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Bone-resorbing Activity from Cholesterol-exposed Macrophages due to Enhanced Expression of Interleukin-1

¹ Departments of Endodontics and
² Oral Cell Biology, Umeå University, SE-901 87, Umeå, Sweden;
³ present address, Department of Maxillofacial Prosthetics, Maxillofacial Reconstruction/Function, Division of Maxillofacial/Neck Reconstruction, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo-ku, Tokyo 113-8549, Japan;

View larger version (14K): [in this window] [in a new window]   Figure 1. 45Ca release from pre-labeled mouse calvarial bones incubated with supernatants from stimulated or unstimulated mouse peritoneal macrophages. (a) The effect on 45Ca release induced by 10% supernatants from macrophages incubated (72 hrs) with different amounts of cholesterol crystals (0.1, 1, 10 mg/mL; chol/Mø mg/mL). For comparison, supernatants (10%) from unstimulated (Mø) and zymosan-stimulated (100 µg/mL; zym/Mø) macrophages were used. (b) 45Ca release induced by different concentrations (1, 3, 10, 30%) of supernatants from both cholesterol-stimulated (10 mg/mL; chol/Mø) and unstimulated macrophages (3, 30%; Mø) is shown. (c) The result of a time-course study on 45Ca release when pre-labeled bones were incubated with 10% supernatants from unstimulated (Mø) or cholesterol-stimulated (10 mg/mL; chol/Mø) macrophages. All values are mean of 5 bones ± SEM. Statistically significant difference in 45Ca release was found for all stimulated macrophage supernatants compared with unstimulated (p < 0.05; [a]); for cholesterol-stimulated (30%) compared with unstimulated (30%) supernatant (p < 0.05; [b]); and in time-course study for stimulated supernatants, even at the first time point studied (24 hrs; p < 0.05; [c]).

View larger version (19K): [in this window] [in a new window]   Figure 2. Neutralizing effect of antisera (a) and interleukin-1 receptor antagonistic protein (IRAP; b) on 45Ca release from pre-labeled mouse calvarial bones incubated with supernatants from cholesterol-stimulated (chol/Mø) and unstimulated (Mø) macrophages. (a) Anti-IL-1 and anti-IL-1β (concentrations 0.5 µg/mL and 2 µg/mL, respectively) were pre-incubated (24 hrs) with supernatants from unstimulated (Mø) and cholesterol-stimulated (chol/Mø) macrophages. The conditioned media were incubated (96 hrs) with pre-labeled calvarial bones, and 45Ca release was measured. For comparison, recombinant IL-1 (80 pg/mL) or IL-1β (80 pg/mL), with or without antisera, was incubated with the calvarial bones. Values shown are mean of 5 bones ± SEM. Differences in 45Ca release between control bones and bones incubated with supernatant from cells stimulated either by cholesterol, IL-1, or IL-1β were statistically significant (p < 0.01). Anti-IL-1 and antisera neutralizing both IL-1 and β significantly (p < 0.01) inhibited the effects of cholesterol-stimulated macrophages. Antisera neutralizing either IL-1 or IL-1β significantly (P < 0.01) inhibited the stimulatory effect of IL-1 and IL-1β, respectively. (b) Effect of interleukin-1 receptor antagonist protein (IRAP) on the release of 45Ca from mouse calvarial bones incubated (96 hrs) either with supernatants from unstimulated macrophages (Mø), from bones stimulated by recombinant mouse IL-1 (100 pg/mL), IL-1β (100 pg/mL), or from cholesterol-stimulated (chol/Mø; 10%) macrophages. Shown are the mean of 5 bones ± SEM. Differences in 45Ca release between unstimulated bones and bones stimulated by IL-1, IL-1β, or supernatants from cholesterol-stimulated macrophages were statistically significant (p < 0.01). IRAP significantly decreased (p < 0.01) 45Ca release augmented by IL-1, IL-1β, or supernatants from cholesterol-stimulated macrophages.

View larger version (67K): [in this window] [in a new window]   Figure 3. Release of IL-1, IL-1β, and PGE2 and gene expression of IL-1, IL-1β, COX-1, COX-2, and GAPDH in mouse peritoneal macrophages incubated without or with cholesterol crystals. (a) Mouse peritoneal macrophages were incubated without or with cholesterol crystals (10 mg/mL) for 72 hrs. The concentrations of IL-1 and IL-1β were measured by ELISA. Values shown represent the mean of 5 cultures ± SEM. (b) Time-course study of IL-1 release from macrophages incubated with or without cholesterol crystals. Small aliquots of the supernatant were withdrawn at indicated time points, and the amounts of cytokine were measured (ELISA). Shown are the mean values of 5 cultures ± SEM. (c) PGE2 in supernatant from mouse peritoneal macrophages incubated (72 hrs) without (Mø) or with cholesterol crystals (Mø/chol; 10 mg/mL). For comparison, macrophages were incubated with particles of zymosan (100 µg/mL), and PGE2 was measured. Bars represent 1 of 3 experiments with similar results. (d) The gene expression of IL-1, IL-1β, COX-1, COX-2, and GAPDH in macrophages incubated with (+) or without (-) cholesterol crystals. RNA from the macrophages was reverse-transcribed into single-stranded cDNA and PCR-amplified with primers for IL-1, IL-1β, COX-1, COX-2, and GAPDH.

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