Convergence of CpG DNA- and BCR-mediated signals at the c-Jun N-terminal kinase and NF-{kappa}B activation pathways: regulation by mitogen-activated protein kinases

Ae-Kyung Yi1,2, Jae-Geun Yoon1 and Arthur M. Krieg3,4,5

1 Children’s Foundation Research Center at Le Bonheur Children’s Medical Center and Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN 38103, USA 2 Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163, USA 3 Interdisciplinary Graduate Program in Immunology and Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242, USA 4 Department of Veteran Affairs Medical Center, Iowa City, IA 52246, USA 5 Coley Pharmaceutical Group Inc., Wellesley, MA 02481, USA

Correspondence to: A.-K. Yi; E-mail: ayi{at}utmem.edu
Transmitting editor: Kenneth Murphy


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Depending on the experimental model, unmethylated CpG motifs in bacterial DNA or synthetic oligodeoxynucleotides (CpG DNA) either augment or antagonize BCR-induced signals in B cells. CpG DNA synergizes with BCR-induced proliferation and Ig production of mature B cells, but blocks BCR-mediated apoptosis of immature B cells. Here, we demonstrate using a murine B lymphoma cell line WEHI-231, which is a model for immature B lymphocytes, that CpG DNA augments BCR-mediated signals for the activation of mitogen-activated protein kinase (MAPK) kinase (MKK)3, MKK4 and MKK6, and their subsequent downstream effectors c-Jun N-terminal kinase (JNK) and p38, but does not enhance MEK1/2 or extracellular signal-regulated kinase (ERK) activation. CpG DNA- and BCR-mediated signals also synergize for the activation of transcription factors AP-1, NFAT and NF-{kappa}B, but not for cAMP-responsive elements binding factor. Synergistic activations of JNK and p38 contribute to the synergistic production of cytokines induced by CpG DNA- and BCR-mediated signals, but have little or no effect on the ability of CpG DNA to protect WEHI-231 cells from anti-IgM-induced growth arrest. In contrast, all three MAPK, JNK, ERK and p38, contribute to the synergistic induction of splenic mature B cell proliferation by CpG DNA and anti-IgM. These results indicate that CpG DNA- and BCR-mediated signals converge at the level of MKK, NF-{kappa}B and NFAT activation, and that MAPK have differential regulatory roles for CpG DNA-mediated cytokine production versus cell proliferation in splenic mature B cells and WEHI-231 cells.

Keywords: apoptosis, B Lymphocyte, BCR, CpG DNA, cytokine, protein kinase


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In addition to recognizing and internalizing antigen, the BCR complex has important functions in transducing a series of biochemical signals through the plasma membrane. BCR engagement triggers a series of biochemical signaling events that lead to either positive (proliferation and differentiation) or negative (anergy and apoptosis) responses in B lymphocytes. These diverse effects of BCR engagement on the B cells depend on the maturation stage of the cell, on avidity and time of BCR engagement, and on additional signals the cell receives [reviewed in (1)]. Cross-linking of BCR in the immature B cells by high-affinity self-antigen results in programmed cell death of B cells in the bone marrow and the germinal centers of peripheral lymphoid organs, while continuous exposure to low-affinity self-antigen results in anergy of B cells in the periphery. The mechanism by which the BCR delivers these diverse signals in B lymphocytes is not completely understood at the present time. Recent studies have uncovered some differences in the BCR signaling events in mature versus immature or self-tolerant B cells. Antigen-induced protein tyrosine phosphorylation of BCR and other associated proteins is greatly reduced in tolerant hen egg lysozyme (HEL)-specific B cells that develop in mice carrying a soluble HEL transgene (MD4 x ML5 double transgenic) or in immature B cells (2,3). In tolerant B cells, antigen cross-linking induces low calcium oscillation instead of the large, transient calcium elevation observed in normal B cells. In addition, only extracellular signal-regulated kinase (ERK) and NFAT, but not c-Jun N-terminal kinase (JNK) and NF-{kappa}B, are activated by antigen in tolerant or immature B cells (46). These alterations in the BCR-mediated activation of mitogen-activated protein kinases (MAPK) and transcription factors in tolerant or immature B cells have been suggested to be responsible for B cell anergy and apoptosis (6). The second signals provided by activated Th cells (e.g. CD40 ligand), activated innate immune cells (e.g. cytokines produced by monocytic cells) or molecular signatures of microorganisms (e.g. lipopolysaccharide and CpG DNA) guide antigen-binding B cells towards immunity rather than tolerance [reviewed in (7)]. However, mechanisms by which second signals provided by Th cells or innate immune stimulators antagonize BCR signals in immature B cells and tolerant B cells are not completely understood.

Animal and bacterial DNA have marked differences in the frequency and methylation of cytosine in the CpG dinucleotides: only vertebrate DNA shows extensive under- representation and methylation of CpG dinucleotides (CpG suppression) (8). We have previously demonstrated that unmethylated CpG dinucleotides in particular sequence contexts (CpG motifs) in bacterial DNA are a conserved molecular signature in microorganisms to which innate immune cells respond as an indication of infection (9). CpG motifs are recognized by the Toll-like receptor (TLR)9, which is required for all of the subsequent immune stimulatory effects (10). The immune stimulatory activity of the CpG motifs in bacterial DNA can be mimicked by synthetic oligodeoxynucleotides (ODN) containing CpG motifs (CpG DNA). CpG DNA directly activates dendritic cells (DC) and macrophages/monocytes to secrete cytokines and chemokines including tumor necrosis factor (TNF)-{alpha}, IFN-{alpha}/ß, IL-6, IL-10, IL-12, MIP1a, MIP1b, IP-10 and RANTES, to express increased levels of co-stimulatory molecules, and to increase antibody-dependent cellular cytotoxicity activity, antigen presentation and cross-priming [(1119) and Yi and Krieg unpublished data]. Cytokines secreted by monocytic cells act in concert with CpG DNA on NK cells to express IFN-{gamma} and to increase NK cells’ lytic activity (13,2023). In addition to its profound effects on monocytic cells, CpG DNA rapidly activates B cells to proliferate, to produce TNF-{alpha}, IL-6, IL-10 and Ig, and to express increased levels of co-stimulatory molecules (9,2426). CpG DNA also rescues mature spleen B cells from spontaneous apoptosis and WEHI-231 cells from BCR-mediated apoptosis by inducing a sustained activation of NF-{kappa}B, and subsequent expression of Bcl-xL and c-Myc (2730).

