1,25 (OH)2 Vitamin D3-Stimulated Osteoclast Formation in Spleen-Osteoblast Cocultures Is Mediated in Part by Enhanced IL-1α and Receptor Activator of NF- κB Ligand This information is current as Production in Osteoblasts of September 28, 2021. Sun-Kyeong Lee, Judy Kalinowski, Sandra Jastrzebski and Joseph A. Lorenzo J Immunol 2002; 169:2374-2380; ; doi: 10.4049/jimmunol.169.5.2374 Downloaded from http://www.jimmunol.org/content/169/5/2374

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

1,25 (OH)2 Vitamin D3-Stimulated Osteoclast Formation in Spleen-Osteoblast Cocultures Is Mediated in Part by Enhanced IL-1␣ and Receptor Activator of NF-␬B Ligand Production in Osteoblasts1

Sun-Kyeong Lee,2 Judy Kalinowski, Sandra Jastrzebski, and Joseph A. Lorenzo

We examined the ability of 1,25 (OH)2 vitamin D3 (Vit D) to stimulate osteoclast-like cell (OCL) formation in cocultures of spleen cells and primary calvarial osteoblasts from wild-type (WT) and IL-1R type 1-deficient (knockout; KO) mice. Vit D dose depen- dently increased OCL in cocultures containing WT osteoblasts. In contrast, there was a 90% reduction in OCL numbers in cocultures containing KO osteoblasts. In cocultures with either WT or KO osteoblasts, treatment with Vit D increased receptor Downloaded from activator of NF-␬B ligand mRNA by 17-, 19-, or 3.5-fold, respectively. Vit D decreased osteoprotegerin mRNA to undetectable in all groups. Intracellular IL-1␣ protein increased after Vit D treatment in cocultures containing WT, but not KO osteoblasts. We also examined direct effects of Vit D, IL-1␣, and their combination on gene expression in primary osteoblasts. In WT cells, Vit D and IL-1 stimulated receptor activator of NF-␬B ligand mRNA expression by 3- and 4-fold, respectively, and their combination produced a 7-fold increase. Inhibition of osteoprotegerin mRNA in WT cells was partial with either agent alone and greatest with ␣

their combination. In KO cells, only Vit D stimulated a response. IL-1 alone increased IL-1 protein expression in WT osteoblasts. http://www.jimmunol.org/ However, in combination with Vit D, there was a synergistic response (100-fold increase). In KO cultures, there were no effects of IL-1, Vit D, or their combination on IL-1␣ protein. These results demonstrate interactions between IL-1 and Vit D in primary osteoblasts that appear important in both regulation of IL-1␣ production and the ability of Vit D to support osteoclastogenesis. The Journal of Immunology, 2002, 169: 2374Ð2380.

nterleukin-1 is a proinflammatory that is a potent been described (9). IL-1R1 binds both IL-1␣ and ␤ and appears to stimulator of bone resorption and inhibitor of bone formation be the principal mediator of IL-1 actions. The type 2 IL-1R (IL- (1). A variety of cells in the bone microenvironment, includ- 1R2) has a short cytoplasmic tail and does not transmit a biologic

