Suppression of Cytokine Production in Mouse Macrophages by TEGDMA

Methods: Murine macrophages (RAW264.7) were exposed to increasing TEGDMA concentrations (0.125-2.0 mmol/l) for 6, 24 and 48h both in the presence and absence of LPS (lipopolysaccharide). LPS from E. coli (25 µg/ml) was used as a positive control, and culture medium was the negative control. Cytotoxicity of TEGDMA and LPS/TEGDMA was detected photometrically using the crystal violet assay. The release of the cytokines TNF-a, IL-6, and IL-10 from cell cultures was determined by enzyme-linked immunosorbent assay (ELISA). Statistical analyses were performed using the Mann-Whitney-U-test (p≤0.05).

Results: LPS alone strongly induced secretion of all cytokines after the various exposure periods. Amounts of TNF-a, IL-6, and IL-10 were increased 200-fold, 600-fold, and 15-fold compared to untreated controls after a 6h exposure. The amounts of TNF-a and IL-6 were further increased about 2-fold after 24h and 48h, and IL-10 levels increased about 100-fold. TEGDMA alone had no influence on cytokine secretion. However, LPS-stimulated cytokine secretion was inhibited by increasing TEGDMA concentrations at all time points. The addition of 0.125mM TEGDMA to LPS-stimulated cell cultures reduced TNF-a, IL-6, and IL-10 to 92%, 87%, and 75%, while 2.0mM TEGDMA even decreased these values to 0.7%, 2.4%, and 1.2% after 6h. Inhibition of cytokine release by TEGDMA was even higher after 24 and 48h. Furthermore, complementary crystal violet staining showed that reduction of cytokine secretion by TEGDMA was only partly caused by reduced cell numbers.

Conclusion: TEGDMA has the ability to suppress LPS-stimulated responses of the cells of the innate immune system.