L-arginine-dependent nitric oxide production of a murine macrophage-like RAW 264.7 cell line stimulated with Porphyromonas gingivalis lipopolysaccharide

The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg-LPS). RAW 264.7 cells were incubated with i) various concentrations of Pg-LPS or Salmonella typhosa LPS (St-LPS), ii) Pg-LPS with or without L-arginine and/or NG-monomethyl-L-arginine (NMMA), an arginine analog or iii) Pg-LPS and interferon-gamma (IFN-gamma) with or without anti-IFN-gamma antibodies or interleukin-10 (IL-10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg-LPS, but was observed after stimulation with St-LPS. Exogenous L-arginine restored the ability of Pg-LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg-LPS with exogenous L-arginine was abolished by NMMA. IFN-gamma induced independent NO production by Pg-LPS-stimulated macrophages and this stimulatory effect of IFN-gamma could be completely suppressed by anti-IFN-gamma antibodies and IL-10. These results suggest that Pg-LPS is able to stimulate NO production in the RAW 264.7 macrophage cell model in an L-arginine-dependent mechanism which is itself independent of the action of IFN-gamma.     

Nitric oxide production by a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

The aim of this study was to determine whether Actinobacillus actinomycetemcomitans lipopolysaccharide (LPS-A. actinomycetemcomitans) could stimulate a murine macrophage cell line (RAW264.7 cells) to produce nitric oxide (NO). The cells were treated with LPS-A. actinomycetemcomitans or Escherichia coli LPS (LPS-Ec) for 24 h. The effects of N(G)-monomethyl-L-arginine (NMMA), polymyxin B and cytokines (IFN-gamma, TNF-alpha, IL-4 and IL-12) on the production of NO were also determined. The role of protein tyrosine kinase, protein kinase C and microtubulin organization on NO production were assessed by incubating RAW264.7 cells with genistein, bisindolylmaleide and colchicine prior to LPS-A. actinomycetemcomitans stimulation, respectively. NO levels from the culture supernatants were determined by the Griess reaction. The results showed that LPS-A. actinomycetemcomitans stimulated NO production by RAW264.7 cells in a dose-dependent manner, but was slightly less potent than LPS-Ec. NMMA and polymyxin B blocked the production of NO. IFN-gamma and IL-12 potentiated but IL-4 depressed NO production by LPS-A. actinomycetemcomitans-stimulated RAW264.7 cells. TNF-alpha had no effects on NO production. Genistein and bisindolylmalemaide, but not colchicine, reduced the production of NO in a dose-dependent mechanism. The results of the present study suggest that A. actinomycetemcomitans LPS, via the activation of protein tyrosine kinase and protein kinase C and the regulatory control of cytokines, stimulates NO production by murine macrophages.     

Kaempferol Inhibits P. intermedia Lipopolysaccharide‐Induced Production of Nitric Oxide Through Translational Regulation in Murine Macrophages: Critical Role of Heme Oxygenase‐1‐Mediated ROS Reduction

Background: Nitric oxide (NO) could be a potential target for the development of new therapeutic approaches to the treatment of periodontal disease because this molecule plays a significant role in the tissue destruction observed in periodontitis. In this study, the authors investigate the effect of kaempferol on the production of NO by murine macrophage‐like RAW264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in periodontal disease, and try to determine the underlying mechanisms of action. Methods: NO production was assayed by measuring the accumulation of nitrite in culture supernatants. Real‐time polymerase chain reaction was performed to quantify inducible NO synthase (iNOS) and heme oxygenase‐1 (HO‐1) mRNA expression. iNOS and HO‐1 protein expression and phosphorylation of c‐Jun N‐terminal kinase and p38 were characterized via immunoblot analysis. Reactive oxygen species (ROS) production was measured using the redox‐sensitive fluorescent probe 2′,7′‐dichlorodihydrofluorescein diacetate. Results: Kaempferol significantly inhibited NO production and expression of iNOS protein in P. intermedia LPS‐stimulated RAW246.7 cells without affecting iNOS mRNA expression. Kaempferol upregulated HO‐1 expression in LPS‐activated cells. Inhibition of HO‐1 activity by tin protoporphyrin IX (SnPP) abolished the suppressive effect of kaempferol on NO production. In addition, kaempferol significantly attenuated P. intermedia LPS‐induced increase of intracellular ROS, and SnPP blocked this reduction. Treatment with antioxidants downregulated the production of LPS‐induced NO. Conclusions: Kaempferol inhibits NO production and iNOS protein expression in P. intermedia LPS‐stimulated RAW264.7 cells at the translational level via HO‐1‐mediated ROS reduction and could be an efficient modulator of host response in the treatment of periodontal disease.     

