CpG motifs in bacterial DNA cause inflammation in the lower respiratory tract.

Since unmethylated CpG motifs are more frequent in DNA from bacteria than vertebrates, and the unmethylated CpG motif has recently been reported to have stimulatory effects on lymphocytes, we speculated that bacterial DNA may induce inflammation in the lower respiratory tract through its content of unmethylated CpG motifs. To determine the role of bacterial DNA in lower airway inflammation, we intratracheally instilled prokaryotic and eukaryotic DNA in C3H/HeBFEJ mice and performed whole lung lavage 4 h after the exposure. Heat denatured, single stranded Escherichia coli genomic DNA (0.06 ng endotoxin/microg DNA) was compared to heat denatured, single stranded calf thymus DNA (0.007 endotoxin/microg DNA). 10 microg of bacterial DNA, in comparison to 10 microg of calf thymus DNA, resulted in a fourfold increase in the concentration of cells (P = 0.0002), a fivefold increase in the concentration of neutrophils (P = 0.0002), a 50-fold increase in the concentration of TNF-alpha (P = 0.001), and a fourfold increase in the concentration of both IL-6 (P = 0.0003) and macrophage inflammatory protein-2 (P = 0.0001) in the lavage fluid. Importantly, instillation of 0.60 ng of E. coli LPS resulted in a negligible inflammatory response. To test whether the stimulatory effects of bacterial DNA are due to its unmethylated CpG dinucleotides, we methylated the bacterial DNA and also prepared 20 base pair oligonucleotides with and without CpG motifs. In comparison to instillation of untreated bacterial DNA, methylation of the bacterial DNA resulted in a significant reduction in the concentration of cells and cytokines in the lower respiratory tract. Moreover, oligonucleotides containing embedded unmethylated CpG motifs resulted in inflammation in the lower respiratory tract that was indistinguishable from that observed with untreated bacterial DNA. In contrast, oligonucleotides without the embedded CpG motifs or with embedded but methylated CpG motifs resulted in significantly less inflammation in the lower respiratory tract. The possible relevance of these data to human disease was shown by extracting and analyzing DNA in sputum from patients with cystic fibrosis (CF). Approximately 0.1 to 1% of this sputum DNA was bacterial. Intratracheal instillation of highly purified CF sputum DNA caused acute inflammation similar to that induced by bacterial DNA. These findings suggest that bacterial DNA, and unmethylated CpG motifs in particular, may play an important pathogenic role in inflammatory lung disease.

The role of CpG dinucleotides in DNA vaccines.

DNA vaccines can induce potent humoral and cellular immune responses without any additional adjuvant. Recent studies indicate that unmethylated CpG dinucleotides within DNA vaccines are immune stimulatory and exert an essential endogenous adjuvant activity. These CpG motifs can be added deliberately to DNA or conventional protein vaccines to enhance the Th1 immune response.

Mechanisms and therapeutic applications of immune stimulatory cpG DNA.

Aside from its function as the "blueprint of life" that encodes genetic information, DNA can have direct immune activities. The immune system has evolved a defense mechanism that is able to distinguish microbial DNA from our own because of differences in the frequency and methylation of CpG dinucleotides in particular base contexts. Within minutes of detecting such "CpG-S DNA," cells of the innate immune system become activated and produce cytokines that promote the generation of antigen specific T-helper-1-like immune responses. Animal studies indicate therapeutic utility for CpG-S DNA as a vaccine adjuvant and for the immunotherapy of cancer and infectious and allergic diseases.

Interleukin-10 functions in vitro and in vivo to inhibit bacterial DNA-induced secretion of interleukin-12.

Bacterial DNA (bDNA) has a number of biologic properties, including the ability to induce interleukin-12 (IL-12) production by macrophages. We studied the role of the regulatory cytokine IL-10 as a potential inhibitor of bDNA-induced IL-12 production. IL-10 concentrations as low as 0.3 ng/ml profoundly inhibited bDNA-induced macrophage IL-12 production as measured by Elispot analysis of IL-12 p40-secreting cells. Additionally, we found that IL-10 inhibited bDNA-induced IL-12 secretion by the macrophage cell lines J774 and RAW 264. Preincubation of splenic adherent cells with IL-10 markedly reduced bDNA-induced transcription of IL-12 p40 mRNA. Interestingly, after 2 h of exposure, bDNA also induces transcription of IL-10 mRNA by splenic adherent cells. The importance of IL-10 in the in vivo regulation of bDNA-induced cytokine secretion was illustrated by the response of mice with disrupted IL-10 genes (IL-10 ko mice) to i.v. bDNA challenge. Compared to +/+ mice, IL-10 knockout (ko) mice exhibited increased numbers of IL-12 and interferon-gamma (IFN-gamma)-secreting cells following either single or repeated challenge with bDNA. These findings indicate that IL-10 plays a key role in regulating bDNA-induced production of inflammatory cytokines.