Depending on the experimental model, CpG DNA can either augment or antagonize BCR-induced signals in B cells. In primary mature B cells, CpG DNA synergizes with BCR-mediated signals for amplifying Ig production and B cell proliferation, indicating its function as a co-stimulatory factor in the presence of a specific antigen (9,24). The TLR9 signaling pathways triggered by CpG DNA in immune complexes also synergize with the BCR to co-stimulate autoreactive B cells, leading to autoantibody production (31). On the other hand, in B cell lines with immature properties, such as WEHI-231 and CH31, CpG DNA blocks BCR-mediated growth arrest and apoptosis [(27,28) and Yi and Krieg, unpublished data]. Those data suggested that CpG DNA may synergize with BCR-induced signals in primary mature B cells, but oppose these in WEHI-231 cells. However, the molecular mechanisms by which CpG DNA interacts with BCR-mediated signals are poorly understood. In this study, we investigated whether CpG DNA blocks all biologic effects induced by BCR engagement in a murine immature B cell line WEHI-231 cells. Here we demonstrate that in WEHI-231 cells, CpG DNA augments BCR-induced signals for cytokine production, but simultaneously antagonizes these signals for the induction of growth arrest and apoptosis. CpG DNA- and BCR-mediated signals are shown to integrate by the level of the MAPK, NF-{kappa}B and NFAT activation pathways. Using specific inhibitors, we show that synergistic activation of JNK and p38 contributes to the production of cytokines induced by CpG DNA- and BCR-mediated signals in WEHI-231 cells, but not to the protection of WEHI-231 cells from BCR-induced growth arrest.


    Methods
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 Abstract
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 Methods
 Results
 Discussion
 References
 
ODN
Nuclease-resistant phosphorothioate ODN (S-ODN) were supplied by the Coley Pharmaceutical Group (Wellesley, MA) and had no detectable endotoxins by Limulus assay. The sequences of S-ODN used were 5'-TCCATGACG TTCCTGACGTT-3' (CpG DNA: 1826) and 5'-TCCAGGACT TCTCTCAGGTT-3' (non-CpG DNA: 1982).

Mice and preparation of splenic mature B cells
BALB/c mice at 5–10 week of age were used as a source of splenic mature B lymphocytes. All mice were obtained from Jackson Laboratory (Bar Harbor, ME) and maintained under specific pathogen-free conditions. Mice were killed by cervical dislocation. Single-cell suspensions were aseptically prepared from the spleens of mice. T cells were depleted by using anti-Thy-1.2 (Cedarlane, Hornby, Ontario, Canada) and complement, and centrifugation over Lympholyte M (Cedarlane) as described (26). Macrophages were stained with anti-CD11c–biotin (PharMingen, San Diego, CA), and depleted using streptavidin-coated paramagnetic beads and MACS isolation (Dynal, Lake Success, NY). This procedure typically yielded 97% B220+ cells.

Cell lines, culture conditions and reagents
Murine splenic mature B cells and murine B lymphoma cell line, WEHI-231 (clone 28) cells (provided by Dr D. Scott, American Red Cross, Rockville, MD), were cultured at 37°C in a 5% CO2 humidified incubator, and maintained in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FCS, 1.5 mM L-glutamine, 50 µM 2-mercaptoethanol, 100 U/ml penicillin and 100 µg/ml streptomycin. All culture reagents were purchased from Life Technologies (Gaithersburg, MD).

Cell proliferation assay
Cell proliferation assays were performed as previously described (26). Briefly, mouse splenic B cells or WEHI-231 cells (5 x 104/ml, 200 µl/well in 96-well culture plates) were stimulated with medium, CpG DNA (0.6–12 µg/ml) or non-CpG DNA (0.6–12 µg/ml) in the presence or absence of anti-IgM (5 or 10 µg/ml) for 24 h. In some experiments, cells were stimulated in the presence of DMSO, SB203580 (5 µM), SP600125 (5 µM) or U0126 (2.5 µM). [3H]Thymidine (1 µCi) was added to each well and the cells were harvested after an additional 4 h of culture. Filters were counted by scintillation counting. Anti-IgM (µ chain specific) was purchased from Sigma (St Louis, MO). SB203580, a p38 kinase inhibitor, SP600125, a JNK inhibitor, and U0126, a MEK inhibitor, were purchased from Calbiochem (La Jolla, CA).

PhiPhiLux
Caspase-3 activation using PhiPhiLux was performed as described previously (32). Briefly, WEHI-231 cells (2 x 105 cells/ml) were placed in 24-well plates and treated with medium, CpG DNA (12 µg/ml) or non-CpG DNA in the presence or absence of anti-IgM (10 µg/ml) for designated time periods. Cells were harvested by centrifugation, washed twice with PBS and then resuspended in 10 µl of PhiPhiLux (OncoImmunin, Gaithersburg, MD) in RPMI 1640. After 1 h incubation at 37°C, the reaction was stopped by the addition of 300 µl of ice-cold FACS buffer and the cells were analyzed immediately by flow cytometry on an Epics XL-MCL using EXPO32 ADC software (Coulter, Miami, FL). The frequency of cells containing active caspase-3 was determined by monitoring increased free fluorescence on FL1.

Annexin-V staining for apoptotic cells and FACS analysis
WEHI-231 cells (2 x 105 cells/ml) were placed in 24-well plates and treated with medium, CpG DNA (12 µg/ml) or non-CpG DNA in the presence or absence of anti-IgM (10 µg/ml) for designated time periods. Cells were harvested by centrifugation, washed twice with PBS and then resuspended in 100 µl of Annexin-V binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl and 2.5mM CaCl2). After the addition of Annexin-V–FITC (5 µl; PharMingen), cells were incubated at room temperature for 15 min and then analyzed by flow cytometry for Annexin-V+ cells as an indication of the plasma membrane transition during the process of apoptosis.

Cytokine-specific ELISA
WEHI-231 cells (2 x 106 cells/ml for TNF-{alpha}, 5 x 105 cells/ml for IL-6 and 1 x 105 cells/ml IL-10) were stimulated with medium, CpG DNA (0.3–6 µg/ml), non-CpG DNA (0.3–6 µg/ml) and/or anti-IgM (1–10 µg/ml) for 6 h (TNF-{alpha}) or 24 h (for IL-6 and IL-10). In some experiments, cells were stimulated in the presence DMSO, SB203580 (5 µM), SP600125 (5 µM) or U0126 (2.5 µM). Culture supernatants were analyzed by ELISA for TNF-{alpha}, IL-6 or IL-10 as described previously (24). All recombinant murine cytokines and antibodies specific for murine cytokines were purchased from PharMingen.

