Biomedical subjects
Publications and source records attributed to A M Krieg.
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.
Bacterial DNA or synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within the context of certain flanking bases (CpG motifs) have potent stimulatory effects on the vertebrate immune system. CpG ODN with a synthetic nuclease-resistant phosphorothioate backbone (S-ODN) can be used as an adjuvant to augment both humoral and cell-mediated immune responses against a protein antigen. It has also been shown that the presence of CpG motifs in DNA vaccines may be responsible, at least in part, for their efficacy. Here we evaluate the possibility of using CpG ODN as an adjuvant with DNA vaccines to further improve their efficacy. We show that it is not possible to directly mix S-ODN with plasmid DNA because this will result in an ODN dose-dependent reduction in gene expression from the plasmid, possibly because of competitive interference at binding sites on the surface of target cells. Although ODN with a phosphorothioate-phosphodiester chimeric backbone (SDS-ODN) do not adversely effect the level of gene expression (except when certain sequences, such as a poly G, are present), this is not useful, as SDS-ODN are apparently also not sufficiently nuclease resistant to exert a strong CpG adjuvant effect. Neither is it possible to augment responses to DNA vaccines by administering the CpG S-ODN at a different time or site than the plasmid DNA. Thus, at least for the present, it appears necessary to clone CpG motifs into DNA vaccine vectors to take advantage of their adjuvant effect.
Synthetic oligodeoxynucleotides (ODN) that contain unmethylated CpG motifs (CpG ODN) induce macrophages to secrete IL-12, which induces interferon (IFN)-gamma secretion by natural killer (NK) cells. Since these cytokines can induce T helper 1 (Th1) differentiation, we examined the effects of coadministered CpG ODN on the differentiation of Th responses to hen egg lysozyme (HEL). In both BALB/c (Th2-biased) and B10.D2 (Th1-biased) mice, immunization with HEL in incomplete Freund's adjuvant (IFA) resulted in Th2-dominated immune responses characterized by HEL-specific secretion of IL-5 but not IFN-gamma. In contrast, immunization with IFA-HEL plus CpG ODN switched the immune response to a Th1-dominated cytokine pattern, with high levels of HEL-specific IFN-gamma secretion and decreased HEL-specific IL-5 production. IFA-HEL plus CpG ODN also induced anti-HEL IgG2a (a Th1-associated isotype), which was not induced by IFA-HEL alone. Control non-CpG ODN did not induce IFN-gamma or IgG2a, excepting lesser increases in B10.D2 (Th1-biased) mice. Thus, CpG ODN provide a signal to switch on Th1-dominated responses to coadministered antigen and are potential adjuvants for human vaccines to elicit protective Th1 immunity.
Recent advances in our understanding of the immune response are allowing for the logical design of new approaches to cancer immunization. One area of interest is the development of new immune adjuvants. Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can induce production of a wide variety of cytokines and activate B cells, monocytes, dendritic cells, and NK cells. Using the 38C13 B cell lymphoma model, we assessed whether CpG ODN can function as immune adjuvants in tumor antigen immunization. The idiotype served as the tumor antigen. Select CpG ODN were as effective as complete Freund's adjuvant at inducing an antigen-specific antibody response but were associated with less toxicity. These CpG ODN induced a higher titer of antigen-specific IgG2a than did complete Freund's adjuvant, suggesting an enhanced TH1 response. Mice immunized with CpG ODN as an adjuvant were protected from tumor challenge to a degree similar to that seen in mice immunized with complete Freund's adjuvant. We conclude that CpG ODN are effective as immune adjuvants and are attractive as part of a tumor immunization strategy.
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.
Bacterial DNA and synthetic oligodeoxynucleotides containing the CpG motif (CpG ODN) can activate various immune cell subsets, including natural killer cells and macrophages. We evaluated whether the combination of CpG ODN and antitumor monoclonal antibody is effective at preventing tumor growth in an immunocompetent murine lymphoma model. CpG ODN-activated murine splenocytes induced lysis of tumor targets more effectively than unactivated splenocytes. These effector cells were also superior to unactivated splenocytes or cells activated with a control methylated ODN at inducing antibody-mediated lysis of 38C13 murine lymphoma cells. In vivo, CpG ODN alone had no effect on survival of mice inoculated with 38C13 cells. However, a single injection of CpG ODN enhanced the antitumor response to antitumor monoclonal antibody therapy. Ninety percent of mice treated with monoclonal antibody alone developed tumor compared with 20% of mice treated with antibody and CpG ODN. These antitumor effects were less pronounced when treatment consisted of an identical ODN containing methylated CpG dinucleotides. A single dose of CpG ODN appeared to be as effective as multiple doses of interleukin-2 at inhibiting tumor growth when combined with antitumor monoclonal antibody. We conclude that immunostimulatory CpG ODN can enhance antibody dependent cellular cytotoxicity and warrant further evaluation as potential immunotherapeutic reagents in cancer.
Bacterial DNA (bDNA) activates B cells and macrophages and can augment inflammatory responses by inducing release of proinflammatory cytokines. We found that bDNA stimulation of mouse spleen cells induced NK cell IFN-gamma production that was dependent upon the presence of unmethylated CpG motifs, and oligonucleotides with internal CpG motifs could also induce splenocytes to secrete IFN-gamma. The bDNA-induced IFN-gamma response was strictly macrophages dependent. While splenocytes from SCID mice secreted IFN-gamma in response to bDNA, depletion of macrophages eliminated this response. Additionally, purified NK cells did not respond to bDNA; however, addition of macrophages restored the NK cell IFN-gamma response. Coculture of NK cells with preactivated macrophages further increased bDNA-induced NK cell IFN-gamma production. Anti-IL-12 or IL-10 inhibited bDNA-induced IFN-gamma response. Treatment of purified macrophages with bDNA resulted in IL-12 secretion accompanied by an increase in IL-12 p40 mRNA level. Although isolated NK cells did not make IFN-gamma in response to bDNA, NK cells costimulated with IL-12 gained the ability to respond to bDNA. These experiments show that bDNA induces macrophage IL-12 production which, in turn, stimulates NK cell IFN-gamma production. Macrophage-derived IL-12 renders NK cells responsive to bDNA permitting an even greater IFN-gamma response to bDNA.
