No abstract

No abstract

A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for human osteoclast differentiation factor (ODF/RANKL/ OPGL/TRANCE) utilizing a polyclonal antibody that recognizes both human soluble ODF and mouse ODF in combination with a osteoclasogenesis inhibitory factor (OCIF/OPG) was developed. We can quantify the ODF level in not only human ODF (detection limit: 0.05 ng/ml), but also mouse ODF by virtue of cross-reactivity. Employing this assay system, we demonstrated that ODF is constitutively present as a membrane-bound form in both the human osteosarcoma cell lines, MG-63, HOS and SaOS-2, and the mouse osteoblastic cell line MC3T3-E1.

Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) has been implicated in the etiology of localized juvenile periodontitis (LJP), and produces a multiplicity of tissue-damaging products. Among those products, the capsular-like polysaccharide antigen (CPA) from A. actinomycetemcomitans is a potent mediator of bone resorption. In fact, this CPA (serotype b) is known to promote osteoclast-like cell formation via interleukin (IL)-1alpha production in mouse marrow cultures. Although osteoblasts complete bone formation, there are few reports focusing on the effect of CPA in bone-forming activity of osteoblasts in inflammatory disease sites. We hypothesized that CPA plays a mediating role in osteoblastic cells. Therefore, the purpose of this study was to examine the effect of CPA from A. actinomycetemcomitans on the mouse osteoblastic cell line MC3T3-E1 and human osteosarcoma SaOS-2 cells. A. actinomycetemcomitans serotype c resulted in a potent dose-dependent inhibition of cell proliferation of both cell lines. Characterization of the antiproliferative activity in the CPA demonstrated that it was not cytotoxic for MC3T3-E1. A 20-hour incubation with CPA-c resulted in a significant increase in apoptotic cell death in the cells, as evaluated by both cellular DNA fragmentation ELISA and FACS analysis. In contrast to the results obtained with a cytokine mixture (tumor necrosis factor-alpha, IL-1beta, and interferon-gamma), no inducible nitric oxide (NO) synthase gene expression or NO release could be detected in MC3T3-E1 after incubation with CPA-c. Further, both CPA-b and -c caused potent induction of apoptosis-related modifiers, e.g., Fas mRNA, whereas bcl-2 mRNA levels were unchanged. Therefore, this study has shown that CPA from A. actinomycetemcomitans contains a potent antiproliferative polysaccharide whose activity is associated with apoptotic cell death in MC3T3-E1, and that CPA per se is an inducer of apoptosis mediated by the Fas system but not by NO.

No abstract