Preparation of whole-cell lysates and Western blot analysis
Mouse splenic mature B cells or WEHI-231 cells (2 x 106 cells/ml) were stimulated with medium, CpG DNA (0.3–12 µg/ml) or non-CpG DNA (12 µg/ml) in the presence or absence of anti-IgM (0.5–20 µg/ml) for designated time periods. In some experiments, cells were stimulated in the presence of DMSO, SB203580 (1–5 µM), SP600125 (2.5–10 µM) or U0126 (2.5–10 µM). Cells were harvested and then whole-cell lysates were prepared as previously described (33). To determine the phosphorylation status of MEK1/2, MAPK kinase (MKK)3/6, MKK4, ERK, JNK, p38 or cAMP-responsive elements binding factor (CREB), equal amounts of whole-cell lysates (50 µg/lane) were subjected to electrophoresis on a 10% polyacrylamide gel containing 0.1% SDS (SDS–PAGE) and then Western blots were performed as previously described (33) using a specific antibody against the phosphorylated form of each protein. Tyrosine-phosphorylated proteins were detected using anti-phosphotyrosine antibody 4G10. Specific antibodies against the phosphorylated form of MEK1/2, MMK3/6, MKK4, ERK, JNK, p38 and CREB were purchased from New England BioLabs (Beverly, MA). 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY). Specific antibodies against p38 and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Transfections, luciferase assay and ß-galactosidase assay
WEHI-231 cells (107 cells/ml) were electroporated with AP-1–ß-galactosidase (40 µg), NFAT–luciferase (40 µg) or NF-{kappa}B–luciferase (20 µg) constructs at 960 µF, 270V using a Gene Pulser II system (Bio-Rad, Hercules, CA). Transfected cells were pooled, washed 3 times with culture media and then incubated for 4 h. Cells (1 x 106 cells/ml) were stimulated with medium, CpG DNA (12 µg/ml) or non-CpG DNA (12 µg/ml) in the presence or absence of anti-IgM (10 µg/ml) for 12 h. ß-Galactosidase and luciferase activities in cell extracts were analyzed according to the manufacturer’s protocol using the Galacto-Light Plus Reporter gene assay for ß-galactosidase (Tropix, Bedford, MA) and the Luciferase Reporter Assay System (Promega, Madison, WI), respectively. Luciferase activity and ß-galactosidase activity was normalized to the equal concentration of cell extracts in each sample. AP-1–ß-galactosidase, NFAT–luciferase and NF-{kappa}B–luciferase constructs were kindly provided by Dr G. Koretzky (University of Pennsylvania).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Agonistic and antagonistic action of CpG DNA on the BCR-mediated signals in WEHI-231 B lymphoma cells
Depend on the maturation stage of B lymphocytes, CpG DNA either augment or antagonize BCR-induced signals (9,24,27,28). CpG DNA synergizes with BCR-mediated signals for Ig production and B cell proliferation in mature B cells, while it blocks BCR-mediated signals for induction of growth arrest and apoptosis in immature B cells. These previous findings were confirmed using splenic B cells and WEHI-231 cells as model systems for mature and immature B cells respectively, and antibody against surface Ig (anti-IgM) as a surrogate for antigen. As expected, CpG DNA synergized with anti-IgM for splenic B cell proliferation, while it prevented anti-IgM-induced caspase-3 activation and subsequent apoptosis of WEHI-231 cells (Fig. 1). In contrast, control non-CpG DNA neither enhanced anti-IgM-mediated splenic B cell proliferation nor prevented anti-IgM-induced caspase-3 activation and subsequent apoptosis of WEHI-231 cells (Fig. 1).



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Fig. 1. CpG DNA synergizes with BCR for spleen B cell proliferation, but inhibits BCR-induced apoptosis of WEHI-231 cells. (A and B) Mouse splenic B cells (5 x 104/ml, 200 µl/well in 96-well culture plates) were stimulated with medium, CpG DNA (0.6 or12 µg/ml) or non-CpG DNA (0.6 or12 µg/ml) in the presence (solid bar) or absence (empty bar) of anti-IgM (5 or 10 µg/ml) for 24 h. [3H]Thymidine (1 µCi) was added to each well and the cells were harvested after an additional 4 h of culture. Data present the mean ± SD of triplicates. The experiment was performed 3 times with similar results. (C and D) WEHI-231 cells (2 x 105 cells/ml) were stimulated with medium, anti-IgM, anti-IgM + CpG DNA or anti-IgM + non-CpG DNA for 24 h (for caspase-3 activation in C) or 36 h (for apoptosis in D). Activated caspase-3 in the cell was detected using PhiPhiLux followed by flow cytometric analysis. Apoptotic cells were detected by flow cytometric analysis after Annexin-V–FITC staining. Numbers represent percent of cells containing active caspase-3 or percent of Annexin-V+ cells. The experiments were performed 3–4 times with similar results.

 
To further investigate whether CpG DNA antagonizes all the aspects of BCR-mediated biological effects in immature B cells, we analyzed the effects of CpG DNA on BCR-induced cytokine production in WEHI-231 cells. As shown in Fig. 2, anti-IgM was a weak inducer of TNF-{alpha} and IL-6, but induced very high levels of IL-10 production in WEHI-231 cells. In contrast, CpG DNA induced very low levels of TNF-{alpha} and IL-10 production, but it strongly induced production of IL-6. Surprisingly, CpG DNA, but not control non-CpG DNA, synergized with anti-IgM for production of all three cytokines, TNF-{alpha}, IL-6 and IL-10, in a dose-dependent manner in WEHI-231 cells. These results indicate the presence of agonistic as well as antagonistic interactions between CpG DNA- and BCR-mediated signals in these B cells.



