CpG ODN activates NO and iNOS production in mouse macrophage cell line (RAW 264.7)

Abstract

Synthetic CpG containing oligodeoxynucleotide (CpG ODN) is recognized for its ability to activate cells to produce several cytokines, such as IL-12 and TNF-alpha. In the present study we have demonstrated that CpG ODN 1826, known for its immunostimulatory activity in the mouse system could, by itself, induce nitric oxide (NO) and inducible nitric oxide synthase (iNOS) production from mouse macrophage cell line (RAW 264.7). Neutralizing antibody against TNF-alpha was not able to inhibit NO or iNOS production from the CpG ODN 1826-activated macrophages, suggesting that although the TNF-alpha was also produced by CpG ODN-activated macrophages, the production of iNOS was not mediated through TNF-alpha. Although both CpG ODN 1826 and lipopolysaccharide (LPS) were able to stimulate NO and iNOS production, the exposure time required for maximum production of NO and iNOS for the CpG ODN 1826-activated macrophages was significantly longer than those activated with LPS. These results were due probably to a delay of NF-kappaB translocation, as indicated by the delay of IkappaBalpha degradation. Moreover, the fact that chloroquine abolished NO and iNOS production from the cells treated with CpG ODN 1826 but not from those treated with LPS suggested that the induction of NO and iNOS production from the cells stimulated with CpG ODN (1826) also required endosomal maturation/acidification.

Fig. 1

NO production by mouse macrophages stimulated with CpG ODN. RAW 264·7 macrophages (1 × 10⁶ cells/well) were incubated with different concentrations of CpG ODN 1826 (•) or 1982 (○) for 24 h. The supernatant was analysed for nitrite by Griess reaction. Data represent the mean and s.d. of three separate experiments, each carried out in triplicate.

Fig. 2

Specificity of CpG ODN sequence in stimulating NO production. RAW 264·7 macrophages (1 × 10⁶ cells/well) were incubated with of CpG ODN 1826, 1982, 1585, 2006 or E. coli DNA at a final concentration of 1 μg/ml for 24 h. Nitrite in the supernatant was determined after 24h of incubation. Unstimulated cells served as control. Data represent means and s.d. of three separate experiments each carried out in triplicate.

Fig. 3

Failure of polymyxin B to inhibit NO production by CpG ODN 1826-activated macrophages. RAW 264·7 macrophages (1 × 10⁶ cells/well) were activated with 1 μg/ml of CpG 1826 (•) or 10 ng/ml of E. coli LPS (○) in the presence of different concentrations of polymyxin B (PxB) for 24 h. The supernatant was analysed for nitrite (a) while the cells were used in the immunoblot for iNOS (b). Control is unstimulated cells. Data (a) represent mean and s.d. of five separate experiments, each carried out in triplicate.

Fig. 4

NO and iNOS production by mouse macrophages activated with CpG ODN 1826 is independent of TNF-α production. RAW 264·7 macrophages (1 × 10⁶ cells/well) were stimulated with CpG ODN 1826 (1 μg/ml) in the presence or absence of neutralizing antibody (10 μg/ml) against TNF-α. After 24 h, the supernatant was analysed for nitrite (a) and the cells were analysed for iNOS by immunoblotting (b). The ability of antibody to neutralize biological activity of TNF-α was also determined by cytolysis of mouse fibroblasts (L929) (c). Data (a and c) represent mean and s.d. of three separate experiments, each carried out in duplicate. *P < 0·05 according to Student’s t-test.

Fig. 5

Time required for maximal stimulation of NO in CpG ODN 1826 or LPS activated macrophages. RAW 264·7 macrophages (1 × 10⁶ cells/well) were activated with CpG ODN 1826 (1 μg/ml) (•) or LPS (10 ng/ml) (○) for different time intervals. At times indicated, the supernatant was discarded, washed three times with PBS before incubating further in fresh media. At 24 h, the supernatant was analysed for nitrite (a). Data represent mean and s.d. for three separated experiments, each carried out in duplicate. The cells were analysed for iNOS by immunoblotting (b).

Fig. 6

Kinetics of IκBα degradation in the CpG ODN 1826 activated macrophages. RAW 264·7 macrophages (1 × 10⁶ cells/well) were activated with 1μg/ml of CpG ODN 1826 (a) or 10 ng/ml of LPS for different time intervals before the cells were lysed in lysis buffer and analysed for IκBα by immunoblotting.

Fig. 7

Endosomal maturation/acidification is required for CpG ODN 1826 to activate NO and iNOS production. RAW 264·7 macrophages (1 × 10⁶ cells/well) were preincubated with or without chloroquine (2·5 μg/ml) for 2 h before activated with CpG ODN 1826 (1 μg/ml) or E. coli LPS (10 ng/ml). After 24 h, the supernatant was analysed for nitrite (a) and the cells were analysed for iNOS (b). Control represents the unstimulated cells. Data (a) represent mean and s.d. of three separate experiments, each carried out in duplicate. *P < 0·05 according to Student’s t-test.