I by guest on September 28, 2021 ing /, osteoblasts, and osteoclasts, can syn- signal. Instead, it is believed to be a decoy receptor, which binds thesize IL-1 (2). Production of IL-1 has been linked to the disease IL-1␣ and ␤ to prevent their binding to IL-1R1. Estrogen can osteoporosis because inhibitors of IL-1 activity block the bone loss regulate IL-1R1 and IL-1R2 on osteoclasts, and this response may in mice that occurs with ovariectomy (3). In addition, mice, which be involved in the effects that estrogen has on bone (10). Binding are insensitive to IL-1 because they lack the bioactive type 1 IL-1R of IL-1 to cells requires interaction between the IL-1R accessory (IL-1R1),3 fail to lose bone mass after ovariectomy (4). In humans, protein and IL-1R1 or IL-1R2 (11). the presence of osteoporotic fractures was linked to a polymor- Osteoclasts form in vitro through the interactions of hemopoi- phism in the gene locus for IL-1R antagonist (IL-1Ra) in two stud- etic cells, which contain osteoclast precursors, and support cells of ies (5, 6), but not in a third (7). mesenchymal origin (stromal cells or osteoblasts; Ref. 12). The Two biologically active isoforms of IL-1 are known. IL-1␣ and latter produce factors that are required for the maturation and ter- IL-1␤ have similar biologic activities and structure and both bind minal differentiation of osteoclast precursor cells. It appears that the same receptors. A third IL-1 isoform, IL-1Ra, is a competitive physical interactions between osteoclast precursor cells and mes- inhibitor of IL-1␣ and ␤. It binds the bioactive IL-1R1 without enchymal support cells are necessary for osteoclasts to form in stimulating downstream events (8). Two receptors for IL-1 have response to signals from most resorption stimuli (13). Among the that are produced by mesenchymal sup- port cells and have been identified as regulating osteoclast devel- Division of Endocrinology, Department of Medicine, University of Connecticut Health Center, Farmington, CT 06030 opment are receptor activator of NF-␬B ligand (RANKL) and os- Received for publication October 11, 2001. Accepted for publication June 24, 2002. teoprotegerin (OPG; Ref. 12). RANKL is a TNF-like protein, The costs of publication of this article were defrayed in part by the payment of page which binds to a receptor on osteoclast precursor cells, named charges. This article must therefore be hereby marked advertisement in accordance receptor activator of NF-␬B (RANK). OPG is a soluble decoy with 18 U.S.C. Section 1734 solely to indicate this fact. receptor for RANKL that is released from cells, binds RANKL, 1 This work was supported by Grant PO1-AR38933 from the U.S. Public Health and prevents it from interacting with RANK. Stimulators of re- Service. sorption enhance RANKL production in mesenchymal support 2 Address correspondence and reprint requests to Dr. Sun-Kyeong Lee, Division of Endocrinology, University of Connecticut Health Center, AM047, MC 1850, 263 cells, and some also inhibit OPG (14, 15). Regulation of RANKL Farmington Avenue, Farmington, CT 06030. E-mail address: [email protected] and OPG in bone is believed to be a critical mechanism for the 3 Abbreviations used in this paper: IL-1R1, type 1 IL-1R; OCL, osteoclast-like cell; precise management of osteoclastogenesis and bone resorption. WT, wild type; KO, knockout; OPG, osteoprotegerin; IL-1Ra, IL-1R antagonist; IL- In the current study, we examined the role of IL-1 in the osteo- 1R2, type 2 IL-1R; HIFBS, heat-inactivated FBS; TRAP, tartrate-resistant acid phos- phatase; PBM, peripheral monocyte; RANK, receptor activator of NF-␬B; RANKL, clastogenic response to 1,25 (OH)2 vitamin D3 (Vit D) by exam- RANK ligand; Vit D, 1,25 (OH)2 vitamin D3. ining the effects that Vit D had on cocultures of spleen cells, which

