Cellular senescence with SASP in periodontal ligament cells triggers inflammation in aging periodontal tissue.

The direct cause of periodontitis is periodontopathic bacteria, while various environmental factors affect the severity of periodontitis. Previous epidemiological studies have shown positive correlations between aging and periodontitis. However, whether and how aging is linked to periodontal health and disease in biological processes is poorly understood. Aging induces pathological alterations in organs, which promotes systemic senescence associated with age-related disease. Recently, it has become evident that senescence at the cellular level, cellular senescence, is a cause of chronic diseases through production of various secretory factors including proinflammatory cytokines, chemokines, and matrix metalloproteinases (MMPs), which is referred to the senescence-associated secretory phenotype (SASP). In this study, we examined the pathological roles of cellular senescence in periodontitis. We found localization of senescent cells in periodontal tissue, particularly the periodontal ligament (PDL), in aged mice. Senescent human PDL (HPDL) cells showed irreversible cell cycle arrest and SASP-like phenotypes in vitro. Additionally, we observed age-dependent upregulation of microRNA (miR)-34a in HPDL cells. These results suggest that chronic periodontitis is mediated by senescent PDL cells that exacerbate inflammation and destruction of periodontal tissues through production of SASP proteins. Thus, miR-34a and senescent PDL cells might be promising therapeutic targets for periodontitis in elderly people.

Long-term exposure to cigarette smoke influences characteristics in human gingival fibroblasts.

BACKGROUND: Periodontal disease is a chronic inflammatory disease caused by periodontopathic bacteria accumulated in the gingival sulcus and periodontal pocket. Cigarette smoking is a well-established risk factor for periodontal disease, and periodontal tissues in smokers are chronically exposed to cigarette smoke on a long-term basis. OBJECTIVE: In this study, we investigated the effects of long-term exposure to nicotine or cigarette smoke condensate (CSC) on cellular functions of human gingival fibroblasts (HGFs). METHODS: In vitro-maintained HGFs were divided into two groups. The HGFs of the short-term and the long-term culture groups were cultured for 4 and 25 days, respectively, in the presence or absence of nicotine, which is one of the main components of cigarette smoke, or CSC. The cellular proliferation and migration capacities of HGFs exposed to nicotine or CSC were evaluated by WST-1 and wound healing assays. The effects of exposure to nicotine or CSC on the expression of various extracellular matrix (ECM) components, inflammatory cytokines, and senescence-related genes were examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The cellular senescence of HGFs exposed to nicotine or CSC was detected by the senescence-associated ß-galactosidase (SA-ß-gal) assay. To explore the senescence-associated microRNA (miRNA), we extracted miRNA from the HGFs and the expression profiles were examined by miRNA array. RESULTS: In short-term culture, no significant changes were observed. Long-term exposure of HGFs to nicotine or CSC significantly suppressed their cellular proliferation and migration and upregulated type Ⅰ collagen, type Ⅲ collagen, interleukin (IL)-6, IL-8, p16, p21, and p53 mRNA expression, and IL-6 and IL-8 protein expression. Furthermore, long-term nicotine or CSC exposure significantly increased the percentage of SA-ß-gal-positive HGFs. In addition, long-term nicotine or CSC exposure reduced miR-29b and miR-199a expression to less than 50% of that in the unstimulated HGFs. CONCLUSION: These data suggest that long-term smoking habits may reduce wound healing ability, modulate ECM protein homeostasis, stimulate the inflammatory response, and accelerate cellular senescence in HGFs, and consequently accelerate the progression of periodontal diseases.

Aromatase inhibitor anastrozole modifies cellular functions in gingival fibroblasts and endothelial cells: possible periodontal complications of aromatase inhibitor treatment.

BACKGROUND: Recent studies have shown that treatment with aromatase inhibitors contributes to an increased prevalence of periodontitis. OBJECTIVE: In this study, we assessed effects of the aromatase inhibitor anastrozole on cellular function of human gingival fibroblasts (HGFs) and endothelial cells. METHODS: Expression levels of collagen, extracellular matrix (ECM) proteins, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) were examined in HGFs exposed to anastrozole. Furthermore, inflammatory responses in HGFs cultured with anastrozole were evaluated in the presence of Porphyromonas gingivalis lipopolysaccharide. We also evaluated the vascular permeability and vascular endothelial (VE)-cadherin expression of endothelial cells exposed to anastrozole. RESULTS: Anastrozole enhanced expression levels of collagen, ECM proteins, TIMPs, and inflammatory cytokines in HGFs, as well as vascular permeability of endothelial cells. In addition, anastrozole reduced expression levels of MMPs in HGFs and VE-cadherin in endothelial cells. CONCLUSION: These results suggest that anastrozole modulates various cellular functions in HGFs and endothelial cells.

