Development of an alveolar bone resorption inhibitor that suppresses interleukin-1

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Periodontitis is characterized by alveolar bone loss. Interleukin-1 (IL-1) produced by macrophages and polymorphonuclear leukocytes is considered to play a central role in the bone resorption at inflammatory regions in periodontitis. In this study, we purified an IL-1 inhibitor from culture supernatants of macrophages and examined whether it suppresses the bone resorption.IL-1 inhibitor was purified from culture supernatants of P388D_1 cell line murine macrophages stimulated with lipopolysaccharide for 72 h to homogeneity by a five-step procedure: acetic acid extraction from culture supernatants; Bio-Gel P-60 gel chromatography; DEAE-Sepharose CL-6B column chromatography; reverse-phase high-performance liquid chromatography on a C_<18> hydrophobic support; and high-performance liquid chromatography on a gel filtration column. The purified IL-1 inhibitor gave a single band of protein with a molecular mass of 67 kDa on SDS-polyacrylamide gel electrophoresis, and had an isoelectric point of 4.8. The IL-1 inhibitor was a heat- and acid-stable protein that was inactivated by digestion with trypsin and reduction with dithiothreitol. The IL-1 inhibitor suppressed murine and human IL-1-induced proliferation of C3H/HeJ mouse thymocytes. However, the IL-1 inhibitor did not affect the proliferation of IL-2-dependent CTLL-2 cells induced by IL-2 and the proliferation of C3H/HeJ mouse thymocytes stimulated with a mitogenic dose of concanavalin A. The inhibitor at final concentrations of 1 to 5 mug/ml inhibited almost completely in vitro IL-1 induced bone resorption of BALB/c mouse calvaria, suggesting that the inhibitor may be useful for control of bone resorption in periodontitis.