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 The Journal of Immunology 2375 are primarily hemopoietic in origin, and primary murine osteo- PCR amplification was done as previously described (19) using gene- blasts, which derive from mesenchymal cells. In these cultures specific primers and Taq polymerase (AmpliTaq; Applied Biosystems, either, both, or neither the hemopoietic and mesenchymal cells Norwalk, CT). The PCR mixture (without enzyme) was overlaid with min- eral oil and heated to 94¡C for 5 min. During the last minute, AmpliTaq were from wild-type (WT) or IL-1R1-deficient mice. was added (hot start) and amplification was allowed to proceed in a thermal cycler (Applied Biosystems). Temperature cycling was as follows: dena- Materials and Methods turation at 94¡C for 1 min, primer annealing at 65¡C for 2 min, and ex- Mice tension at 72¡C for 3 min for 10 cycles. In subsequent cycles, the primer annealing temperature was decreased stepwise (step-down method) by 5¡C IL-1R1-deficient (knockout; KO) mice were a gift from Dr. J. Peschon every five cycles. After the last cycle, the mixture was incubated at 72¡C (Immunex, Seattle, WA; Refs. 4 and 16). Animals were in a mixed C57BL/ for 7 min. To verify that amplification was in the linear range for each PCR 6 ϫ 129Sv background. WT controls were also C57BL/6 ϫ 129Sv mice analysis, we performed PCR amplification between 24 and 36 cycles at that were derived from the same heterozygous breeding pair, which have three cycle intervals, and measured product yield as previously described been used to generate the homozygous KO mice. (19). Specific amplimer sets are designed from published cDNA sequences: murine RANKL (27) spanning all five exons (antisense: 5Ј-TCCCGAT Materials GTTTCATGATGC-3Ј, sense: 5Ј-TGTACTTTCGAGCGCAGATG-3Ј), Ј Recombinant human IL-1␣ and IL-1Ra were from R&D Systems (Minne- murine OPG (28) spanning exons II to V (antisense: 5 -TCAAGTGCTT Ј Ј Ј apolis, MN) and ELISA kits for murine IL-1␣ was from Endogen GAGGGCATAC-3 ; sense: 5 -TGGAGATCGAATTCTGCTTG-3 ), mu- ␤ Ј Ј (Woburn, MA). Vit D was a gift from Roche Pharmaceuticals (Nutley, NJ). rine -actin (29) (antisense: 5 -CTCTTTGATGTCACGCACGATTTC-3 ; Ј Ј ␣ Unless otherwise stated, all other reagents were from Sigma-Aldrich (St. sense: 5 -GTGGGCCGCTCTAGGCACCAA-3 ), murine IL-1 (30) (anti- Ј Ј Ј Louis, MO). sense: 5 -AGGTCGGTCTCACTACCTGTGATGAGTTTTGG-3 ; sense: 5 - AAGATGTCCAACTTCACCTTCAAGGAGAGCCG-3Ј), murine IL-1␤ Primary osteoblastic cells (31) (antisense: 5Ј-CAGGACAGGTATAGATTCTTTCCTTT-3Ј, sense: 5Ј- Downloaded from ATGGCAACTGTTCCTGAACTCAACT-3Ј), and murine G3PDH (32) (anti- Calvaria were removed from 3-day-old neonatal mice, dissected free of sense: 5Ј-CATGTAGGCCATGAGGTCCACCAC-3Ј, sense: 5Ј-TGAAGG loose connective tissue, and rinsed in PBS as previously described (17). TCGGTGTGAACGGATTTGGC-3Ј). Cells were liberated by five sequential 15-min incubations of calvaria with bacterial collagenase (Collagenase P; Boehringer Mannheim, Indianapolis, ELISAs 2ϩ 2ϩ IN), 0.1% trypsin, and 0.8 mM Na2EDTA in Ca ,Mg free PBS. Cells were collected by centrifugation after each digestion and washed with Cells were incubated for the indicated times with stimuli before the me- DMEM (Life Technologies, Gaithersburg, MD) and 10% heat-inactivated dium was removed and replaced with 250 ␮lof␣-MEM without serum per http://www.jimmunol.org/ FBS (HIFBS; HyClone, Logan, UT). Cells obtained from digestions three well in a 12-well plate. Cells were then frozen and thawed three times, and to five were pooled and used as primary osteoblasts. Cells were first cul- the cell extracts removed for assay by commercial ELISA according to the tured to confluence in 100-mm dishes with DMEM and 10% HIFBS in a manufacturer’s recommendations, because most IL-1␣ remains in the cy- ϫ 4 tosol of producing cells in its precursor form (33). Data were expressed as humidified incubator with 5% CO2, and then plated at 2 10 cells per well of a 24-well plate. For coculture experiments, cells were incubated picograms of protein per milliliters of cell extract. We also assayed the overnight to allow the osteoblasts to attach before spleen cells were added levels of IL-1␣ and ␤ in the conditioned medium from both cocultures and to the wells. osteoblasts, but were unable to detect either protein under any condition. Spleen cells Statistics

