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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;

*corresponding author, Ulf.Sjogren{at}odont.umu.se

	ABSTRACT

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The presence of cholesterol crystals, macrophages, and foreign giant cells has been associated with impaired bone healing of periapical lesions. Therefore, we investigated whether macrophages exposed to cholesterol crystals can release factors changing the activity of bone-resorbing osteoclasts. Mouse peritoneal macrophages treated with cholesterol crystals in vitro produced factor(s) that stimulated the release of ⁴⁵Ca and ³H from mouse calvariae pre-labeled with ⁴⁵Ca(CaCl₂) or [³H]-proline, respectively. No bone-resorbing activity was released by epithelial cells, fibroblasts, or osteoblasts exposed to cholesterol crystals. Interleukin-1 receptor antagonist protein and antiserum neutralizing mouse interleukin-1 (IL-1) inhibited ⁴⁵Ca release induced by cholesterol-activated macrophages. The addition of cholesterol to the macrophages augmented the release of IL-1 protein and the expression of IL-1 mRNA. These findings indicate that frustrated phagocytosis by macrophages exposed to cholesterol crystals results in release of factors stimulating osteoclastic bone resorption, primarily due to increased transcription of the IL-1 gene.

KEY WORDS: cholesterol • macrophages • bone • interleukin-1 • osteitis

	INTRODUCTION

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In the oral cavity, accumulation of cholesterol crystals may occur in periapical lesions of endodontically involved teeth (Nair et al., 1993). The reported incidence of cholesterol crystals in such lesions varies from 18 to 44% (Shear, 1963; Browne, 1971; Trott et al., 1973). The dense accumulation of macrophages and giant cells around the cholesterol crystals suggests that the crystals induce a typical foreign-body reaction (Nair et al., 1993). It is of clinical interest to know to what extent these cells are able to eliminate the cholesterol crystals. Naturally occurring cholesterol granulomas are characterized by a persisting dense accumulation of macrophages and giant cells in the vicinity of cholesterol crystals (Nair et al., 1993). Recently, we showed that cholesterol crystals experimentally implanted into animals caused an accumulation of macrophages and giant cells which were unable to eliminate the crystals during an observation period of 8 mos (Nair et al., 1998). Thus, analyses of both clinical and experimental data show that macrophages are unable to phagocytose cholesterol crystals. The most likely reason is the size of the cholesterol crystals (Schumacher, 1998). The dimensions of such crystals can be up to 300-400 x 10-20 µm (Nair et al., 1993), which is too large for macrophages or giant cells to engulf, resulting in enclosure by the host cells without successful phagocytosis.

The presence of cholesterol crystals has been suggested to be a negative factor interfering with periapical healing after conventional endodontic treatment (Nair et al., 1993). We hypothesize that cholesterol-containing periapical lesions may be in part a crystal-deposition disease with pathogenetic similarities to other microcrystal-associated conditions, such as gout and pseudogout (Doherty and Dieppe, 1998). It is well-known that crystals of monosodium urate monohydrate and calcium pyrophosphate dihydrate can activate different cells to secrete several inflammatory mediators, including osteotropic cytokines, e.g., tumor necrosis factor- (TNF-) and interleukin-1ß (Hachicha et al., 1995; Matsukawa et al., 1998). It is not known, however, if exposure of macrophages to cholesterol crystals, being too large to be phagocytosed, results in release of inflammatory mediators that could adversely affect the post-therapeutic healing of jaw bones. In the present study, we show that macrophages encountering cholesterol crystals release factor(s), most likely due to frustrated phagocytosis, that stimulate bone resorption. Activation of IL-1 transcription and IL-1 release by cholesterol is primarily responsible for the bone-resorbing effect.

