDTC   pyrrolidinedithiocarbamate
RT   reverse transcription
TE   Tris-ethylenediaminetetraacetic acid
TLR   Toll-like receptor
TNF   tumor necrosis factor

    Notes
 
Transmitting editor: K. Takatsu

Received 13 April 2005, accepted 4 October 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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