Spleens from 8- to 10-wk-old male mice were macerated with a sterile Differences between groups were assessed by ANOVA and then by the by guest on September 28, 2021 needle. Cells were collected by allowing the splenic tissue to settle at unit Bonferroni post hoc test if significant differences were identified. All ex- gravity before removing the supernatant, which contained the liberated periments were repeated at least once with similar results. cells. Cells were pelleted by centrifugation and washed three times with PBS. The spleen cells (2 ϫ 106 cells/well of a 24-well plate) were then Results cocultured with osteoblasts for 5Ð6 days. Osteoclast-like cells (OCL) were Coculture of osteoblasts and spleen cells alone produced relatively identified by their characteristic multinucleation and tartrate-resistant acid Ͻ phosphatase (TRAP) staining. Osteoblast layer was trypsinized before cells few OCLs ( 10 per well; Fig. 1), which were defined as multinu- were fixed with 2.5% glutaraldehyde in PBS for 30 min and TRAP stained cleated and having positive staining for TRAP. In contrast, treat- with a commercial (Sigma-Aldrich). ment of the cultures with Vit D caused a dose-dependent increase To determine cells that possess calcitonin receptors, cultures were in- in the number of OCL in cultures that contained both WT osteo- cubated with 125I-labeled salmon calcitonin (1 ϫ 106 dpm/ml; Amersham Pharmacia Biotech, Piscataway, NJ) in medium for2hatroom tempera- blasts and spleen cells (Fig. 1). In cultures that contained IL-1R1 Ϫ8 ture, washed two times in PBS, and developed by autoradiography (18). deficient (KO) osteoblasts, Vit D (10 M) stimulated osteoclast Specificity of the 125I-labeled calcitonin binding was determined by adding excess cold salmon calcitonin (100-fold, 10Ϫ7 M; Bachem, Torrence, CA) to the reaction mixture before incubating it with the cells. Slides were stained with Giemsa. Bone marrow cells Bone marrow cells from C57BL/6 mice (Charles River Farms, Wilming- ton, MA) were isolated by a modification of previously published methods (19Ð26). Mouse bone marrow cells from femur, tibia, and humerus were flushed, collected into tubes, washed twice with ␣-MEM, and cultured (1 ϫ 106 cells/cm2)in␣-MEM containing 10% HIFBS. Cultures were fed every 3 days with fresh medium. IL-1Ra (100 ng/ml), indomethacin (10Ϫ6 M), and/or Vit D (10Ϫ9 or 10Ϫ8 M) was added to cultures as indicated in each experiment. Cells were fixed on day 6 of culture with 2.5% glutaraldehyde in PBS for 30 min at room temperature before being stained for TRAP. Enzyme histochemistry for TRAP was performed with a commercial kit (Sigma-Aldrich).

PCR amplification FIGURE 1. Dose-response effects of Vit D on osteoclast formation in WT osteoblast-spleen cell cocultures. Cells from WT mice were cultured Total RNA was extracted from the cells with TRI REAGENT (Molecular ϩ Research Center, Cincinnati, OH). Total RNA was converted to cDNA by for 6 days with vitamin D. OCL were identified as TRAP multinucleated reverse transcriptase (Superscript II; Life Technologies) using random hex- (Ͼ3 nuclei) giant cells. Values are mean Ϯ SEM for six determinations per Significant effect of vitamin D, p Ͻ 0.01 ,ء .amer primers. Aliquots of the first-strand cDNA were amplified by PCR. group 2376 VITAMIN D, IL-1, AND RANKL ON OSTEOCLASTOGENESIS

