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.     

Neutrophils in chronic and aggressive periodontitis in interaction with Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans

Background and Objective: This study analyzed the interaction of Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 with peripheral blood polymorphonuclear neutrophils taken from patients with aggressive periodontitis and chronic periodontitis. Material and Methods: Peripheral blood polymorphonuclear neutrophils obtained from 12 patients with chronic periodontitis, six patients with aggressive periodontitis and 12 healthy controls were exposed to P. gingivalis and A. actinomycetemcomitans following opsonization of the bacteria using the patient’s own serum. Serum immunoglobulin G (IgG) levels against both periodontopathogens were measured. Phagocytosis and killing of the bacteria, as well as the extracellular human neutrophil elastase activity, were quantified. The total amount and the extracellular release of reactive oxygen species were measured using luminol‐dependent and isoluminol‐dependent chemiluminescence. Results: Polymorphonuclear neutrophils from patients with chronic (62.16 ± 19.39%) and aggressive (43.26 ± 26.63%) periodontitis phagocytosed more P. gingivalis than the healthy controls (24.43 ± 19.87%) at the 30‐min time point after exposure to the bacteria (p < 0.05). High serum IgG levels against P. gingivalis and A. actinomycetemcomitans were detected in subjects with periodontitis. Polymorphonuclear neutrophils from subjects with chronic and aggressive periodontitis released significantly more reactive oxygen species and demonstrated greater human neutrophil elastase activity in the absence of any stimulus than polymorphonuclear neutrophils from healthy controls (p < 0.05). Polymorphonuclear neutrophils in chronic periodontitis released significantly more reactive oxygen species when exposed to P. gingivalis and A. actinomycetemcomitans than polymorphonuclear neutrophils in aggressive periodontitis. Conclusion: High serum IgG levels against P. gingivalis and A. actinomycetemcomitans promote phagocytosis in periodontitis. The extracellular release of reactive oxygen species and neutrophil elastase by polymorphonuclear neutrophils may also contribute to damage of the surrounding periodontal tissues.     

Azithromycin Enhances Phagocytic Killing of Aggregatibacter actinomycetemcomitans Y4 by Human Neutrophils

Background: Aggregatibacter actinomycetemcomitans resists killing by neutrophils and is inhibited by azithromycin (AZM) and amoxicillin (AMX). AZM actively concentrates inside host cells, whereas AMX enters by diffusion. The present study is conducted to determine whether AZM is more effective than AMX at enhancing phagocytic killing of A. actinomycetemcomitans by neutrophils. Methods: Killing assays were conducted in the presence of either 2 μg/mL AZM or 16 μg/mL AMX (equipotent against A. actinomycetemcomitans). Neutrophils were loaded by incubation with the appropriate antibiotic. Opsonized A. actinomycetemcomitans strain Y4 was incubated with the indicated antibiotic alone, with loaded neutrophils and antibiotic, or with control neutrophils (without antibiotic) at multiplicities of infection (MOIs) of 30 and 90 bacteria per neutrophil. Results: Neutrophil incubation with 2 μg/mL AZM yielded an intracellular concentration of 10 μg/mL. At an MOI of 30, neutrophils loaded with AZM failed to kill significantly more bacteria than control neutrophils during the 60‐ and 90‐minute assay periods. At an MOI of 90, neutrophils loaded with AZM killed significantly more bacteria than either AZM alone or control neutrophils during 60‐ and 90‐minute incubations (P <0.05), and killed significantly more bacteria after 90 minutes than the sum of the killing produced by AZM alone or neutrophils alone. Neutrophils incubated with AMX under identical conditions also killed significantly more bacteria than either AMX alone or control neutrophils, but there was no evidence of synergism between AMX and neutrophils. Conclusions: Neutrophils possess a concentrative transport system for AZM that may enhance killing of A. actinomycetemcomitans. Its effects are most pronounced when neutrophils are greatly outnumbered by bacteria.     

