Chihomi Kato

Nitric Oxide Production and iNOS mRNA Expression in Mice Induced by Repeated Stimulation with Live Fusobacterium nucleatum

There have been few studies on the detection of direct nitric oxide (NO) production and interferon‐gamma (IFN‐γ) in vivo without using animal cell culture. We questioned whether NO and IFN‐γ could be produced at the site of infection. The peritoneal cavity of mice was used as the local infection model. NO and IFN‐γ in abdominal washings from these mice were measured directly at various times after injection of Fusobacterium nucleatum, a gram‐negative rod periodontal pathogen. The mice were divided into three groups: those treated with live bacteria (LB), those treated with heat‐killed bacteria (HKB) and those untreated: normal (N). These mice were compared on the basis of cell filtration, NO and IFN‐γ production by injection of live bacteria (LFn) or heat‐killed bacteria (HKFn). In the LB group, the total cell number increased corresponding to an increase in neutrophils after injection of both LFn and HKFn. A low level of NO was constantly produced in abdominal washings, but a significant amount of NO was synthesized in the LB group only 12 hr to 24 hr after injection of LFn. At the same time iNOS enzyme activity and iNOS mRNA expression were detected. IFN‐γ, which may contribute to enhance NO production, was also secreted at a high level from peritoneal exudate cells (PEC) at 12 hr and 24 hr in the LB group by stimulation of LFn. At 12 hr and 24 hr, iNOS positive cells in the LB group by infection of LFn were identified and shown to contain mostly macrophages. These findings indicate that live bacteria play important roles in NO production by macrophages. It is suggested that NO may contribute to the inflammatory response during F. nucleatum infection in periodontitis.

Comparison of the Immunological Responses in Mice Sensitized with Live Bacteria of Either Fusobacterium nucleatum or Porphyromonas gingivalis and Its Bactericidal Effect

Abstract The immunological responses induced by Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn) in mice were compared. SPF ICR immune mice were obtained by 3 injections with either Pg or Fn. Two weeks later each bacteria was administered to immunized mice and mice were assayed for cell numbers in exudates, antibody titer, cytokine production, expression of cytokine receptors, and the elimination of the bacteria. The total cell numbers in Fn immunized, then Fn challenged, mice were 1.6 times that of mice challenged with Pg. High IgG antibody titers were detected in Pg immunized mice and IgM antibody predominated in Fn immunized mice. TNFα production was higher in the abdomen of Pg immunized mice than in serum. IL-1β levels were the same in both Fn and Pg challenged mice. IFNγ production was significantly higher in Fn than Pg-immunized mice. Expression of CXCR-1 and CXCR-3 mRNA was significantly higher in Fn than Pg-immunized mice. The elimination effect of the bacteria in the abdominal cavity was remarkable after Fn but not in Pg immunization. Fn immunized mice were more effective in eliminating Pg in the abdomen, suggesting the macrophage activation via Th1 may play a part in protection from periodontitis.

The Participation of Nitric Oxide in Peritoneal Exudate Cell Cytotoxicity of Mice by Fusobacterium nucleatum

Previously we reported that mice infected recurrently with live Fusobacterium nucleatum (Fn) synthesize a significant amount of NO between 12 hr and 24 hr after Fn injection. Fn is a gram‐negative rod periodontal pathogen. NO could not be induced by heat‐killed Fn or in untreated mice. This NO, derived from the iNOS after infection of live Fn, was not involved in the Fn reduction because Fn clearance occurs within 6 hr. We investigated in this study whether this NO was involved in cytotoxicity in peritoneal exudate cells (PEC) in vivo. The mice were divided into two groups: those treated with live Fn (immune) and those left untreated (normal). PEC number, NO production, detection of apoptosis or death cells, and lactate dehydrogenase (LDH) release activity after injection of live Fn were compared in these groups. In the immune group, the increase of the total cell numbers caused by an increase in neutrophils, a significant NO production only after injection of live Fn at 24 hr and identification of iNOS positive macrophages were confirmed. The apoptotic rate was very low and did not increase at 24 hr in vivo. Therefore, apoptosis was seldom relevant to the NO. In the immune group, LDH activity was remarkable high at 24 hr, and dead cells and macrophages phagocytizing cell fragments increased at the same time. Pretreatment of L‐NMMA, an inhibitor of iNOS, suppressed LDH activity and cell death. Therefore, the NO derived from the iNOS is involved in the cytotoxicity. These results suggest that NO may contribute to the inflammatory response during Fn infection in periodontitis.