Epithelial Cells Secrete Interferon‐γ Which Suppresses Expression of Receptor Activator of Nuclear Factor Kappa‐B Ligand in Human Mandibular Osteoblast‐Like Cells

Background: Prostaglandin (PG)E₂ accumulates in inflamed periodontal tissue and induces receptor activator of nuclear factor kappa‐B ligand (RANKL)‐RANK‐osteoprotegerin (OPG) signaling associated with bone resorption. Although oral epithelial cells maintain tissue homeostasis, the role of these cells in RANKL regulation remains unknown. Methods: To mimic an inflamed condition, RANKL upregulation in human mandibular osteoblast‐like cells (HMOBs) were stimulated with PGE₂. Effect of recombinant human interferon (IFN)‐γ or epithelial‐derived IFN‐γ in constitutively released or Porphyromonas gingivalis lipopolysaccharide (PgLPS)‐stimulated epithelial supernatant was investigated in HMOBs. Some HMOBs were pretreated with an anti‐IFN‐γ antibody before PGE₂ stimulation. THP‐1 human monocytes and HMOBs were cocultured in a transwell system to investigate RANKL‐driven THP‐1 osteoclastic activity. Results: PGE₂ significantly increased RANKL messenger RNA (mRNA) and protein in HMOBs in a dose‐dependent manner, while OPG protein remained similar to baseline. Epithelial cells constitutively released IFN‐γ, which was substantially increased by PgLPS. HMOBs treated with epithelial supernatant or recombinant IFN‐γ, concurrently with PGE₂ stimulation, reduced RANKL, but not OPG, expression. In contrast, anti‐IFN‐γ antibody reversed the effect of epithelial mediators on RANKL expression. When cocultured with THP‐1, RANKL released by PGE₂‐stimulated HMOBs is adequate to drive THP‐1 differentiation as osteoclastogenic gene expression and bone resorption pit are increased. However, recombinant IFN‐γ, or IFN‐γ derived from oral epithelial cells, suppressed RANKL expression at both the mRNA and protein level, resulting in decreased THP‐1‐derived osteoclastic activity. Conclusion: Oral epithelial cells interact with HMOBs by releasing IFN‐γ to regulate RANKL expression and contribute to osteoclastogenesis.     

Production of IL‐10 and IL‐12 by antigen‐presenting cells in periapical lesions

J Oral Pathol Med (2010) 39 : 690–696 Background: Interferon‐γ (IFN‐γ) plays an important role in the pathogenesis of periapical lesions. Its expression is up‐regulated by interleukin (IL)‐12) and down‐regulated by IL‐10. The aim of this work was to study the cellular source of these cytokines and their mutual interactions in human periapical lesions. Methods: Mononuclear cells, macrophages and dendritic cells were isolated from periapical lesions using plastic adherence and osmotic gradients. Cytokines were measured in culture supernatants by a microbeads fluorescence assay. Phenotypic characteristics of cells were studied by immunocytochemistry, whereas allostimulatory activity of antigen‐presenting cells was tested using a mixed leukocyte reaction. Results: We observed the positive correlations between the levels of IL‐12 and IFN‐γ as well as IL‐12 and IL‐10 in cultures of mononuclear cells. As IL‐10 and IL‐12 are produced by dendritic cells and activated macrophages, we examined their contribution to the production of these cytokines. Macrophages, CD14⁺ adherent cells, produced high levels of IL‐10 and very low levels of IL‐12. In contrast, non‐adherent, strongly HLA‐DR⁺ dendritic cells, potent stimulators of the alloreactive T‐cell response, produced low levels of IL‐10 and moderate levels of IL‐12. Dendritic cells stimulated the production of IFN‐γ by allogeneic CD4⁺ T cells. In contrast, the level of IFN‐γ was significantly decreased and the production of IL‐10 was enhanced by addition of macrophages to the culture system. Conclusion: Our results suggest that a fine balance between the production of IL‐10 and IL‐12 by different antigen‐presenting cells, through IFN‐γ, may control the course of chronic inflammation in periapical lesions.     