FIGURE 2. Effects of IL-1 responsiveness on the ability of Vit D to induce osteoclast formation in mixed osteoblast-spleen cell cocultures. Cells were from either WT or IL-1R1-deficient (KO) mice and were cul- FIGURE 4. Dose-response effects of Vit D on RANKL, OPG, and Ϫ8 ϩ tured for 6 days with vitamin D (10 M). OCL were identified as TRAP IL-1␣ mRNA expression in osteoblast-spleen cell cocultures. Cells were multinucleated (Ͼ3 nuclei) giant cells. Values are mean Ϯ SEM for four from WT mice, were cultured for 6 days without or with Vit D, and then Downloaded from Significantly different from respective group extracted for RNA. Levels of RANKL, OPG, IL-1␣, and G3PDH mRNA ,ء .determinations per group using WT osteoblasts, p Ͻ 0.01 were determined by RT-PCR. Values below each band are the ratio of the band intensity normalized to that of G3PDH. formation, but their numbers were only 10% of those in cocultures that contained WT osteoblasts (Fig. 2). To confirm that the OCL in cocultures that contained IL-1R1 KO osteoblasts. Inhibition of http://www.jimmunol.org/ that formed in these cultures had characteristics of authentic oste- OPG mRNA expression with Vit D treatment in all cocultures was oclasts, we demonstrated that they contained large numbers of cal- similar and almost complete. citonin receptor as determined by specific binding of 125I-labeled Levels of intracellular IL-1␣ protein were also measured in the calcitonin to the OCL (Fig. 3). cocultures (Fig. 6). In cocultures that were not treated with Vit D, ␣ To further characterize the effects of 1,25 (OH)2 vitamin D3 on little IL-1 protein was detectable in the cells. In contrast, in co- this system, we measured mRNA levels of RANKL, OPG, and cultures that were stimulated with Vit D (10Ϫ8 M), there was a IL-1␣ in the cocultures (Fig. 4). Relatively little RANKL and marked increase in IL-1␣ protein in those cocultures that contained IL-1␣ and high levels of OPG mRNA were present in control WT osteoblasts, and this response was similar with either WT or ␣ culture. Vit D treatment increased RANKL and IL-1␣ mRNA and KO spleen cells. In contrast, little intracellular IL-1 protein was by guest on September 28, 2021 decreased OPG mRNA expression dose dependently. Treatment detected in Vit D-stimulated cocultures that contained KO osteo- with Vit D (10Ϫ8 M) also increased RANKL and inhibited OPG blasts and either WT or KO spleen cells. mRNA levels in the cocultures that contained KO osteoblasts (Fig. These results suggested that differences existed in the osteoclas- 5). However, the increase in RANKL expression was 17- to 19- togenic signal that were produced by osteoblasts from WT and KO fold in cocultures that contained WT osteoblasts, but only 3.5-fold mice, and that these differences were related to the ability of Vit D to stimulate IL-1 production. To further examine these differences, we measured the effects of Vit D treatment on cultures of either WT or IL-1R1 KO osteoblasts. In these experiments, cultures were

FIGURE 3. OCL formed in osteoblast-spleen cell cocultures have spe- cific calcitonin receptors. Cells from WT osteoblasts and WT spleen cells were incubated with Vit D (10Ϫ8 M) for 7 days. At the conclusion of the experiment, cells were then incubated with radiolabeled calcitonin and FIGURE 5. Effects of IL-1 responsiveness on the ability of Vit D to without (A and B) or with 100-fold excess cold calcitonin (C and D). Cells regulate RANKL and OPG mRNA expression in osteoblast-spleen cell were stained for TRAP and then developed for autoradiography. A and C cocultures. Cells were from either WT or IL-1R1-deficient (KO) mice, are brightfield. B and D are darkfield. Silver grains, indicating radiolabeled were cultured for 7 days without or with Vit D (10Ϫ8 M), and then ex- calcitonin binding, appear as black dots in brightfield images and white tracted for RNA. Levels of RANKL, OPG, and ␤-actin mRNA were de- dots in darkfield images. Note white reticular pattern in B, overlying OCL termined by RT-PCR. Values below each band are the ratio of the band which is not present in D, demonstrating specific OCL calcitonin binding. intensity normalized to that of ␤-actin. The Journal of Immunology 2377

