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Bone-resorbing Activity from Cholesterol-exposed Macrophages due to Enhanced Expression of Interleukin-1
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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-1β (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.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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-1β, 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-1β (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-1β Production
We determined the amounts of IL-1 and IL-1β 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-1β, 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-1β, 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-1β, 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-1β, 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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).
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Conditioned media (diluted to 30%) from cholesterol-treated macrophages also caused a 2.1-fold stimulation of 3H release from calvarial bones pre-labeled with [3H]-proline (data not shown).
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).
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Effects of Antisera-neutralizing Interleukin-1 and of Interleukin-1 Receptor Antagonistic Protein
Antiserum-neutralizing IL-1
added to supernatants from cholesterol-stimulated macrophages significantly reduced the stimulatory effect on 45Ca release from calvarial bones (Fig. 2a). This inhibition could be further enhanced by the use of antibodies neutralizing both IL-1 and IL-1β (Fig. 2a). Anti-IL-1β alone, however, did not cause any significant inhibition (Fig. 2a). The bone-resorbing effects of recombinant mouse IL-1 or IL-1β were specifically abolished when corresponding neutralizing antibodies were added to the test media prior to incubation with the calvarial culture (Fig. 2a).
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The stimulatory effect on 45Ca release from mouse calvariae by supernatants from cholesterol-stimulated macrophages was significantly inhibited when these supernatants were added to a culture of mouse calvariae pre-incubated with IL-1 receptor antagonistic protein (IRAP; Fig. 2b
). Similarly, IRAP abolished the stimulatory effects of mouse IL-1 or IL-1β (100 pg/mL) on 45Ca release from mouse calvarial bones (Fig. 2b). IRAP does not inhibit 45Ca release stimulated by PTH (Ohlin et al.,1999).
Effects of Cholesterol Crystals on Macrophage Release of Interleukin-1, Interleukin-1β, 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-1β (Fig. 3a). The stimulation of IL-1 release induced by cholesterol was time-dependent, with an effect already seen at 24 hrs (Fig. 3b).
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In three independent experiments, cholesterol crystals added to peritoneal macrophages caused a five- to six-fold increase of PGE2 (data from one experiment shown in Fig. 3c
). 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-1β, 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-1β (Fig. 3d). The expression of COX-1 or GAPDH was not affected by cholesterol (Fig. 3d).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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-1β 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.
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ACKNOWLEDGMENTS |
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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 for publication July 10, 2000. Revision received October 29, 2001. Accepted for publication November 7, 2001.
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REFERENCES |
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Journal of Dental Research, Vol. 81, No. 1,
11-16 (2002)
DOI: 10.1177/154405910208100104
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