Effect of exogenous nitric oxide on murine immune response induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide

Background and Objective: Elevated nitric oxide (NO) has been associated with destructive periodontal disease. The aim of the present study was to test the hypothesis that exogenous NO may inhibit a protective immune response to Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) in a murine model. Material and Methods: Mice of the BALB/c strain were sham immunized, immunized with A. actinomycetemcomitans LPS, treated with S‐nitroso‐N‐acetyl penicillamine (SNAP; a NO donor) and immunized with A. actinomycetemcomitans LPS or treated with SNAP plus 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (carboxy‐PTIO) and immunized with A. actinomycetemcomitans LPS. All animals were then challenged subcutaneously with viable A. actinomycetemcomitans. The serum‐specific immunoglobulin G (IgG) subclasses and both interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4) as well as splenic inducible nitric oxide synthase (iNOS) activity before and after bacterial challenge were assessed. The diameter of skin lesions was determined. Groups of mice were treated with l ‐N⁶‐(1 ‐iminoethyl)‐lysine (l ‐NIL), an iNOS inhibitor, or 1H‐(1,2,4)oxadiazolo(4,3‐a)quinoxalin‐1‐one (ODQ), a guanylyl cyclase inhibitor, prior to injections with SNAP and/or A. actinomycetemcomitans LPS, and the skin lesions were assessed. Results: Treatment with SNAP increased the iNOS activity, suppressed both serum‐specific IgG2a and IFN‐γ levels, and delayed the healing of the lesions. These SNAP‐induced immune alterations were restored by treatment with carboxy‐PTIO. Pretreatment with l ‐NIL resulted in partial healing, whereas pretreatment with ODQ induced a delayed healing of the lesions. Conclusion: The present study suggests that exogenous NO may suppress a protective T helper 1‐like murine immune response to A. actinomycetemcomitans LPS by an endogenous NO‐independent but a cyclic GMP‐dependent mechanism.     

The role of Toll‐like receptors 2 and 4 on reactive oxygen species and nitric oxide production by macrophage cells stimulated with root canal pathogens

Introduction: Periapical lesions arise as a result of the activation and interaction of the host immune responses against root canal infection. Recently identified Toll‐like receptors (TLR) seem to be involved in the recognition and development of immune responses against a myriad of microorganisms. However, very little information is available on the role of TLR in the induction of periapical lesions. Method: The role of TLR‐2 and TLR‐4 in the activation of murine macrophages stimulated using Fusobacterium nucleatum and Peptostreptococcus anaerobius was investigated. The production of nitric oxide (NO) and reactive oxygen species (ROS) was assessed. Results: The results demonstrate that TLR‐2 and TLR‐4 are involved in the production of ROS by activated macrophages. The microorganisms induced similar levels of NO production by TLR‐2‐competent and TLR‐2‐deficient macrophages, regardless of the addition of interferon‐γ (IFN‐γ), ruling out a role for TLR‐2 in the NO production induced by these bacteria. Only P. anaerobius induced NO production by TLR‐4‐competent macrophages without the addition of IFN‐γ. However, after IFN‐γ addition, F. nucleatum induced macrophage NO production. Therefore, NO production stimulated by IFN‐γ and these microorganisms seems to be TLR‐4‐independent. Conclusion: TLR‐2 seems to be involved in the induction of ROS production by macrophages in response to prevalent root canal bacteria, while only F. nucleatum induced ROS production by TLR‐4‐competent macrophages. Both microorganisms significantly induced large amounts of NO independent of TLR‐2 and TLR‐4. We conclude that microorganisms may participate in the induction and progression of periapical lesions through NO and ROS production by activated macrophages.     