FIGURE 6. Effects of Vit D on intracellular IL-1␣ protein levels in osteoblast-spleen cell cocultures. Cells were from either WT or IL-1R1- FIGURE 8. Effects of Vit D and IL-1␣ on intracellular IL-1␣ protein deficient (KO) mice, and were cultured for 7 days without or with Vit D levels in osteoblast cultures. Primary murine osteoblastic cells from either Ϫ8 ␣ WT or IL-1R1-deficient (KO) mice were cultured for 5 days without or (10 M). Levels of intracellular IL-1 were determined by ELISA. Val- Downloaded from Ϫ8 Significantly with Vit D (10 M), IL-1, or their combination. Levels of intracellular ,ء .ues are mean Ϯ SEM for four determinations per group different from respective control group, p Ͻ 0.01 IL-1␣ were determined by ELISA. Values are mean Ϯ SEM for four de- Significantly different from respective control ,ء .terminations per group ␣Significantly different from cells treated with IL-1 ,ءء .group, p Ͻ 0.01 Ͻ treated with doses of IL-1 (10 ng/ml) and/or Vit D (10Ϫ8 M), alone, p 0.01 which produced maximal responses. In WT osteoblasts, only low levels of RANKL mRNA were present in controls and both Vit D http://www.jimmunol.org/ and IL-1 increased the levels of RANKL mRNA by 3- or 4-fold, els, which was enhanced in WT osteoblast cultures that were respectively (Fig. 7). There was an additive effect of the combi- treated with both Vit D and IL-1. IL-1 alone had no effect on OPG nation of Vit D and IL-1 on RANKL mRNA expression, which mRNA expression in WT osteoblasts. In KO osteoblasts, only Vit produced a 7-fold increase in RANKL mRNA expression. In KO D treatment partially inhibited OPG mRNA expression. In addi- ␣ osteoblasts, the response to Vit D was preserved but, as expected, tion, Vit D had no effect on IL-1 mRNA expression in WT os- there was no effect of IL-1␣ treatment on RANKL mRNA levels. teoblast cultures, while IL-1 treatment induced a small signal. ␣ In contrast to the spleen cell osteoblast cocultures, there was less Combined treatment with both Vit D and IL-1 increased IL-1 mRNA expression by 3-fold over that seen in WT osteoblast cul-

regulation of OPG mRNA expression in WT osteoblast or KO by guest on September 28, 2021 osteoblast cultures that were treated with either Vit D or IL-1. Vit tures that were treated with IL-1 alone. There was no detectable ␣ D treatment produced a partial inhibition of the OPG mRNA lev- IL-1 mRNA expression in any group of KO osteoblasts cells. There was also little regulation of the relatively low level of IL-1␤ mRNA by treatment of the cultures with Vit D, IL-1, or their combination in either WT osteoblasts or KO osteoblasts. Expression of IL-1␣ protein in osteoblast cultures was similar to the effects on mRNA expression. IL-1␣ was not detectable in WT osteoblast controls or cultures that were treated with Vit D (Fig. 8). However, there was a small but significant production of IL-1␣ in

