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(Journal of Leukocyte Biology. 2002;71:686-694.)
© 2002 by Society for Leukocyte Biology

Inhibition of murine macrophage nitric oxide production by synthetic oligonucleotides

Fu-Gang Zhu, Charles F. Reich and David S. Pisetsky

Medical Research Service, Durham Veterans Administration Hospital and Division of Rheumatology, Allergy and Clinical Immunology, Department of Medicine, Duke University Medical Center, Durham, North Carolina

Correspondence: Dr. David S. Pisetsky, Durham VA Medical Center, Box 151G, 508 Fulton Street, Durham, NC 27705. E-mail: dpiset{at}acpub.duke.edu


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ABSTRACT
 
Synthetic 30-mer phosphorothioate (Ps) oligonucleotides (ODN) comprised of single bases (SdA30, SdC30, SdG30, and SdT30) were assessed for their effects on nitric oxide (NO) production by murine bone marrow macrophages (BMMC) and macrophage cell lines J774 and RAW264.7. Pretreatment of these cells with any of the four Ps ODN inhibited NO production induced by CpG ODN, E. coli DNA (EC DNA), or LPS. This inhibition was time- and dose-dependent and was observed even if the Ps ODN were added as long as 12 h after stimulation. As in the case of stimulatory ODN, inhibition was dependent on backbone structure and length. Thus, all four 30-mer, single-base Ps ODN were inhibitory, and only dG30 among phosphodiester ODN was inhibitory. Together, these observations indicate that Ps ODN can inhibit macrophage production of inflammatory mediators, suggesting a role of these compounds as immunomodulatory agents.

Key Words: DNA • CpG • lipopolysaccharide • inflammatory mediator


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INTRODUCTION
 
DNA is a complex macromolecule whose immunological activities vary with sequence and backbone structure [1 2 3 4 5 ]. Although bacterial DNA can stimulate immune responses via CpG motifs, mammalian DNA and certain viral DNA can counteract these effects and inhibit macrophage responses [6 7 8 9 ]. As shown in in vitro experiments, calf thymus DNA (CT DNA) can block the production of interleukin (IL)-12 by murine spleen cells stimulated by plasmid DNA [6 ]. CT DNA can also block the production of IL-12 by murine bone marrow macrophages (BMMC) as well as J774 cells stimulated with Escherichia coli DNA (EC DNA) [7 ]. Although the sequences in CT DNA causing inhibition are not defined yet, studies on neutralizing effects of viral DNA suggest that CpG repeats or a CpG motif preceded by C or followed by G are inhibitory [8 ].

In previous studies, we investigated the structural requirement for inhibition by DNA by assessing the effects of synthetic oligonucleotides (ODN) on the in vitro production of IL-12 by murine macrophages [7 ]. Using a series of phosphodiester (Po) and phosphorothioate (Ps) ODN, we showed that among single-base, 30-mer Po compounds, only dG30 was inhibitory. In contrast, among Ps ODN, all single-base, 30-mer compounds could inhibit the production of IL-12 stimulated with EC DNA, a CpG ODN, and lipopolysaccharide (LPS). Because these compounds could block responses induced by DNA and LPS, these findings suggest a more generalized, inhibitory effect of certain nucleic acids on macrophage activation.

In the present study, we have investigated the inhibitory effects of Ps ODN further using nitric oxide (NO) production as a measure of macrophage activation. NO is a small, gaseous molecule that mediates a variety of physiological responses and plays a key role in inflammation [10 ]. NO is generated by distinct isoforms of NO synthase (NOS) enzymes with inducible NOS (iNOS) or NOS2, a major source of NO produced by macrophages during inflammation [11 ]. Although IL-12 and NO are produced following macrophage activation, their induction involves different regulatory elements [12 , 13 ]. Previous studies have shown that CpG DNA can stimulate macrophage iNOS expression as well as NO production [14 , 15 ].

