Presented in part at the Eighteenth Annual Meeting of the American Society for Bone and Mineral Research, September 7–11, 1996, Seattle, WA, U.S.A. (J Bone Miner Res 11 (Suppl 1):S151).
Alendronate/Interleukin-1β Cotreatment Increases Interleukin-6 in Bone and UMR-106 Cells: Dose Dependence and Relationship to the Antiresorptive Effect of Alendronate†
Article first published online: 1 MAY 1998
Copyright © 1998 ASBMR
Journal of Bone and Mineral Research
Volume 13, Issue 5, pages 786–792, May 1998
How to Cite
Sanders, J. L., Tarjan, G., Foster, S. A. and Stern, P. H. (1998), Alendronate/Interleukin-1β Cotreatment Increases Interleukin-6 in Bone and UMR-106 Cells: Dose Dependence and Relationship to the Antiresorptive Effect of Alendronate. J Bone Miner Res, 13: 786–792. doi: 10.1359/jbmr.19126.96.36.1996
- Issue published online: 4 DEC 2009
- Article first published online: 1 MAY 1998
- Manuscript Revised: 19 DEC 1997
- Manuscript Accepted: 19 DEC 1997
- Manuscript Received: 29 MAY 1997
Aminobisphosphonates inhibit bone resorption but have been shown to elicit acute-phase-like elevations in interleukin-6 (IL-6) in bone in vitro. The current studies were carried out to determine the relationship between the antiresorptive effects of the aminobisphosphonate alendronate and its effects on IL-6. Resorption was elicited in cultured 19-day fetal rat limb bones by 72 h treatment with interleukin-1β (IL-1β). Bone mass was quantitated at the end of the culture period to assess resorption. IL-6 was determined by bioassay (7TD1 cell proliferation). IL-1β (18 and 180 pM) stimulated bone resorption and increased IL-6. Alendronate (70 μM) inhibited the IL-1β–stimulated resorption. Alendronate alone did not affect IL-6 production by the bones. The IL-6 production from bones stimulated with 18 pM IL-1β was not significantly affected by alendronate, but the IL-6 production from bones stimulated with 180 pM IL-1β plus alendronate (21 and 70 μM) was higher than with IL-1β alone. Indomethacin (1 mM) inhibited the IL-6 increase elicited by 180 pM IL-1β and the enhanced IL-6 production elicited by cotreatment with IL-1β and alendronate. Since bone cultures contain multiple cell types, further experiments were carried out to determine whether alendronate could increase IL-1β–stimulated IL-6 production in an osteoblast cell line, UMR-106. Alendronate alone did not affect IL6 in UMR-106 cells. Alendronate (70 μM) in combination with IL-1β (180, 1.8, or 8 nM), or 7 μM alendronate, in combination with 8 nM IL-1β, significantly increased IL-6 in 48 h cell cultures. The results from the bone organ cultures show that alendronate can enhance IL-6 production elicited by higher concentrations of the cytokine IL-1β in bone, but that this effect on IL-6 does not prevent the inhibitory actions of alendronate on bone resorption. The results with the UMR106 cells indicate that one cellular site at which this enhancement of IL-6 production can occur is the osteoblast.
THE AMINOBISPHOSPHONATE ALENDRONATE inhibits bone resorption. As a result of this suppressive effect, alendronate has emerged as a powerful pharmacologic tool for the prevention and treatment of a variety of metabolic bone diseases. Alendronate is effective in preventing the bone loss associated with both immobilization1 and estrogen deficiency.2–5 In addition, alendronate is useful in the management of Paget's disease of bone6,7 and in limiting the skeletal complications of cancer; alendronate has been shown to lower serum calcium in humoral hypercalcemia of malignancy8–10 and to decrease the pain and complications of bone metastases.11
Aminobisphosphonates are more potent antiresorptive agents than nonaminobisphosphonates.12 When an aminobisphosphonate is administered to a patient for the first time, however, it often induces an acute-phase response6,13,14; nonaminobisphosphonates, even at much greater dosages, do not elicit such an effect.14 The acute-phase response, characterized by transient hematological changes including decreased lymphocyte count, decreased serum zinc, and increased C reactive protein, may result in fever and/or other flu-like symptoms (e.g., muscle pain and weakness). These effects subside within a few days in the absence of any specific treatment.