FIGURE 7. Effects of Vit D and IL-1␣ on RANKL, OPG, IL-1␣, and IL-1␤ mRNA expression in osteoblast cultures. Cells were from either WT FIGURE 9. Effects of Vit D and IL-1Ra on osteoclast formation in bone or IL-1R1-deficient (KO) mice, were cultured for 5 days without or with marrow cell cultures. Cells from C57BL/6 mice were cultured for 6 days Vit D (10Ϫ8 M), and then extracted for RNA. Levels of RANKL, OPG, with vitamin D (10Ϫ9 M) and/or IL-1Ra (100 ng/ml). OCL were identified IL-1␣, IL-1␤, and ␤-actin mRNA were determined by RT-PCR. Values as TRAPϩ multinucleated (Ͼ3 nuclei) giant cells. Values are mean Ϯ SEM ,Significant effect of vitamin D; ؉ ,ء .below each band are the ratio of the band intensity normalized to that of for six determinations per group ␤-actin. significant effect of IL-1Ra, p Ͻ 0.01 2378 VITAMIN D, IL-1, AND RANKL ON OSTEOCLASTOGENESIS

WT osteoblasts that were treated with IL-1. In WT osteoblast cul- ulate IL-1␣ production. Instead, it markedly augmented the ability of tures that were treated with both Vit D and IL-1, there was a exogenously added IL-1 to increase endogenous production of this synergistic increase in intracellular IL-1␣ protein, with levels be- cytokine in primary murine osteoblast cultures. ing 100-fold greater than those seen in cultures that were treated There have been varying effects of Vit D on IL-1 production in with IL-1 alone. IL-1␣ protein was not detectable in control KO other models. In the human monocyte cell line U-937, Vit D had osteoblast cultures, and treatment with Vit D, IL-1, or their com- no effect alone on IL-1 production, but augmented the ability of T bination failed to produce detectable IL-1␣ protein levels in KO lymphocyte-produced cytokines, PMA, or LPS to stimulate IL-1 osteoblast cells. (37Ð39). However, LPS directly supported survival and fusion of To examine the role of IL-1 production in the effects of Vit D on preosteoclast in inflammatory bone loss and this phenomenon was OCL formation in another in vitro model of osteoclastogenesis, we independent of IL-1 and RANKL action (40). In the murine mac- treated WT murine bone marrow cultures with or without Vit D rophage cell line P388D1 and in human peripheral and with or without IL-1Ra, a competitive inhibitor of IL-1␣ and (PBMs), Vit D was found to be a direct stimulator of IL-1 (41, 42). IL-1␤ (Fig. 9). As with cocultures that contained IL-1R1 KO os- In addition, Vit D stimulated IL-1 in PBM and ST-2 cell cocultures teoblastic cells, IL-1Ra inhibited osteoclast formation that was (43, 44). However, another group found no effect of Vit D alone on stimulated by Vit D (10Ϫ9 M) in the bone marrow cultures. Be- IL-1 production by PBM, but did find it to augment IL-1 produc- cause increased osteoclast formation by IL-1 has been shown to be tion that was stimulated by hydroxyapetite and collagen (45). In ␤ mediated by PGE2 (34, 35), we investigated whether prostaglandin contrast to our results, which found no effects of Vit D on IL-1 synthesis was involved in the responses of bone marrow cultures to expression in primary osteoblasts, Wang et al. (46) demonstrated Vit D. Bone marrow cells were stimulated with or without Vit D that Vit D increased IL-1␤ mRNA in osteoblastic cells from 5Ð12- Downloaded from and with or without indomethasin, a selective inhibitor of prosta- wk-old mice, but not in osteoblastic cells from 10Ð12-mo-old glandin synthesis. We found that indomethacin had no effect on mice. Hence, the age of the mice from which the osteoblasts are OCL formation in Vit D-stimulated bone marrow cultures (data derived may affect their ability to be regulated by Vit D. not shown). In some models, Vit D inhibits IL-1 activity. Muller et al. (47) found that Vit D inhibited the ability of LPS-stimulated human