Photoreactivation rescue and dark repair demonstrated in UV-irradiated embryos of the self-fertilizing fish Rivulus ocellatus marmoratus (Teleostei; Aplocheilidae).

The effects of photoreactivation (PR) rescue and dark repair on the survival of UV-irradiated embryos of the hermaphroditic fish (Rivulus ocellatus marmoratus) are reported. When UV-irradiated embryos were illuminated by photoreactivating light (PRL) from fluorescent lamps, survival at the hatching stage was markedly increased. The maximum recovery to UV damage was shown by embryos that were exposed to PRL for at least 6 h after UV irradiation. The effect of PRL decreased 30 min after UV irradiation and no PR rescue was detected beyond 96 h. Treatment with 2 mM caffeine for 48 h after UV irradiation increased the sensitivity of the embryos in the dark. The above results demonstrate that Rivulus embryos have an efficient PR system and a caffeine-sensitive dark repair capacity.

Rapid induction of mitogen-activated protein kinases by immune stimulatory CpG DNA.

Unmethylated CpG motifs in bacterial DNA or synthetic oligodeoxynucleotides (CpG DNA) rapidly activate B cells and monocyte-derived cells; however, the intracellular signaling pathways involved in this process are unclear. Here, we demonstrate that CpG DNA induces the activation of c-Jun NH2-terminal kinase and p38 but does not activate the extracellular receptor kinase in murine B and monocyte-like cell lines. CpG DNA also induces the phosphorylation of activating transcription factor-2, c-Jun, and mitogen-activated protein kinase (MAPK)-activated protein kinase 2 as well as the activation of activator protein-1 (AP-1) DNA binding. Inhibition of p38 led to the suppression of CpG DNA-induced AP-1 DNA-binding activity and cytokine production, indicating that the p38 pathway is required for mediating these immune stimulatory effects of CpG DNA. Chloroquine, an endosomal acidification inhibitor, selectively abolished CpG DNA-mediated MAPK activation. Our results indicate that CpG DNA activates the p38 and c-Jun NH2-terminal kinase MAPK and leads to the activation of AP-1 via a pathway which is sensitive to chloroquine.

CpG DNA rescue from anti-IgM-induced WEHI-231 B lymphoma apoptosis via modulation of I kappa B alpha and I kappa B beta and sustained activation of nuclear factor-kappa B/c-Rel.

Unmethylated CpG dinucleotides in particular base contexts in oligonucleotides (CpG DNA) rescue WEHI-231 cells from anti-IgM-induced cell cycle arrest and apoptosis. Anti-IgM rapidly elevated the levels of NFkappaB p50/c-Rel heterodimers followed by a decline of p50/c-Rel heterodimers by 3 h and a concomitant increase of p50/p50 homodimers. In contrast, CpG DNA induced and maintained the levels of p50/c-Rel heterodimers in the presence or absence of anti-IgM, while control non-CpG DNA failed to induce NFkappaB activation. Anti-IgM induced IkappaB alpha degradation followed by increased IkappaB alpha protein levels. The levels of IkappaB beta were increased after anti-IgM treatment. In contrast, CpG DNA, but not non-CpG DNA, induced sustained IkappaB alpha and IkappaB beta degradation in the presence or absence of anti-IgM. Inhibition of IkappaB degradation blocked CpG DNA-induced NFkappaB activation and expression of c-myc. Prevention of NFkappaB activation by inhibiting IkappaB degradation also suppressed the ability of CpG DNA to rescue WEHI-231 cells from anti-IgM-induced apoptosis. These results indicate that CpG DNA-mediated sustained activation of NFkappaB depends on the degradation of IkappaB alpha and IkappaB beta and is required for the CpG DNA-mediated anti-apoptosis gene expression and the protection against anti-IgM-induced apoptosis of WEHI-231 cells.