Autophagy facilitates type I collagen synthesis in periodontal ligament cells.

Autophagy is a lysosomal protein degradation system in which the cell self-digests its intracellular protein components and organelles. Defects in autophagy contribute to the pathogenesis of age-related chronic diseases, such as myocardial infarction and rheumatoid arthritis, through defects in the extracellular matrix (ECM). However, little is known about autophagy in periodontal diseases characterised by the breakdown of periodontal tissue. Tooth-supportive periodontal ligament (PDL) tissue contains PDL cells that produce various ECM proteins such as collagen to maintain homeostasis in periodontal tissue. In this study, we aimed to clarify the physiological role of autophagy in periodontal tissue. We found that autophagy regulated type I collagen synthesis by elimination of misfolded proteins in human PDL (HPDL) cells. Inhibition of autophagy by E-64d and pepstatin A (PSA) or siATG5 treatment suppressed collagen production in HPDL cells at mRNA and protein levels. Immunoelectron microscopy revealed collagen fragments in autolysosomes. Accumulation of misfolded collagen in HPDL cells was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. E-64d and PSA treatment suppressed and rapamycin treatment accelerated the hard tissue-forming ability of HPDL cells. Our findings suggest that autophagy is a crucial regulatory process that facilitates type I collagen synthesis and partly regulates osteoblastic differentiation of PDL cells.

Useful immunochromatographic assay of calprotectin in gingival crevicular fluid for diagnosis of diseased sites in patients with periodontal diseases.

BACKGROUND: Calprotectin, an inflammation-related protein, is present in gingival crevicular fluid (GCF), and the determination of calprotectin is useful for diagnosing periodontal diseases. The authors have recently developed a novel immunochromatographic (IC) chip system to determine calprotectin levels in GCF. In the present study, the usefulness of this diagnostic system is investigated in patients with periodontal diseases. METHODS: Thirty-six patients with periodontal diseases participated in this clinical test at multiple centers. Periodontitis sites (n = 118) and non-periodontitis (healthy) sites (n = 120) were selected after periodontal examination. GCF collection and periodontal examination were performed at baseline, after supragingival and subgingival scaling and root planing. Calprotectin levels in GCF were determined using a novel IC chip system and evaluated as a visual score and an IC reader value. Correlations between GCF calprotectin levels, clinical indicators, and changes in calprotectin levels by periodontal treatments were investigated. Receiver operating characteristic (ROC) analysis of IC reader value for GCF calprotectin was performed to predict periodontal diseases. RESULTS: The visual score of GCF calprotectin was highly correlated with the IC reader value. IC reader values of GCF calprotectin in the periodontitis group were higher than those of the healthy group at three dental examination stages, and they significantly decreased with periodontal treatments. Visual scores and IC reader values of GCF calprotectin were correlated to levels of clinical indicators. ROC analysis for GCF calprotectin showed an optimal cutoff value to predict periodontal diseases. CONCLUSION: Determination of GCF calprotectin using a novel IC chip system is useful for diagnosis of periodontal diseases.

The Effects of Cigarette Smoke Condensate and Nicotine on Periodontal Tissue in a Periodontitis Model Mouse.

Cigarette smoking is a major lifestyle-related risk factor for periodontal diseases. However, the pathophysiological role of cigarette smoking in periodontal disease has yet to be fully elucidated. Here we report that the systemic administration of cigarette smoke condensate or nicotine, which is the major ingredient of cigarette smoke, augmented alveolar bone loss. Concomitantly, the number of osteoclasts in periodontal tissues increased and the expression of receptor activator of nuclear factor κB ligand was upregulated at the ligated side in mice with periodontitis. Nicotine also attenuated alveolar bone repair after ligature removal. These observations highlight the destruction of periodontal tissue by smoking and the unfavorable clinical course of periodontal disease in patients with a cigarette smoking habit. The present study demonstrates that periodontal disease models are useful for elucidating the pathogenesis of cigarette smoking-related periodontal diseases.

[Periodontal destruction and trends in periodontal treatment for patients].

