Nicotine, LPS, TNF?, and EGCG Differentially Affect Interleukin Secretion

Shango, Jennifer, Michael Byars, and Michelle Wheater

Nuclear factor kappa B (NFkB), a transcription factor that plays a central role in inflammatory mechanisms, can be activated by pro-inflammatory mediators including tumor necrosis factor alpha (TNFa). We have previously shown that epigallocatechin gallate (EGCG), a major catechin component of green tea, suppressed TNFa-induced interleukin secretion in nicotine-treated human oral epithelial cells. Objective: To determine if EGCG inhibits TNFa-induced phosphorylation and activation of NFkB in nicotine-treated cultured human oral epithelial cells. Methods: Commercially available human oral epithelial cells (ScienCell, Carlsbad, CA) were treated for 24 hours with 0.1 mM nicotine, then pre-treated or not with 10 mg/ml EGCG. Cells were additionally treated with 10 mg/ml TNFa for 10, 20, or 30 minutes. The cells were then processed in situ to determine levels of NFkB serine468 and serine536 phosphorylation as well as total levels of NFkB (FACE NFkB p65 profiler in-cell phosphorylation ELISA, Active Motif, Carlsbad, CA). Data were calculated as relative percentages of total cellular NFkB phosphorylation. Whole-cell extracts of similarly treated cells subsequently were assayed for NFkB subunit activation (TransAM NFkB Family ELISA). Statistical analysis was completed using ANOVA and Tukey post-test with probability set at p < 0.05. Results: TNFa-induced stimulation of NFkB phosphorylation and activation was similar in nicotine- and non-nicotine-treated cells. Serine468 levels were increased in cells treated with nicotine, EGCG and TNFa compared to cells treated with nicotine and TNFa. In contrast, at 30 minutes of TNFa stimulation, serine536 phosphorylation was reduced in cells treated with EGCG. Of the five NFkB family members analyzed (RelA/p65, p50, c-Rel, p52, and RelB), only p65 and p50 showed effects of EGCG treatment. EGCG significantly inhibited the activation of p65 and p50 in nicotine and TNFa treated oral epithelial cells. Conclusions: EGCG appears to suppress the activation of the p65/p50 heterodimer of NFkB in nicotine and TNFa-stimulated cells. Suppression of NFkB may explain, at least in part, why EGCG can inhibit the secretion of pro-inflammatory interleukins such as IL-8. Continued examination of the cellular function of EGCG may lead to its use as a natural inhibitor of oral inflammation in patients who use nicotine.