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Fig. 2. CpG DNA synergizes with anti-IgM for cytokine production in WEHI-231 cells. (A, C and E) WEHI-231 cells (2 x 106 cells/ml for TNF-{alpha}, 5 x 105 cells/ml for IL-6 and 1 x 105 cells/ml IL-10) were treated with medium, CpG DNA (3 µg/ml; diamonds) or non-CpG DNA (3 µg/ml; triangles) in the presence of anti-IgM (0–10 µg/ml) for 6 h (TNF-{alpha}) or 24 h (for IL-6 and IL-10). (B, D and F) WEHI-231 cells (2 x 106 cells/ml for TNF-{alpha}, 5 x 105 cells/ml for IL-6 and 1 x 105 cells/ml for IL-10) were treated with medium (solid symbols) or anti-IgM (5 µg/ml; open symbols) in the presence of CpG DNA (0–6 µg/ml; diamonds) or non-CpG DNA (0–6 µg/ml; triangles) for 6 h (TNF-{alpha}) or 24 h (for IL-6 and IL-10). The levels of TNF-{alpha}, IL-6 or IL-10 in culture supernatants were determined by ELISA. Of note, typical viabilities of WEHI-231 cells determined by Trypan blue vital staining at the end of 24 h culture were >=96% in all samples. Typical apoptosis rates of WEHI-231 cells determined by hypodiploid nuclei staining at the end of 24 h culture were 7% in the cells treated with anti-IgM or anti-IgM + non-CpG DNA and <2% in the cells treated with medium, CpG DNA or anti-IgM + CpG DNA. Data present the mean ± SD of triplicates. The experiment was performed 3–6 times with similar results.

 
CpG DNA does not alter the anti-IgM-induced protein tyrosine phosphorylation
The BCR utilizes sequential activation of Src family protein tyrosine kinases (PTK), Syk and Btk to regulate downstream effectors [reviewed in (1)]. Since previous studies have demonstrated that antigen-induced protein tyrosine phosphorylation of BCR and other associated proteins in tolerant and immature B cells is greatly reduced (2,3), we investigated whether CpG DNA enhances anti-IgM-induced tyrosine phosphorylation in WEHI-231 cells. As shown in Fig. 3(A and B), neither CpG DNA nor control non-CpG DNA induced a detectable level of tyrosine phosphorylation within 30 min in WEHI-231 cells. Furthermore, addition of neither CpG DNA nor control non-CpG DNA altered levels of tyrosine phosphorylation of the proteins activated by anti-IgM in WEHI-231 cells (Fig. 3A and B). These results indicate that CpG DNA may act on and interact with the BCR-mediated signals at a more distal step than the level of PTK activation. Both CpG DNA and control non-CpG DNA non-specifically induced tyrosine phosphorylation of some high-mol.-wt (>120 kDa) proteins at later time points (data not shown).



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Fig. 3. CpG DNA does not alter anti-IgM-induced phosphorylation of protein tyrosines and MEK1/2, but potentiates activation of MKK3, MKK4 and MKK6 in WEHI-231 cells. WEHI-231 cells (2 x 106 cells/ml) were stimulated with medium, CpG DNA (12 µg/ml) or non-CpG DNA (12 µg/ml) in the presence or absence of anti-IgM (10 µg/ml) for 3, 7, 15 or 30 min. (A and B) Equal amounts of whole-cell lysates (50 µg/lane) were subjected to SDS–PAGE and then Western blots were performed to detect tyrosine phosphorylation using anti-phosphotyrosine antibody 4G10. (C) Equal amounts of whole-cell lysates (50 µg/lane) were subjected to SDS–PAGE and then Western blots were performed using a specific antibody against the phosphorylated form of MEK1/2 (pMEK1/2), MKK3/6 (pMKK3/6) or MKK4 (pMKK4). Actin was used as a loading control. The experiments were performed 3 times with similar results. (D) The density of pMKK3, pMKK4 and pMKK6 bands in (C) was quantitated by densitometry and normalized to the density of actin band in the same sample. Number represents the fold induction from the normalized densitometric value of each protein band in the unstimulated control sample.

 
CpG DNA and anti-IgM synergistically induce phosphorylation of MKK3, MKK4 and MKK6, but not MEK1/2
In the classical MAPK pathway, Raf1 activates MEK1/2 that in turn activates its direct downstream effector ERK. On other hand, activation of MEKK1 leads to the activation of p38 by activating MKK3 and MKK6, and activates JNK through MKK4 (3438). Previously we have demonstrated that CpG DNA induces activation of JNK and p38, but not ERK, in WEHI-231 cells (33). However, it has not been studied whether CpG DNA induces activation of MKK3, MKK4 or MKK6 in WEHI-231 cells. In addition, it has been suggested previously that activation of ERK induced by anti-IgM may contribute to the induction of growth arrest and apoptosis in WEHI-231 cells (39). Therefore, we investigated whether CpG DNA induces activation of one or more MKK, and whether CpG DNA- and anti-IgM-induced signals can interact at the level of MKK. As shown in Fig. 3(C), CpG DNA alone induced phosphorylation of MKK3, MKK4 and MKK6. However, CpG DNA failed to induce phosphorylation of MEK1/2, the upstream modulators in the classical ERK activation pathway. In contrast, anti-IgM strongly induced phosphorylation of MEK1/2, but induced very weak phosphorylation of MKK3 and MKK6, which are the upstream modulators of p38 (Fig. 3C). However, anti-IgM did not induce any detectable level of phosphorylation of MKK4, an upstream activator of JNK, at the time point we examined. Interestingly, addition of anti-IgM substantially enhanced phosphorylation of MKK3, MKK4 and MKK6 induced by CpG DNA (Fig. 3C). In contrast, CpG DNA did not alter the anti-IgM-induced phosphorylation of MEK1/2.

Interaction of CpG DNA- and BCR-mediated signals at the MAPK level
Since our results suggest that CpG DNA- and BCR-induced signals might converge by the levels of MKK activation (Fig. 3C), we further investigated whether CpG DNA alters anti-IgM-mediated activation of MAPK. As previously demonstrated (33), CpG DNA alone induced phosphorylation of JNK and p38 in a dose- and time-dependent manner, but did not induce ERK phosphorylation in WEHI-231 cells (Fig. 4A and 4C). In contrast, anti-IgM induced phosphorylation of ERK and p38 in a dose- and time-dependent manner (Fig. 4A and D). However, JNK activation by anti-IgM was very weak in WEHI-231 cells (Fig. 4). Strikingly, addition of CpG DNA substantially enhanced the anti-IgM-induced phosphorylation of JNK and p38 as well as their downstream effector ATF-2 (Fig. 4–C and data not shown). However, CpG DNA did not alter anti-IgM-induced activation of ERK at the level of phosphorylation or at the level of enzyme activity (Fig. 4 and data not shown). Control non-CpG DNA did not show any effect on the anti-IgM-induced ERK and p38 activation (Fig. 4B).