IFN-gamma promotes IL-6 and IgM secretion in response to CpG motifs in bacterial DNA and oligodeoxynucleotides.

Lymphocyte recognition of characteristic structural features in microbial DNA may contribute to immune defense by promoting protective immune responses. The dinucleotide CpG is significantly under-represented in vertebrate DNA and is usually methylated. In contrast, CpG dinucleotides are generally present at the expected frequency in bacterial DNA and are unmethylated. These unmethylated CpG motifs induce B cells to secrete IL-6 and IgM, and can induce NK and CD4+ T cells to produce the immunoregulatory Th1 cytokine, IFN-gamma. IFN-gamma inhibits IgM secretion that is triggered by a different bacterial product, LPS. The present study demonstrates that in contrast to its antagonistic interaction with LPS, IFN-gamma causes a dose-dependent increase in the level of IgM secretion induced by CpG DNA. Like IgM secretion, B cell secretion of IL-6 more than doubles after the addition of exogenous IFN-gamma. Mice with disrupted IFN-gamma genes produced less than half as much IL-6 and IgM in response to CpG DNA, supporting the hypothesis that CpG-induced IFN-gamma production contributes to the B cell response. In contrast to its promotion of IL-6 and IgM secretion, IFN-gamma did not significantly affect the spleen cell proliferation activated by CpG motifs. These results indicate that IFN-gamma produced by T and NK cells after CpG DNA stimulation contributes to the B cell production of IL-6 and the subsequent Ig production. These studies provide further evidence that the immune system responds to CpG motifs in bacterial DNA by activating a coordinated set of humoral and cellular responses.

Rapid immune activation by CpG motifs in bacterial DNA. Systemic induction of IL-6 transcription through an antioxidant-sensitive pathway.

Unmethylated CpG dinucleotides (CpG motif) in bacterial DNA or synthetic oligodeoxynucleotides (CpG DNA) rapidly activate murine B cells to secrete IL-6 and IgM, as well as to proliferate. Within 30 min after CpG DNA stimulation in vivo, IL-6 mRNA levels were increased in liver, spleen, and thymus cells. Serum IL-6 protein was markedly increased within 1 h of stimulation. Treatment of a B cell line with CpG DNA led to an increase in the transcriptional activity of the IL-6 promoter. This CpG DNA-induced IL-6 production was not mediated via either a protein kinase C (PKC)-, protein kinase A (PKA)-, or nitric oxide (NO.)-dependent pathway but was inhibited by an antioxidant. In addition, the level of intracellular reactive oxygen species was increased within 20 min after CpG DNA, but not control non-CpG DNA, treatment. These results suggest that CpG DNA-induced IL-6 production is mediated through a reactive oxygen intermediate-dependent pathway. CpG DNA-mediated IL-6 production was enhanced by simultaneous signals delivered through the Ag receptor. The addition of neutralizing Abs against IL-6 to B cell cultures along with CpG oligodeoxynucleotides essentially abolished the CpG DNA-induced increased IgM secretion but had no significant effect on the B cell proliferation induced by the CpG motif. Our results suggest that the induction of IL-6 expression in response to CpG motifs in bacterial DNA may be an important immune defense mechanism that facilitates a rapid response to microbial infection.

CpG DNA rescue of murine B lymphoma cells from anti-IgM-induced growth arrest and programmed cell death is associated with increased expression of c-myc and bcl-xL.

One of the principal mechanisms thought to maintain B cell tolerance to self Ags is deletion of cells bearing functional IgM receptors for self Ag via apoptosis in the bone marrow. Because of its characteristic growth arrest and apoptosis in response to surface IgM cross-linking, the B cell line WEHI-231 has been a useful model system for studies of Ag receptor-mediated apoptosis. Unmethylated CpG dinucleotides in oligonucleotides (CpG DNA) can be strong B cell mitogens. In the present study we evaluated whether CpG DNA can rescue WEHI-231 cells from anti-IgM-induced cell cycle arrest and apoptosis. The addition of CpG DNA protected WEHI-231 cells from anti-IgM-mediated apoptosis as well as growth arrest. The protective effect of CpG DNA was dependent on the presence of unmethylated CpG dinucleotides. Kinetic analyses showed that the addition of CpG DNA can be delayed for up to 3 h after anti-IgM treatment with no decrease in the protection. CpG DNA reversed anti-IgM-induced down-regulation of c-myc expression in WEHI-231 and up-regulated myn, bcl2, and bcl-xL mRNA expression. Our results suggest that CpG DNA protection of WEHI-231 cells from anti-IgM-induced apoptosis may be mediated by specific and/or cooperative interactions of multiple genes and that CpG DNA could be a useful tool for studies of B cell tolerance.

