No abstract

Human gingival fibroblasts (HGFs), a predominant cell type in tooth-supporting structure, are presently recognized for their active role in the innate immune response. They produce a variety of inflammatory cytokines in response to microbial components such as LPS from the key periodontal pathogen, Porphyromonas gingivalis. In this study, we demonstrated that HGFs expressed mRNA of TLRs 1, 2, 3, 4, 5, 6, and 9, but not TLRs 7, 8, and 10. Stimulation of HGFs with highly purified TLR2 ligand (P. gingivalis LPS), TLR3 ligand (poly(I:C)), TLR4 ligand (Escherichia coli LPS), and TLR5 ligand (Salmonella typhimurium flagellin) led to expression of IL-8 and IDO. A potent TLR 9 ligand, CpG oligodeoxynucleotide 2006 had no effect, although HGFs showed a detectable TLR9 mRNA expression. No significant enhancement on IL-8 or IDO expression was observed when HGFs were stimulated with various combinations of TLR ligands. Surprisingly, the TLR9 ligand CpG oligodeoxynucleotide 2006 was able to specifically inhibit poly(I:C)-induced IL-8 and IDO expression. TNF-α enhanced TLR ligand-induced IL-8 production in HGFs, whereas IFN-γ enhanced TLR ligand-induced IDO expression. HGF production of IDO in response to P. gingivalis LPS, IFN-γ, or the two in combination inhibited T cell proliferation in MLRs. The observed T cell inhibition could be reversed by addition of either 1-methyl-dl-tryptophan or l-tryptophan. Our results suggest an important role of HGFs not only in orchestrating the innate immune response, but also in dampening potentially harmful hyperactive inflammation in periodontal tissue.

SUMMARYPeriodontitis is an inflammatory bone disease caused by Gram-negative anaerobic bacteria, but the precise mechanism of bone destruction remains unknown. Activated T lymphocytes secrete receptor activator of NF-k B ligand (RANKL) and support the differentiation of monocytes into mature osteoclasts. The purpose of this study was to examine the expression of RANKL and its inhibitor, osteoprotegerin (OPG), in inflamed gingival tissue and to clarify the role of human gingival fibroblasts (HGFs) in osteoclastogenesis regulated by RANKL. HGFs and gingival mononuclear cells (GMCs) were obtained from chronic periodontitis patients during routine periodontal surgery. Expression of OPG and RANKL mRNA in gingival tissue and HGFs was examined with RT-PCR. OPG production was measured using ELISA. Expression of RANKL, CD4, CD8 and CD69 on GMCs was determined by flow-cytometry using RANK-Fc fusion protein and the respective monoclonal antibodies. Osteoclastogenesis by RANKL was assayed by counting the number of tartarate-resistant acid phosphatase (TRAP)-positive cells after culturing human peripheral blood monocytes with recombinant human RANKL and macrophage-colony stimulating factor (M-CSF) for 10 days. OPG and RANKL mRNA were expressed in 80% (16/20) and 25% (5/20) of periodontitis lesions, respectively. OPG, but not RANKL, mRNA was expressed within HGFs. OPG mRNA expression and production by HGFs was augmented by LPS stimulation. All GMC samples expressed CD69, and two of five GMC samples expressed RANKL. The culture supernatant of LPS-stimulated gingival fibroblasts significantly reduced the number of TRAP positive cells generated by culturing monocytes with RANKL and M-CSF. The present study suggests that LPS-stimulated HGFs inhibit monocyte differentiation into osteoclasts through the production of OPG.

Human gingival epithelial cells play a critical role in orchestrating the innate immune responses of periodontal tissue via TLR signalling. Our results represent the first demonstration that CSE can modulate HGEC function by suppressing hBD-2 and enhancing IL-8 production, and this may be, in part, a possible mechanism which promotes periodontal disease.

The presence of inflammatory infiltrates with B cells, specifically plasma cells, is the hallmark of periodontitis lesions. The composition of these infiltrates in various stages of homeostasis and disease development is not well documented. Human tissue biopsies from sites with gingival health (n = 29), gingivitis (n = 8), and periodontitis (n = 21) as well as gingival tissue after treated periodontitis (n = 6) were obtained and analyzed for their composition of B cell subsets. Ag specificity, Ig secretion, and expression of receptor activator of NF-κB ligand and granzyme B were performed. Although most of the B cell subsets in healthy gingiva and gingivitis tissues were CD19+CD27+CD38− memory B cells, the major B cell component in periodontitis was CD19+CD27+CD38+CD138+HLA-DRlow plasma cells, not plasmablasts. Plasma cell aggregates were observed at the base of the periodontal pocket and scattered throughout the gingiva, especially apically toward the advancing front of the lesion. High expression of CXCL12, a proliferation-inducing ligand, B cell–activating factor, IL-10, IL-6, and IL-21 molecules involved in local B cell responses was detected in both gingivitis and periodontitis tissues. Periodontitis tissue plasma cells mainly secreted IgG specific to periodontal pathogens and also expressed receptor activator of NF-κB ligand, a bone resorption cytokine. Memory B cells resided in the connective tissue subjacent to the junctional epithelium in healthy gingiva. This suggested a role of memory B cells in maintaining periodontal homeostasis.