The cytokine interleukin-6 (IL-6) is an important mediator of the acute-phase response15 and is transiently increased in some Paget's disease patients shortly after their initial treatment with the nitrogen-containing bisphosphonate (3-dimethyl-amino-1-hydroxypropylidene)-1,1-bisphosphonate (dimethyl-APD). Dimethyl-APD also stimulates IL-6 release from fetal mouse bone explants and enhances parathyroid hormone (PTH)-stimulated IL-6 production.16 Such an increase in IL-6 following administration of an aminobisphosphonate presents a seemingly contradictory situation. Aminobisphosphonates inhibit bone resorption yet can increase IL-6, which promotes bone resorption. IL-6, which is produced by a number of cell types, including monocytes,17 osteoblasts,18,19 and fibroblasts,20 promotes resorption through its effects on osteoclast differentiation. IL-6 is a potent stimulator of osteoclast formation,21 and it elicits bone resorption both in vivo22 and in in vitro models containing early osteoclast precursors.18,19 Little is known, however, about the relationship between the antiresorptive effects of aminobisphosphonates and their effects on IL-6.
The current study was designed to determine the effects of alendronate on IL-6 production in control and interleukin-1β (IL-1β)–stimulated fetal rat limb bone cultures, and the relationship of changes in IL-6 production to the inhibition of bone resorption by alendronate. In addition, because bone cultures consist of multiple cell types, including osteoblasts, further studies were carried out to determine if alendronate affects IL-6 production in UMR-106 osteoblastic cells.
MATERIALS AND METHODS
Fetal rat limb bone culture
Fetuses were obtained on day 19 of gestation from pregnant Holtzman rats (Harlan, Indianapolis, IN, U.S.A.). Limb bones were dissected from the fetuses and cultured as previously described.23 For the studies reported here, treatments were added at the start of culture and were present throughout. IL-1β (recombinant human IL-1β; Calbiochem, La Jolla, CA, U.S.A.) was dissolved in phosphate-buffered saline containing 1% bovine serum albumin (Sigma Chemical Co., St. Louis, MO, U.S.A.), alendronate (Merck & Co., West Point, PA, U.S.A.) was dissolved directly in the culture medium, and indomethacin (Sigma) was dissolved in ethanol. Solvents were added to control cultures; the final concentration of each solvent in the culture medium did not exceed 0.1%. Bones were cultured for 72 h at 37°C in a humidified atmosphere of 5% CO2 in air. Four replicates were run per treatment.
At the end of the treatment period, the bones were removed from the culture medium. The culture medium from the bones was stored at −20°C until assayed for IL-6. The bones were air dried on the filters for at least 1 week, and bone mass was then quantitated with a Cahn C-30 microbalance (Cahn Instruments, Cerritos, CA, U.S.A.).
UMR-106 rat osteoblastic osteosarcoma cells (American Type Culture Collection [ATCC], Rockville, MD, U.S.A.) were grown in 75 cm2 cell culture flasks at 37°C in a humidified 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% heat-inactivated horse serum and 100 U/ml K-penicillin G. Cells were passaged every 5–7 days with medium changes every 3 days.
For experiments, UMR-106 cells were seeded in 24-well plates in the DMEM culture medium described above. After a 48 or 72 h culture period, the cells were treated for 48 h with IL-1β in the presence or absence of alendronate. At the end of the treatment period, the culture medium was removed from each well and stored at −20°C until assayed for IL-6.