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Fig. 4. CpG DNA synergizes with anti-IgM for induction of JNK and p38, but not ERK, phosphorylation in WEHI-231 cells. (A and B) WEHI-231 cells (2 x 106 cells/ml) were stimulated with medium, CpG DNA (3 µg/ml) or non-CpG DNA (3 µg/ml) in the presence or absence of anti-IgM (10 µg/ml) for 15 min to 4 h. (C) WEHI-231 cells (2 x 106 cells/ml) were stimulated with various concentrations of CpG DNA (0–6 µg/ml) in the presence or absence of anti-IgM (10 µg/ml) for 30 min. (D) WEHI-231 cells (2 x 106 cells/ml) were stimulated with various concentrations of anti-IgM (0–20 µg/ml) in the presence or absence of CpG DNA (3 µg/ml) for 30 min. Equal amounts of whole-cell lysates (15 µg/lane) were subjected to SDS–PAGE and then Western blots were performed using a specific antibody against actin or the phosphorylated form of ERK (pERK), JNK (pJNK) or p38 (pp38). Actin was used as a loading control. Experiments were performed 3 times with similar results. (E) The density of pJNKp52, pJNKp46 and pp38 bands in (A), (C) and (D) was quantitated by densitometry and normalized to the density of actin band in the same sample. Number represents the fold induction from the normalized densitometric value of each protein band in the unstimulated control sample.

 
As in the WEHI-231 cells, CpG DNA synergizes with BCR-mediated signals for cytokine production in mature B cells (data not shown). In addition, CpG DNA synergistically enhanced BCR-induced splenic mature B cell proliferation (Fig. 1A and B). Therefore, we investigated whether CpG DNA induces activation of different MAPK and whether CpG DNA synergizes with BCR-induced signals for MAPK activation in these cells. Purified mouse splenic B cells were stimulated with medium, CpG DNA or non-CpG DNA in the presence or absence of anti-IgM for 1 h. As shown in Fig. 5, CpG DNA induced phosphorylation of JNK and p38, but not ERK, in the mature B cells, consistent with the results in the WEHI-231 B cells. Anti-IgM strongly induced phosphorylation of ERK. However, anti-IgM weakly induced phosphorylation of JNK and p38 in splenic B cells at the time point we analyzed. Interestingly, CpG DNA substantially enhanced the anti-IgM-induced phosphorylation of JNK and p38, but did not alter anti-IgM-induced activation of ERK. Taken together, these results indicate that CpG DNA- and BCR-mediated signals may converge at or before the p38 and JNK activation pathways, but not at the ERK activation pathway, in both mature B cells and WEHI-231 cells.



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Fig. 5. CpG DNA substantially enhances anti-IgM-mediated activation of JNK and p38, but not ERK, phosphorylation in splenic mature B cells. (A) Splenic B cells (2 x 106 cells/ml) were stimulated with medium, CpG DNA (12 µg/ml) or non-CpG DNA (12 µg/ml) in the presence or absence of anti-IgM (10 µg/ml) for 1 h. Equal amounts of whole-cell lysates (15 µg/lane) were subjected to SDS–PAGE and then Western blots were performed using a specific antibody against actin or the phosphorylated form of ERK (pERK), JNK (pJNK) or p38 (pp38). Actin was used as a loading control. Experiments were performed 3 times with similar results. (B) The density of pJNKp52, pJNKp46 and pp38 bands in (A) was quantitated by densitometry and normalized to the density of actin band in the same sample. Number represents the fold induction from the normalized densitometric value of each protein band in the unstimulated control sample.

 
CpG DNA and BCR-mediated signals lead to the synergistic activation of transcription factors NFAT, NF-{kappa}B and AP-1
The transcription factors NFAT, NF-{kappa}B and AP-1 regulate B cell survival and cytokine production (6,24,33). Our previous studies demonstrated that MAPK activated after CpG DNA stimulation may stimulate AP-1 and CREB in B and monocytic cells (12,33). Therefore we investigated whether CpG DNA can enhance or suppress anti-IgM-induced activation of transcription factors in WEHI-231 cells. WEHI-231 cells were transiently transfected with constructs encoding NFAT– luciferase, NF-{kappa}B–luciferase or AP-1–ß-galactosidase and then stimulated with medium, CpG DNA or non-CpG DNA in the presence or absence of anti-IgM. CpG DNA alone strongly induced transcriptional activities of NF-{kappa}B, while anti-IgM weakly activated NF-{kappa}B in WEHI-231 cells (Fig. 6A). In contrast, anti-IgM strongly induced transcriptional activities of AP-1, while CpG DNA-mediated AP-1 activation was minimal (Fig. 6B). In addition, anti-IgM weakly induced activation of NFAT (about a fold increase from basal activity in the unstimulated cells), but CpG DNA did not induce activation of NFAT in WEHI-231 cells (Fig. 6C). Interestingly, CpG DNA and anti-IgM synergistically induced transcriptional activities of all three transcription factors, AP-1, NFAT and NF-{kappa}B, in WEHI-231 cells (Fig. 6A–C). In contrast, CpG DNA neither induced CREB phosphorylation nor altered the anti-IgM-induced CREB phosphorylation (Fig. 6D). These results suggest that in addition to the MAPK activation pathways that contribute to AP-1 activation, CpG DNA- and BCR-mediated signals also converge in the NF-{kappa}B activation pathway and the NFAT activation pathway.



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Fig. 6. CpG DNA and BCR signals lead to the synergistic activation of transcription factor NF-{kappa}B, AP-1 and NFAT, but not CREB. (A–C) WEHI-231 cells (107 cells/ml/sample) were transfected with NF-{kappa}B–luciferase (A), AP-1–ß-galactosidase (B) or NFAT–luciferase (C). The transfected cells (1 x 106 cells/ml) were stimulated with medium, CpG DNA (12 µg/ml) or non-CpG DNA (12 µg/ml) in the presence (solid bar) or absence (empty bar) of anti-IgM (10 µg/ml) for 12 h. Luciferase or galactosidase activities in equal concentrations of cell extracts were analyzed by Luciferase Reporter Assay System or Galacto-Light Plus Reporter gene assay. The experiment was performed 3 times with similar results. (D) WEHI-231 cells (2 x 106 cells/ml) were stimulated with medium, CpG DNA (3 µg/ml) or non-CpG DNA (3 µg/ml) in the presence or absence of anti-IgM (10 µg/ml) for 1 h. Equal amounts of whole-cell lysates (15 µg/lane) were subjected to SDS–PAGE and then Western blots were performed using a specific antibody against actin or the phosphorylated form of CREB (pCREB). Actin was used as a loading control. The experiment was performed 3 times with similar results.

