Article Figures & Data
Figures
- FIGURE 1.
Induction of NO by E. coli DNA, S. aureus DNA, synthetic ODNs, and LPS. RAW 264.7 macrophages were subjected to various stimuli for 20 h before culture supernatants were taken for nitrite assay. A, Macrophages were stimulated with various concentrations of E. coli DNA (○), S. aureus DNA (▴), and CpG-containing T3 ODNs (▵) in the presence of 0.3 ng/ml of LPS. Controls (not completely listed in the figure due to space limit) include various concentrations of salmon sperm DNA plus 0.3 ng/ml of LPS, non-CpG C3 ODN plus LPS, DNase I-treated E. coli DNA plus LPS, DNase I-treated S. aureus DNA plus LPS, E. coli DNA, S. aureus DNA, salmon sperm DNA, C3 ODNs, and DNase I-digested E. coli DNA or S. aureus DNA. The nitrite levels induced by controls were <0.5 μM. ∗, Concentrations of T3 and C3 are expresses as micromolar concentrations. B, Macrophages were stimulated with different concentrations of LPS (•), LPS plus 1 μg/ml of E. coli DNA (○), LPS plus DNase I-treated E. coli DNA (▵), or LPS plus salmon sperm DNA (▴).
- FIGURE 2.
Effects of neutralizing Abs against IFN on E. coli DNA plus LPS-induced NO production. RAW 264.7 macrophages were treated as indicated in the figure. In samples involving neutralizing Abs, anti-IFN-αβ (0.3 μg/ml), anti-IFN-γ (1 μg/ml), or a combination of both were incubated with macrophages for 30 min before the addition of stimuli. IFN-γ and E. coli DNA were used at 100 U/ml and 3 μg/ml, respectively. LPS was used at 0.3–100 ng/ml. The data indicated with an asterisk were obtained from C57BL/6 peritoneal macrophages exclusively to demonstrate that anti-αβ is biologically functional. Control Abs showed no effect on NO induction by E. coli DNA plus LPS (data not shown).
- FIGURE 3.
Induction of mouse iNOS mRNA by E. coli DNA and LPS. RAW 264.7 macrophages were incubated with culture medium alone, medium containing 1 μg/ml of E. coli DNA, DNase I-treated E. coli DNA, or salmon sperm DNA either in the absence or in the presence of 0.3 ng/ml of LPS for 2.5 h before RNA isolation for RT-PCR analysis (30 PCR cycles), as described in Materials and Methods. The top panel of the figure shows the densitometric scanning data from the amplified iNOS mRNA normalized against β-actin controls.
- FIGURE 4.
Effects of E. coli DNA and LPS pretreatment on induction of NO and iNOS mRNA in macrophages in response to E. coli DNA plus LPS. A, RAW 264.7 macrophages were preincubated with 3 μg/ml of E. coli DNA or 0.3 ng/ml of LPS for various amounts of time as shown in the figure. They were then washed twice with culture medium and continually incubated with 3.0 μg/ml of E. coli DNA plus 0.3 ng/ml of LPS for 20 h before assaying nitrite from the supernatants. B, RAW 264.7 macrophages were either unpretreated or pretreated with 3 μg/ml of E. coli DNA or 0.3 ng/ml of LPS for 24 h before exposure to E. coli DNA (3 μg/ml) plus LPS (0.3 ng/ml) for 2 h. Total RNA was then isolated for RT-PCR analysis of iNOS mRNA as described in Fig. 3, except that 24 PCR cycles were used in this experiment.
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