Using BMMC, J774, and RAW264.7 cell lines as models, we have investigated the inhibitory capacity of ODN for NO, the structure-function relationships for inhibition, and potential mechanisms of action. In results presented herein, we show that ODN can inhibit DNA- and LPS-induced NO production in a dose- and time-dependent way and that these inhibitory effects are influenced by the backbone structure and chain length of the synthetic ODN. These findings confirm the inhibitory potential of Ps ODN and suggest that nucleic acids have anti-inflammatory and immunosuppressive effects that may be useful clinically.


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MATERIALS AND METHODS
 
Synthetic ODN and bacterial and mammalian DNA
Ps and Po oligodeoxynucleotides were purchased from Midland Certified Reagent Company (Midland, TX) and have the sequence and structures shown in Table 1 . A CpG-containing oligodeoxynucleotide (CpG ODN) and a non-CpG ODN have been described previously [16 ]. E. coli DNA and calf thymus DNA were purchased from Sigma Chemical Co. (St. Louis, MO). The DNA products were purified further in the laboratory by extraction with phenol/chloroform/isoamyl alcohol, and the aqueous phase was precipitated and resuspended in distilled water with the final A260/A280 >= 1.8.


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  		Table 1. Synthetic ODN Used in This Study

To prepare fluorescently labeled ODN, those containing an amino terminus were mixed with fluorescein isothiocyanate (FITC; Sigma Chemical Co.) in a ratio of 0.1 mg FITC:10 mg ODN in carbonate/bicarbonate buffer (pH 9.5). After 2 h incubation at room temperature, unreacted FITC was removed by gel filtration through a Sephadex G-25 column (NAP10 column; Phamarcia, Piscataway, NJ), followed by ethanol precipitation.

Culture of murine macrophages
Murine J774 and RAW264.7 macrophage cell lines were maintained in culture medium consisting of RPMI-1640 medium (Life Technologies, Grand Island, NY), supplemented with 10% fetal calf serum (FCS; HyClone, Logan, UT) and 50 µg/ml gentamicin. BMMC were cultured as described previously [17 ]. Briefly, intact femurs and tibias were removed from BALB/c mice (Jackson Laboratory, Bar Harbor, ME). The bone marrow cells were collected by repeated flushing of the bone shaft with RPMI-1640 medium. Bone marrow cells were cultured at a concentration of 1 x 106 cells/ml in RPMI-1640 medium supplemented with 10% FCS, 50 µg/ml gentamicin, and 20% L929-conditioned medium. After culture for 5–7 days, cells were removed with a cell scraper, washed, and resuspended in culture medium. BMMC or J774 and RAW264.7 were plated as 5 x 105 cells/100 µl/well in 96-well cell-culture plates the day before experiments.

Activation of murine macrophages
Murine BMMC or J774 and RAW264.7 cells were pretreated for 1 h at 37°C with 30-mer Ps ODN (SdA30, SdC, SdG30, and SdT30) at a dose range up to 50 µg/ml. The cells were then stimulated with 5 µg/ml CpG ODN or EC DNA or 0.01–0.1 µg/ml LPS (E. coli 0111:B4; Sigma Chemical Co.) for up to 48 h, and the culture supernatants were collected at designated time points for nitrite assays. For controls, cells were incubated with medium alone or a non-CpG ODN or CT DNA.

To assess the time course of NO production, culture supernatant samples were taken at different time points and kept at -20°C for later nitrite assays. To assess possible toxicity of the synthetic ODN, cell viability was measured by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay on macrophages exposed to different synthetic ODN [18 ]. All synthetic ODN showed minimal effects on the viability of these cells at the highest dose (50 µg/ml) used in the current study (unpublished results).

Nitrite assay
NO production in the culture supernatants was quantified by using the Griess method to measure nitrite (NO2-), a stable breakdown product of NO [19 ]. Briefly, 50 µl culture supernatant was transferred to a well in a 96-well microtiter plate, followed by addition of 100 µl Griess reagent I (1% sulfanilamide in 2.5% phosphoric acid) and 100 µl Griess reagent II (0.1% naphthylenediamine in 2.5% phosphoric acid). The absorbency was read within 5 min at 550 nm on an automated microtiter plate reader, and nitrite concentrations were calculated by comparison to a standard curve generated with sequential dilutions of sodium nitrite.