Cyclosporin A enhances IL-12 production by CpG motifs in bacterial DNA and synthetic oligodeoxynucleotides.

Certain sequences of nucleotides (CpG motifs) in bacterial DNA or synthetic oligonucleotides (CpG DNA) promote the production of proinflammatory cytokines, including TNF-alpha, IFN-gamma, IL-6, and IL-12. Here we demonstrate that the immunosuppressant cyclosporin A (CsA) unexpectedly enhanced CpG DNA-induced IL-12 production in murine splenocytes. CsA did not inhibit CpG DNA-induced TNF-alpha or IL-6 production, but decreased the production of IFN-gamma by CpG DNA. Upon examining mechanisms by which CsA increases IL-12 production, we found that CpG DNA can also induce IL-10 production in B cells and that this production was sensitive to CsA. IL-10 has anti-inflammatory effects and can reduce the production of IL-12. To determine the possible role of CsA-modulated IL-10 production in mediating the increased IL-12 levels, splenocytes from IL-10 gene-disrupted mice (IL-10 -/-) and splenocytes cultured in anti-IL-10 Ab were studied. CpG DNA-stimulated IL-10 (-/-) splenocytes demonstrated no increase in IL-12 levels in the presence of CsA. Anti-IL-10 Ab treatment of normal splenocytes increased the magnitude of CpG DNA-induced IL-12 production to that seen with CsA. These results suggest that CpG DNA induces CsA-sensitive IL-10 production in B cells and that IL-10 acts as a negative feedback regulator of CpG DNA-induced IL-12 production.

CpG DNA induces sustained IL-12 expression in vivo and resistance to Listeria monocytogenes challenge.

Vertebrates have evolved innate immune defense mechanisms that recognize and respond to structural patterns that are specific to microbial molecules. One such pattern recognition system is based on unmethylated CpG dinucleotides in particular sequence contexts (CpG motifs); these motifs are common in bacterial DNA but are under-represented ("CpG suppression") and methylated in vertebrate DNA. Mice that are injected with bacterial DNA or synthetic oligodeoxynucleotides (ODNs) containing CpG motifs respond with a rapid production of IL-12 and IFN-gamma. The serum levels of IL-12 were increased for at least 8 days after a single injection of CpG ODNs, but IFN-gamma levels returned to baseline within 24 h. This Th1-like cytokine response to CpG motifs induces a state of resistance to infection by Listeria monocytogenes in susceptible specific pathogen-free BALB/c mice. Resistance developed within 48 h of pretreatment with CpG ODNs, persisted for at least 2 wk, and was dependent upon IFN-gamma secretion. These data support the hypothesis that CpG DNA motifs are a "danger signal" that activates protective innate immune defenses and may have therapeutic potential.

CpG oligodeoxyribonucleotides rescue mature spleen B cells from spontaneous apoptosis and promote cell cycle entry.

Isolated murine splenic B cells undergo spontaneous apoptosis. Motifs containing unmethylated CpG dinucleotides in bacterial DNA or in synthetic oligodeoxynucleotides (ODN) are known to activate murine B cells. Now we show that ODN that induce spleen B cell cycle entry also inhibit spontaneous apoptosis in a sequence-specific fashion. Reversal of the CG to GC abolished activity. Methylation of the central cytosine decreased activity. When CpG is preceded by a cytosine or followed by a guanine, activity was abolished. Other substitutions at the same positions had no effect. Dose-response curves for apoptosis protection and G1 entry suggested that a uniform population of ODN recognition sites controlled downstream ODN effects. A CpG ODN with a nuclease-resistant phosphorothioate backbone (S-ODN) was also active, and increased the levels of c-myc, egr-1, c-jun, bclXL, and bax mRNA and c-Myc, c-Jun, Bax, and BclXL protein in spleen B cells. Levels of c-myb, myn, c-Ki-ras, and bcl2 mRNA remained unchanged. When protein synthesis was inhibited, at 16 h ODN-induced cell cycle entry was abolished and apoptosis protection was partially preserved. Under these conditions, c-Myc was still present, but c-Jun and BclXL were not detected. Our results suggest that CpG containing ODN motifs provide signals for both survival and cell cycle entry. Single base changes determine whether this signal proceeds through a rate-limiting step governing at least two steps in apoptosis (plasma membrane transition, DNA cleavage) and two phases of the cell cycle (G1 and S phase entry). This biologic action is associated with increased c-Myc, c-Jun, and BclXL expression.