Periodontal diseases are chronic inflammatory disorders caused by the accumulation of a bacterial biofilm, characterized by the destruction of periodontal tissues, and result in loss of tooth. Recently, a part of mechanism of alveolar bone destruction has been revealed with advances in osteoimmunology. Here, we review the possible mechanisms of periodontal destruction. In addition, we described future outlook of our translational research for periodontal regenerative medicine.

TGF-Beta Negatively Regulates the BMP2-Dependent Early Commitment of Periodontal Ligament Cells into Hard Tissue Forming Cells.

Transforming growth factor beta (TGF-ß) is a multi-functional growth factor expressed in many tissues and organs. Genetic animal models have revealed the critical functions of TGF-ß in craniofacial development, including the teeth and periodontal tissue. However, the physiological function of TGF-ß in the periodontal ligament (PDL) has not been fully elucidated. In this study, we examined the roles of TGF-ß in the cytodifferentiation of PDL cells using a TGF-ß receptor kinase inhibitor, SB431542. Mouse PDL cell clones (MPDL22) were cultured in calcification-inducing medium with or without SB431542 in the presence or absence of various growth factors, such as bone morphogenetic protein (BMP)-2, TGF-ß and fibroblast growth factor (FGF)-2. SB431542 dramatically enhanced the BMP-2-dependent calcification of MPDL22 cells and accelerated the expression of ossification genes alkaline phosphatase (ALPase) and Runt-related transcription factor (Runx) 2 during early osteoblastic differentiation. SB431542 did not promote MPDL22 calcification without BMP-2 stimulation. The cell growth rate and collagen synthesis during the late stage of MPDL22 culture were retarded by SB431542. Quantitative reverse transcription polymerase chain reaction analysis revealed that the expressions of Smurf1 and Smad6, which are negative feedback components in the TGF-ß/BMP signaling pathway, were downregulated in MPDL22 cells with SB431542 treatment. These results suggest that an endogenous signal from TGF-ß negatively regulates the early commitment and cytodifferentiation of PDL cells into hard tissue-forming cells. A synthetic drug that regulates endogenous TGF-ß signals may be efficacious for developing periodontal regenerative therapies.

Immunomodulation of dendritic cells differentiated in the presence of nicotine with lipopolysaccharide from Porphyromonas gingivalis.

Tobacco smoking is a significant risk factor for periodontal diseases. Nicotine, one of the most studied constituents in cigarette smoke, is thought to modify immune responses. Dendritic cells (DCs), which are key mediators between innate and adaptive immunity, stimulate naive T cells to differentiate to effector T-cell subsets that may be actively involved in the immunopathogenesis of periodontal diseases. In this study, we evaluated the effects of nicotine and lipopolysaccharide (LPS) from Porphyromonas gingivalis, alone and in combination, on the functions of human monocyte-derived DCs to elucidate the mechanism of tissue destruction of smoking-associated periodontal diseases. P. gingivalis LPS-stimulated DCs differentiated with nicotine (NiDCs) induced lower T-cell proliferation and human leukocyte antigen (HLA)-DR expression, but elevated expression of programmed cell death ligand 1. Additionally, NiDCs impaired interferon-γ production but maintained interleukin (IL)-5 and IL-10 production in co-cultured T cells. Furthermore, NiDCs produced lower levels of proinflammatory cytokines compared with DCs differentiated in the absence of nicotine. Interestingly, NiDCs preferentially produced the T helper 2 (Th2)-type chemokines macrophage chemotactic protein-1 and macrophage-derived chemokine. These results suggest that the presence of nicotine during differentiation of DCs modulates the immunoregulatory functions of P. gingivalis LPS-stimulated DCs.

Nicotine modulates the immunological function of dendritic cells through peroxisome proliferator-activated receptor-γ upregulation.

We examined the effects of nicotine on differentiation and function of monocyte-derived human dendritic cells (DCs). NiDCs, which were the DCs differentiated in the presence of nicotine, showed lower levels of CD1a. Secretion of IL-12 and TNF-α by lipopolysaccharide (LPS)-stimulated NiDCs was significantly suppressed compared to monocyte-derived DCs grown without nicotine. NiDCs displayed a diminished capacity to induce allogeneic T cell proliferation with a reduced production of IFN-γ, and maintained/enhanced LPS-mediated expression of coinhibitory molecules. Interestingly, NiDCs enhanced the expression of nuclear receptor peroxisome proliferator-activated receptors γ (PPAR γ), which has immunomodulatory properties. Expression of PPAR γ and PPAR γ-target genes was significantly inhibited by pretreatment with d-tubocurarine, antagonist of non-selective nicotinic acetylcholine receptors (nAChR). In addition, reduction of Th1 responses was inhibited after blocking nAChR-mediated signal. These data suggest the effect of nicotine on altering DC immunogenicity by impeding Th1 immunity is partially mediated by upregulation of PPAR γ.