Participation of glutathione in the elimination of Porphyromonas gingivalis in vivo

INTRODUCTION Glutathione is involved in immune responses such as cell proliferation and bactericidal activity. The aim of this study was to see whether glutathione influences the intraperitoneal elimination of Porphyromonas gingivalis in Fusobacterium nucleatum-immunized mice. METHODS Mice were immunized with P. gingivalis or F. nucleatum, and then P. gingivalis was inoculated into the peritoneal cavity of the mice. After various lengths of time, the numbers of bacteria were determined by a colony-forming assay and by polymerase chain reaction. The effect of glutathione on the elimination of P. gingivalis was explored by changing the intracellular glutathione level. Furthermore, we examined the effects of glutathione on the peritoneal levels of interferon-gamma, a macrophage activator, and of nitrite, a derivative of nitric oxide that acts as an antimicrobial agent when produced by macrophages and neutrophils. RESULTS Inoculated P. gingivalis was eliminated more rapidly from F. nucleatum-immunized mice than from P. gingivalis-immunized mice. Interferon-gamma levels in peritoneal lavage fluid and glutathione levels in peritoneal exudate cells were higher in F. nucleatum-immunized mice than in P. gingivalis-immunized mice. When P. gingivalis-immunized mice were given glutathione monoethylester (a derivative of glutathione that is converted to glutathione intracellularly through hydrolysis) into the peritoneal cavity, the elimination of P. gingivalis was accelerated. On the other hand, when F. nucleatum-immunized mice were given L-buthionine-[S,R]-sulfoximine (an inhibitor of glutathione synthesis) into the peritoneal cavity, the elimination of P. gingivalis was suppressed. CONCLUSION In F. nucleatum-immunized mice, glutathione may have a key role in the defense against P. gingivalis infections.

The reduction of Fusobacterium nucleatum in mice is irrelevant to the nitric oxide induced by iNOS.

Previously we reported that mice infected recurrently with live Fusobacterim nucleatum (Fn) synthesize a significant amount of NO between 12 hr and 24 hr after the Fn injection. We now investigated whether the NO has the capability of killing Fn, a gram‐negative rod periodontal pathogen. The mice were divided into three groups: treated with live bacteria (LB), treated with heat‐killed bacteria (HKB) and untreated: normal (N). The Fn reduction, NO production and cell number after Fn injection were then compared in these mice. In the LB group, no Fn was detected at 6 hr, whereas it was still detected in the HKB and N groups at 24 hr as assessed by both colony counts and PCR assays. A significant amount of NO was synthesized in the LB group at 24 hr after the Fn injection. Fn is not killed by SNAP‐generated NO in vitro. An increase in the total cell number was accompanied by an increase of the neutrophil numbers in the LB group. Intracellular O2– generation (including ONOO–) was visualized using dihydrorhodamine (DHR)‐123. The peak of O2– generation by PEC was shown to be at 3 hr in all 3 groups. The number of O2– positive cells in the LB group at 3 hr was remarkably high, and most of them were likely to be neutrophils. The Fn reduction would be performed cooperatively via oxygen dependent and oxygen independent mechanisms. Thus reactive oxygen species (ROS) included in the oxygen dependent mechanism appear to be important for Fn reduction. However the significant amounts of NO derived from the iNOS synthesized in the LB group between 12 hr and 24 hr after injection of LFn were not involved in the Fn reduction.

Macrophages Contribute to the Elimination of Porphyromonas gingivalis More Strongly Than Neutrophils in Vivo

Abstract We previously reported that the elimination of Porphyromonas gingivalis from Fusobacterium nucleatum -immunized mice was more rapid than that of P. gingivalis -immunized mice, and that antibodies against P. gingivalis had no effect on the killing of P. gingivalis by neutrophils and macrophages. It remains unclear which phagocytes are mainly involved in the killing of P. gingivalis . To elucidate the activation of neutrophils and/or macrophages in the two types of immunized mice, we compared the elimination of P. gingivalis in a neutrophil-dominant with that in a macrophage-dominant state. Both states were induced in the mouse peritoneal cavity by casein injection. In the macrophage-dominant state, the elimination was faster in F. nucleatum -immunized than in P. gingivalis -immunized mice. Peritoneal levels of both interferon-γ (IFN-γ) and nitric oxide significantly increased in F. nucleatum-immunized mice, whereas only the IFN-γ level rose in P. gingivalis -immunized mice. In the neutrophil-dominant state, elimination was comparable between the two types of immunized mice. The peritoneal IFN-γ level slightly increased and nitric oxide level did not change in this state. No differences in the total cell number and rate of neutrophils or macrophages to the total peritoneal exudate cells in each state were comparable between the two immunized mouse types. These results suggest that macrophages, rather than neutrophils, are activated in F. nucleatum -immunized mice, and that nitric oxide produced by macrophages is important for the killing of P. gingivalis .

