Lipopolysaccharide of Aggregatibacter actinomycetemcomitans up‐regulates inflammatory cytokines, prostaglandin E2 synthesis and osteoclast formation in interleukin‐1 receptor antagonist‐deficient mice

Abstract

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). Peritoneal macrophages (Mφs) were obtained from 13-wk-old IL-1Ra KO and WT mice. Peritoneal Mφs were cultured with or without 10μg/mL of Aggregatibacter actinomycetemcomitans LPS for 24h. 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 9d, 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. 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. 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.