Nicotine up-regulates IL-8 expression in human gingival epithelial cells following stimulation with IL-1ß or P. gingivalis lipopolysaccharide via nicotinic acetylcholine receptor signalling.

OBJECTIVE: Cigarette smoking is an important risk factor for periodontal disease. The aim of this study is to evaluate the effect of nicotine, a major component of cigarette smoke, on interleukin-8 (IL-8) production and cellular signalling via nicotinic acetylcholine receptors (nAChRs) in human gingival epithelial cells (HGECs). DESIGN: Messenger RNA (mRNA) expression of nAChR subunits in three different HGEC lines (epi 4, Tfx and E6E7) was assessed using reverse transcription-polymerase chain reaction (RT-PCR). HGECs were stimulated by 1×10(-3)M nicotine in the presence or absence of IL-1ß or Porphyromonas gingivalis lipopolysaccharide (LPS). IL-8 production was then examined using real-time PCR and enzyme-linked immunosorbent assay. Nicotine-mediated signalling in the epi 4 cell line was also evaluated by Western blotting. RESULTS: HGECs expressed several nAChR subunits. Nicotine increased the secretion of IL-8 from HGECs that were cultured in the presence of IL-1ß or P. gingivalis LPS and also induced the phosphorylation of extracellular signal-regulated kinase (ERK) in epi 4. Pretreatment with non-selective nAChR antagonist or intracellular calcium chelator reduced the nicotine-induced phosphorylation of ERK. Furthermore, nicotine-induced IL-8 secretion was decreased by pretreatment with non-selective nAChR antagonist, ERK1/2 inhibitor or intracellular calcium chelator. CONCLUSION: These findings indicate that nicotine increases IL-8 production in gingival epithelial cells via ERK phosphorylation following Ca(2+) signalling after nAChR activation.

Osteoinductive and anti-inflammatory effect of royal jelly on periodontal ligament cells.

Royal jelly (RJ) has been reported to possess several physiological and pharmacological properties such as the ability to prevent osteoporosis in rats and anti-inflammatory effects. We hypothesized that RJ could have beneficial effects on the prevention or treatment of periodontal diseases, which are chronic inflammatory diseases caused by bacterial infection that result in resorption of the tooth-supporting bone. We assessed the effect of RJ on mineralization in mouse periodontal ligament cell clone 22 (MPDL22 cells), which are of an osteogenic and cementogenic lineage. The mRNA expression of osteopontin, osteocalcin and osterix, and mineralized nodule formation were significantly enhanced in RJ-treated MPDL22 cells. In addition, we investigated the effects of RJ on the production of inflammatory cytokines from MPDL22 cells stimulated with lipopolysaccharide (LPS) of Porphyromonas gingivalis, a periodontopathic bacterium. RJ suppressed LPS-induced interleukin-6 and CXC chemokine ligand 10 production from MPDL22 cells. Furthermore, RJ suppressed the expression of CD54 in MPDL22 cells: CD54 is the adhesion molecule involved in the accumulation of leukocytes in periodontal lesions. These findings suggest that the osteoinductive and anti-inflammatory effects of RJ can provide benefits for the treatment and prevention of periodontal diseases.

Periodontal disease in a patient with Prader-Willi syndrome: a case report.

