Advanced glycation end-products increase lipocalin 2 expression in human oral epithelial cells.

BACKGROUND AND OBJECTIVES: Diabetes mellitus (DM), a risk factor of periodontal diseases, exacerbates the pathological condition of periodontitis. A major factor for DM complications is advanced glycation end-products (AGEs) that accumulate in periodontal tissues and cause inflammatory events. Lipocalin 2 (LCN2) is an antimicrobial peptide and inflammation-related factor, and LCN2 levels increase in DM. In this study, the effects of AGEs and lipopolysaccharide of Porphyromonas gingivalis (P g-LPS) on LCN2 expression in human oral epithelial cells (TR146 cells) and the role of secreted LCN2 in periodontitis with DM were investigated. MATERIAL AND METHODS: TR146 cells were cultured with AGEs (AGE2) and control BSA and cell viability was estimated, or with P g-LPS. Conditioned medium and cell lysates were prepared from cultures of epithelial cells and used for Western blotting and ELISA to analyze LCN2, RAGE, IL-6, MAPK, and NF-κB. RNA was isolated from AGE-treated TR146 cells and differentiated HL-60 (D-HL-60) cells and used for quantitative real-time PCR to examine the expression of LCN2 and interleukin-6 (IL-6) mRNAs. RAGE- and LCN2-siRNAs (siRAGE, siLCN2) were transfected into epithelial cells, and AGE-induced LCN2 expression was investigated. D-HL-60 cells were co-cultured with TR146 cells that were transfected with siLCN2 and treated with AGEs, and IL-6 mRNA expression in D-HL-60 cells and cell migration was investigated. RESULTS: AGEs increased the expression levels of LCN2 and IL-6 in oral epithelial cells. siRAGE and a neutralizing antibody for RAGE inhibited AGE-induced LCN2 expression. AGEs stimulated the phosphorylation of ERK, p38, and NF-κB in epithelial cells, and their inhibitors suppressed AGE-induced LCN2 expression. In contrast, P g-LPS did not show a significant increase in LCN2 level in TR146 cells that expressed Toll-like receptor 2. In co-culture experiments, AGE-induced LCN2 inhibited IL-6 mRNA expression in D-HL-60 cells, and LCN2 knockdown in epithelial cells suppressed HL-60 cell migration. CONCLUSION: These results suggested that AGEs increase LCN2 expression via RAGE, MAPK, and NF-κB signaling pathways in oral epithelial cells, and secreted LCN2 may influence the pathological condition of periodontitis with DM.

High Glucose-Mediated Cytokine Regulation in Gingival Fibroblasts and THP-1 Macrophage: a Possible Mechanism of Severe Periodontitis with Diabetes.

BACKGROUND/AIMS: Diabetic patients are susceptible to severe periodontitis, but the precise mechanism is not fully understood. Aim of this study was to explore the biological pathogenesis of severe periodontitis in diabetic patients focusing on the crosstalk of human gingival fibroblasts (HGFs) and macrophages. METHODS: A total of 70 periodontitis patients with or without diabetes mellitus (DM) were enrolled, and the statistical relationships of diabetic conditions to the periodontal inflammatory parameters were examined by cross-sectional study. In in vitro study, HGFs cell line CRL-2014® (ATCC) and differentiated THP-1 macrophages were cultured with normal glucose (NG: 5.5 mM) or high glucose (HG: 25 mM) condition, and treated with indicated inflammatory factors such as calprotectin (CPT), interleukin (IL)-1ß and IL-6. To examine the effects of HG on soluble IL-6 receptor (sIL-6R) production in THP-1 macrophages, the supernatants were collected and the sIL-6R levels were measured by ELISA. To examine the effects of HG on IL-1ß or IL-6-induced matrix metalloproteinase (MMPs) production in HGFs, the supernatants were collected. Levels of MMP-1 and tissue inhibitor of MMP-1 (TIMP-1) were measured by ELISA. Finally, after conditioned medium (CM) from THP-1 macrophages cultured with NG or HG conditions was collected, HGFs were treated with the CM. The supernatants were collected 24 hours later and the levels of MMP-1 and TIMP-1 were measured. To examine the specific effects of IL-1ß contained in CM on MMP-1 and TIMP-1 production in HGFs, IL-1 receptor antagonist (IL-1ra) was used. RESULTS: There were statistical correlation between IL-1ß and sIL-6R levels in gingival crevicular fluid (GCF) and HbA1c in periodontitis patients with DM (IL-1ß: P=0.035, sIL-6R: P=0.040). HG and CPT significantly induced sIL-6R production in THP-1 macrophages. HG significantly enhanced IL-1ß or IL-6/sIL-6R-induced MMP-1 production in HGFs. The increase of MMP-1 by both IL-1ß and IL-6/sIL-6R was significantly inhibited by specific ERK or IκB inhibitors. Corresponding to the regulation of MMP-1 production, HG condition increased the phosphorylation of p44/42 MAPK and IκBα in HGFs treated with IL-1ß or IL-6/sIL-6R. Finally, MMP-1 production in HGFs cultured with HG increased significantly by CM from THP-1 macrophages cultured with HG. The induction of MMP-1 by the CM from THP-1 macrophages cultured with HG was significantly inhibited by dose dependent of IL-1ra in HGFs cultured with HG. CONCLUSION: Diabetic conditions such as HG induce IL-1ß and sIL-6R production from macrophages in inflammatory periodontal tissues and may exacerbate the periodontitis synergistically via MMP-1 production from HGFs.