Bacterial DNA induces NK cells to produce IFN-gamma in vivo and increases the toxicity of lipopolysaccharides.

Microbial products released during bacterial infection induce cytokine-mediated inflammatory responses that can be protective, but excessive release of inflammatory cytokines may promote development of the sepsis syndrome. We examined the ability of bacterial DNA to induce in vivo cytokine release and to potentiate the toxicity of LPS. Intravenous treatment of mice with Escherichia coli (EC) DNA, but not calf thymus (CT) DNA, induced a rapid (within 4 h) dose-dependent increase in serum IFN-gamma and splenic IFN-gamma-forming cells. Over 90% of splenic IFN-gamma-producing cells were identified by surface phenotype as NK cells. Mice also mounted an IFN-gamma response following challenge with 20-base oligonucleotide that contained an internal CG motif (but did not respond to a control oligonucleotide). Treatment of mice with EC DNA followed by a sublethal LPS challenge resulted in a 3-fold increase in the peak serum level of TNF-alpha and a 10-fold increase in the peak level of IL-6 compared with mice that received CT DNA followed by LPS. Mice treated with EC DNA followed by LPS showed 75% mortality, compared with no deaths in mice treated with CT DNA followed by LPS. EC DNA/LPS treatment of mice with disrupted IFN-gamma genes resulted in a 5% mortality while 59% of similarly treated +/+ mice died. Thus, bacterial DNA induces in vivo release of IFN-gamma which, in turn, is associated with an increase in LPS-induced TNF-alpha and IL-6 release, and with increased sensitivity to the toxic effects of LPS.

CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma.

Bacterial infection stimulates the host to mount a rapid inflammatory response. A 6-base DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines was shown to contribute to this response by inducing polygonal B-cell activation. This stimulatory motif is 20 times more common in the DNA of bacteria than higher vertebrates. The current work shows that the same motif induces the rapid and coordinated secretion of interleukin (IL) 6, IL-12, and interferon gamma (but not IL-2, IL-3, IL-4, IL-5, or IL-10) in vivo and in vitro. Stimulatory CpG DNA motifs induced B, T, and natural killer cells to secrete cytokine more effectively than did lipopolysaccharide. Thus, immune recognition of bacterial DNA may contribute to the cytokine, as well as the antibody production characteristic of an innate inflammatory response.

CpG motifs in bacterial DNA activate leukocytes through the pH-dependent generation of reactive oxygen species.

B cells and monocytes endocytose DNA into an acidified intracellular compartment. If this DNA contains unmethylated CpG dinucleotides in particular base contexts (CpG motifs), these leukocytes are rapidly activated. We now show that both B cell and monocyte-like cell line responses to DNA containing CpG motifs (CpG DNA) are sensitive to endosomal acidification inhibitors; they are completely blocked by bafilomycin A, chloroquine, and monensin. The specificity of these inhibitors is demonstrated by their failure to prevent responses to LPS, PMA, or ligation of CD40 or IgM. Acidification of endosomal CpG DNA is coupled to the rapid generation of intracellular reactive oxygen species. The CpG DNA-induced reactive oxygen species burst is linked to the degradation of IkappaB and the activation of NFkappaB, which induces leukocyte gene transcription and cytokine secretion. These studies demonstrate a novel pathway of leukocyte activation triggered by CpG motifs.

CpG DNA rescues B cells from apoptosis by activating NFkappaB and preventing mitochondrial membrane potential disruption via a chloroquine-sensitive pathway.