INTRODUCTION: Prader-Willi syndrome is a complex genetic disease caused by lack of expression of paternally inherited genes on chromosome 15q11-q13. The prevalence of Prader-Willi syndrome is estimated to be one in 10,000 to 25,000. However, descriptions of the oral and dental phenotype are rare. CASE PRESENTATION: We describe the clinical presentation and periodontal findings in a 20-year-old Japanese man with previously diagnosed Prader-Willi syndrome. Clinical and radiographic findings confirmed the diagnosis of periodontitis. The most striking oral findings were anterior open bite, and crowding and attrition of the lower first molars. Periodontal treatment consisted of tooth-brushing instruction and scaling. Home care involved recommended use of adjunctive chlorhexidine gel for tooth brushing twice a week and chlorhexidine mouthwash twice daily. Gingival swelling improved, but further treatment will be required and our patient's oral hygiene remains poor. The present treatment of tooth-brushing instruction and scaling every three weeks therefore only represents a temporary solution. CONCLUSIONS: Rather than being a direct result of genetic defects, periodontal diseases in Prader-Willi syndrome may largely result from a loss of cuspid guidance leading to traumatic occlusion, which in turn leads to the development of periodontal diseases and dental plaque because of poor oral hygiene. These could be avoided by early interventions to improve occlusion and regular follow-up to monitor oral hygiene. This report emphasizes the importance of long-term follow-up of oral health care by dental practitioners, especially pediatric dentists, to prevent periodontal disease and dental caries in patients with Prader-Willi syndrome, who appear to have problems maintaining their own oral health.

Fibroblast growth factor-2 stimulates directed migration of periodontal ligament cells via PI3K/AKT signaling and CD44/hyaluronan interaction.

Fibroblast growth factor-2 (FGF-2) regulates a variety of functions of the periodontal ligament (PDL) cell, which is a key player during tissue regeneration following periodontal tissue breakdown by periodontal disease. In this study, we investigated the effects of FGF-2 on the cell migration and related signaling pathways of MPDL22, a mouse PDL cell clone. FGF-2 activated the migration of MPDL22 cells and phosphorylation of phosphatidylinositol 3-kinase (PI3K) and akt. The P13K inhibitors, Wortmannin and LY294002, suppressed both cell migration and akt activation in MPDL22, suggesting that the PI3K/akt pathway is involved in FGF-2-stimulated migration of MPDL22 cells. Moreover, in response to FGF-2, MPDL22 showed increased CD44 expression, avidity to hyaluronan (HA) partly via CD44, HA production and mRNA expression of HA synthase (Has)-1, 2, and 3. However, the distribution of HA molecular mass produced by MPDL22 was not altered by FGF-2 stimulation. Treatment of transwell membrane with HA facilitated the migration of MPDL22 cells and an anti-CD44 neutralizing antibody inhibited it. Interestingly, the expression of CD44 was colocalized with HA on the migrating cells when stimulated with FGF-2. Furthermore, an anti-CD44 antibody and small interfering RNA for CD44 significantly decreased the FGF-2-induced migration of MPDL22 cells. Taken together, PI3K/akt and CD44/HA signaling pathways are responsible for FGF-2-mediated cell motility of PDL cells, suggesting that FGF-2 accelerates periodontal regeneration by regulating the cellular functions including migration, proliferation and modulation of extracellular matrix production.

Fibroblast growth factor-2 regulates the cell function of human dental pulp cells.

INTRODUCTION: Homeostasis and tissue repair of dentin-pulp complex are attributed to dental pulp tissue and several growth factors. Dental pulp cells play a pivotal role in homeostasis of dentin-pulp complex and tissue responses after tooth injury. Among these cytokines, fibroblast growth factor (FGF)-2 has multifunctional biologic activity and is known as a signaling molecule that induces tissue regeneration. In this study, we examined the effects of FGF-2 on growth, migration, and differentiation of human dental pulp cells (HDPC). METHODS: HDPC were isolated from healthy dental pulp. Cellular response was investigated by [(3)H]-thymidine incorporation into DNA. Cytodifferentiation was examined by alkaline phosphatase (ALPase) assay and cytochemical staining of calcium by using alizarin red. Migratory activity was determined by counting the cells migrating into cleared area that had introduced with silicon block. RESULTS: FGF-2 activated HDPC growth and migration but suppressed ALPase activity and calcified nodule formation. Interestingly, HDPC, which had been pretreated with FGF-2, showed increased ALPase activity and calcified nodule formation when subsequently cultured without FGF-2. These results suggest that FGF-2 potentiates cell growth and accumulation of HDPC that notably did not disturb cytodifferentiation of the cells later. Thus, FGF-2 is a favorable candidate for pulp capping agent. CONCLUSIONS: These results provide new evidence for the possible involvement of FGF-2 not only in homeostasis but also in regeneration of dentin-pulp complex.

Nicotine can skew the characterization of the macrophage type-1 (MPhi1) phenotype differentiated with granulocyte-macrophage colony-stimulating factor to the MPhi2 phenotype.