 
Specific effect of selective inhibitors of MEK1/2, JNK and p38 on MAPK activation induced by CpG DNA or anti-IgM
MAPK p38 has been demonstrated to play a critical role in CpG DNA-mediated cytokine production in B cells and monocytic cells (12,18,33,40). To investigate the role of MAPK in CpG DNA-mediated cytokine production and apoptosis protection in WEHI-231 cells, we employed selective inhibitors of MEK1/2, JNK and p38 in our study. To insure specificity of a MEK1/2 inhibitor (U0126), a JNK inhibitor (SP600125) and a p38 inhibitor (SB203580), WEHI-231 cells were stimulated with media, anti-IgM or CpG DNA in the presence or absence of U0126, SP600125 or SB203580 for 45 min. Phosphorylation of ERK, JNK and p38 was analyzed by phospho-specific Western blot. The MEK1/2 inhibitor U0126 at 2.5 µM inhibited the anti-IgM-induced ERK phosphorylation without affecting phosphorylation of JNK or p38 induced by CpG DNA (Fig. 7A). These results indicate the specific action of U0126 on MEK1/2 activity and MEK1/2-dependent ERK activation by anti-IgM in WEHI-231 cells. JNK-specific inhibitor SP600125 (41) substantially suppressed CpG DNA-induced JNK phosphorylation in a dose-dependent manner without affecting phosphorylation of ERK or p38 induced by anti-IgM or CpG DNA, thus indicating the specificity of SP600125 for inhibiting JNK activation (Fig. 7B). As shown in Fig. 7(C), the p38 kinase inhibitor SB203580 inhibited CpG DNA-induced phosphorylation of p38 in a dose-dependent manner without affecting phosphorylation of ERK induced by anti-IgM. However, CpG DNA-induced JNK phosphorylation was dramatically enhanced by SB203580 in WEHI-231 cells. SB202190, another specific p38 inhibitor, also showed the same results (data not shown).



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Fig. 7. Specific effects of a MEK1/2 inhibitor (U0126), a p38 inhibitor (SB203580) and a JNK inhibitor (SP600125). WEHI-231 cells (2 x 106 cells/ml) were stimulated with medium, CpG DNA (3 µg/ml) or anti-IgM (10 µg/ml) in the presence or absence of U0126 (0–10 µM: A), SP600125 (0–10 µM: B) or SB203580 (0–5 µM: C) for 45 min. Equal amounts of whole-cell lysates (15 µg/lane) were subjected to SDS–PAGE and then Western blots were performed using a specific antibody against actin or the phosphorylated form of ERK (pERK), JNK (pJNK) or p38 (pp38). Actin was used as a loading control. The experiment was performed 3 times with similar results.

 
Role of MAPK on the cytokine production induced by CpG DNA and anti-IgM
To investigate the possible regulatory roles of ERK, JNK and p38 activation on the synergistic production of cytokines induced by CpG DNA and anti-IgM, WEHI-231 cells were stimulated with CpG DNA and/or anti-IgM in the presence or absence of U0126, SP600125 or SB203580. Levels of TNF-{alpha}, IL-6 and IL-10 in culture supernatants were measured by cytokine-specific ELISA. As demonstrated in Fig. 8(A), CpG DNA-induced TNF-{alpha} production was almost completely abolished by SB203580, the p38 inhibitor, and was partially suppressed by SP600125, the JNK inhibitor. However, CpG DNA-induced TNF-{alpha} production was not affected by UO126, the MEK1/2 inhibitor. The synergistic production of TNF-{alpha} induced by CpG DNA and anti-IgM was almost completely inhibited by the p38 inhibitor and was partially suppressed by the MEK1/2 or JNK inhibitors. These results indicate that p38 is essential for TNF-{alpha} production induced by both CpG DNA and anti-IgM, and that CpG DNA-induced JNK activation and anti-IgM-induced ERK activation at least partially contribute to the synergistic production of TNF-{alpha} in WEHI-231 cells. IL-6 production induced by CpG DNA alone or by CpG DNA and anti-IgM was substantially suppressed by either SB203580 or SP600125, indicating an essential role for p38 and JNK in CpG DNA-induced IL-6 production (Fig. 8B). CpG DNA-mediated IL-6 production was also partially inhibited by U0126, suggesting that the basal activity of ERK in WEHI-231 cells may contribute to CpG DNA-mediated IL-6 production. In the presence of U0126, the synergistic production of IL-6 induced by CpG DNA and anti-IgM went down to the level induced by CpG DNA alone, indicating that ERK might play an essential role in anti-IgM-mediated IL-6 production. In contrast to their dramatic suppressive effects on TNF-{alpha} and IL-6 production, all three MAPK inhibitors showed only partial inhibitory effects on anti-IgM-induced IL-10 production (Fig. 8C). Interestingly, the CpG DNA-mediated enhancement on anti-IgM-mediated IL-10 production was completely abolished in the presence of either SB203580 or SP600125, indicating an essential role of p38 and JNK on CpG DNA-mediated IL-10 production.



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Fig. 8. Effects of MAPK on the production of cytokines induced by CpG DNA and/or anti-IgM. WEHI-231 cells (2 x 106 cells/ml for TNF-{alpha}, 5 x 105 cells/ml for IL-6 and 1 x 105 cells/ml for IL-10) were treated with medium, anti-IgM (10 µg/ml), CpG DNA (3 µg/ml) or anti-IgM + CpG DNA in the presence or absence (empty bars) of U0126 (2.5 µM; solid bars), SB203580 (5 µM; hatched bars) or SP600125 (5 µM; crossed bars) for 6 h (TNF-{alpha}) or 24 h (for IL-6 and IL-10). The levels of TNF-{alpha}, IL-6 or IL-10 in culture supernatants were determined by ELISA. Data present the mean ± SD of triplicates. The experiment was performed 3 times with similar results.