Isolated murine splenic B cells gradually undergo spontaneous apoptosis while WEHI-231 B lymphoma cells undergo activation-induced apoptosis. Unmethylated CpG dinucleotides in a particular sequence context (CpG motif) in bacterial DNA or in synthetic oligodeoxynucleotides (CpG DNA) rescue both splenic B cells and WEHI-231 cells from apoptosis, an effect which could potentially contribute to autoimmune disease. Chloroquine has been used as an effective therapeutic agent for some autoimmune diseases, although the mechanism of action is not clearly understood. Low concentrations of chloroquine (<5 microM) selectively abolished CpG DNA-mediated protection against spontaneous apoptosis of splenic B cells and against anti-IgM-induced apoptosis of WEHI-231 cells without affecting anti-apoptotic activities of anti-CD40 or lipopolsaccharide. CpG DNA effectively prevented mitochondrial membrane potential disruption through a chloroquine-sensitive pathway in splenic B cells. Apoptosis protection by CpG DNA was also associated with increased expression of several proto-oncogenes and oncoproteins directly and/or indirectly through a rapid and sustained activation of NFkappaB in splenic B cells and WEHI-231 cells. These effects were also suppressed by chloroquine. Our results suggest that despite the difference in maturation phenotype of splenic B cells and WEHI-231 cells, CpG DNA rescues both from apoptosis by similar pathway, which is blocked at an early step by chloroquine.

CpG stimulation of primary mouse B cells is blocked by inhibitory oligodeoxyribonucleotides at a site proximal to NF-kappaB activation.

Bacterial DNA and CpG-oligodeoxyribonucleotides (ODN) are powerful B cell activators, inducing apoptosis protection, cell cycle entry, proliferation, costimulatory molecule expression, immunoglobulin (Ig) and interleukin-6 (IL-6) secretion. However, proximal events in B cell activation by ODN are only partially characterized, including the translocation of NF-kappaB to the nucleus. In this paper, we provide evidence that CpG-ODN-induced cell cycle entry and apoptosis protection are blocked by SN50 or gliotoxin and thus require NF-kappaB activation. NF-kappaB activation occurred within 30 minutes of stimulation of murine B cells with a phosphorothioate (S) CpG-ODN and persisted for up to 40 hours, with p50, p65, and c-Rel as the major components. Similar to other NF-kappaB inducers, CpG-ODN caused an early IkappaBalpha and IkappaBbeta degradation plus cleavage of the p50 precursor and subsequent NF-kappaB nuclear translocation. A group of closely related S-ODN, which specifically blocked CpG-induced B cell activation at submicromolar concentrations, also prevented NF-kappaB DNA binding and transcriptional activation. These inhibitory S-ODN differed from stimulatory S-ODN by having 2-3 G substitutions in the central motif. As inhibitory S-ODN did not directly interfere with the NF-kappaB DNA binding but prevented CpG-induced NF-kappaB nuclear translocation of p50, p65, and c-Rel and blocked p105, IkappaBalpha, and IkappaBbeta degradation, we concluded that their putative target must lie upstream of inhibitory kinase (IKK) activation.

Lipopolysaccharide and CpG DNA synergize for tumor necrosis factor-alpha production through activation of NF-kappaB.

Unmethylated CpG motifs in bacterial DNA (CpG DNA) activate host innate immune responses synergistically with some other microbial products, such as endotoxins, and may contribute to disease pathogenesis through excessive production of proinflammatory cytokines. Because monocyte-derived tumor necrosis factor (TNF)-alpha is an important mediator of disease, we investigated whether CpG DNA and lipopolysaccharide (LPS) synergize for inducing TNF-alpha biosynthesis. CpG DNA and LPS synergistically induce TNF-alpha production in RAW264.7 cells and J774 cells through activation of NF-kappaB. Furthermore, transient transfection with a super-repressive mutant of IkappaBalpha (IkappaBalpha-AA) demonstrated that NF-kappaB plays a critical role in CpG DNA-mediated TNF-alpha expression. Like NF-kappaB activation, CpG DNA-induced activation of mitogen-activated protein kinases (MAPK) regulates TNF-alpha production. Both extracellular receptor kinase (ERK) and p38 can regulate TNF-alpha gene transcription induced by CpG DNA. Although CpG DNA at the higher concentration slightly enhanced LPS-mediated phosphorylation of ERK, it did not alter the LPS-mediated activation of c-Jun N-terminal kinase and p38. In addition, CpG DNA showed little or no enhancement of LPS-mediated AP-1 activation. These results suggest that CpG DNA- and LPS-mediated signals converge at or above the level of NF-kappaB and ERK, and that there are distinct, as well as common, signaling pathways which are utilized by both CpG DNA and LPS for activating various transcription factors and MAPK.