Discussion PBM to produce IL-1␣. Vit D also inhibited IL-1 production by a http://www.jimmunol.org/ These results demonstrate that the ability of Vit D to stimulate human leukemia cell line OCIM2 (48), human monocyte/macro- OCL formation in mixed spleen cell osteoblast cocultures is de- phages (49), and human corneal epithelial cells (50). pendent on the ability of the osteoblasts in the cultures to respond We found that the regulation of OPG mRNA by Vit D in pri- to IL-1. This effect appears dependent on changes in the expression mary osteoblast cultures was less in cells that were incubated alone of RANKL and OPG as increases in OCL formation correlated than in cells that were cocultured with spleen cells. This result is with increased expression of RANKL mRNA and inhibition of similar to our previous finding of decreased OPG regulation in OPG mRNA levels in the cocultures. primary murine osteoblasts with parathyroid hormone treatment In these cocultures, we found both additive and synergistic in- compared with the responses of whole murine marrow cultures teractions between Vit D and IL-1 in their ability to stimulate (19). These observations suggest that interaction between osteo- by guest on September 28, 2021 RANKL and IL-1␣ production by osteoblasts. Additive effects oc- blasts and hemopoietic cells modulate the OPG responses of os- curred between IL-1 and Vit D with regards to their ability to teoblasts. It has previously been demonstrated that there are bidi- stimulate RANKL and inhibit OPG mRNA levels. Synergistic ef- rectional interactions between mesenchymal and hemopoietic cells fects were demonstrated in the ability of IL-1 and Vit D to stim- with Vit D stimulation (43, 44), and similar effects may regulate ulate IL-1␣ protein and mRNA. As previously demonstrated by OPG production. Pfeilschifter et al. (36), IL-1 enhanced osteoclast formation in Vit The ability of IL-1 and Vit D to stimulate RANKL production D-treated human marrow cultures. PGs have previously been appears to involve different pathways because inhibitors of STAT3 shown to mediate some of their effects on osteoclast formation block responses to IL-1 but not to Vit D (51). NF-␬B, which is a through IL-1 (34, 35). However, it is unlikely that Vit D induced mediator of the response of cells to IL-1 (52), is regulated by Vit PG, which in turn, induced IL-1 in our models because the specific D in human MRC-5 fibroblasts (53), and this effect may be in- PG synthesis inhibitor, indomethacin, had no effect on Vit D or volved in the interactions that we observed between these agents. IL-1-mediated actions in our studies. The ability of IL-1 to stimulate IL-6 in MC3T3-E1 osteoblastic It is likely that endogenous IL-1 production in our cultures is cells was also enhanced by treatment with Vit D through a mech- involved in the ability of Vit D to stimulate an osteoclastic re- anism that was dependent on the IL-1R1 (54). sponse because Vit D enhanced the ability of IL-1 to stimulate Direct actions of IL-1 on osteoclast precursor and osteoclast IL-1␣ production and the combination of IL-1 and Vit D enhanced have recently been demonstrated (55). However, it is unlikely that RANKL and inhibited OPG production in primary murine osteo- such effects were involved in the responses that we observed be- blasts. In addition, we found that IL-1Ra, which specifically blocks cause we found no difference in OCL formation between cocul- IL-1 activity, inhibited Vit D-mediated osteoclastogenesis in WT tures that used WT and KO spleen cells (Fig. 2). Instead, it seems murine bone marrow cultures. that the differences in OCL formation in the cocultures depended We have previously demonstrated a tight correlation between solely on the ability of osteoblasts to express the IL-1R1. It has increases in RANKL mRNA, decreases in OPG mRNA, and in- been shown that IL-1 stimulates bone resorption through a primary creases in OCL formation with parathyroid hormone in murine action on osteoblast that was induced by IL-1 to transmit a signal marrow cell cultures (19); and it appears that similar relationships that stimulates osteoclastic bone resorption (56). exist for Vit D stimulation of OCL formation in murine spleen cell It is tempting to speculate on the role that the interaction of IL-1 osteoblast cocultures. and Vit D may have in vivo. IL-1 production occurs in bone (57) The ability of Vit D to regulate IL-1 production in primary osteo- and is believed involved in the role that estrogen plays in post- blast cultures appears restricted to IL-1␣ because we found no effect menopausal bone loss (58). 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