The concentration of IL-6 in the culture medium was determined by bioassay utilizing the IL-6–dependent 7TD1 mouse hybridoma cell line24 (ATCC). Fifty microliter aliquots of serial 2-fold dilutions of the culture medium or 2-fold dilutions of a recombinant mouse IL-6 standard (rmIL-6; Genzyme, Cambridge, MA, U.S.A.) were prepared in 96-well culture plates with RPMI 1640 containing 10% heat-inactivated fetal calf serum, 10 mM HEPES, 50 mM 2-mercaptoethanol, and 80 μg/ml gentamicin. Next, 2000 7TD1 cells were added to each well, and the cultures were incubated for 4 days at 37°C in a humidified atmosphere of 5% CO2 in air. The resulting proliferation was determined using a colorimetric assay.25 One unit of IL-6 was defined as the reciprocal of the medium dilution giving 50% of the maximal stimulation of proliferation by rmIL-6. IL-1β, alendronate, or indomethacin did not have a direct effect on the proliferation of the 7TD1 cells. An anti–IL-6 antibody (Upstate Biotechnology Inc., Lake Placid, NY, U.S.A.) completely blocked 7TD1 cell proliferation induced by conditioned medium from normal mouse osteoblasts, which are able to produce IL-11. This study does not definitively exclude a role for IL-11, but it suggests that IL-11 is not a major factor in this bioassay.
Data presentation and statistical analysis
Values were expressed as mean ± SEM. Statistical significance was determined by analysis of variance and subsequent Fisher's least significant difference multiple-comparison test.
Effect of alendronate on IL-1β–stimulated resorption and IL-6 production in fetal rat limb bone cultures
Figure 1 demonstrates the effect of alendronate on bone mass and IL-6 production in fetal rat limb bone cultures in the absence (Fig. 1A) and presence (Figs. 1B and 1C) of IL-1β. IL-1β–stimulated resorption at both doses (18 and 180 pM) employed, as indicated by the lower bone weights for the IL-1β–treated bones compared with control bones. These doses of IL-1β elicited approximately the same degree of resorption, suggesting that this was the maximal resorptive effect of this cytokine. IL-1β (18 and 180 pM) also stimulated IL-6 production in the limb bone cultures, as reported previously.26 The two doses of IL-1β stimulated IL-6 to approximately the same extent.
Alendronate (21 and 70 μM) alone had no effect on bone mass or basal IL-6 production (Fig. 1A). The resorptive effect of 18 pM IL-1β was significantly inhibited by 21 or 70 μM alendronate (Fig. 1B). The resorptive effect of 180 pM IL-1β was significantly inhibited by 70 μM but not 21 μM alendronate (Fig. 1C). In contrast, alendronate (21 and 70 μM) had no significant effect on the IL-6 response elicited by 18 pM IL-1β (Fig. 1B). However, when bones were treated with both 180 pM IL-1β and alendronate (21 and 70 μM), IL-6 production was significantly higher than that produced by IL-1β alone (Fig. 1C).
Since IL-1 can increase prostaglandin production in bone,27–29 we used the cyclo-oxygenase inhibitor indomethacin to determine the possible role of prostaglandins in IL-1β–stimulated IL-6 production and the enhancement by alendronate. Indomethacin alone had no significant effect on IL-6 in fetal rat limb bone cultures (Table 1). IL-1β stimulated IL-6, as shown in Fig. 1, and this response was inhibited by indomethacin. Indomethacin also inhibited the enhanced IL-6 response elicited by cotreatment with IL-1β and alendronate.
Effect of alendronate on IL-1β–stimulated IL-6 production in UMR-106 osteoblastic cells
IL-1β stimulated IL-6 production in a dose-dependent manner (18 pM–1.8 nM) in the UMR-106 cells (Fig. 2A), as shown previously.30 Alendronate (0.7 and 7 μM) had no effect on IL-1β–stimulated IL-6; however, 70 μM alendronate increased IL-1β–stimulated IL-6. This enhancement was observed only with the two highest concentrations of IL-1β used in this experiment, i.e., 180 pm and 1.8 nM.
In further studies, the UMR-106 cells were treated with a higher concentration of IL-1β (8 nM) in the absence or presence of alendronate (7 and 70 μM). At this higher dose of IL-1β, alendronate enhanced IL-6 production at both 7 and 70 μM doses (Fig. 2B). The treatments had no significant effects on cell number (data not shown).