Clinical significance of GCF sIL-6R and calprotectin to evaluate the periodontal inflammation.

Background Periodontitis is an inflammatory disease. The aim of this study was to investigate whether the soluble form of interleukin-6 receptor (sIL-6R) and calprotectin concentrations in gingival crevicular fluid are useful biomarkers in the evaluation of periodontitis. Methods First, a cross-sectional study was performed. A total of 34 periodontitis patients were enrolled and the gingival crevicular fluid samples were collected from the healthy and inflamed sites of periodontal pockets in each patient. The relationship between periodontal condition and gingival crevicular fluid sIL-6R and calprotectin concentrations was analysed statistically. The cut-off values of gingival crevicular fluid sIL-6R and calprotectin concentrations for the evaluation of periodontitis were determined using a receiver operating characteristic curve. Next, by using enzyme-linked immunosorbent assay, it was examined whether calprotectin induces sIL-6R production in THP-1 macrophages. Results Both gingival crevicular fluid sIL-6R and calprotectin concentrations were significantly higher in the inflamed sites than in the healthy sites ( P < 0.0001). The cut-off values of gingival crevicular fluid sIL-6R and calprotectin concentrations for the evaluation of periodontal inflammation were as follows: sIL-6R: 43.5 pg/site; calprotectin: 134.3 ng/site. In the in vitro study, calprotectin significantly induced sIL-6R production in THP-1 macrophages ( P < 0.01). Conclusions Both gingival crevicular fluid sIL-6R and calprotectin concentrations are significant biomarkers in the evaluation of periodontal inflammation.

Gene polymorphism of ß-defensin-1 is associated with susceptibility to periodontitis in Japanese.

Periodontitis is a multifactorial disease associated with genetic and environmental factors. Single-nucleotide polymorphisms (SNPs) are associated with susceptibility to common diseases such as diabetes and periodontitis. Although the oral cavity is exposed to various organisms, the conditions are well controlled by innate and acquired immune systems. Antimicrobial peptides (AMPs) play an important role in the innate immune system; however, the association of AMP-SNPs with periodontitis has not been fully elucidated. This study investigated the relationship between AMP-SNPs and periodontitis in Japanese. One hundred and five Japanese subjects were recruited, which included patients with aggressive, severe, moderate and mild periodontitis, and age-matched healthy controls. Genomic DNA was isolated from peripheral blood and genotypes of SNPs of ß-defensin-1 and lactoferrin genes (DEFB1: rs1799946, rs1800972 and rs11362; and LTF: rs1126478) were investigated using the PCR-Invader assay. Protein level of AMPs in gingival crevicular fluid (GCF) was quantified by ELISA. Case-control studies revealed that the -44 CC genotype of DEFB1 (rs1800972) was associated with periodontitis (OR 2.51), particularly with severe chronic periodontitis (OR 4.15) and with combined severe and moderate chronic periodontitis (OR 4.04). No statistical differences were found in other genotypes. The ß-defensin-1 concentrations in GCF were significantly lower in subjects with the -44 CC genotype of DEFB1 than in those without this genotype. No significant differences between GCF concentrations of AMPs and other genotypes were detected. The -44 CC genotype of the ß-defensin-1 gene (DEFB1 rs1800972) may be associated with susceptibility to chronic periodontitis in Japanese.