Nitric oxide production by a human osteoblast cell line stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide

Background/aim:  Human osteoblasts induced by inflammatory stimuli express an inducible nitric oxide synthase (iNOS). The aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans lipopolysaccharide stimulates the production of nitric oxide (NO) by a human osteoblast-like cell line (HOS cells).
Methods:  Cells were stimulated directly with A. actinomycetemcomitans lipopolysaccharide or pretreated with the following l -NIL (an iNOS inhibitor), anti-CD14, Toll-like receptor 2 (TLR2), or TLR4 antibody before stimulation with A. actinomycetemcomitans lipopolysaccharide. The role of the cyclic nucleotides was assessed by pretreating the cells with the following; ODQ (a guanylyl cyclase inhibitor); SQ22536 (an adenylyl cyclase inhibitor); db-cAMP (a cyclic adenosine monophosphate analog); br-cGMP (a cyclic guanosine monophosphate analog); forskolin (an adenylyl cyclase activator), IBMX [a non-specific phosphodiesterase (PDE) inhibitor], or KT5720 [a protein kinase A (PKA) inhibitor]. The cells were also preincubated with genistein [a protein tyrosine kinase (PTK) inhibitor], bisindolylmaleimide [a protein kinase C (PKC) inhibitor], BPB [a phospholipase A2 (PLA2) inhibitor], and NDGA (a lipoxygenase inhibitor). The iNOS activity and nitrite production in the cell cultures were determined spectrophotometrically.
Results:  The results showed that A. actinomycetemcomitans lipopolysaccharide stimulated both iNOS activity and nitrite production by HOS cells; this was reduced by l -NIL, anti-CD14, or anti-TLR4 antibody, SQ22536, KT5720, genistein, bisindolylmaleimde, BPB, and NDGA, but was enhanced by db-cAMP, IBMX, and forskolin.
Conclusion:  These results therefore suggest that A. actinomycetemcomitans lipopolysaccharide may induce the production of NO by HOS cells via a CD14–TLR4 molecule complex, a cAMP–PKA pathway, as well as by a PTK, PKC, PLA2, and lipoxygenase-dependent mechanism.     

Effect of L-N6-(1-iminoethyl)-lysine, an inducible nitric oxide synthase inhibitor, on murine immune response induced by Actinobacillus actinomycetemcomitans lipopolysaccharide

BACKGROUND AND OBJECTIVES: Inducible nitric oxide synthase (iNOS) activity is known to regulate the immune response. The present study was carried out to determine the effect of L-N6-(1-iminoethyl)-lysine (L-NIL), an iNOS inhibitor, on the induction of immune response to Actinobacillus actinomycetemcomitans lipopolysaccharide in mice. MATERIAL AND METHODS: BALB/c mice were sham-immunized (group I), immunized with A. actinomycetemcomitans lipopolysaccharide (group II) or treated with L-NIL and immunized with A. actinomycetemcomitans lipopolysaccharide (group III). All animals were then challenged with viable A. actinomycetemcomitans. The levels of serum nitric oxide (NO), specific immunoglobulin G (IgG) isotypes and both interferon-gamma and interleukin-4, as well as spleen cell-derived iNOS activity, before and after bacterial challenge, were assessed. The diameter of skin lesions was also determined. Serum and spleen cells from the above groups were adoptively transferred to the recipients that were then subsequently challenged with live bacteria. RESULTS: Treatment with L-NIL suppressed serum NO and splenic iNOS activity, but enhanced serum-specific IgG2a antibody and interferon-gamma levels. The lesions in L-NIL-treated mice healed much more rapidly. Transfer with serum and cells from L-NIL-treated and A. actinomycetemcomitans lipopolysaccharide-immunized donors resulted in rapid healing of the lesions in the recipients. CONCLUSION: It is suggested that treatment with L-NIL in mice immunized with A. actinomycetemcomitans lipopolysaccharide may shift the immune response towards a protective T helper 1-like immunity against A. actinomycetemcomitans-induced infection.     

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.     

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.     