Macrophages (MPhis) exhibit functional heterogeneity and plasticity in the local microenvironment. Recently, it was reported that MPhis can be divided into proinflammatory MPhis (MPhi1) and anti-inflammatory MPhis (MPhi2) based on their polarized functional properties. Here, we report that nicotine, the major ingredient of cigarette smoke, can modulate the characteristics of MPhi1. Granulocyte-macrophage colony-stimulating factor-driven MPhi1 with nicotine (Ni-MPhi1) showed the phenotypic characteristics of MPhi2. Like MPhi2, Ni-MPhi1 exhibited antigen-uptake activities. Ni-MPhi1 suppressed IL-12, but maintained IL-10 and produced high amounts of MCP-1 upon lipopolysaccharide stimulation compared with MPhi1. Moreover, we observed strong proliferative responses of T cells to lipopolysaccharide-stimulated MPhi1, whereas Ni-MPhi1 reduced T cell proliferation and inhibited IFN-gamma production by T cells. These results suggest that nicotine can change the functional characteristics of MPhi and skew the MPhi1 phenotype to MPhi2. We propose that nicotine is a potent regulator that modulates immune responses in microenvironments.

Blood-derived inflammatory dendritic cells in lymph nodes stimulate acute T helper type 1 immune responses.

T helper type 1 (T(H)1)-polarized immune responses, which confer protection against intracellular pathogens, are thought to be initiated by dendritic cells (DCs) that enter lymph nodes from peripheral tissues. Here we found after viral infection or immunization, inflammatory monocytes were recruited into lymph nodes directly from the blood to become CD11c(+)CD11b(hi)Gr-1(+) inflammatory DCs, which produced abundant interleukin 12p70 and potently stimulated T(H)1 responses. This monocyte extravasation required the chemokine receptor CCR2 but not the chemokine CCL2 or receptor CCR7. Thus, the accumulation of inflammatory DCs and T(H)1 responses were much lower in Ccr2(-/-) mice, were preserved in Ccl2(-/-) mice and were relatively higher in CCL19-CCL21-Ser-deficient plt mutant mice, in which all other lymph node DC types were fewer in number. We conclude that blood-derived inflammatory DCs are important in the development of T(H)1 immune responses.

Nicotine inhibits mineralization of human dental pulp cells.

Nicotine is a major component of tobacco smoke, and signals via nicotinic acetylcholine receptors (nAChR). However, little is known about the effects of nicotine on human dental pulp cells (HDPCs). In this study, we assessed the effects of nicotine on mineralization in HDPCs. We confirmed messenger RNA expression of nAChR subunits and examined the effects of nicotine on expression of extracellular matrices (ECMs), alkaline phosphatase (ALP) activity, and mineralized nodule formation by HDPCs. Gene expression of nAChR subunits alpha1, alpha2, alpha 4, alpha 5, alpha 6, alpha 7, beta1, beta2, and beta 4 was detected in HDPCs. Interestingly, the messenger RNA expression of dentin matrix acidic phosphoprotein-1, bone sialoprotein, and ALP activity were significantly reduced in nicotine-treated HDPC. In addition, mineralized nodule formation, which was examined by alizarin red staining, was also inhibited in HDPCs by the same treatment. These results indicate that nicotine suppresses the cytodifferentiation and mineralization of HDPCs, possibly via nAChR.

Thrombin regulates the function of human blood dendritic cells.

Thrombin is the key enzyme in the coagulation cascade and activates endothelial cells, neutrophils and monocytes via protease-activated receptors (PARs). At the inflammatory site, immune cells have an opportunity to encounter thrombin. However little is known about the effect of thrombin for dendritic cells (DC), which are efficient antigen-presenting cells and play important roles in initiating and regulating immune responses. The present study revealed that thrombin has the ability to stimulate blood DC. Plasmacytoid DC (PDC) and myeloid DC (MDC) isolated from PBMC expressed PAR-1 and released MCP-1, IL-10, and IL-12 after thrombin stimulation. Unlike blood DC, monocyte-derived DC (MoDC), differentiated in vitro did not express PAR-1 and were unresponsive to thrombin. Effects of thrombin on blood DC were significantly diminished by the addition of anti-PAR-1 Ab or hirudin, serine protease inhibitor. Moreover, thrombin induced HLA-DR and CD86 expression on DC and the thrombin-treated DC induced allogenic T cell proliferation. These findings indicate that thrombin plays a role in the regulation of blood DC functions.

IL-15 up-regulates iNOS expression and NO production by gingival epithelial cells.

To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.