	MATERIALS & METHODS

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Isolation of Macrophages and Preparation of Macrophage-conditioned Media
Mouse peritoneal macrophages were isolated and cultured in -Minimal Essential Medium (-MEM) containing 10% fetal calf serum (FCS) as previously described (Ohlin et al., 1999). The macrophages were incubated in 2-cm² multi-well dishes (1 x 10⁶ cells/cm²) at 37°C in 5% CO₂ in air and exposed to cholesterol crystals (median size, 30 x 19 µm, range 5 x 2 µm to 356 x 256 µm; 87% of the crystals were more than 10 µm long; Merck, CH-8953 Dieticon, Switzerland) by the addition of 2 mL of serum-free -MEM containing cholesterol crystals, at different concentrations, to the multi-well dishes. In parallel, macrophages were incubated in medium without cholesterol crystals. The conditioned media were harvested 72 hrs later by aspiration of the supernatants, which then were clarified by centrifugation and subsequently frozen. These supernatants were later analyzed for the presence of IL-1, IL-1ß, or PGE₂ or tested for bone-resorbing activity. In some experiments, RNA was isolated from cells exposed to cholesterol crystals for 24 hrs, and subsequently used for RT-PCR.

Bone Resorption Bioassays
Mineral mobilization was assessed by analysis of the release of ⁴⁵Ca from cultured pre-labeled neonatal mouse calvarial bones, and bone matrix degradation was assessed by determination of the release of ³H from bones pre-labeled with [³H]-proline, as previously described (Lerner, 1987; Ljunggren et al., 1991). Supernatants from conditioned macrophages were diluted (1-30%) in -MEM. Calvarial bones were then individually incubated in 2 mL of the diluted media and cultured for 96 hrs. In all experiments, one group of bones was cultured in -MEM without macrophage-conditioned media. Animal use protocols for macrophage isolation and bone resorption bioassays were approved by the Institutional Ethical Committee for Animal Care and Use at Umeå University.

Inhibition Experiments with Interleukin-1 Receptor Antagonist Protein
The bone resorption activity of conditioned macrophage supernatant was assayed in the presence or absence of interleukin-1 receptor antagonist protein (IRAP; Genzyme, Cambridge, MA, USA). Calvarial bones were pre-incubated with IRAP (100 ng/mL) for 3 hrs, and then the conditioned macrophage supernatant was added and the bones were incubated for another 93 hrs (Ohlin et al., 1999).

Antibody Neutralization Experiments
Antibodies neutralizing mouse IL-1 (0.5 µg/mL) or IL-1ß (2 µg/mL; Genzyme, Cambridge, MA, USA) were added to -MEM with or without macrophage-conditioned media and kept at 4°C for 24 hrs prior to bone culture experiments. The specificity and efficiency of these antibodies have previously been determined (Ohlin et al., 1999).

Measurement of Prostaglandin E₂ Concentration
The concentration of PGE₂ in media was measured before and at the end of the culture period by means of a commercially available radioimmunoassay kit (DuPont/New England Nuclear, Boston, MA, USA) following the instructions supplied by the manufacturer.

Neutralization Experiments with Polymyxin B
Macrophages were incubated for 72 hrs with cholesterol crystals (10 mg/mL) or lipopolysaccharide (LPS; 10 ng/mL; Sigma Chemical Co., St. Louis, MO, USA) in the presence or absence of polymyxin B sulphate (10 µg/mL; Sigma Chemical Co., St. Louis, MO, USA). The supernatants were analyzed for PGE₂ concentration and their ability to activate the bone-resorbing system (as described above).

Isolation and Culture of Epithelial Cells, Dental Pulp Fibroblasts, and Osteoblasts
Epithelial cells were isolated from the palatal gums of Sprague-Dawley rats. Fibroblasts were isolated from human pulp tissue explants. The mouse calvarial osteoblastic cell line MC3T3-E1 was cultured in 2-cm² multi-well dishes containing -MEM with 10% FCS. At confluence, cholesterol crystals (different concentrations) were added. Seventy-two hrs later, supernatants were harvested.

Measurements of Interleukin-1 and Interleukin-1ß Production
We determined the amounts of IL-1 and IL-1ß synthesized by analyzing the concentrations of these two cytokines in the media using enzyme-linked immunosorbent assays (Amersham Internat, plc., Buckinghamshire, UK). The sensitivity of both assays was 6 pg/mL.

