Geranylgeranylacetone ameliorates inflammatory response to lipopolysaccharide (LPS) in murine macrophages: inhibition of LPS binding to the cell surface

Abstract

We investigated whether pretreatment with geranylgeranylacetone (GGA), a potent heat shock protein (HSP) inducer, could inhibit proinflammatory cytokine liberation and nitric oxide (NO) production in lipopolysaccharide (LPS)-treated murine macrophages. The levels of NO and tumor necrosis factor-alpha (TNF-alpha) released from murine macrophage RAW 264 cells were increased dose- and time-dependently following treatment with LPS (1 microg/ml). GGA (80 microM) treatment 2 h before LPS addition significantly suppressed TNF-alpha and NO productions at 12 h and 24 h after LPS, respectively, indicating that GGA inhibits activation of macrophages. However, replacement by fresh culture medium before LPS treatment abolished the inhibitory effect of GGA on NO production in LPS-treated cells. Furthermore, GGA inhibited both HSP70 and inducible NO synthase expressions induced by LPS treatment despite an HSP inducer. When it was examined whether GGA interacts with LPS and/or affects expression of Toll-like receptor 4 (TLR4) and CD14 on the cell surface, GGA inhibited the binding of LPS to the cell surface, while GGA did not affect TLR4 and CD14 expressions. These results indicate that GGA suppresses the binding of LPS to the cell surface of macrophages, resulting in inhibiting signal transduction downstream of TLR4.

Keywords: Geranylgeranylacetone; lipopolysaccharide; macrophage; nitric oxide; tumor necrosis factor-α.

Fig. 1

Dose-dependent production of NO (A) and TNF-α (B) in RAW 264 cells by LPS treatment. Cells were incubated with LPS (10, 100, or 1000 ng/ml) for 24 h (A) or 12 h (B) at 37°C under a 5% CO₂ and 95% air atmosphere. The levels of NO and TNF-α were measured as described in Materials and Methods. Data points represent the means ± SE (n = 3). ^ap<0.01 vs 0 ng/ml, ^bp<0.01 vs 10 ng/ml, ^cp<0.01 vs 100 ng/ml in (A). ^ap<0.01 vs 0 ng/ml, ^bp<0.05 vs 10 ng/ml, ^cp<0.01 vs 10 ng/ml, ^dp<0.01 vs 100 ng/ml in (B).

Fig. 2

Time-dependent production of NO (A) and TNF-α (B) in RAW 264 cells by LPS treatment. Cells were incubated with LPS (1 µg/ml) for 4, 8, 12 or 24 h at 37°C under a 5% CO₂ and 95% air atmosphere. The levels of NO and TNF-α were measured as described in Materials and Methods. Data points represent the means ± SE (n = 3). ^ap<0.01 vs 0 h, ^bp<0.01 vs 12 h.

Fig. 3

Inhibitory effect of GGA on LPS-mediated production of NO (A) and TNF-α (B). Cells were incubated in the presence or absence of LPS (1 µg/ml) for 24 h (A) or 12 h (B) at 37°C under a 5% CO₂ and 95% air atmosphere. GGA (0, 20, 40, or 80 µM) was added 2 h before the addition of LPS. The levels of NO and TNF-α were measured as described in Materials and Methods. Data points represent the means ± SE (n = 3). ^ap<0.05, ^bp<0.01 vs LPS alone-treated cells.

Fig. 4

Effect of GGA on LPS-mediated iNOS induction in RAW 264 cells. Cells were incubated in the presence or absence of LPS (1 µg/ml) for 24 h at 37°C under a 5% CO₂ and 95% air atmosphere. GGA (0, 20, 40, or 80 µM) was added 2 h before the addition of LPS. iNOS level was analyzed by Western blotting, quantified densitometrically and expressed as relative density to control cells. Data points represent the means ± SE (n = 3). ^ap<0.01 vs control, ^bp<0.01 vs LPS alone, ^cp<0.05 vs LPS alone, ^dp<0.05 vs LPS (1 µg/ml) + GGA (80 µM).

Fig. 5

Effect of GGA on HSP70 induction in RAW 264 cells. (A) Cells were incubated in the presence or absence of LPS (1 µg/ml) for 24 h at 37°C under a 5% CO₂ and 95% air atmosphere. GGA (0, 20, 40, or 80 µM) was added 2 h before the addition of LPS. (B) Cells were incubated in the presence or absence of GGA (80 µM) for 2 h at 37°C under a 5% CO₂ and 95% air atmosphere. HSP70 level was analyzed by Western blotting, quantified densitometrically and expressed as relative density to control cells. Data points represent the means ± SE (n = 3). ^ap<0.01 vs control, ^bp<0.01 vs LPS alone-treated cells.

Fig. 6

Effect of replacement by fresh culture medium before LPS administration on inhibition by GGA of NO production in LPS-treated RAW 264 cells. GGA (0, 20, 40, or 80 µM) was added to the medium 2 h before LPS administration and subsequently culture medium was replaced by fresh one without GGA just before LPS addition. Cells were further incubated in the presence of LPS (1 µg/ml) for 24 h at 37°C under a 5% CO₂ and 95% air atmosphere. The level of NO in the medium was measured as described in Materials and Methods. Data points represent the means ± SE (n = 3).

Fig. 7

Effects of GGA on binding of LPS to the cell surface in RAW 264 cells. Cells were incubated with 80 µM GGA for 2 h at 37°C and for further 30 min after addition of LPS (1 µg/ml). Cells were washed, and stained with anti-LPS antibody, followed by goat anti-mouse IgG-FITC. The LPS fluorescence resulting from LPS binding to cells was measured by using Epics cytofluorometer. Representative data from three separate experiments are shown.

Fig. 8

Inhibitory effect of GGA on lipid A-mediated NO production in RAW 264 cells. Cells were incubated in the presence or absence of Lipid A (LA) (100 ng/ml) for 24 h at 37°C under a 5% CO₂ and 95% air atmosphere. GGA (80 µM) was added 2 h before the addition of LA. NO level was measured as described in Materials and Methods. Data points represent the means ± SE (n = 3). ^ap<0.01 vs LA alone-treated cells.

Fig. 9

TNF-α release from RAW 264 cells after LPS stimulation in the presence or absence of serum and its inhibition by GGA. Cells were incubated with LPS (1 µg/ml) in the presence (closed column) or absence (open column) of serum for 4 h at 37°C under a 5% CO₂ and 95% air atmosphere. GGA (80 µM) was added 2 h before the addition of LPS. TNF-α level was measured as described in Materials and Methods. Data points represent the means ± SE (n = 3). ^ap<0.01 vs LPS alone-treated cells in the presence of serum. ^bp<0.05 vs LPS alone-treated cells in the absence of serum.