Aggregatibacter actinomycetemcomitans‐Induced AIM2 Inflammasome Activation Is Suppressed by Xylitol in Differentiated THP‐1 Macrophages

Background: Aggressive periodontitis is characterized by rapid destruction of periodontal tissue caused by Aggregatibacter actinomycetemcomitans. Interleukin (IL)‐1β is a proinflammatory cytokine, and its production is tightly regulated by inflammasome activation. Xylitol, an anticaries agent, is anti‐inflammatory, but its effect on inflammasome activation has not been researched. This study investigates the effect of xylitol on inflammasome activation induced by A. actinomycetemcomitans. Methods: The differentiated THP‐1 macrophages were stimulated by A. actinomycetemcomitans with or without xylitol and the expressions of IL‐1β and inflammasome components were detected by real time PCR, ELISA, confocal microscopy and Immunoblot analysis. The effects of xylitol on the adhesion and invasion of A. actinomycetemcomitans to cells were measured by viable cell count. Results : A. actinomycetemcomitans increased pro IL‐1β synthesis and IL‐1β secretion in a multiplicity of infection‐ and time‐dependent manner. A. actinomycetemcomitans also stimulated caspase‐1 activation. Among inflammasome components, apoptosis‐associated speck‐like protein containing a CARD (ASC) and absent in melanoma 2 (AIM2) proteins were upregulated by A. actinomycetemcomitans infection. When cells were pretreated with xylitol, proIL‐1β and IL‐1β production by A. actinomycetemcomitans infection was significantly decreased. Xylitol also inhibited ASC and AIM2 proteins and formation of ASC puncta. Furthermore, xylitol suppressed internalization of A. actinomycetemcomitans into differentiated THP‐1 macrophages without affecting viability of A. actinomycetemcomitans within cells. Conclusions: A. actinomycetemcomitans induced IL‐1β production and AIM2 inflammasome activation. Xylitol inhibited these effects, possibly by suppressing internalization of A. actinomycetemcomitans into cells. Thus, this study proposes a mechanism for IL‐1β production via inflammasome activation and discusses a possible use for xylitol in periodontal inflammation caused by A. actinomycetemcomitans.     

Antibacterial activity of polysaccharide gel extract from fruit rinds of Durio zibethinus Murr. against oral pathogenic bacteria

Aim: The polysaccharide gel (PG) extract from durian fruit rinds (Durio zibethinus Murr. “Monthong”) is a pectic polysaccharide with antibacterial activity. This study aimed to investigate the in vitro antibacterial activity of PG against oral pathogens, Streptococcus mutans (S. mutans) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans). Methods: The inhibitory activity of PG at 50, 100, and 150 mg/mL against S. mutans (American Tissue Culture Collection 25175) and A. actinomycetemcomitans (American Tissue Culture Collection 43718) was determined after 1‐ and 5‐min exposure by broth macrodilution susceptibility test and scanning electron microscopy. Normal saline or culture broth medium and 0.1% chlorhexidine were used as negative and positive controls, respectively. Results: For 1‐min exposure, 150 mg/mL PG or 0.1% chlorhexidine significantly possessed bactericidal activity against both tested bacteria (P = 0.037), while PG at 100 mg/mL possessed significant bactericidal activity against S. mutans (P = 0.037) and inhibitory activity against A. actinomycetemcomitans (P = 0.05). Blebs, irregular‐shaped cells, and disrupted cells were found in bacteria treated with either 0.1% chlorhexidine or 50–150 mg/mL PG under scanning electron microscopy. Conclusion: The bactericidal activity of PG at 150 mg/mL against oral bacteria at 1‐min exposure suggests its possibility to be used as a natural antibacterial ingredient in oral hygiene products.     