 
Differential role of MAPK on CpG- and BCR-induced proliferation of splenic mature B cells and WEHI-231 cells
As demonstrated previously and in Fig. 1, CpG DNA synergizes with anti-IgM for splenic mature B cell proliferation, but prevents anti-IgM induced growth arrest and apoptosis of WEHI-231 cells (9,27). In addition, there are contradictory reports on the role of MAPK in anti-IgM-mediated growth arrest and apoptosis of WEHI-231 cells (39,42,43). Therefore, we investigated whether MAPK activated by CpG DNA and anti-IgM play a differential role in the proliferation of splenic mature B cells and WEHI-231 cells. Splenic mature B cells or WEHI-231 cells were stimulated with medium, CpG DNA and/or anti-IgM in the presence or absence of U0126, SB203580 or SP600125 and then cellular uptake of [3H]thymidine was measured as an indication of cell proliferation. As demonstrated in Fig. 9(A), splenic B cell proliferation induced by CpG DNA with or without anti-IgM was substantially inhibited in the presence of SB203580 or SP600125, but not in the presence of U0126, indicating a critical role of p38 and JNK in CpG DNA-induced splenic B cell proliferation. Interestingly, anti-IgM-mediated enhancement of CpG DNA-mediated splenic B cell proliferation was almost completely inhibited by the MEK1/2 inhibitor U0126, indicating a critical contribution of ERK to anti-IgM-mediated splenic B cell proliferation. In contrast, all three MAPK inhibitors neither prevented anti-IgM-induced growth arrest nor inhibited the ability of CpG DNA to block anti-IgM-induced growth arrest of WEHI-231 cells (Fig. 9B). Furthermore, neither the pro-apoptotic effect of anti-IgM nor the anti-apoptotic ability of CpG DNA in WEHI-231 cells was inhibited by these MAPK inhibitors (data not shown). These results indicate that MAPK may play an essential role in CpG DNA- and anti-IgM-induced splenic mature B cell proliferation, but do not mediate their effects on the growth and apoptosis of WEHI-231 cells.



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Fig. 9. Effects of MAPK on the proliferation of splenic mature B cells and WEHI-231 cells induced by CpG DNA and anti-IgM. (A) Mouse splenic B cells (5 x 104/ml, 200 µl/well in 96 well culture plate) were stimulated with medium, anti-IgM (10 µg/ml), CpG DNA (12 µg/ml) or anti-IgM + CpG DNA in the presence or absence (empty bars) of U0126 (2.5 µM; solid bars), SB203580 (5 µM; hatched bars) or SP600125 (5 µM; crossed bars) for 24 h. (B) WEHI-231 cells (5 x 104/ml, 200 µl/well in 96-well culture plate) were stimulated with medium (empty bars), anti-IgM (10 µg/ml; solid bars), CpG DNA (3 µg/ml; hatched bars) or anti-IgM + CpG DNA (crossed bars) in the presence of DMSO, U0126 (2.5 µM), SB203580 (5 µM) or SP600125 (5 µM) for 24 h. [3H]Thymidine (1 µCi) was added to each well and the cells were harvested after an additional 4 h of culture. Data present the mean ± SD of triplicates. The experiments were performed 3 times with similar results.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In addition to rapidly activating innate immune cells including macrophages/monocytes and DC, CpG DNA also directly activates B lymphocytes. Moreover, CpG DNA can either augment or antagonize BCR-induced signals depending on the experimental models. Agonistic effects of CpG DNA on BCR-mediated signals were initially observed in the primary mature B cells and in CH12.LX cells, a B cell line with mature B cell characteristics (9). In mature B cells, CpG DNA synergizes with BCR-mediated signals for B cell proliferation and production of various cytokines (e.g. TNF-{alpha} and IL-6) and Ig, including autoantibody production [(9,31) and Yi and Krieg, unpublished data]. In contrast, CpG DNA opposes the BCR-induced signals leading to growth arrest and apoptosis in neonatal B cells and B cell lines with immature B cell characteristics including BKS-2, CH31 and WEHI-231 [(27,28,30,44,45) and Yi and Krieg, unpublished data]. Our results in the WEHI-231 system unexpectedly demonstrate that CpG DNA simultaneously synergizes with BCR-induced signals for cytokine production, but blocks the BCR-mediated signals from inducing the usual growth arrest and apoptosis. The present studies were performed to elucidate the signaling pathways mediating these complex interactions between CpG DNA- and BCR-induced signals. We show that signal integration between these signals takes place in the JNK, p38, NFAT and NF-{kappa}B activation pathways.

BCR engagement in WEHI-231 cells induced robust production of IL-10, but minimal production of TNF-{alpha} or IL-6 (Fig. 2). In contrast, CpG DNA induced both TNF-{alpha} and IL-6 production, but had little effect on IL-10 production in WEHI-231 cells (Fig. 2). Surprisingly, anti-IgM and CpG DNA synergized for induction of all three cytokines, indicating the presence of agonistic as well as the antagonistic interactions through which CpG DNA blocks BCR-mediated apoptosis in WEHI-231 cells (Fig. 1). Both IL-6 and IL-10 have previously been reported to play a functional role in B cell differentiation, growth and survival (46,47). In addition, IL-6 plays a key role in CpG DNA-mediated IgM production in primary mature B cells (24). The possible role of these cytokines in regulating anti-IgM-induced growth arrest and apoptosis is currently unknown, and requires further investigation.

Our initial studies of the interactions between the BCR and CpG DNA-induced signaling pathways focused on the membrane-proximal signals induced by BCR ligation. Cross-linking of BCR by antigen or anti-IgM triggers activation of a series of PTK including the Src family PTK (Lyn, Fyn, Lck, Blk), Syk and Btk, and other downstream signaling adapters and modulators such as BLNK, phospholipase C{gamma}2, Vav, Grb2 and Nck [reviewed in (1)]. Recent studies have demonstrated that BLNK, Btk, phospholipase C{gamma}2 and Rac1 play a critical role in BCR-mediated NF-{kappa}B activation, and B cell development and survival (4851). Antigen-induced protein tyrosine phosphorylation of BCR and other associated proteins is greatly reduced in tolerant B cells and immature B cells, which may account for their marked reduction in BCR-induced responses (2,3). It has been reported previously that CpG DNA does not activate PTK detectably within 15 min in primary mature B cells or CH12.LX cells (9). Our data in WEHI-231 cells were in agreement with those results (Fig. 3A). Furthermore, the addition of CpG DNA did not alter levels of tyrosine phosphorylation of the proteins activated by anti-IgM in WEHI-231 cells (Fig. 3A). Non-specific sequence-independent tyrosine phosphorylation of some high-mol.-wt (>120 kDa) proteins was detected in the cells stimulated with control non-CpG DNA or CpG DNA for longer periods (data not shown), but the biologic significance of this is uncertain, as non-CpG DNA does not affect BCR-induced cytokine production or apoptosis (Fig. 1) (27,28,43). In addition, CpG DNA still effectively blocked anti-IgM-induced growth arrest and apoptosis of WEHI-231 cells even in the presence of PTK inhibitors (data not shown) (52). Taken together, these results indicate that CpG DNA most likely interacts with the BCR-mediated signals at a more distal step than PTK activation.

