Mouse interleukin-1 receptor antagonist induced by Actinobacillus actinomycetemcomitans lipopolysaccharide blocks the effects of interleukin-1 on bone resorption and osteoclast-like cell formation.
We have reported that P388D1 cell line murine macrophages stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans release interleukin-1 (IL-1) inhibitor. The IL-1 inhibitor was purified from conditioned media of P388D1 cells stimulated with A. actinomycetemcomitans LPS for 72 h to homogeneity by a four-step procedure: acetic acid extraction from conditioned media; Bio-Gel P-60 gel filtration chromatography; DEAE-Sepharose CL-6B column chromatography; and reverse-phase high-performance liquid chromatography on a C18 hydrophobic support. The purified IL-1 inhibitor gave a single band of protein with a molecular mass of 26 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified IL-1 inhibitor was a heat- and acid-stable protein that was inactivated by digestion with trypsin and reduction with dithiothreitol. This inhibitory factor suppressed the proliferation of C3H/HeJ mouse thymocytes and the proliferation of IL-1-dependent cell lines, D10.G4.1 and RPMI 1788, induced by IL-1. However, this inhibitor did not affect the proliferation of IL-2-dependent CTLL-2 cells induced by IL-2, the proliferation of C3H/HeJ mouse thymocytes stimulated with a mitogenic dose of concanavalin A, and the proliferation of IL-6-dependent B9 cells induced by IL-6. Furthermore, the IL-1 inhibitor significantly blocked stimulation of bone resorption in organ cultures of newborn mouse calvaria and inhibited the osteoclast-like cell formation in mouse marrow cultures. A monoclonal antibody prepared against the purified IL-1 inhibitor reacted with mouse recombinant IL-1 receptor antagonist (rIL-1ra), and a polyclonal antibody to mouse rIL-1ra reacted with the IL-1 inhibitor by Western blot (immunoblot) analysis. These results indicate that the IL-1 inhibitor is an identical molecule to rIL-1ra, suggesting that the IL-1 inhibitor (IL-1ra) released by macrophages stimulated with LPS from A. actinomycetemcomitans may play an important mediative role in the development of periodontal disease.
Nishihara
T
T
Department of Oral Science, National Institute of Health, Tokyo, Japan.
Ohsaki
Y
Y
Ueda
N
N
Saito
N
N
Mundy
G R
GR
D016976:Aggregatibacter actinomycetemcomitans / Q000276:immunology; D000818:Animals; D001862:Bone Resorption / Q000209:etiology / Q000473:pathology / Q000517:prevention & control*; D002460:Cell Line; D017077:Culture Media, Conditioned; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1 / Q000037:antagonists & inhibitors / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008809:Mice, Inbred C3H; D009924:Organ Culture Techniques; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects*; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors*; D012795:Sialoglycoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*
D017077:Culture Media, Conditioned; C507764:Il1rn protein, mouse; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1; D008070:Lipopolysaccharides; D017472:Receptors, Interleukin-1; D012795:Sialoglycoproteins