Human salivary cystatin SA exhibits antimicrobial effect against Aggregatibacter actinomycetemcomitans

Ganeshnarayan K, Velliyagounder K, Furgang D, Fine DH. Human salivary cystatin SA exhibits antimicrobial effect against Aggregatibacter actinomycetemcomitans. J Periodont Res 2012; 47: 661–673. © 2012 John Wiley & Sons A/S Background and Objective: Healthy subjects who do not have Aggregatibacter actinomycetemcomitans in their oral cavity may possess factors in saliva that might demonstrate antibacterial activity against the bacterium. The aim of this study was to identify and purify proteins from saliva of healthy subjects that might demonstrate antibacterial activity against A. actinomycetemcomitans and test the same against the bacteria. Material and Methods: Saliva from 10 healthy volunteers was tested individually for its anti‐A. actinomycetemcomitans activity. Among the 10 subjects, eight demonstrated anti‐A. actinomycetemcomitans activity. Saliva was collected from one healthy volunteer who demonstrated the highest antimicrobial activity against A. actinomycetemcomitans. After clarifying the saliva, it was subjected to an affinity chromatography column with A. actinomycetemcomitans. The proteins bound to A. actinomycetemcomitans were eluted from the column and identified using mass spectrometry (MALDI‐TOF/TOF MS). Among other proteins that bound to A. actinomycetemcomitans, which included lactoferrin, immunoglobulin A and kallikrein, cystatin SA was observed in significantly higher concentrations, and this was purified from the eluate. The purified cystatin SA was tested at different concentrations for its ability to kill A. actinomycetemcomitans in a 2 h cell killing assay. The bacteria were also treated with a proteinase inhibitor, leupeptin, to clarify whether the antimicrobial effect of cystatin SA was related to its protease inhibitory function. Cystatin SA was also tested for its ability to prevent binding of A. actinomycetemcomitans to buccal epithelial cells (BECs) in an A. actinomycetemcomitans–BEC binding assay. Results: Cystatin SA (0.1 mg/mL) demonstrated a statistically significant antimicrobial activity against A. actinomycetemcomitans. The effect of cystatin SA decreased with lower concentrations, with 0.01 mg/mL showing no effect. The addition of monoclonal cystatin SA antibodies to the purified sample completely negated the antimicrobial effect. Treatment of A. actinomycetemcomitans with leupeptin resulted in no antimicrobial effect, suggesting that the antimicrobial activity of cystatin SA is independent of its protease inhibitory function. A. actinomycetemcomitans pretreated with cystatin SA showed reduced binding to BECs, suggesting a potential role for cystatin SA in decreasing the colonization of A. actinomycetemcomitans. Conclusion: The present study shows that cystatin SA demonstrates antimicrobial activity against the periodontopathogen A. actinomycetemcomitans, and future studies determining the mechanism of action are necessary. The study also shows the ability of cystatin SA to reduce significantly the binding of A. actinomycetemcomitans to BECs.     

Inhibition of interferon‐γ‐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.     

Macrophage tolerance response to Aggregatibacter actinomycetemcomitans lipopolysaccharide induces differential regulation of tumor necrosis factor-alpha, interleukin-1 beta and matrix metalloproteinase 9 secretion

Background and Objective: The lipopolysaccharide of Aggregatibacter actinomycetemcomitans, a potent stimulator of the immune system, induces the secretion of inflammatory mediators that modulate periodontal tissue destruction. In this study, we investigated the tolerance response of human macrophages to stimulation with A. actinomycetemcomitans lipopolysaccharide. Material and Methods: U937 monocytes were differentiated into adherent macrophage‐like cells by treatment with phorbol myristic acid. Macrophage‐like cells were then pretreated for 24 h with either 0.01 or 0.1 μg/mL LPS A. actinomycetemcomitans. Culture medium supernatants were removed and cells were restimulated with LPS at 1 μg/mL. Cell‐free supernatants were collected after 24 h of stimulation and analyzed by ELISA for TNF‐α, IL‐1β, IL‐6, IL‐8, PGE₂ and MMP‐9. Results: Phorbol myristic acid‐differentiated U937 macrophages treated with low doses of lipopolysaccharide developed tolerance to subsequent lipopolysaccharide treatments, resulting in significantly reduced secretion of tumor necrosis factor‐α. However, this tolerance response was associated with increased secretion of interleukin‐1β and matrix metalloproteinase 9, whereas the secretion of interleukin‐6, interleukin‐8 and prostaglandin E₂ was unaffected. Phosphatidylinositol‐3′‐kinase inhibitors added during the tolerance‐induction period markedly attenuated the increase in interleukin‐1β secretion but had no effect on tumor necrosis factor‐α. Conclusion: This study showed that A. actinomycetemcomitans lipopolysaccharide can induce a tolerance response in macrophages that alters the secretion of two important inflammatory mediators as well as of the tissue‐degrading enzyme matrix metalloproteinase‐9. This phenomenon may play a role in modulating the host inflammatory response and the progression of periodontitis.     