Immunoblotting analysis
J774 or RAW264.7 cells (5x106/well) in a six-well culture plate were pretreated with medium or 50 µg/ml SdA30, SdC30, SdG30, and SdT30 for 1 h, followed by stimulation with 10 µg/ml CpG ODN and EC DNA or 1 µg/ml LPS for 30 min. The stimulation was stopped by adding 3 ml/well cold phosphate-buffered saline (PBS) containing 1 mM Na3VO4. The cells were lysed with 300 µl/well lysis buffer (25 mM HEPES, 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM 0.5 M ethylenediaminetetraacetate, 0.1% Triton X-100, 5 mM Na3VO4, 10 mM NaF, 50 µg/ml leupeptin, 50 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol). The lysates were cleared by centrifugation at 16,000 g for 15 min, and protein concentration was determined with the Bradford method (Bio-Rad Laboratories, Hercules, CA). The lysates (10 µg protein/lane) were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to HybondTMECLTM nitrocellulose membranes (Amersham Pharmacia Biotech, Little Chalfont, UK). The membranes were blocked in 5% nonfat dry milk/0.1% Triton X-100/Tris-buffer saline (TBS), pH 6.8, followed by staining with monoclonal anti-mouse phosphorylated ERK1/2 (1:200), purchased from Santa Cruz Biotech (Santa Cruz, CA). After washing with 0.1% Tween 20/TBS, pH 6.8, the membranes were incubated with a 1:5000 dilution of horseradish peroxidase-conjugated anti-mouse immunoglobulin G (Sigma Chemical Co.). The specific bands were detected with an enhanced chemiluminescence (ECL) Western blotting detection kit and Hyperfilm-ECL, according to the manufacturer’s instructions (Amersham Pharmacia Biotech).

Flow cytometry
For DNA or LPS uptake assays, 1 x 106 BMMC or J774 cells were pretreated with different doses (indicated in figure legends) of Ps ODN for 1 h at 37°C, followed by incubation with 5 µg/ml FITC-labeled CpG ODN or LPS (Sigma Chemical Co.) in RPMI 1640 with 5% FCS. After 10 min incubation, unbound ODN or LPS were stripped by incubating cells in 0.2 M acetic acid (pH 2.5) on ice for 10 min and were washed three times in cold PBS/2% bovine serum albumin/0.1% NaN3 by centrifugation at 400 g for 5 min. Cells were fixed in 1% paraformaldehyde/PBS/0.1% NaN3 and examined by FACScan flow cytometer (Becton Dickinson, Mansfield, MA). The acquisition was performed with 10,000 events per sample. The list mode data were analyzed by using LYSYSTM II software (Becton Dickinson Immunocytometry Systems, San Jose, CA).


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RESULTS
 
Inhibition of DNA-induced NO production by ODN
The inhibitory effects of Ps ODN on NO production were tested first to determine whether the activity of these compounds on macrophages extends to the production of this inflammatory mediator as well as IL-12. For this purpose, J774 macrophages were incubated with medium with or without 2–50 µg/ml SdA30, SdC30, SdG30, and SdT30, respectively, at 37°C for 1 h, followed by stimulation with 5 µg/ml CpG ODN or EC DNA for 24 h. As shown in Figure 1 , the cells produced NO as reflected in nitrite production in response to these stimulants. Similar to results demonstrated with IL-12 production, Ps ODN inhibited nitrite production in a dose-dependent manner (Fig. 1) . Although SdC30 at 50 µg/ml reduced CpG ODN-induced NO production by about 50%, at the same dose, SdA30, SdG30, and SdT30 abolished NO production induced by EC DNA or CpG ODN almost completely. SdA30, SdC30, SdG30, and SdT30 displayed similar levels of inhibition of NO production by BMMC and RAW264.7 cells, and none of the four Ps ODN at the above dose range induced NO by BMMC or J774 and RAW264.7 cells (unpublished results).



