High mobility group box 1 (HMGB1), known widely as a nuclear protein, acts as a proinflammatory cytokine extracellularly. HMGB1 can be actively secreted by monocytes stimulated with lipopolysaccharides (LPS), TNF-a or IL-1. It can also diffuse passively from necrotic cells. In periodontitis, HMGB1 was strongly detected in the gingival crevicular fluid and gingival epithelial cells were able to secrete it after being stimulated with TNF-a, suggesting a possible contribution of HMGB1 in the progression of periodontitis. In this study, we investigated whether human gingival fibroblasts (HGF) can produce HMGB1 after cell stress such as stimulation with LPS from periodontopathogens and necrotic and apoptotic cell death.
Methods:
HGF from healthy periodontal tissue were cultured and stimulated with LPS from Aggregatibacter actinomycetemcomitans (A.a), Porphyromonas gingivalis (P.g), and Escherichia coli (E.coli). We also initiated apoptotic and necrotic cell death in HGF. The amounts of HMGB1 released from stimulated or dying cells were measured by ELISA. Immunocytochemical staining against HMGB1 was performed. The cell viability and the activity of lactate dehydrogenase (LDH) were tested.
Results:
A significantly higher amount of HMGB1 diffused from necrotic and apoptotic fibroblasts compared to control. LPS from A.a, P.g and E.coli significantly induced the production of HMGB1 in a dose and time dependent manner. After 6 hours of stimulation with A.a LPS, HMGB1 was visible in the cytoplasm of cells. However, its location was mainly nuclear after 24 hours. The cytotoxicity assays showed that the amounts of LPS used were not cytotoxic indicating that HMGB1 can be actively secreted by stimulated cells.
Conclusions:
LPS from two major periodontopathogens A.a and P.g induced HMGB1 secretion from HGF. Apoptotic and necrotic cell deaths resulted as well in the enhancement of HMGB1. Our results suggest that HMGB1 may be an important factor in the aggravation of periodontal disease.