title: Lipopolysaccharide of Aggregatibacter actinomycetemcomitans up-regulates inflammatory cytokines, prostaglandin E2 synthesis and osteoclast formation in interleukin-1 receptor antagonist-deficient mice.
abstract: OBJECTIVE
  The interleukin (IL)-1 receptor antagonist (Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is unclear whether the IL-1Ra plays a protective role in periodontal disease. The purpose of this study was to compare IL-1Ra knockout (KO) and wild-type (WT) mice in regard to proinflammatory cytokine production, osteoclast formation and bone resorption in response to periodontal bacterial lipopolysaccharide (LPS).
  
  
  METHODS
  Peritoneal macrophages (Mφs) were obtained from 13-wk-old IL-1Ra KO and WT mice. Peritoneal Mφs were cultured with or without 10 μg/mL of Aggregatibacter actinomycetemcomitans LPS for 24 h. The levels of IL-1alpha (IL-1α), IL-1beta (IL-1β), tumor necrosis factor-α (TNF-α) and IL-6 were measured in periotoneal Mφs supernatant fluid (PM-SF) using an ELISA. Bone marrow cells were obtained from the mice and stimulated with PM-SF for 9 d, then stained with TRAP. The frequency of TRAP-positive multinucleated giant cell formation was calculated based on a fusion index. PM-SF-stimulated calvarial bone resorption was analyzed using micro-computed tomography, and calvarial histological analysis was performed using hematoxylin and eosin and TRAP staining. The expression of cyclooxygenase-2 (Cox2), prostanoid receptor EP4 (Ep4) and Rank mRNAs in bone marrow cells were measured using real-time quantitative PCR, while prostaglandin E2 (PGE2 ) production was determined by ELISA.
  
  
  RESULTS
  The levels of IL-1α, IL-1β, TNF-α and IL-6 in IL-1Ra KO mice PM-SF stimulated with A. actinomycetemcomitans LPS were significantly increased by approximately 4- (p < 0.05), 5- (p < 0.05), 1.3- (p < 0.05) and 6- (p < 0.05) fold, respectively, compared with the levels in WT mice. Moreover, osteoclast formation, expression of Rank, Ep4 and Cox2 mRNAs and production of PGE2 were significantly increased by approximately 2- (p < 0.05), 1.6- (p < 0.05), 2.5- (p < 0.05), 1.6- (p < 0.05) and 1.9- (p < 0.05) fold, respectively, in IL-1Ra KO mice stimulated with A. actinomycetemcomitans LPS compared with WT mice.
  
  
  CONCLUSIONS
  IL-1Ra regulates IL-1 activity and appears to reduce the levels of other inflammatory cytokines, including TNF-α and IL-6, while it also reduces expression of the EP4 receptor related to prostanoid sensitivity and osteoclast formation. These results suggest that IL-1Ra is an important molecule for inhibition of inflammatory periodontal bone resorption.
authors.affiliation: Department of Periodontology, School of Dentistry, Aichi Gakuin University, Nagoya, Aichi, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Mizutani
authors.forename: Y
authors.initials: Y
authors.lastname: Ishihara
authors.forename: A
authors.initials: A
authors.lastname: Izawa
authors.forename: Y
authors.initials: Y
authors.lastname: Fujihara
authors.forename: S
authors.initials: S
authors.lastname: Kobayashi
authors.forename: H
authors.initials: H
authors.lastname: Gotou
authors.forename: E
authors.initials: E
authors.lastname: Okabe
authors.forename: H
authors.initials: H
authors.lastname: Takeda
authors.forename: Y
authors.initials: Y
authors.lastname: Ozawa
authors.forename: Y
authors.initials: Y
authors.lastname: Kamiya
authors.forename: H
authors.initials: H
authors.lastname: Kamei
authors.forename: T
authors.initials: T
authors.lastname: Kikuchi
authors.forename: G
authors.initials: G
authors.lastname: Yamamoto
authors.forename: A
authors.initials: A
authors.lastname: Mitani
authors.forename: T
authors.initials: T
authors.lastname: Nishihara
authors.forename: T
authors.initials: T
authors.lastname: Noguchi
mesh_terms: D000135:Acid Phosphatase / Q000032:analysis; D016976:Aggregatibacter actinomycetemcomitans / Q000502:physiology*; D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects; D001862:Bone Resorption / Q000276:immunology; D018929:Cell Culture Techniques; D051546:Cyclooxygenase 2 / Q000187:drug effects; D016207:Cytokines / Q000187:drug effects*; D015232:Dinoprostone / Q000378:metabolism*; D015726:Giant Cells / Q000187:drug effects; D053590:Interleukin 1 Receptor Antagonist Protein / Q000235:genetics / Q000276:immunology*; D053582:Interleukin-1alpha / Q000032:analysis; D053583:Interleukin-1beta / Q000187:drug effects; D015850:Interleukin-6 / Q000032:analysis; D007527:Isoenzymes / Q000032:analysis; D008070:Lipopolysaccharides / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D010010:Osteoclasts / Q000187:drug effects*; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000187:drug effects; D058309:Receptors, Prostaglandin E, EP4 Subtype / Q000187:drug effects; D012886:Skull / Q000276:immunology; D000071681:Tartrate-Resistant Acid Phosphatase; D014409:Tumor Necrosis Factor-alpha / Q000187:drug effects; D015854:Up-Regulation
chemical_list: D016207:Cytokines; D053590:Interleukin 1 Receptor Antagonist Protein; D053582:Interleukin-1alpha; D053583:Interleukin-1beta; D015850:Interleukin-6; D007527:Isoenzymes; D008070:Lipopolysaccharides; D053246:Receptor Activator of Nuclear Factor-kappa B; D058309:Receptors, Prostaglandin E, EP4 Subtype; C505687:Tnfrsf11a protein, mouse; D014409:Tumor Necrosis Factor-alpha; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase; D015232:Dinoprostone
keywords: cyclooxygenase-2; periodontal disease; prostaglandin E2; prostanoid receptor EP4