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  		Figure 1. Dose-dependent inhibition of macrophage NO production by Ps ODN. J774 cells were pretreated with medium or indicated concentrations of SdA30, SdC30, SdG30, and SdT30 for 1 h and then were stimulated with 5 µg/ml CpG ODN (A) or EC DNA (B) in the presence or absence of the Ps ODN for 24 h. Nitrite levels in the culture supernatants were determined by the Griess reaction assay. The control group treated with medium alone displayed nitrite levels of 0.511 ± 0.238 µM (mean±SD). Each value represents mean of triplicates. Error bars depict the standard deviations. The data represent one of three identical experiments.

We next examined the time course for the effects of the Ps ODN on macrophage NO production. J774 cells were treated with 50 µg/ml Ps ODN for 1 h and then stimulated with 5 µg/ml CpG ODN or EC DNA for up to 48 h. Culture supernatants were collected at different time points for nitrite assays. All four ODN displayed a time-dependent inhibition of NO production by J774 cells stimulated with EC DNA or CpG ODN (Fig. 2 ). Although CpG ODN-induced nitrite levels at 48 h were reduced by 97%, 37%, 61%, and 91% in cells pretreated with SdA30, SdC30, SdG30, and SdT30, respectively, EC DNA-induced nitrite levels at the same time point were abrogated to the levels in unstimulated cells. Furthermore, the inhibitory effects by the ODN were greater on J774 cells treated with EC DNA than with CpG ODN (Figs. 1 and 2) .



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  		Figure 2. Time course of inhibition of macrophage NO production by Ps ODN. J774 cells were pretreated with medium or 50 µg/ml SdA30, SdC30, SdG30, and SdT30 for 1 h, followed by stimulation with 5 µg/ml CpG ODN (A) or EC DNA (B) for 48 h. Supernatants were collected at different time intervals and assayed for nitrite levels. Each value represents mean of triplicates. Error bars depict the standard deviations. The data are representative of three repeats.

Inhibition of LPS-induced NO production by ODN
To determine whether Ps ODN could inhibit induction of NO by other stimulants, the effects of Ps ODN on LPS-induced NO production were investigated. LPS (0.01–0.1 µg/ml) stimulated NO production in a dose-dependent manner by J774 cells. The induced NO production was blocked to various extents by the four Ps ODN at a concentration of 50 µg/ml (Fig. 3 ). Similar inhibitory effects on LPS-induced NO production were also observed in murine BMMC and RAW264.7 cells, although the inhibition of BMMC was less marked than in J774 and RAW264.7 cells (unpublished results). However, the Ps ODN failed to suppress NO production by the three types of murine macrophages stimulated with a dose of LPS 10 times higher (1 µg/ml; unpublished results).



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  		Figure 3. Inhibition of LPS-induced NO production. J774 cells were pretreated with medium or 50 µg/ml SdA30, SdC30, SdG30, or SdT30 for 1 h, followed by stimulation with 0.01, 0.05, and 0.1 µg/ml LPS for 48 h. Supernatants were collected at 48 h and assayed for nitrite levels. Each value represents mean of triplicates. Error bars depict the standard deviations. The data represent one of three identical experiments.

To confirm the inhibitory effects of the ODN, J774 cells were treated with 50 µg/ml SdA30, SdC30, SdG30, or SdT30 and then stimulated with CpG ODN, EC DNA, or LPS. As shown in Figure 4 , the ODN displayed varying levels of inhibition of NO production induced by the stimulants tested. In these experiments, SdG30 and SdT30 appeared more effective than SdA30 and SdC30 in blocking macrophage NO production induced by the three stimulants.