Because of its antiresorptive properties, the aminobisphosphonate alendronate is effective in treating a variety of metabolic bone diseases. In recent years, a great deal of effort has been directed toward understanding the mechanisms underlying this inhibition. At the cellular level, the data suggest that the antiresorptive effects of alendronate are due to direct actions of this agent on both osteoclasts and osteoblasts. Alendronate inhibits the bone-resorbing activity of the osteoclast by altering at least two important membrane functions: alendronate disrupts the ruffled border of the osteoclast31,32 and it inhibits proton extrusion, which is necessary for acidification of the subosteoclastic space.33
Alendronate has also been reported to inhibit osteoclast formation,34 but this issue is controversial since negative results have also been described.32 Recent studies, however, have identified a molecular target for alendronate and support a suppressive effect on osteoclast formation. Alendronate inhibits a protein tyrosine phosphatase, PTP-ϵ, that is highly expressed in osteoclastic cells. Inhibition of PTP activity in vitro suppresses both the formation of multinucleated osteoclasts (from precursors) and the resorptive activity of isolated osteoclasts.35 Elucidation of the mechanism(s) by which alendronate modulates the function of PTP-ϵ and other putative molecular targets is of great interest.
Alendronate can also inhibit osteoclasts indirectly through an osteoblast-mediated mechanism.36 This inhibitory effect is thought to be mediated through one or more soluble factors released from the osteoblast. Alendronate could potentially inhibit the secretion of an osteoclast-stimulating factor (s) and/or induce osteoblasts to secrete an inhibitor of osteoclast development or osteoclast-mediated resorption. The work of Vitté et al.36 and Nishikawa et al.34 provide evidence that bisphosphonates stimulate the production of an osteoclast inhibitor. Vitté et al.36 have determined that this inhibitor acts by repressing osteoclast formation and survival rather than resorptive activity. The identity of this inhibitory factor is not yet known.
Both osteoblasts and osteoclasts are present in the bone organ cultures used in the current study. Alendronate produced a dose-dependent inhibition of resorption in the fetal rat limb bone organ culture model (Fig. 1). Alendronate (21 and 70 μM) inhibited the resorption elicited by 18 pM IL-1β; however, when a higher concentration of IL-1β (180 pM) was used, only 70 μM alendronate was able to inhibit resorption. Thus, although both doses of IL-1β produced a maximal response by 72 h, the effect of the lower concentration was more effectively antagonized by alendronate, possibly because it was slower to develop.
Alendronate alone did not affect IL-6 production in the fetal rat limb bone cultures (Fig. 1A). Our results contrast with those of Schweitzer et al.,16 who found that low concentrations (50 nM) of the bisphosphonate dimethyl-APD alone stimulate IL-6 production in fetal mouse bone explants (metacarpal/metatarsal bones). This discrepancy may reflect differences in the aminobisphosphonates employed, the concentrations tested, the differentiation state of the bones used in the experiments, and/or the different cytokine environments of the bone cultures.
In the present study, we demonstrated that alendronate enhances the IL-6 production elicited by a relatively high (180 pM) concentration of IL-1β (Fig. 1C). These results are similar to those of Schweitzer et al.16 who found a synergistic effect of dimethyl-APD with PTH on IL-6 production. In contrast to the findings from our studies, however, dimethyl-APD (≤500 nM) was also shown to act synergistically with PTH to enhance bone resorption.37
Our findings demonstrate a dissociation between the effects of alendronate on resorption and those on IL-6. At lower doses of IL-1β (18 pM), alendronate inhibited resorption but had no effect on IL-6 (Fig. 1B). With a higher dose of IL-1β (180 pM), there was also a dissociation between these responses; the dissociation was particularly striking in this case because of its dependence on the concentration of alendronate. Alendronate, 70 μM, inhibited IL-1β–stimulated resorption while actually increasing IL-6. However, 21 μM alendronate, although failing to inhibit resorption, also significantly increased the IL-6 response (Fig. 1C). These dissociations suggest at least two possibilities: the direct antiresorptive effect of high doses of alendronate can override any effects of increased IL-6, or IL-6 is not the major factor in IL-1β–stimulated resorption in this bone culture model. One or both of these factors may contribute to the results described here.