The Role of Complement Receptors in Chemiluminescence Response and Phagocytic Activity of Human Neutrophils Against Periodontopathic Bacteria.

A. actinomycetemcomitans (Aa), Pgingivalis (Pg), F. nucleotum (Fn) に対するヒト好中球の活性酸素産生 (Chemiluminescence反応: CL反応) と食菌における補体の役割, および関与する補体レセプター型について明らかにすることを目的とした。CL反応はルミホトメターを使用し, 補体レセプター型の検討にはヒト抗CR1と抗CR3抗体を利用した。食菌は染色標本で測定した。Aa, Fn, Pgの補体に依存するCL反応の値はそれぞれ約90%, 75%, 50%であり, 菌種による補体依存性の差が示された。抗CR1, 抗CR3抗体添加時のCL反応の阻害作用から利用したレセプター型を求めると, Aa, Fnでは抗CR1抗体と抗CR3抗体によるCL反応の阻害比は1対3であった。またPgではCR3のみが阻害に関係していた。Aa, Fn, Pgの食菌に関しては補体レセプターCR3のみの関与が認められた。活性酸素産生と食菌での補体およびc3biレセプターの重要性を示した。

Fluctuations in Antibody Titers to Periodontopathic Bacteria According to Age and Comparison of These Titers with Antibodies of Age-matched Periodontal Patients.

本研究では10歳未満から60歳代までの医学部外来患者164名, 各病型歯周炎患者51名を対象として, 各年代間における歯周病原菌種に対する抗体価の推移を検討するために, ELISA法を用いて血清抗体価の検索を行った。抗体価は2方法 (End-point法, EU) で算定した。抗原にはA. actinomycetemcomitans (A. a.), P. intermedia (P. g.), P . intermedia (P. i.) を用い, IgA, IgM, IgGについて検索した。その結果, 医学部外来g患ingivalis (者各年代間での抗体価の推移では, 3菌種に対して同様に20歳・30歳代でピークを示した。またA. a. に対しては, P. g., P. i. に比較して若い混合歯列期から上昇を認めた。歯周炎患者については, 年齢相応となる医学部外来患者各年代と比較してJPおよびAP患者において上昇している傾向が認められた。Post-JP患者については細菌学的検索も行い, 患者病巣局所からはP. i. の分離頻度が高かったが, 抗体価との関連性は低かった。

Chemiluminescence and Phagocytic Activity of Murine Macrophages in Response to Various Species of Oral Bacteria.

マクロファージの口腔細菌貧食時に放出される活性酸素およびリゾチームを測定し, 歯周組織への為害作用を検討した。活性酸素はルミノール依存による化学発光 (CL反応) により, またリゾチームはMicrococcus lysodeikticusの溶菌活性により測定した。CL反応では, T. denticola, F. nucJeatum, S. epidermidisがlys 高い値を示したが, 多形核白血球に比べると, 1/4-1/30と低いものであった。S. mutansとS . salivariusが, 良く食べ込まれていた。歯周病原細菌は, 食菌率は比較的良好なのに, マクロファージ100個内総菌数が少なかった。CL反応と食菌能には, 歯周病原細菌を除くと良好な相関が見られた。無刺激時のリゾチーム量は全体量の40%にもあたり, 各種細菌刺激による増減は23%以下であった。以上の結果より, マクロファージが産生する活性酸素およびリゾチームによる歯周組織への為害作用は, 多形核白血球より小さいと考えられる。

Peripheral polymorphonuclear leukocyte function in periodontal disease. 1. Chemiluminescence response and phagocytosis.

最近, 歯周疾患も宿主の防御機能, 特に, 好中球との関係がいろいろ調べられているが, Chemiluminescence (CL) 反応の報告がない。そこでJpとRPP患者9名, AP患者18名について, 末梢血好中球のS. epidermidisによるCL反応と食菌能を健常者と比較, 検討した。JPとRPP, AP患者のCL値は, 健常者の標準偏差域を超えた高い群と低い群の二群に分かれた。正常の範囲に入るものは軽症例であった。食菌能は, JPとRPP患者が, 食菌率も好中球100個内総菌数も低下していた。患者では高い群と低い群に分かれた。CLと好中球100個総菌数との問には良いい相関が見つかった。fMLP刺激によるCLは, 正常かむしろ亢進する値を示した。AP患者好中球が, 機能亢進型と低下型の二群に分かれることは, 機能亢進による歯肉組織への影響と, 低下に伴なう感染防御力の低下の両面から, 病態を考える必要がある。