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  		Figure 4. Ps ODN inhibition of NO induction by different stimulants. J774 cells pretreated with medium or 50 µg/ml SdA30, SdC30, SdG30, and SdT30 for 1 h, followed by stimulation with CpG ODN (5 µg/ml), EC DNA (5 µg/ml), or LPS (0.1 µg/ml) for 24 h. Nitrite levels in the culture supernatants were evaluated with the Griess assay. Each value represents mean of triplicates. Error bars depict the standard deviations. The data are representative of three repeats.

To test the time dependence of the inhibitory effects by the ODN, J774 cells were stimulated with 5 µg/ml CpG ODN and EC DNA or 0.1 µg/ml LPS for different periods of time (1–24 h) before exposure to 50 µg/ml SdT30. After a total of 48 h of stimulation, culture supernatants were collected for nitrite assay. As shown in Figure 5 , SdT30 could block NO production in cells stimulated for 1–12 h before inhibitor addition, although it failed to block NO production in cells stimulated for 24 h before exposure. These observations indicate that ODN are active even after initial activation by the stimulants and that inhibition does not require simultaneous addition of inhibitor with the stimulants.



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  		Figure 5. Time-dependent inhibition of macrophage NO production by Ps ODN. J774 cells were stimulated with 5 µg/ml CpG ODN (A) and EC DNA (B) or 0.1 µg/ml LPS (C), and 50 µg/ml SdT30 was added at 1, 3, 6, 12, and 24 h, respectively, after the stimulation. Supernatants were collected at a 48-h time point and assayed for nitrite levels. Each value represents mean of triplicates. Error bars depict the standard deviations. The experiment represents one of three identical experiments.

Influence of ODN backbone chemistry on inhibition of NO production
ODN can be been synthesized as Po or Ps molecules, with the latter containing a sulfur substitution for one of the nonbridging oxygen atoms in the Po backbone. Previous studies have demonstrated that Ps and Po ODN differ in their effects on macrophages as well as other cells [7 , 20 21 22 ]. To determine the influence of ODN backbone structure on their activities, we tested SdA30, SdC30, SdG30, and SdT30 and their Po versions dA30, dC30, dG30, and dT30 in in vitro culture of J774 cells and BMMC. All four Ps ODN inhibited NO production by J774 cells stimulated with CpG ODN, EC DNA, and LPS, and only dG30 among Po ODN inhibited NO production induced by EC DNA (Fig. 6 ). Thus, backbone structure can influence inhibition by ODN.



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  		Figure 6. Influence of backbone structure on ODN inhibition of NO production. J774 cells were pretreated with medium or 50 µg/ml 30-mer Ps ODN (SdA, SdC, SdG, and SdT) or 30-mer Po ODN (dA, dC, dG, and dT) for 1 h, followed by stimulation with 5 µg/ml CpG ODN (A) and EC DNA (B) or 0.1 µg/ml LPS (C) for 48 h. Nitrite levels in the culture supernatants were evaluated with nitrite assay. Each value represents mean of triplicates. Error bars depict the standard deviations. The data shown are from a representative experiment that was repeated with similar results.

Influence of ODN chain length on inhibition of NO production
Previous studies have shown that ODN length can influence its activity [20 , 22 ]. To determine the influence of chain length on inhibitory activity, Ps ODN with 5, 11, 20, and 30 bases of thymidine (dT), respectively, were tested in in vitro culture of BMMC and J774 cells. Because of the differences in molecular weight of the ODN, the concentrations are expressed in molarity instead of micrograms to compare potency. BMMC were treated with 0.08–2 µM dT Ps ODN for 1 h followed by stimulation with 5 µg/ml CpG ODN and EC DNA and 0.1 µg/ml LPS, respectively, for 48 h. SdT30 and SdT20 displayed a dose-dependent inhibition of the NO induction by all three stimulants, and SdT11 and SdT5 were less effective (Fig. 7 ). Similar results were obtained with J774 cells (unpublished results). These results indicate a relationship between ODN size and activity.