RNA Extraction
Macrophages were incubated in -MEM/10% FCS in 24 multi-well plates at a density of 1 x 10⁶ cells/cm². After attachment overnight, the cells were incubated in serum-free -MEM with or without cholesterol crystals (10 mg/mL) for 24 hrs. RNA was isolated by means of an mRNA Capture Kit with streptavidin-coated tubes (Boehringer-Mannheim, Mannheim, Germany) for the capture of poly (A⁺) mRNA hybridized with biotin-labeled oligo(dt)₂₀.

Reverse Transcription Polymerase Chain-reaction
Poly (A⁺) mRNA in the streptavidin-coated tubes was reverse-transcribed into single-stranded cDNA with the 1st Strand cDNA Synthesis Kit (Boehringer-Mannheim, Mannheim, Germany) and oligo-p(dt)₂₀ primers. The cDNA was amplified in polymerase chain-reactions (PCR) by means of a PCR Core Kit (Boehringer-Mannheim, Mannheim, Germany) and a PC-960G Gradient Thermal Cycler (Corbett Research, Mortlake 2137, Australia). For PCR of IL-1 and IL-1ß, the reactions were performed in a total volume of 100 µL with the use of a 1-µL template, 1 µmol/L of each primer (Life Technology Ltd, Paisley, UK), 2.5 U taq DNA polymerase, 1 x PCR buffer, 0.2 mmol/L dNTPs, and 2.5 mmol/L MgCl₂ for 10 cycles consisting of denaturing at 94°C for 45 sec, annealing at 67°C for 45 sec, and 90 sec of extension at 72°C. In subsequent cycles, the primer annealing temperature was decreased stepwise by 5°C every 5 cycles. After the last cycle, the mixtures were incubated at 72°C for 6 min. For the PCR of COX-1 and COX-2, the reactions were performed with the 1-µL template, 0.4 µmol/L of each primer (Life Technology Ltd, Paisley, UK), 2.5 U taq DNA polymerase, 1 x PCR buffer, 0.2 mmol/L dNTPs, and 2.0 mmol/L MgCl₂ for 29-33 cycles consisting of denaturation at 94°C for 45 sec, annealing at 52°C (COX-1) or 61°C (COX-2) for 45 sec, and 90 sec of extension at 72°C. After the last cycle, the mixtures were incubated at 72°C for 6 min. The PCRs for IL-1, IL-1ß, COX-1, and COX-2 were hot-started at 95°C for 15 min (Hotstar Taq polymerase; Qiagen GmbH, Hilden, Germany). PCR for GAPDH was performed with the 1-µL template, 0.4 µmol/L of each primer (Life Technology Ltd, Paisley, UK), 2.5 U taq DNA polymerase, 1 x PCR buffer, 0.2 mmol/L dNTPs, and 1.5 mmol/L MgCl₂ for 21-27 cycles consisting of denaturing at 94°C for 5 min, annealing at 57°C for 45 sec, and extension at 72°C for 90 sec. The amplification of all PCR products was compared at the logarithmic phase of the PCRs. The PCR products were fractionated by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. The sequences of the specific primers used were as follows. For IL-1, sense primer 5'-ATGGCCAAAGTTCCTGACTTGTTT-3', antisense primer 5'-CCTTCAGCAACACGGGCTGGT-3'; for IL-1ß, sense primer 5'-ATGGCAACTGTTCCTGAACTCAACT-3', antisense primer 5'-CAGGACAGGTATAGATTCTTTCCTTT-3'; for COX-1, sense primer 5'- CTCACAGTGCGGTCCAAC-3', antisense primer 5'-CCAGCACCTGGTACTTAAG-3'; for COX-2, sense primer 5'-CAAGCAGTGGCAAAGGCCTCCA-3', antisense primer 5'- GGCACTTGCATTGATGGTGGCT-3'; and for GAPDH, sense primer 5'-ACTTTGTCAAGCTCATTTCC-3', antisense primer 5'-TGCAGCGAACTTTATTGATG-3'. The estimated sizes of the PCR products were: 625 bp for IL-1, 563 bp for IL-1ß, 424 bp for COX-1, 459 bp for COX-2, and 270 bp for GAPDH.