Detection of a single bacterial cell using a 16S ribosomal RNA‐specific oligonucleotide probe designed to investigate periodontal pathogens

Introduction: The current detection methods for periodontopathogens mainly use polymerase chain reactions. However, there are few methods available for visualizing the bacteria that impact on patients with periodontal disease for use in health education. The purpose of this study was to develop a specific detection method to visualize periodontopathogenic bacteria. Methods: Fluorescently‐labeled oligonucleotide probes directed to specific 16S ribosomal RNA (rRNA) sequences of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were synthesized. Cultured individual bacterial species were fixed with 4% paraformaldehyde and smeared on glass slides. Fluorescein isothiocyanate‐labeled oligonucleotide probes were hybridized under stringent conditions with smeared whole cells, and then probe specificity was investigated by epifluorescence microscopy. Results: Comparatively long (50‐mer) oligonucleotide probes for P. gingivalis and A. actinomycetemcomitans were designed. These probes clearly hybridized with 16S rRNA of the target species in situ and single bacterial cells were detectable visually. The probes exhibited no cross‐hybridization against the additional organisms that were closely related to the target species. Conclusions: The fluorescence in situ hybridization technique is a specific and reliable method by which to visually identify the target organisms. The oligonucleotide probes designed in this study will be useful for detecting P. gingivalis and A. actinomycetemcomitans populations.     

Binding of Actinobacillus actinomycetemcomitans lipopolysaccharides to Peptostreptococcus micros stimulates tumor necrosis factor α production by macrophage‐like cells

Peptostreptococcus micros is a gram‐positive bacterium that has been associated with periodontitis and endodontic infections. In this study, we hypothesized that P. micros binds the immunomodulating component lipopolysaccharide derived from gram‐negative bacteria to increase its capacity to stimulate cytokine production by host cells. The ability of P. micros to bind Actinobacillus actinomycetemcomitans lipopolysaccharide was demonstrated by an enzyme‐linked immunosorbent assay and by immunoelectron microscopy. Pretreatment of P. micros cells with A. actinomycetemcomitans lipopolysaccharide was associated with a 49‐fold increase in tumor necrosis factor alpha production by human monocytic cells U937 differentiated into adherent macrophages, compared to the stimulation with untreated P. micros. This effect was suppressed by incorporating polymyxin B, a lipid A‐binding substance, during treatment of macrophage‐like cells with lipopolysaccharide‐coated P. micros cells. This is the first study reporting a binding interaction between lipopolysaccharide and a gram‐positive bacterium. This interaction represents a new mechanism that could promote the inflammatory response during periodontitis.     