Locally administered interferon-γ accelerates lipopolysaccharide-induced osteoclastogenesis independent of immunohistological RANKL upregulation

Ayon Haro ER, Ukai T, Yokoyama M, Kishimoto T, Yoshinaga Y, Hara Y. Locally administered interferon‐γ accelerates lipopolysaccharide‐induced osteoclastogenesis independent of immunohistological RANKL upregulation. J Periodont Res 2011; 46: 361–373. © 2011 John Wiley & Sons A/S Background and Objective: Interferon‐γ (IFN‐γ) potently inhibits RANKL‐induced osteoclastogenesis in vitro. In contrast, previous studies have shown that an increase in IFN‐γ expression is correlated with an increase in lipopolysaccharide (LPS)‐induced bone loss in vivo. However, it is not clear whether local IFN‐γ accelerates osteoclastogenesis or not in vivo. Therefore, the aim of this study was to clarify the role of local IFN‐γ in LPS‐induced osteoclastogenesis. Material and Methods: We induced bone loss in calvaria by injecting LPS. One group of mice received an IFN‐γ injection together with LPS injection, while another group received IFN‐γ 2 d after LPS injection. Bone resorption was observed histologically. Next, we stimulated murine bone marrow macrophages with macrophage‐colony stimulating factor and RANKL in vitro. We added different doses of IFN‐γ and/or LPS at 0 or 48 h time points. Cells were stained with tartrate‐resistant acid phosphatase at 72 h. Results: Local administration of IFN‐γ together with LPS injection did not affect osteoclast formation. However, IFN‐γ injected after LPS injection accelerated osteoclast formation. Also, we confirmed that IFN‐γ added at 0 h inhibited RANKL‐induced osteoclastogenesis in vitro. However, inhibition by IFN‐γ added at 48 h was reduced compared with that by IFN‐γ added at 0 h. Interestingly, IFN‐γ together with a low concentration of LPS accelerated osteoclast formation when both were added at 48 h compared with no addition of IFN‐γ. Conclusion: The results suggest that local IFN‐γ accelerates osteoclastogenesis in certain conditions of LPS‐induced inflammatory bone loss.     

The role of cyclic‐AMP on arginase activity by a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

Aims: The aim of the present study was to determine the role of cyclic adenosine monophosphate (cAMP) on arginase activity in a murine macrophage cell line (RAW264.7 cells) stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. Materials and methods: The cells were treated with A. actinomycetemcomitans LPS for 24 h. The effects of SQ22536 (an adenylyl cyclase inhibitor), ODQ (a guanylyl cyclase inhibitor), dibutyryl cAMP (a cAMP analog), 8‐bromo cyclic guanosine monophosphate (a cGMP analog), forskolin (an adenylyl cylase activator), and cycloheximide (a protein synthesis inhibitor) on arginase activity in A. actinomycetemcomitans LPS‐stimulated RAW264.7 cells were also determined. Arginase activity was assessed in LPS‐stimulated cells in the presence of 3‐isobutyl‐1‐methylxanthine (IBMX), siguazodan and rolipram [phosphodiesterase (PDE) inhibitors] as well as KT5720 [a protein kinase A (PKA) inhibitor]. Results: Arginase activity in A. actinomycetemcomitans LPS‐stimulated RAW264.7 cells was suppressed by SQ22536 but not ODQ. Enhancement of arginase activity was observed in the presence of cAMP analog or forskolin but not cGMP analog. Cycloheximide blocked arginase activity in the cells in the presence of cAMP analog or forskolin with or without A. actinomycetemcomitans LPS. IBMX augmented arginase activity in A. actinomycetemcomitans LPS‐stimulated cells. Rolipram (a PDE4 inhibitor) increased the levels of arginase activity higher than siguazodan (a PDE3 inhibitor) in the antigen‐stimulated cells. The effect of cAMP analog or forskolin on arginase activity in the presence or absence of A. actinomycetemcomitans LPS was blocked by the PKA inhibitor (KT5720). Conclusion: The results of the present study suggest that A. actinomycetemcomitans LPS may stimulate arginase activity in murine macrophages (RAW264.7 cells) in a cAMP‐PKA‐dependent pathway.     