Statistics
Statistical analysis was performed by Student's t test.

	RESULTS

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Effects of Cholesterol-treated Macrophages on Bone Resorption
Conditioned media from macrophages exposed to cholesterol crystals (10 mg/mL), added at a concentration of 10% to mouse calvarial bone cultures, reproducibly stimulated ⁴⁵Ca release. The stimulatory effect was dependent on the concentration of cholesterol (Fig. 1a), the amount of conditioned media (Fig. 1b), and the time of bone cultures (Fig. 1c). The degree of ⁴⁵Ca release response was similar to that caused by conditioned media from zymosan-treated macrophages (Fig. 1a), or that caused by a maximally effective concentration of PTH (10 nmol/L, data not shown). Addition of calcitonin (1 nmol/L) to the bone cultures abolished the bone-resorbing effect caused by cholesterol-activated macrophage supernatant (data not shown).

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]).

Cholesterol crystals (0.1-10 mg/mL) added to cultures of rat epithelial cells, human pulp fibroblasts, or mouse osteoblastic MC3T3-E1 cells did not induce any release of bone-resorbing activity (data not shown).

Assessment of the Molecular Weight of the Cholesterol-induced Bone-resorbing Activity
Ultrafiltration of the supernatant from cholesterol-stimulated cells, by means of filters with molecular-weight cut-offs of 3000 D and 30,000 D, showed that the bone-resorbing effect was retained with the smaller-pore filter and completely lost with the larger-pore filter (Table).

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-1ß (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-1ß (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-1ß were statistically significant (p < 0.01). Anti-IL-1 and antisera neutralizing both IL-1 and ß significantly (p < 0.01) inhibited the effects of cholesterol-stimulated macrophages. Antisera neutralizing either IL-1 or IL-1ß significantly (P < 0.01) inhibited the stimulatory effect of IL-1 and IL-1ß, 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-1ß (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-1ß, 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-1ß, or supernatants from cholesterol-stimulated macrophages.

Effects of Cholesterol Crystals on Macrophage Release of Interleukin-1, Interleukin-1ß, and PGE₂
In two experiments with similar results, the addition of cholesterol crystals (10 mg/mL) for 72 hrs to mouse peritoneal macrophages resulted in a markedly enhanced release of IL-1 (data from one experiment shown in Fig. 3a). The release of IL-1 from cholesterol-treated macrophages was considerably higher than that of IL-1ß (Fig. 3a). The stimulation of IL-1 release induced by cholesterol was time-dependent, with an effect already seen at 24 hrs (Fig. 3b).

View larger version (67K): [in this window] [in a new window]   Figure 3. Release of IL-1, IL-1ß, and PGE2 and gene expression of IL-1, IL-1ß, 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-1ß 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-1ß, 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-1ß, COX-1, COX-2, and GAPDH.

The specific endotoxin-neutralizing compound polymyxin B (10 µg/mL) abolished the ability of endotoxin to induce PGE₂ formation in the macrophages (as expected), but had no effect on PGE₂ biosynthesis induced by cholesterol crystals (data not shown). In accordance with this, polymyxin B (10 µg/mL) did not affect the release of bone-resorbing activity from macrophages induced by cholesterol crystals (10 mg/mL; data not shown). These observations demonstrate that contamination by endotoxin is not responsible for the effects of cholesterol crystals on bone resorption and prostaglandin formation.

Effects of Cholesterol Crystals on mRNA Expression of Interleukin-1, Interleukin-1ß, Cyclo-oxygenase-1, and Cyclo-oxygenase-2
In three independent experiments, cholesterol clearly stimulated the mRNA expression of IL-1 and COX-2, and in two of the three experiments, cholesterol enhanced mRNA expression of IL-1ß (Fig. 3d). The expression of COX-1 or GAPDH was not affected by cholesterol (Fig. 3d).