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  		Figure 7. Influence of chain length on ODN inhibition of NO production. BMMC were pretreated with 0.08–10 µM dT Ps ODN (SdT5, SdT11, SdT20, and SdT30) for 1 h, followed by stimulation with 5 µ/ml CpG ODN (A) and EC DNA (B) or 0.1 µg/ml LPS (C) for 48 h. Nitrite levels in the culture supernatants were evaluated with nitrite assay. Each value represents mean ± standard deviation of triplicates. The data are representative of three separate experiments.

Effects of Ps ODN on DNA and LPS uptake
Because cell-binding and/or internalization are necessary for cell activation by CpG DNA and LPS [23 24 25 ], we tested whether the Ps ODN can inhibit these processes. For this purpose, we used flow cytometry to determine whether the ODN affected cellular uptake of fluorescently labeled CpG ODN and LPS. J774 cells were incubated with 50 µg/ml ODN 1 h before incubation with 5 µg/ml FITC-labeled CpG ODN or LPS at 37°C for 1 h. Flow cytometric analysis revealed that although the four Ps ODN blocked CpG ODN uptake, they had less effects on LPS uptake (Fig. 8 ).



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  		Figure 8. Effects of Ps ODN on uptake of CpG ODN and LPS. BMMC were pretreated with 50 µg/ml of Ps ODN (SdA, SdC, SdG, and SdT) for 1 h, followed by incubation with 5 µg/ml FITC-labeled CpG ODN or LPS at 37°C for another hour. Internalized CpG ODN or LPS was examined by flow cytometry. The levels of CpG or LPS uptake are represented as percentage of labeled cells for each group. The data are representative of three separate experiments.

To assess the influence of ODN chain length on their inhibition of DNA and LPS uptake, the effects of dT Ps ODN of different lengths on the cellular uptake of fluorescently labeled CpG ODN and LPS were examined using flow cytometry. BMMC were incubated with 10 µM SdT5, SdT11, SdT20, and SdT30 for 1 h followed by incubation with 5 µg/ml FITC-labeled CpG ODN or LPS for another hour. Although the SdT30 and SdT20 blocked the uptake (presented as mean fluorescence intensity) of CpG ODN by more than 60%, SdT11 and SdT5 were much less (17.0% and 17.3%, respectively). In contrast to inhibition of CpG ODN uptake, the dT Ps ODN, including SdT30 and SdT20, showed little effect on the uptake of labeled LPS (less than 20% for all the dT Ps ODN).

Effects of Ps ODN on intracellular signaling pathway activated by CpG DNA
To elucidate the mechanism of action of inhibitory ODN, we began characterization of signal transduction pathways that may be blocked by these compounds. ERK1/2 constitute members of the mitogen-activated protein (MAP) kinase family that are downstream-effector kinases in a signaling pathway activated by a wide range of extracellular stimulants, including LPS and CpG ODN [26 27 28 ]. In the current study, the four Ps ODN inhibited phosphorylation of ERK1/2 in J774 macrophages triggered by CpG ODN to various extents, with SdG as the least-potent inhibitor of the activation of the MAP kinase (Fig. 9 ).



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  		Figure 9. Ps ODN on activation of MAP kinases ERK1/2. J774 cells were pretreated with medium or 50 µg/ml SdA30, SdC30, SdG30, and SdT30 for 1 h and then were stimulated with 10 µg/ml CpG ODN for 30 min. Cell lysates were analyzed for the presence of phosphorylated ERK1/2 by Western blotting. Membranes were exposed using ECL, the images were scanned, and optical density (OD) was determined. Values are presented as the folds of OD readings against that of medium control without Ps ODN treatment. The data shown are from a representative experiment that was repeated with identical results.


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DISCUSSION
 
The results presented herein provide further insight into the immunological effects of Ps ODN and their ability to inhibit as well as stimulate immune responses. Thus, we have shown that four single-base, 30-mer Ps ODN can block NO production by murine macrophages stimulated with bacterial DNA, CpG ODN, or LPS. This inhibition was time- and dose-dependent and was observed even when the ODN were added as long as 12 h after the stimulation. Coupled with studies on the activities of mammalian and viral DNA, these studies suggest that natural and synthetic DNA can down-regulate inflammatory responses and attenuate responses to a variety of bacterial products.