Interferon‐Gamma and Fas Are Involved in Porphyromonas gingivalis–Induced Apoptosis of Human Extravillous Trophoblast‐Derived HTR8/SVneo Cells via Extracellular Signal‐Regulated Kinase 1/2 Pathway

Background: A number of studies recently revealed a link between periodontal disease and preterm birth (PTB). PTB can be induced by dental infection with Porphyromonas gingivalis (Pg), a periodontopathic bacterium. This study aims to investigate responses of human extravillous trophoblast‐derived HTR8/SVneo cells to Pg infection. Methods: Cell apoptosis, cell viability, protein expression, and cytokine production in HTR8 cells were measured via: 1) flow cytometry, 2) CCK‐8 assay, 3) western blot, and 4) enzyme‐linked immunosorbent assay methods, respectively. Results: Pg decreased cell viability and increased cell apoptosis, active caspase‐3 and Fas expression, and interferon‐gamma (IFN‐γ) secretion in HTR8 cells. Extracellular signal‐regulated kinase (ERK) 1/2 inhibitor U0126 and FasL neutralizing antibody NOK1 that blocks FasL/Fas interaction both significantly suppressed Pg‐induced apoptosis. U0126 also inhibited IFN‐γ secretion and Fas expression close to control levels. Moreover, treatment with recombinant IFN‐γ also significantly decreased number of viable HTR8 cells and increased Fas expression, suggesting IFN‐γ may play an important role in Pg‐induced apoptosis of HTR8 cells, at least partially through regulation of Fas expression. Conclusions: To the best of the authors’ knowledge, this is the first study to demonstrate Pg induces IFN‐γ secretion, Fas expression, and apoptosis in human extravillous trophoblast‐derived HTR8/SVneo cells in an ERK1/2‐dependent manner, and IFN‐γ (explored by recombinant IFN‐γ) and Fas are involved in Pg‐induced apoptosis. The finding that Pg infection abnormally regulates inflammation and apoptosis of human trophoblasts may give new insights into the possible link of PTB with maternal periodontal disease and periodontal pathogens.     

Anti‐phosphorylcholine‐opsonized low‐density lipoprotein promotes rapid production of proinflammatory cytokines by dendritic cells and natural killer cells

Kikuchi T, El Shikh MM, El Sayed RM, Purkall DB, Elaasser MM, Sarraf A, Barbour SE, Schenkein HA, Tew JG. Anti‐phosphorylcholine opsonized low‐density lipoprotein promotes rapid production of proinflammatory cytokines by dendritic cells and natural killer cells. J Periodont Res 2010; 45: 720–730. © 2010 John Wiley & Sons A/S Background and Objective: Epidemiological and animal studies suggest that periodontal infections increase atherosclerosis risk. Periodontitis patients have elevated levels of anti‐phosphorylcholine (anti‐PC) reactive not only with numerous periodontal organisms but also with minimally modified low‐density lipoprotein (mmLDL). Dendritic cells (DCs) reside in arterial walls and accumulate in atherosclerotic lesions. The ability of anti‐PC to bind mmLDL prompted the hypothesis that opsonized mmLDL would stimulate DCs and enhance the production of proinflammatory cytokines that promote atherogenic plaque development. Material and Methods: Monocyte‐derived DCs (mDCs) were generated using granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)‐4, then stimulated with mmLDL or with anti‐PC‐opsonized mmLDL. The anti‐PC effect was determined using flow cytometry, cofocal microscopy and cytokine assays. The production of CD83, IL‐12p35 mRNA, IL‐12p40 mRNA, IL‐12p70 and IL‐10 by DCs was monitored. Results: Dendritic cells stimulated with mmLDL expressed little CD83 and produced little IL‐12p70. However, anti‐PC‐opsonized mmLDL enhanced DC maturation, as indicated by upregulated CD83 and rapid (≤ 48 h) production of IL‐12p70 if a source of interferon‐γ (IFN‐γ) was available. In leukocyte cultures, natural killer (NK) cells rapidly produced IFN‐γ (≤ 48 h) when interacting with IL‐12‐producing DCs activated by anti‐PC‐opsonized mmLDL. Moreover, IFN‐γ promoted DC IL‐12 responses that were further augmented when mmLDL was opsonized with anti‐PC. Conclusion: Minimally modified LDL‐stimulated DCs and NK cells were mutually stimulatory, with DC IL‐12p70 needed by NK cells and with NK cell IFN‐γ needed by DCs. Moreover, production of these proinflammatory cytokines was markedly enhanced when LDL was opsonized by anti‐PC. In short, the data suggest that the elevated anti‐PC levels in periodontitis patients could promote a mechanism that facilitates atherosclerosis.     