	DISCUSSION

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In the present study, mouse peritoneal macrophages, exposed to cholestrol crystals, were demonstrated to release factors that induce bone resorption in mouse calvariae. This process was related to the amount of cholesterol crystals added to the macrophages. Conditioned media from cholesterol-treated macrophages stimulated the release of both ⁴⁵Ca and ³H from bones pre-labeled with [⁴⁵Ca]CaCl₂ and [³H]-proline, respectively, indicating that both the inorganic and the organic components of the calvariae were degraded, a characteristic feature of osteoclastic bone resorption. Calcitonin completely inhibited the enhanced ⁴⁵Ca release, further supporting that the effect was a result of osteoclastic bone resorption.

Prostaglandins can be produced by macrophages activated by different stimuli. Since prostaglandins are well-known stimulators of bone resorption (Pilbeam et al.,1996), we addressed the question whether the bone-resorbing activity released by cholesterol-treated macrophages could be due to prostaglandins. Cholesterol crystals added to mouse peritoneal macrophages induced a five- to six-fold increased release of PGE₂. The enhanced release was probably caused by stimulation of cyclo-oxygenase, since we could detect enhanced mRNA expression of the inducible form of cyclo-oxygenase (COX-2) in macrophages exposed to cholesterol crystals, without seeing any effect on the mRNA expression of the constitutively expressed COX-1 or the housekeeping gene GAPDH. The enhanced release of ⁴⁵Ca from the calvarial culture caused by supernatants from cholesterol-treated macrophages was not likely to be a prostanoid-mediated effect, however, since ultrafiltration of the supernatants, with the use of filters with a molecular-weight cut-off of 3000 D, did not abolish the release of ⁴⁵Ca when the retentates were added to the bone cultures. Furthermore, the concentration of PGE₂ in the cholesterol-conditioned macrophage media was only 2-3 ng/mL (data not shown). It is therefore unlikely that the enhancement of PGE₂ in the supernatants could be responsible for the bone-resorbing activity released by the cholesterol-treated macrophages.

The observation that the size of the active molecule was between 3000 and 30,000 D indicates that bone-resorbing cytokines may be responsible for the bone-resorbing activity released by cholesterol-exposed macrophages. Since IL-1 (M ~ 17,000 D) is the most potent bone-resorbing cytokine (Horowitz and Lorenzo, 1996), these observations prompted us to analyze whether IL-1 could be involved.

The IL-1 receptor antagonistic protein (IRAP) was found to efficiently abolish the release of ⁴⁵Ca induced by cholesterol-stimulated macrophage supernatant. Antisera neutralizing IL-1 significantly reduced the bone-resorbing effect of cholesterol-stimulated macrophage supernatants, whereas antisera to IL-1ß only slightly impaired ⁴⁵Ca release from the calvarial bones, indicating that the bone-resorbing activity is mainly mediated by IL-1. This view is further supported by our finding that cholesterol stimulated the release of IL-1 protein from the macrophages in a time-dependent manner. The enhancement of IL-1 release was most likely due to induced transcription of the IL-1 gene, since cholesterol stimulated the expression of IL-1 mRNA in the macrophages.

Analysis of our data demonstrates that cholesterol crystals, although being too large to be phagocytosed by macrophages, activate the transcriptional level of IL-1, subsequently resulting in enhanced release of this bone-resorbing cytokine. We suggest that the stimulation of macrophages is a consequence of frustrated phagocytosis. Thus, the presence of cholesterol crystals in periapical lesions may, similar to other microcrystal-associated processes, result in the release of cytokines from macrophages. The release of osteotropic cytokines such as IL-1 from such cholesterol-activated macrophages may prevent the healing of endodontically treated teeth.

	ACKNOWLEDGMENTS

 
This study was supported by grants from the Swedish Medical Research Council, the Royal 80 Year Fund of King Gustav V, the Swedish Association Against Rheumatic Diseases, and the County Council of Västerbotten.

Received July 10, 2000; Last revision October 29, 2001; Accepted November 7, 2001

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This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (8) Citing Articles via Google Scholar Google Scholar Articles by Sjögren, U. Articles by Lerner, U.H. Search for Related Content PubMed PubMed Citation Articles by Sjögren, U. Articles by Lerner, U.H.