Secreted adenosine triphosphate from Aggregatibacter actinomycetemcomitans triggers chemokine response

Extracellular ATP (eATP) is an important intercellular signaling molecule secreted by activated immune cells or released by damaged cells. In mammalian cells, a rapid increase of ATP concentration in the extracellular space sends a danger signal, which alerts the immune system of an impending danger, resulting in recruitment and priming of phagocytes. Recent studies show that bacteria also release ATP into the extracellular milieu, suggesting a potential role for eATP in host–microbe interactions. It is currently unknown if any oral bacteria release eATP. As eATP triggers and amplifies innate immunity and inflammation, we hypothesized that eATP secreted from periodontal bacteria may contribute to inflammation in periodontitis. The aims of this study were to determine if periodontal bacteria secrete ATP, and to determine the function of bacterially derived eATP as an inducer of inflammation. Our results showed that Aggregatibacter actinomycetemcomitans, but not Porphyromonas gingivalis, Prevotella intermedia, or Fusobacterium nucleatum, secreted ATP into the culture supernatant. Exposure of periodontal fibroblasts to filter sterilized culture supernatant of A. actinomycetemcomitans induced chemokine expression in an eATP‐dependent manner. This occurred independently of cyclic adenosine monophosphate and phospholipase C, suggesting that ionotrophic P2X receptor is involved in sensing of bacterial eATP. Silencing of P2X7 receptor in periodontal fibroblasts led to a significant reduction in bacterial eATP‐induced chemokine response. Furthermore, bacterial eATP served as a potent chemoattractant for neutrophils and monocytes. Collectively, our findings provide evidence for secreted ATP of A. actinomycetemcomitans as a novel virulence factor contributing to inflammation during periodontal disease.     

Molecular mimicry of Aggregatibacter actinomycetemcomitans with β2 glycoprotein I

Introduction: β2‐Glycoprotein I (β2GPI) is important in the suppression of coagulation, and antibodies against TLRVYK peptides on the β2GPI molecule are related to thrombosis. According to the Swiss‐Prot database, Aggregatibacter actinomycetemcomitans leukotoxin c has sequences (SIRVYK) that are homologous to the TLRVYK peptides. The aim of this study was to investigate the effects of A. actinomycetemcomitans infection on the antibody response against SIRVYK peptides in patients with periodontitis. Methods: Serum immunoglobulin G (IgG) antibody and IgG subclass antibody titers against SIRVYK or TLRVYK peptides were measured by enzyme‐linked immunosorbent assay in 46 patients with aggressive periodontitis (eight with localized disease, 38 with generalized disease), 28 patients with chronic periodontitis, and 20 periodontally healthy subjects. The presence of A. actinomycetemcomitans in plaque and saliva samples was determined using polymerase chain reaction. Results: The level of anti‐SIRVYK antibodies was significantly higher in patients who were A. actinomycetemcomitans‐positive than in A. actinomycetemcomitans‐negative patients (P < 0.05) in the chronic periodontitis group. A similar trend was found in the antibody response to TLRVYK peptide; however, no statistically significant difference was seen between A. actinomycetemcomitans‐positive and ‐negative patients. The A. actinomycetemcomitans‐positive patients displayed significantly higher levels of anti‐SIRVYK IgG2 and IgG3 antibodies than A. actinomycetemcomitans‐negative patients (P < 0.05 and P < 0.05, respectively). The level of IgG2 was highest among the four IgG subclasses and it predominantly increased in patients who were A. actinomycetemcomitans‐positive. Anti‐TLRVYK antibody levels were significantly correlated with anti‐SIRVYK IgG antibody levels. Conclusion: The results suggest that A. actinomycetemcomitans infection may elicit anti‐SIRVYK IgG antibodies and modify the anti‐TLRVYK antibody response in patients with periodontitis by molecular mimicry with β2GPI.     