The effect of parachlorophenol and camphorated parachlorophenol on nitric oxide production by a murine macrophage cell line,RAW264.7

The aim of this study was to determine the effect of parachlorophenol (PCP) and camphorated parachlorophenol (CMCP) on nitric oxide (NO) production by a murine macrophage cell line, RAW264.7. The cells were incubated on plastic disks with either PCP or CMCP. Plastic adherent and nonadherent cells were subsequently stimulated with recombinant mouse IFN-gamma or bacterial lipopolysaccharide (LPS). Nitric oxide (NO) levels detected from the culture supernatants were determined by the Griess reaction. The results showed that PCP and CMCP diluted at 10(-1) but not at 10(-3) suppressed NO production by both plastic adherent and nonadherent cells, suggesting that both phenolic compounds may suppress NO production by murine macrophages in a dose-dependent manner.     

Whole‐Blood Cultures From Patients With Chronic Periodontitis Respond Differently to Porphyromonas gingivalis but not Escherichia coli Lipopolysaccharide

Background: Porphyromonas gingivalis lipid A heterogeneity modulates cytokine expression in human cells. This study investigates the effects of two lipid A isoforms of P. gingivalis, lipopolysaccharide (LPS)_1435/1449 and LPS₁₆₉₀, on the secretion of proinflammatory and regulatory cytokines in total blood cultures from patients with and without chronic periodontitis (CP). Methods: A cross‐sectional study was conducted in 38 systemically healthy individuals divided in two groups: 1) the CP group (n = 19), in which patients were diagnosed with CP; and 2) the no periodontitis (NP) group (n = 19), which included control patients without CP. Blood samples were collected from all patients, and whole‐blood cell cultures (WBCCs) were stimulated for 48 hours with P. gingivalis LPS_1435/1449 and LPS₁₆₉₀ and Escherichia coli LPS. Unstimulated WBCCs served as negative controls. The secretion of interferon‐γ (IFN‐γ), interleukin‐10 (IL‐10), and transforming growth factor‐β (TGF‐β) was detected in WBCC supernatants by enzyme‐linked immunosorbent assays. Results: E. coli LPS significantly increased the expression of all cytokines in WBCCs from both the NP and CP groups when compared to non‐stimulated cells (control treatment). P. gingivalis LPS preparations increased IFN‐γ levels in the CP group but not in the NP group when compared with controls (P <0.05). P. gingivalis LPS preparations also increased IL‐10 and TGF‐β levels in both CP and NP groups, but P. gingivalis LPS₁₆₉₀ showed a three‐fold increase on IL‐10 production in the NP group (P <0.05) when compared to P. gingivalis LPS_1435/144. Conclusions: These data demonstrate that WBCC cell populations obtained from healthy individuals and patients with CP may differ in the cytokine response to P. gingivalis but not E. coli LPS. This is consistent with the notion that CP alters the systemic WBCC response and that this can be detected by the different P. gingivalis LPS structures.     

Involvement of adhesion molecule in in vitro plaque‐like formation of macrophages stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide

Takeshi T, Keisuke N, Takaaki I, Makoto Y, Tatsuji N. Involvement of adhesion molecule in in vitro plaque‐like formation of macrophages stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide. J Periodont Res 2010; 45: 550–556. © 2010 John Wiley & Sons A/S Background and Objective: Inflammatory agents, such as lipopolysaccharide (LPS), in periodontal pockets may promote atherogenesis by activating leukocytes. In our previous study, we developed a microchannel chip to observe the cell adhesion process in a fluid system. The objective of this investigation was to examine the mechanism by which periodontopathic bacterial LPS enhances plaque‐like formation on a microchannel chip. Material and Methods: To evaluate the effect of Aggregatibacter actinomycetemcomitans LPS on the expression of adhesion molecules, e.g. intercellular adhesion molecule 1 (ICAM‐1), lymphocyte function‐associated antigen 1 (LFA‐1) and L‐selectin, on the surface of murine macrophage RAW264.7 cells, the expression of each adhesion molecule was examined by flow cytometry and western blot analysis. Moreover, a flow test on the microchannel chip involving anti‐adhesion molecule antibodies was conducted to clarify which adhesion molecule is related to plaque‐like formation of RAW264.7 cells. Results: The expressions of ICAM‐1 and LFA‐1 on the surface of RAW 264.7 cells increased following 12 h culture with LPS; L‐selectin expression was unaffected. An increase in ICAM‐1 expression was also confirmed by western blot analysis. The flow test revealed that anti‐ICAM‐1 antibody inhibited plaque‐like formation of LPS‐stimulated macrophages on the micropillars of the microchannel chip. Conclusion: These findings indicate that ICAM‐1 plays an important role in plaque‐like formation of LPS‐stimulated macrophages. Our microchannel chip is a suitable tool for the investigation of etiological factors of atherosclerosis, including periodontitis, in vitro.     

Distribution of carcinoembryonic antigen‐related cellular adhesion molecules in human gingiva

Carcinoembryonic antigen‐related cellular adhesion molecules (CEACAMs) are glycoproteins produced in epithelial, endothelial, lymphoid, and myeloid cells. Carcinoembryonic antigen‐related cellular adhesion molecules mediate cell–cell contact and host–pathogen interactions. The aims of this study were to map the distribution and examine the regulation of CEACAMs in human gingival sites. Quantitative real‐time PCR performed on human gingival biopsies from periodontitis sites revealed mRNA coding for CEACAM1, ‐5, ‐6, and ‐7. Immunohistochemistry showed that CEACAMs were not found in oral gingival epithelium, except for CEACAM5 in periodontitis. Carcinoembryonic antigen‐related cellular adhesion molecules 1, 5, and 6 were present in the oral sulcular epithelium of periodontitis but not in that of healthy gingiva. In junctional epithelium, all three molecules were present in healthy gingiva, but in periodontitis only CEACAM1 and ‐6 were detected. Staining for CEACAM1 and ‐6 was also seen in the inflammatory cell infiltrate in periodontitis. No staining for CEACAM7 was found. Proinflammatory mediators, including lipopolysaccharide (LPS), tumour necrosis factor‐α (TNF‐α)/interleukin‐1β (IL‐1β), and interferon‐γ (IFN‐γ), increased the expression of CEACAM1 and CEACAM6 mRNAs in cultured human oral keratinocytes. CEACAM1 and CEACAM6 mRNAs were also strongly up‐regulated upon stimulation with lysophosphatidic acid. In conclusion, the distribution of different CEACAMs was related to specific sites in the gingiva. This might reflect different functional roles in this tissue.     

Distinct Th1, Th2 and Treg cytokines balance in chronic periapical granulomas and radicular cysts

J Oral Pathol Med (2010) 39 : 250–256 Background: Periapical lesions are a host response that involves immune reaction to prevent dissemination of bacteria from an infected root canal. The purpose of this study was to evaluate the levels of nitric oxide (NO), IL‐4, TGF‐β, tumor necrosis factor‐α (TNF‐α), and interferon‐γ (IFN‐γ) in chronic periapical lesions and to determine their possible association with clinical and radiographic parameters. Methods: Seventeen human radicular cysts and 30 periapical granulomas were used in this study. Cytokines and NO were assessed by enzyme‐linked immunosorbent assay and by the Griess reaction respectively confirmed by immunohistochemical. Results: TNF‐α and IFN‐γ were detected in 10% of granulomas and in 41.2% and 70% of radicular cysts. IL‐4 was reactive in 24% of cysts, and TGF‐β was positive in all samples. Patients with tenderness showed significantly higher levels of IFN‐γ and IL‐4 (P < 0.05). Swelling was associated with high levels of TNF‐α, IFN‐γ, and IL‐4 (P < 0.05). Lesions presenting bone resorption were associated with high levels of NO (P < 0.05). Conclusions: Periapical granulomas display a regulatory environment characterized by high TGF‐β and low inflammatory cytokine levels, while radicular cysts has mist Th1 and Th2 inflammatory reaction with the presence of IFN‐γ, TNF‐α, and IL‐4.