Inhibition of interferon-gamma-induced nitric oxide production in endotoxin-activated macrophages by cytolethal distending toxin

Introduction:  Cytolethal distending toxin (CDT) is a DNA-targeting agent produced by certain pathogenic gram-negative bacteria such as the periodontopathogenic organism Aggregatibacter actinomycetemcomitans . CDT targets lymphocytes and other cells causing cell cycle arrest and apoptosis, impairing the host immune response and contributing to the persistence of infections caused by this microorganism. In this study we explored the effects of CDT on the innate immune response, by investigating how it affects production of nitric oxide (NO) by macrophages.
Methods:  Murine peritoneal macrophages were stimulated with Escherichia coli sonicates and NO production was measured in the presence or not of active CDT.
Results:  We observed that CDT promptly and significantly inhibited NO production by inducible nitric oxide synthase (iNOS) in a dose-dependent manner. This inhibition is directed towards interferon-γ-dependent pathways and is not mediated by either interleukin-4 or interleukin-10.
Conclusion:  This mechanism may constitute an important aspect of the immunosuppression mediated by CDT and may have potential clinical implications in A. actinomycetemcomitans infections.     

Role of Aggregatibacter actinomycetemcomitans in glutathione catabolism

Introduction:  Our previous studies demonstrated that three enzymes, γ-glutamyltransferase (GGT), cysteinylglycinase (CGase) and cystalysin, are required for the catabolism of glutathione to produce hydrogen sulfide (H₂S) in Treponema denticola . In this study, we examined glutathione catabolism in Aggregatibacter actinomycetemcomitans .
Methods:  The GGT and CGase of A. actinomycetemcomitans were determined by biological methods and GGT was characterized using a molecular biological approach.
Results:  A. actinomycetemcomitans showed GGT and CGase activity, but could not produce H₂S from glutathione. The addition of recombinant T. denticola cystalysin, an l -cysteine desulfhydrase, to whole cells of A. actinomycetemcomitans resulted in the production of H₂S from glutathione. Subsequently, we cloned A. actinomycetemcomitans GGT gene ( ggt ) and overexpressed the 63 kDa GGT protein. The recombinant A. actinomycetemcomitans GGT was purified and identified. The K _cat/ K _m of the recombinant GGT from N -γ- l -glutamyl-4-nitroaniline as substrate was 31/μ m /min. The activity of GGT was optimum at pH 6.9–7.1 and enhanced by thiol-containing compounds.
Conclusion:  The results demonstrated that A. actinomycetemcomitans had GGT and CGase activities and that the GGT was characterized. The possible role of A. actinomycetemcomitans in glutathione metabolism and H₂S production from oral bacteria was discussed.     

Protective effects of grape seed proanthocyanidins against oxidative stress induced by lipopolysaccharides of periodontopathogens

BACKGROUND: During phagocytosis or stimulation with bacterial components, macrophages activate various cell processes, including the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are critical for successful defense against invading organisms. Increased levels of ROS/RNS create oxidative stress that results in tissue and bone destruction. Grape seed proanthocyanidins have been reported to possess a wide range of biologic properties against oxidative stress. In the present study, we investigated the effects of a grape seed proanthocyanidin extract (GSE) and commercial polyphenols on the production of ROS and RNS and on the protein expression of inducible nitric oxide synthase (iNOS) by murine macrophages stimulated with lipopolysaccharides (LPS) of periodontopathogens. METHODS: Macrophages (RAW 264.7) were treated with non-toxic concentrations of either GSE or commercial polyphenols (gallic acid [GA] and [-]-epigallocatechin-3-gallate [EGCG]) and stimulated with LPS of Actinobacillus actinomycetemcomitans or Fusobacterium nucleatum, and iNOS expression was evaluated by immunoblotting. Nitric oxide (NO) production was quantified using the colorimetric Griess assay, whereas ROS production was measured with the fluorescent 123-dihydrorhodamine dye. RESULTS: GSE strongly decreased NO and ROS production and iNOS expression by LPS-stimulated macrophages. GA also revealed a strong inhibitory effect on NO production without affecting iNOS expression but slightly increasing ROS production. EGCG showed an inhibitory effect on NO and ROS production and on iNOS expression by macrophages. CONCLUSION: Our findings demonstrate that proanthocyanidins have potent antioxidant properties and should be considered a potential agent in the prevention of periodontal diseases.     

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.     