Porphyromonas gingivalis promotes murine abdominal aortic aneurysms via matrix metalloproteinase-2 induction

Aoyama N, Suzuki J, Wang D, Ogawa M, Kobayashi N, Hanatani T, Takeuchi Y, Izumi Y, Isobe M. Porphyromonas gingivalis promotes murine abdominal aortic aneurysms via matrix metalloproteinase‐2 induction. J Periodont Res 2011; 46: 176–183. © 2010 John Wiley & Sons A/S Background and Objective: Abdominal aortic aneurysm (AAA) is a common and lethal disorder, and MMPs are highly expressed in AAA lesions. Large numbers of periodontopathic bacteria have been reported to be present in specimens obtained from the aortic walls of patients with an AAA. The purpose of this study was to analyze the influence of periodontopathic bacteria on AAA dilatation. Material and Methods: AAAs were produced in mice by the periaortic application of 0.25 m CaCl₂, and NaCl was used as a control. The mice were inoculated once weekly with live Porphyromonas gingivalis, live Aggregatibacter actinomycetemcomitans or vehicle. Results: Four weeks after the periaortic application of either CaCl₂ or NaCl, a significant increase was observed in the aortic diameter of P. gingivalis‐challenged mice compared with the vehicle control mice (p < 0.05), whereas there was no statistically significant increase in the aortic diameter of the A. actinomycetemcomitans‐challenged mice. Immunohistochemical analysis found significantly higher numbers of CD8‐positive and MOMA2‐positive cells and significantly higher levels of MMP‐2 in the aneurysmal samples of P. gingivalis‐challenged mice compared with control mice. Live P. gingivalis promoted a significant proliferation of splenocytes in comparison with P. gingivalis‐lipopolysaccharide and live A. actinomycetemcomitans (p < 0.05). Conclusion: These findings demonstrate that challenge with P. gingivalis, but not with A. actinomycetemcomitans, can accelerate, or even initiate, the progression of experimental AAA through the increased expression of MMPs.     

Clarithromycin accumulation by phagocytes and its effect on killing of Aggregatibacter actinomycetemcomitans

Background: Clarithromycin inhibits several periodontal pathogens and is concentrated inside gingival fibroblasts and epithelial cells by an active transporter. We hypothesized that polymorphonuclear leukocytes (PMNs) and less mature myeloid cells possess a similar transporter for clarithromycin. It is feasible that clarithromycin accumulation inside PMNs could enhance their ability to kill Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans). Methods: To test the first hypothesis, purified PMNs and cultured HL‐60 cells were incubated with [³H]‐clarithromycin. Clarithromycin transport was assayed by measuring changes in cell‐associated radioactivity over time. The second hypothesis was examined with PMNs loaded by incubation with clarithromycin (5 μg/ml). Opsonized bacteria were incubated at 37°C with control and clarithromycin‐loaded PMNs. Results: Mature human PMNs, HL‐60 cells differentiated into granulocytes, and undifferentiated HL‐60 cells all took up clarithromycin in a saturable manner. The kinetics of uptake by all yielded linear Lineweaver‐Burk plots. HL‐60 granulocytes transported clarithromycin with a K_m of ≈250 μg/ml and a V_max of 473 ng/min/10⁶ cells, which were not significantly different from the values obtained with PMNs. At steady state, clarithromycin levels inside HL‐60 granulocytes and PMNs were 28‐ to 71‐fold higher than extracellular levels. Clarithromycin‐loaded PMNs killed significantly more A. actinomycetemcomitans and achieved shorter half‐times for killing than control PMNs when assayed at a bacteria‐to‐PMN ratio of 100:1 (P <0.04). At a ratio of 30:1, these differences were not consistently significant. Conclusions: PMNs and less mature myeloid cells possess a transporter that takes up and concentrates clarithromycin. This system could help PMNs cope with an overwhelming infection by A. actinomycetemcomitans.     

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.     