As in the case of immunostimulatory DNA, inhibition by ODN appears related to backbone structure, sequence, and length. In these studies, although dG30 was active as a Ps and Po structure, other ODN showed activity only with the Ps backbone. These observations are consistent with studies on the structure-function relationships of stimulatory ODN that demonstrate the enhanced potency of the Ps backbone [7 , 29 , 30 ]. Ps ODN have the substitution of a sulfur for one of the nonbridging oxygens in the Po backbone. This substitution, which creates a chiral center, affects cell uptake, nuclease resistance, and protein binding [31 ]. Which of these properties is most relevant for enhanced activity is not known.

In addition to establishing the importance of the Ps backbone for activity, these studies demonstrate the effect of ODN length on inhibitory capacity. Among dT ODN tested, SdT30 and SdT20 were much more effective inhibitors of NO production than SdT11 and SdT5 on a molar basis. In a previous study on the effects of Ps ODN containing deoxyguanosine sequences, we also showed that longer compounds are more effective inhibitors of interferon-{gamma} than compounds of 5 and 10 bases [22 ]. Similarly, among stimulatory ODN, longer compounds are more effective in cell activation [23 , 32 ]. These effects may relate to the extent of binding to a target molecule or cell uptake and trafficking. In this regard, we tested single-base ODN as inhibitors; it is possible that mixed-base compounds would produce a different profile with respect to length requirements for activity.

Among mechanisms by which Ps ODN may inhibit responses, we tested effects on cell uptake of CpG ODN and LPS. In these studies, the Ps ODN inhibited CpG ODN but not LPS uptake. These findings could suggest that blockade of uptake can contribute to inhibition, although it is important to note the differences in doses of Ps ODN required for inhibition of uptake compared with those for inhibition of NO production. Although 10 µM SdT30 and SdT20 essentially blocked stimulation by a CpG ODN at 5 µg/ml (approximately 1 µM), they inhibited the CpG ODN uptake by only 60% (see Fig. 7 ). These findings suggest that inhibition of uptake can, at most, account partially for the effects of Ps ODN on macrophage stimulation by ODN.

In other studies on the inhibitory effects of Ps ODN, their effects on uptake of DNA were tested [28 , 33 ]. Although these studies involved compounds that differed in sequence from the ones we studied, a similar conclusion was reached (i.e., inhibition of stimulant uptake does not account fully for inhibition of activation). Furthermore, in our studies, Ps ODN did not affect the uptake of LPS, although they inhibited NO production significantly by this stimulant as well. Although some polyanions may affect the binding of LPS to CD14 [34 ], the Ps ODN we studied do not appear to act via this mechanism.

We note that other studies on inhibitory ODN have failed to demonstrate an effect on stimulation by LPS [28 , 33 ]. The differences between our studies and others may relate to the sequences of the inhibitory ODN tested. We have used single-base compounds, whereas other investigators have used mixed-base compounds. These mixed-based compounds were generally derived from an active ODN, in which a CpG sequence was switched to a GpC sequence. Although such inactive compounds could block activation by CpG DNA, they did not affect activation by LPS. It is possible that the single-base ODN have structural features that allow a broader range of activity. This issue is now under investigation with a larger number of ODN.

In assessing effects of inhibitory ODN, it is important to consider nonspecific, cellular toxicity. As we have shown previously, some of the compounds that inhibit macrophage activation are in fact B-cell mitogens [35 ], arguing against such an activity. Furthermore, we did not observe effects on cell growth or viability as measured using the MTT assay. These findings indicate that the inhibitory activity is not the result of cytotoxicity but rather affects specific signal-transduction pathways.