Human gingival fibroblasts release high-mobility group box-1 protein through active and passive pathways

Introduction:  The nuclear protein high-mobility group box-1 (HMGB1) acts as a late mediator of inflammation when secreted in the extracellular milieu. In this study, we examined the effect of lipopolysaccharides from periodontal pathogens and apoptotic and necrotic cell death on HMGB1 production in human gingival fibroblasts (HGF).
Methods:  HGF from healthy periodontal tissue were cultured and stimulated with lipopolysaccharides (LPS) from Aggregatibacter actinomycetemcomitans , Porphyromonas gingivalis , and Escherichia coli . We also initiated apoptotic and necrotic cell deaths in HGF. The HMGB1 released in the supernatants from stimulated or dying cells was measured. Immunocytochemical staining against HMGB1 was performed in LPS-stimulated HGF.
Results:  A significantly higher amount of HMGB1 was detected from necrotic and apoptotic HGF. LPS from A. actinomycetemcomitans , P. gingivalis , and E. coli significantly induced the production of HMGB1 in a time-dependent manner. After 6 h of LPS stimulation, HMGB1 was present in the cytoplasm of cells whereas its location was mainly nuclear after 24 h.
Conclusions:  LPS from two major periodontal pathogens, A. actinomycetemcomitans and P. gingivalis , induced HMGB1 secretion from HGF. Apoptotic and necrotic cell deaths resulted in the enhancement of HMGB1. Our results suggest that HGF can be a source of HMGB1 by both active secretion and passive release, and that HMGB1 from HGF may contribute to periodontal tissue destruction.     

Gene targeting demonstrates that inducible nitric oxide synthase is not essential for resistance to oral candidiasis in mice, or for killing of Candida albicans by macrophages in vitro

Introduction:  Oral candidiasis is caused by opportunistic infections with the yeast Candida albicans . Previous studies have demonstrated important roles for innate immunity and T helper type 1-mediated inflammatory reactions in recovery from infection, with macrophages and neutrophils as key effector cells. Both effector cell types use the inducible isoform of nitric oxide synthase (iNOS) to generate candidacidal molecules, but it is not clear whether nitric oxide (NO) is an absolute requirement for candidacidal effector activity.
Methods:  In this study we directly investigated the role of iNOS-derived NO in resistance to murine experimental oral candidiasis, using iNOS knockout mice.
Results:  Knockout mice were no more susceptible to oral candidiasis than wild-type controls. Bone marrow-derived macrophages from the knockout mice killed C. albicans yeasts efficiently in vitro , and were still able to produce nitrites in an iNOS-independent manner, albeit less efficiently than wild-type controls. There were no significant differences in local mucosal production of interleukins 6, 12, 17A, or 23, interferon-γ, or transforming growth factor-β 24 h after oral challenge with C. albicans .
Conclusion:  These data suggest that iNOS-derived NO is not required for resistance to oral candidiasis in vivo , and that bone marrow-derived macrophages may have iNOS-independent means of generating reactive nitrogen species.     

脂多糖和白细胞介素-1β对人牙周膜细胞中诱导型一氧化氮合酶表达的影响

目的 观察脂多糖(LPS)和白细胞介素-1β(IL-1β)对人牙周膜细胞(hPDLCs)表达诱导型一氧化氮合酶(iGNOS)和一氧化氮(NO)的影响.方法 应用LPS和IL-1β刺激hPDLCs后,通过实时定量PCR检测iNOS基因的表达情况,收集细胞上清液,酶联免疫吸附试验(ELISA)测定诱导后细胞中iNOS的含量...     

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.     

A novel selective medium for isolation of Aggregatibacter (Actinobacillus) actinomycetemcomitans

Background and Objective:  Conventional selective media have been used for the selection of Aggregatibacter ( Actinobacillus ) actinomycetemcomitans in clinical samples. The proportion of A. actinomycetemcomitans grown on the selective media in vitro may not reflect the true counts in vivo because of the low selectivity. A novel selective medium, designated AASM, was developed for the isolation of A. actinomycetemcomitans .
Material and Methods:  AASM was prepared by adding of 200 μg/mL of vancomycin and 10 U/mL of bacitracin to AAGM, which contains dextrose, sodium bicarbonate, trypticase soy, yeast extract and agar. Clinical efficacy was evaluated by the recovery, on AASM, of A. actinomycetemcomitans from subgingival samples of 44 periodontally healthy subjects and 76 patients with chronic periodontitis.
Results:  All serotypes (a–f) of A. actinomycetemcomitans strains grew well, and the average growth recovery of A. actinomycetemcomitans on AASM medium was 94.4% (80.0–109.7%) of that on AAGM. The exclusive rate of other bacteria was 99.9% in clinical samples cultured on AASM. A. actinomycetemcomitans was not detected in periodontally healthy persons but was detected in 25 (32.9%) patients with chronic periodontitis. The predominant serotype was c, detected in 11 subjects.
Conclusion:  The new selective medium, AASM, was highly selective for A. actinomycetemcomitans , eliminated possible false-positive results and was useful for the isolation of A. actinomycetemcomitans from clinical samples.     