Modulation of Matrix Metalloproteinase and Cytokine Production by Licorice Isolates Licoricidin and Licorisoflavan A: Potential Therapeutic Approach for Periodontitis

Background: Inflammatory cytokines and matrix metalloproteinases (MMPs) produced by resident and inflammatory cells in response to periodontopathogens play a major role in the tissue destruction observed in periodontitis, which is a disease that affects tooth‐supporting structures. In the present study, we investigate the effects of licorice‐derived licoricidin (LC) and licorisoflavan A (LIA) on the secretion of various cytokines and MMPs by human monocyte‐derived macrophages stimulated with Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) lipopolysaccharide (LPS). Methods: Macrophages were treated with non‐toxic concentrations of LC or LIA before being stimulated with A. actinomycetemcomitans LPS. The secretion of cytokines and MMPs and the activation of nuclear factor‐kappa B (NF‐κB) p65 and activator protein (AP)‐1 were assessed by enzyme‐linked immunosorbent assays. Results: LC and LIA inhibited the secretion of interleukin (IL)‐6 and chemokine (C‐C motif) ligand 5 in a concentration‐dependent manner but did not affect the secretion of IL‐8 by LPS‐stimulated macrophages. LC and LIA also inhibited the secretion of MMP‐7, ‐8, and ‐9 by macrophages. The suppression of cytokine and MMP secretion by LC and LIA was associated with the reduced activation of NF‐κB p65 but not that of AP‐1. Conclusion: The present study suggests that LC and LIA have potential for the development of novel host‐modulating strategies for the treatment of cytokine and/or MMP‐mediated disorders such as periodontitis.     

Protease‐activated receptor 2 mediates interleukin‐8 and intercellular adhesion molecule‐1 expression in response to Aggregatibacter actinomycetemcomitans

Introduction:  We investigated the mechanisms by which extracts of Aggregatibacter actinomycetemcomitans affect the inflammatory response in gingival epithelial cells. Methods:  Human gingival cells (Ca9‐22) were cultured in bacterial extracts prepared from A. actinomycetemcomitans ATCC 29522. The cells were pretreated with protease inhibitors or transfected with small interfering RNA (siRNA) specific for protease‐activated receptor 2 (PAR‐2). Results:  The pretreatment of cells with serine protease inhibitors significantly inhibited A. actinomycetemcomitans extract‐induced expression of interleukin‐8 (IL‐8) and intercellular adhesion molecule‐1 (ICAM‐1) at both the messenger RNA and protein levels. In addition, A. actinomycetemcomitans extract‐induced IL‐8 and ICAM‐1 expression was significantly decreased in PAR‐2/siRNA‐transfected cells. Conclusions:  A. actinomycetemcomitans extract‐induced IL‐8 and ICAM‐1 expression in gingival epithelial cells is mediated by PAR‐2.     

Activation of toll‐like receptors 2 and 4 by gram‐negative periodontal bacteria

Background/aims: Periodontitis is a chronic infectious disease associated with a gram‐negative subgingival microflora. Bacterial components stimulate, among other receptors, Toll‐like receptor (TLR) 2 and/or TLR4. Accumulating evidence indicates that both qualitatively and quantitatively distinct immune responses result from the triggering of TLR2 as compared to TLR4 triggering. The aim was to study the interaction of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Tannerella forsythensis, Prevotella intermedia, Prevotella nigrescens, Fusobacterium nucleatum and Veillonella parvula with TLR2 and TLR4. We investigated all known serotypes (K⁻, K1–K6) of P. gingivalis and A. actinomycetemcomitans serotype a–e strains for their potency to stimulate cytokine production. Methods: Human embryonic kidney (HEK) cells, stably transfected with CD14, CD14‐TLR2, or CD14‐TLR4 and whole blood were stimulated with bacterial sonicates. Cytokine production (interleukin‐6, ‐8, ‐10 and ‐12) was measured in the supernatant by enzyme‐linked immunosorbent assay. Results: All test bacteria stimulated HEK‐CD14‐TLR2, but only A. actinomycetemcomitans and V. parvula stimulated HEK‐CD14‐TLR4. No differences were found in the activation of HEK‐CD14‐TLR2/4, or cytokine production in whole blood between serotypes of P. gingivalis and A. actinomycetemcomitans. Conclusion: Gram‐negative periodontal bacteria predominantly stimulated TLR2, which may be of importance for the Th1/Th2 cell orientation of the immune response in periodontitis.