Certain oligodeoxynucleotides (ODN) containing cytosine followed by guanosine (CpG) protect B cells from apoptosis, and induce B-cell proliferation and cytokine production. We investigated the effect of phosphorothioate CpG-containing ODNs (5'-ATAATCGACGTTCAAGCAAG-3' or 5'-TCCATGACGTTCCTGACGTT-3') and control ODNs (which did not contain CpG) on apoptosis and cell growth in WEHI 231 murine B lymphoma cells. Anti-surface (alpha-s)IgM antibody induces 40-60% DNA degradation and growth arrest of WEHI 231 cells in 24 h. Both of these effects were substantially reversed by 30 ng/ml CpG-ODN added up to 8 hr after alpha-sIgM. Control ODNs not containing the CpG motif were without effect. We explored various hypotheses to account for these effects. The phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate, inhibits apoptosis induced by alpha-sIgM, but the anti-apoptotic effect of CpG-ODN was not affected by inhibitors of protein kinase C, indicating that CpG-ODN does not act via protein kinase C. CpG-ODN inhibited apoptosis and growth arrest induced by C2- and C8-ceramide, sphingomyelinase and an intracellular Ca2+ pump inhibitor thapsigargin, indicating that inhibition is not mediated via suppression of the ceramide cycle or suppression of Ca2+ mobilization. CpG-ODN partially inhibited apoptosis induced by okadaic acid, a protein phosphatase inhibitor, and by menadione, a free radical generator. CpG-ODN also inhibited apoptosis and growth arrest induced by ultraviolet-irradiation, glucocorticoid, vinca alkaloids, and doxorubicin. CpG-ODN significantly protected cells from DNA fragmentation induced by alpha-sIgM in the presence of cycloheximide, but cycloheximide itself induces apoptosis which was unaffected by CpG-ODN. These results suggest that CpG-ODNs powerfully modulate the process by which immune cells are committed to death or proliferation by a mechanism acting on distal cell signalling events. CpG-ODNs may be able to decrease immunosuppression in patients undergoing cancer chemotherapy.
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.
Protein tyrosine kinases (PTKs) have central roles in cellular signal transduction. We have identified a sequence motif (CGT[C]GA) in phosphorothioate-modified oligodeoxynucleotides (ODNs) that specifically inhibits the enzymatic activity of recombinant or immunoprecipitated PTK in vitro. Hexamer ODNs containing this motif block both substrate and autophosphorylation of at least four different PTKs but have no apparent effect on the enzymatic activity of a serine/threonine protein kinase. These data suggest possible new applications for ODNs and have implications for the design and interpretation of experiments using antisense or triplex ODNs.
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.
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.
We have recently shown that oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides (CpG motif) can induce B cells to proliferate, differentiate, and secrete cytokines. In this study we demonstrate that CpG motifs contained in ODN as short as 15 bases in length were quite effective at inducing NK cell lytic activity in vitro in both human and murine lymphocytes. Such ODN were also effective at inducing NK lytic activity, in vivo, in mice. Experiments designed to determine the cellular and cytokine requirements for NK cell induction revealed that B and T cells are not necessary, that the ODN do not augment the activity of highly purified NK cells, and that the ODN augment NK cell activity indirectly by inducing the secretion of IL-12, IFN-alpha beta, and TNF-alpha. Various ODN sequences were prepared to determine the optimal ODN length, motif, palindrome, backbone modification, and dose requirements. We found no requirement for a palindromic sequence but a definite requirement for an unmethylated CpG motif. While necessary, however, a CpG motif was not sufficient for NK cell induction. Instead, there appeared to be stringent requirements for the immediate flanking bases at the 5' and 3' ends as well as for flanking sequences outside the immediate 5' and 3' bases. In particular poly(G) ends seemed to exert a complex qualitative and quantitative effect which could be up- or down-modulating depending on whether the ODN backbone was phosphorothioate modified or not.
The use of antisense oligonucleotides as tools for modulating gene expression represents a novel strategy for designing drugs to treat a variety of diseases. Several factors, including cellular uptake and internalization of the oligonucleotides, are important parameters in determining the effectiveness of antisense agents such as therapeutic drugs. We have studied oligonucleotides uptake in normal and leukemic human hematopoietic cells, such as peripheral blood, bone marrow (BM), and HL-60 cell line; and have found that, in normal human blood and BM, myeloid cells and B cells preferably took up more oligonucleotides than T cells. There was no marked difference in oligonucleotide uptake between CD4+ helper T cells and CD8+ cytolytic T cells. Leukemic cells had greater oligonucleotide uptake than their normal counterparts. Furthermore, oligonucleotide uptake was closely related to cell activation status and can be modulated by growth factors or inhibitors. These studies provide a basis for using oligonucleotides as therapeutic drugs both in vitro and in vivo.
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.
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.
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.
CpG dinucleotides are present at the expected frequency in prokaryotic DNA, but are underrepresented ('CpG suppression') and methylated in vertebrate DNA. The vertebrate immune system has apparently evolved the ability to recognize these unmethylated CpG motifs and respond with a rapid and coordinated cytokine response leading to the induction of humoral and cell-mediated immunity.