Chemical composition and immunobiological properties of lipopolysaccharide and lipid-associated proteoglycan from Actinobacillus actinomycetemcomitans

Lipopolysaccharide (LPS) and lipid-associated proteoglycan (LPG) were isolated from Actinobacillus actinomycetemcomitans ATCC 29523 by the phenol-water and butanol-water procedures. The LPS was composed of 41 % neutral sugar. 8% hexosamine, 31 % fatty acid, 2% protein, and 2% phosphorus, while the butanol-water-extracted LPG was composed of 13% neutral sugar, 2% hexosamine, 14% fatty acid. 56% protein, and 1% phosphorus. The major fatty acids of LPS and LPG were β-hydroxymyristic, myristic, and palmitic acids. These preparations induced mitogenic and polyclonal responses of LPS-responsive C3H/HeN mouse spleen cells and enhanced in vitro immune responses of the spleen cells to sheep erythrocytes. Spleen cells of C3H/HeJ mouse that were nonresponsive to enterobacteriaceal LPS responded to the butanol-water-extracted LPG, but not to the phenol-water-extracted LPS. The Limulus amebocyte lysate clotting activity of A. actinomycetemcomitans LPS was comparable to that of Escherichia coli K235 LPS. These results indicate that A. actinomycetemcomitans LPS is biologically and immunochemically of Enterobacteriaceae type.     

Influence of proinflammatory cytokines on Actinobacillus actinomycetemcomitans specific IgG responses

OBJECTIVE: High levels of serum anti-Actinobacillus actinomycetemcomitans immunoglobulin G (IgG) correlate with reduced extent and severity of periodontal disease and the present study was undertaken to begin testing the hypothesis that proinflammatory cytokines are important in the induction of optimal anti-A. actinomycetemcomitans IgG responses. BACKGROUND: Studies with pokeweed mitogen indicate that interleukin-1alpha (IL-1alpha) and IL-1beta are necessary for optimal IgG1 and IgG2 production and that prostaglandin E(2) (PGE(2)) and interferon-gamma (IFN-gamma) selectively promote IgG2, which is a major component of the anti-A. actinomycetemcomitans response in vivo. The pokeweed mitogen results suggest that these proinflammatory cytokines would also be necessary for optimal production of IgG specific for A. actinomycetemcomitans. METHODS: Peripheral blood mononuclear cells from A. actinomycetemcomitans-seropositive subjects with localized aggressive periodontitis were stimulated with A. actinomycetemcomitans in immune complexes capable of binding follicular dendritic cells that participate in the induction of recall responses in vivo. Cultures were manipulated with anti-IL-1alpha, anti-IL-1beta, anti-IFN-gamma, anti-IL-12, anti-CD21, indomethacin, and PGE(2). Actinobacillus actinomycetemcomitans specific IgG production was monitored by enzyme-linked immunosorbent assay (ELISA). RESULTS: Addition of follicular dendritic cells to peripheral blood mononuclear cells cultures resulted in follicular dendritic cell-lymphocyte clusters and increased anti-A. actinomycetemcomitans IgG responses (3-40-fold increases) compared with controls lacking follicular dendritic cells. Anti-IL-1alpha, anti-IL-1beta, anti-IFN-gamma, anti-IL-12, anti-CD21 and indomethacin suppressed anti-A. actinomycetemcomitans IgG production by half or more. PGE(2) restored IgG responses suppressed by indomethacin. CONCLUSIONS: The cytokines IL-1alpha, IL-1beta, IFN-gamma, IL-12, and PGE(2) were all necessary for optimal production of human anti-A. actinomycetemcomitans and the need for proinflammatory cytokines including the T helper 1 (Th1) cytokines is consistent with a response with a significant IgG2 component.     

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.