Other clues for the mechanisms of ODN inhibition come from consideration of their spectrum of action and ability to block activation by diverse stimulants. Despite similarities in macrophage activation by LPS and CpG DNA, these stimulants involve different signaling molecules and pathways, with LPS using Toll-like receptor (TLR) 4 [36 ] and DNA using TLR9 and DNA-dependent protein kinase for initial activation [37 , 38 ]. Subsequent steps such as activation of MAP kinases and activation of nuclear factor-{kappa}B (NF-{kappa}B) are similar [39 ]. These findings suggest that the activity of the Ps ODN occurs distal to the TLRs and proximal to activation of NF-{kappa}B or MAP kinases, as our results with ERK1/2 suggest.

Because the MAP kinase cascade plays an important role in macrophage activation by CpG DNA and LPS [27 , 28 ], we tested whether the Ps ODN can block this pathway of signaling transduction. As our data indicate, the ODN inhibited activation of the MAP kinase family members ERK1/2 in murine macrophages stimulated with CpG ODN, suggesting that blockade of this signaling pathway contributes to the inhibition of NO production. In this regard, although SdG at 50 µg/ml blocked most, if not all, CpG ODN-induced NO production by J774 cells, this molecule inhibited CpG ODN-induced ERK1/2 phosphorylation only partially. This finding suggests that other mechanisms may be involved in inhibition by this compound.

As shown in a variety of systems, the presence of runs of dG in ODN is associated with effects not observed among compounds with extended runs of other bases with Ps or Po backbone [35 , 40 ]. These activities result most likely from the structure of dG compounds and their ability to form stable, higher order conformations [41 ]. Because dG can base-pair by itself, ODN containing contiguous guanosines can form four stranded DNA structures known as quadruplexes or G quartets [42 ]. These compounds, which have increased surface binding, are mitogenic for murine B cells and bone marrow macrophages [35 , 43 ]. Therefore, it is possible that dG30 compounds can inhibit macrophage activation by mechanisms that differ from other ODN. This possibility is under investigation.

In macrophages, the synthesis of NO is catalyzed by iNOS, which is induced in activated cells but not detectable in resting cells [44 ]. Although important aspects of the production of IL-12 are regulated at the transcriptional level [45 ], the regulation of iNOS occurs at multiple levels, including gene transcription, mRNA translation and stability, and protein stability [46 ]. The MAP kinase family is involved in mediating signaling transduction that leads to LPS-induced IL-12 and iNOS in macrophages. However, there is evidence that different members of the MAP kinase family may regulate the induction of these two molecules differentially [47 ]. Therefore, studies are underway to define the pathways that mediate the inhibitory effects of the Ps ODN on the production of NO and IL-12.

Although the activities of inhibitory ODN require further elucidation, the findings presented herein are relevant to the role of self-DNA in inhibiting immune responses and the potential use of ODN as anti-inflammatory and immunosuppressive agents. As we have shown previously, mammalian DNA is poorly immunogenic and can block immune activation by bacterial DNA [7 ]. We have suggested that just as foreign DNA can induce cell activation ("danger"), self-DNA may have the opposite effect—a property that can be called "safety". In this scenario, at sites of cell and tissue death, self-DNA may limit inflammation and other processes that predispose to autoreactivity. A similar anti-inflammatory role for apoptotic cells has also been proposed [48 ].

Our findings also have implications for the possible therapeutic use of ODN to treat clinical conditions associated with increased NO production. Excessive NO occurs in a wide variety of infectious inflammatory and autoimmune diseases [10 , 11 ]. As shown using other inhibitors of NO production, reduction in NO levels can be therapeutic [49 , 50 ]. Ps ODN could find use as such agents because Ps ODN in general have few side-effects when used as antisense agents and immunomostimulators [31 , 51 ]. Therefore, we are exploring the potential of Ps ODN to inhibit inflammation in animal models and test their activities on human cells.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the VA Merit Review, the Arthritis Foundation, and NIH. We thank Dr. J. Brice Weinberg for helpful discussion and comments on the manuscript.

Received September 10, 2001; revised November 26, 2001; accepted November 30, 2001.


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