Because limb bone cultures consist of multiple cell types, we sought to determine whether alendronate increases IL-1β–stimulated IL-6 in osteoblasts, which are known to produce IL-6 in response to IL-1β.30 We determined that the enhancing effect of alendronate on IL-1β–stimulated IL-6 is also evident in UMR-106 osteoblastic cells (Fig. 2). As in the limb bones, the concentrations of alendronate and IL-1β both appear to be factors determining the occurrence and magnitude of this response. The dissociation between the effects of alendronate on resorption and on IL-6 may be a consequence of the involvement of two different cell types, the potentiating effects on IL-6 production being mediated at the osteoblast and the overriding inhibitory effects on resorption being on the osteoclasts in the bone organ cultures.
In addition to its effects to promote osteoclastogenesis, IL-6 has been found to have varying effects on proliferation38 and on markers of differentiation in bone cells, producing increased38 or decreased39 alkaline phosphatase and increased osteocalcin38 and insulin-like growth factor binding protein-5.40 These responses could potentially be affected by alendronate through its effects to increase IL-6.
The results of the UMR-106 studies described above contrast with those of Passeri et al.,41 who found that alendronate inhibited IL-6 production induced by the combination of IL-1 and tumor necrosis factor (TNF) in MG63 human osteoblastic cells. There are several differences between the studies reported in their abstract and in our present results that could account for this apparent inconsistency. First, their study included TNF treatment, whereas ours did not. Their study used a 4 h pretreatment with alendronate, whereas in the present study we used a 48 h IL-1β/alendronate cotreatment. The doses of alendronate used in the two studies were quite different. Their study employed alendronate doses of 10−11 to 10−6 M, while our study utilized only concentrations in the micromolar range. Their study and ours used different osteoblastic cell lines. This may be an important factor since these two cell lines produce different amounts of IL-6, the production being much higher in the MG63 cells.42 The factors of cell lines and the presence of TNF are likely to be more critical than the pretreatment protocol, as we have found, in a preliminary experiment employing a 4 h pretreatment of the cells with alendronate, results that were not different from those that we obtained with cotreatments (data not shown).
The IL-6 response elicited by IL-1β alone or in combination with alendronate was decreased by indomethacin in the limb bone cultures, indicating the involvement of prostaglandins in mediating this response (Table 1). This observation raises the possibility that inhibitors of prostaglandin production may prevent a systemic increase in IL-6 and the resultant acute-phase response. Few studies have been carried out to explore this possibility, and those that have been done provide mixed results. Endotoxemia increases serum and intestinal IL-6 and IL-6 mRNA in mice, and pretreatment with indomethacin blocks this effect in the intestine.43 However, in rheumatoid arthritis patients, who have elevated serum IL-6 levels, nonsteroidal anti-inflammatory drugs are ineffective in decreasing IL-6 levels.44 Similarly, indomethacin does not abolish the effect of centrally administered (intracerebroventricular injection) IL-1 on serum IL-6 levels in rats.45 Additional studies are necessary to explore this concept further.
In the present study, concentrations of alendronate ranging from 0.7 to 70 μM were used. Regarding the relevance of these concentrations, it is difficult to assess what the local concentrations of alendronate in the vicinity of the stromal and osteoblastic cells that produce IL-6 are likely to be under in vivo conditions. It is possible that they could be similar to the circulating concentrations attained after oral administration of the drug. Alternatively, they could be approximated by the concentrations achieved locally at sites where resorption is occurring. These have been estimated to be as high as 0.1 to 1 mM.46
The authors thank Merck & Co., Inc., the National Institutes of Health (NIH), the Chicago Chapter of the Arthritis Foundation, and the U.S. Army Medical Research and Materiel Command for support that enabled this work to be carried out. This work was supported by grants from Merck & Co., Inc., the NIH (AR-11262), and the Chicago Chapter of the Arthritis Foundation. J.L.S. is a recipient of a fellowship from the U.S. Army Medical Research and Materiel Command (DAMD-17–94-J-4466).
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