MAPK and the transcription factor NF-{kappa}B play a critical role in various cellular responses including cell survival and cytokine production (4,24,27,28,30,33,39,42,43). In monocytic cells CpG DNA appears to recruit MyD88 to TLR9, initiating a signaling pathway that sequentially involves IL-1 receptor-associated kinase 1 (IRAK1) and TNF-{alpha} receptor-associated factor 6 (TRAF6), which activate MAPK and NF-{kappa}B, and are essential for the subsequent production of cytokines (10,40,5356). Our data demonstrate that in WEHI-231 cells, CpG DNA and BCR engagement activate different sets of MAPK and transcription factors through distinct signaling pathways. BCR-mediated signals strongly activate MEK1/2 and its downstream target, ERK, and also activate MKK 3 and MKK6, and their downstream target p38, but have little effect on the effector of MKK4, JNK (Figs 3C and 4) (5,33). In addition, BCR engagement leads to the activation of the downstream transcription factors AP-1 and NFAT, but initially activates and then subsequently suppresses NF-{kappa}B activity (Fig. 6) (4,28). In contrast to these BCR effects in WEHI-231 cells, CpG DNA induces activation of JNK, p38, NF-{kappa}B and AP-1, but not ERK or NFAT (Figs 4 and 6) (33).

Our studies reveal complex interactions between the BCR and CpG DNA signaling pathways in mature splenic B cells, as well as in WEHI-231 cells. Addition of CpG DNA substantially enhanced anti-IgM-induced activation of MKK3, MKK4 and MKK6 (Fig. 3C). Furthermore, CpG DNA and anti-IgM synergized for the activation of p38 and JNK, and their downstream effector ATF2 and transcription factor AP-1 (Figs 4 and 5, and data not shown). However, CpG DNA did not alter the anti-IgM-induced activation of MEK1/2 and its downstream effector ERK1/2 (Figs 3–5).

In WEHI-231 cells, BCR engagement induces transient activation of the transcription factor NF-{kappa}B and expression of c-myc, followed by a rapid decrease in activity that leads to growth arrest and apoptotic death (4,27,28). CpG DNA protects WEHI-231 cells from anti-IgM-induced growth arrest and apoptosis by inducing phosphorylation of both I{kappa}B{alpha} and I{kappa}Bß, leading to sustained NF-{kappa}B p50/c-Rel activation, and c-myc and bcl-xL expression (27,28,30). Our present results extend those data to show that CpG DNA also synergizes with anti-IgM for transcriptional activation of NF-{kappa}B, which might contribute to the synergistic expression of TNF-{alpha} and IL-6, as well as to the protection against apoptosis (Fig. 6).

Under our experimental conditions, inhibition of ERK or p38 neither prevented anti-IgM-mediated growth arrest and apoptosis of WEHI-231 cells nor suppressed the CpG DNA-mediated protection, despite a clear role for the p38 and JNK pathways in mediating the proliferative effects of CpG DNA in mature splenic B cells (Fig. 9). However, inhibition of p38 greatly suppressed the TNF-{alpha} and IL-6 production induced by CpG DNA and/or anti-IgM in WEHI-231 cells, although it had little effect on BCR-induced IL-10 production (Fig. 8). The JNK pathway appeared to contribute little to the regulation of CpG DNA-induced TNF-{alpha} synthesis, but was required for that of IL-6 (Fig. 8). These data demonstrate that independent MAPK pathways regulate the effects of CpG DNA and BCR ligation on WEHI-231 apoptosis and cytokine production. Moreover, our results point to the different roles of the CpG DNA-induced MAPK signaling pathways in regulating proliferation/growth arrest in WEHI-231 cells compared to splenic mature B cells.

In monocytic cells, CpG DNA induces IL-10 expression by activating ERK and p38 (12). However, in WEHI-231 cells, CpG DNA did not activate ERK or potentiate anti-IgM-induced ERK activation (Figs 4 and 5). Interestingly, inhibition of p38 or JNK partially suppressed the IL-10 production synergistically induced by CpG DNA and anti-IgM (Fig. 8). This indicates that IL-10 production in WEHI-231 cells stimulated with CpG DNA and BCR-ligation is controlled through a pathway independent of ERK, but partially dependent on p38 and JNK. In addition, we have previously demonstrated that CpG DNA-mediated IL-10 production in WEHI-231 cells is effectively suppressed by the potent immunosuppressant cyclosporine A (CsA) (57). CsA binds to the intracellular protein cyclophilin and creates a complex that inhibits the phosphatase calcineurin. Blockade of calcineurin enzymatic activity prevents the translocation of NFAT (58,59). Under our experimental conditions, we could not detect NFAT activation in WEHI-231 cells after CpG DNA stimulation (Fig. 6). However, surprisingly, CpG DNA- and BCR-mediated signals synergistically activate NFAT, the downstream effector for calcineurin (Fig. 6). Further studies will be required to determine the role of this transcription factor in mediating the effects of CpG DNA.

In conclusion, our data demonstrate that CpG DNA- and BCR-mediated signals are integrated in the p38, JNK, NF-{kappa}B and NFAT activation pathways in WEHI-231 cells. Our results using specific MAPK inhibitors demonstrate that these signaling modulators regulate the agonistic interaction of CpG DNA and BCR engagement for inducing cytokine production, but not the antagonistic effects of CpG DNA on BCR-induced apoptosis in WEHI-231 cells. Finally, these data demonstrate distinct differences in the CpG DNA-induced signaling pathways between mature B cells, immature-like WEHI-231 cells and previous studies in monocyte-like cell lines.


    Acknowledgements
 
A.-K. Y. was supported by Children’s Foundation Research Center at Le Bonheur Children’s Hospital, and Center of Excellence for Diseases of Connective Tissue and Rheumatic Disease Research Core Center at the University of Tennessee, and grants from NIH 1R03AR47757, Leukemia Research Foundation and Le Bonheur Children’s Medical Center. A. M. K. was supported through a Career Development Award from the Department of Veterans Affairs and by grants from the Coley Pharmaceutical Group Inc. and NIH P01CA60570.


    Abbreviations
 
DC—dendritic cell

CREB—cAMP-responsive elements binding factor

CsA—cyclosporine A

ERK—extracellular signal-regulated kinase

HEL—hen egg lysozyme

JNK—c-Jun N-terminal kinase

MAPK—mitogen-activated protein kinase

MKK—mitogen-activated protein kinase kinase

ODN—oligodeoxynucleotides

PTK—protein tyrosine kinases

S-ODN—phosphorothioate oligodeoxynucleotides

TLR—Toll-like receptor

TNF—tumor necrosis factor


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 Abstract
 Introduction
 Methods
 Results
 Discussion
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