Signaling 2+ Ca B and Κ Mature Osteoclasts by Affecting NF- IL-4

IL-4 Inhibits Bone-Resorbing Activity of Mature Osteoclasts by Affecting NF- κB and Ca 2+ Signaling

This information is current as Latha S. Mangashetti, Shruti M. Khapli and Mohan R. Wani of September 26, 2021. J Immunol 2005; 175:917-925; ; doi: 10.4049/jimmunol.175.2.917 http://www.jimmunol.org/content/175/2/917 Downloaded from

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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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-4 Inhibits Bone-Resorbing Activity of Mature Osteoclasts by Affecting NF-␬B and Ca2؉ Signaling1

Latha S. Mangashetti, Shruti M. Khapli, and Mohan R. Wani2

IL-4 is an important immune cytokine that regulates bone homeostasis. We investigated the molecular mechanism of IL-4 action on bone-resorbing mature osteoclasts. Using a highly puriﬁed population of mature osteoclasts, we show that IL-4 dose-dependently inhibits receptor activator of NF-␬B ligand (RANKL)-induced bone resorption by mature osteoclasts. We detected the existence of IL-4R mRNA in mature osteoclasts. IL-4 decreases TRAP expression without affecting multinuclearity of osteoclasts, and inhibits actin ring formation and migration of osteoclasts. Interestingly, IL-4 inhibition of bone resorption occurs through prevention of RANKL-induced nuclear translocation of p65 NF-␬B subunit, and intracellular Ca2؉ changes. Moreover, IL-4 rapidly decreases RANKL-stimulated ionized Ca2؉ levels in the blood, and mature osteoclasts in IL-4 knockout mice are sensitive

to RANKL action to induce bone resorption and hypercalcemia. Furthermore, IL-4 inhibits bone resorption and actin ring Downloaded from formation by human mature osteoclasts. Thus, we reveal that IL-4 acts directly on mature osteoclasts and inhibits bone resorption .by inhibiting NF-␬B and Ca2؉ signaling. The Journal of Immunology, 2005, 175: 917–925

steoclasts, the multinuclear cells (MNCs)3 responsible had been limited because of difficulties in obtaining a sufficient for bone resorption, play a crucial role in bone remod- number of mature osteoclasts. Induction of mature osteoclasts by eling. The bone loss in many important skeletal disor- RANKL in mouse and human osteoclast precursors is an excellent O http://www.jimmunol.org/ ders such as osteoporosis, rheumatoid arthritis, hypercalcemia of in vitro model to investigate the novel mechanisms of cytokines malignancy, and bone metastases occurs mainly because of in- that regulate bone resorption. creased osteoclast activity (1). A major breakthrough in under- IL-4 is a 19-kDa pleiotropic type I cytokine secreted by acti- standing the regulation of osteoclastogenesis occurred after the vated TH2 lymphocytes, mast cells, eosinophils, and basophils discovery of novel molecules such as receptor activator of NF-␬B (15). IL-4, an important immune cytokine that regulates function (RANK), RANK ligand (RANKL), and osteoprotegerin (2–7). of lymphocytes and macrophages, also regulates osteoclastogen- RANKL, in the presence of M-CSF, mediates osteoclastogenesis esis and bone resorption (16, 17). Recent work has clarified the through binding to its receptor RANK on osteoclast precursors (5, role and molecular mechanisms by which IL-4 inhibits RANKL-

7). Transgenic and gene knockout studies in mice established the induced osteoclast differentiation in osteoclast precursors (18–21). by guest on September 26, 2021 absolute dependency of osteoclast differentiation and activation on However, the mechanism of IL-4 action on mature osteoclasts and the expression of RANKL and RANK (7, 8). The distinct signaling its function is not fully delineated. Moreno et al. (21) have reported pathways such as NF-␬B, JNK, p38, ERK, and Src pathways me- the inhibitory effect of IL-4 on mouse mature osteoclast function diated by protein kinases are activated by RANKL during oste- and showed that the effect requires STAT6. In this study, we pre- oclastogenesis and bone resorption (9). pared a large number of highly purified mature osteoclasts induced RANKL also plays an important role in survival and activation by RANKL using both mice and human osteoclast precursors, and of mature osteoclasts and rapidly induces actin ring formation provide further advances that clarify in detail the mechanisms of (10–12). In vivo studies have shown that RANKL increases blood IL-4 action on bone-resorbing mature osteoclasts. We show here ionized Ca2ϩ levels within 1 h suggesting its direct effect on pre- that IL-4 acts directly on mature osteoclasts and significantly in- existing mature osteoclasts (11). Stimulation of RANK on mature hibits bone resorption and tartrate-resistant acid phosphatase osteoclasts by RANKL results in activation of transcriptional fac- (TRAP) expression. IL-4 inhibits bone resorption by disruption of tor NF-␬B and Ca2ϩ signaling (13, 14). The study of molecular RANKL-induced actin ring formation in mouse and as well as mechanisms by which cytokines secreted by T cells or other im- human mature osteoclasts. Furthermore, IL-4 prevents RANKL- mune cells regulate bone-resorbing activity of mature osteoclasts induced nuclear translocation of p65 NF-␬B subunit, and intracellular Ca2ϩ changes in mature osteoclasts. In addition, RANKL- National Center for Cell Science, Pune, India induced hypercalcemia in vivo is attenuated by IL-4 and accentuated by IL-4 deficiency. In conclusion, IL-4 acts directly on Received for publication February 18, 2005. Accepted for publication May 10, 2005. mature osteoclasts and inhibits bone resorption by inhibiting The costs of publication of this article were defrayed in part by the payment of page ␬ 2ϩ charges. This article must therefore be hereby marked advertisement in accordance NF- B and Ca signaling. with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Department of Biotechnology, Government of India. Materials and Methods L.S.M. is the recipient of Senior Research Fellowship from Department of Biotech- Chemicals and animals nology (India). S.M.K. is the recipient of Senior Research Fellowship from the Coun- cil for Scientific and Industrial Research (New Delhi, India). Mouse and human rIL-4, anti-mouse IL-4 Ab, and human M-CSF were 2 Address correspondence and reprint requests to Dr. Mohan R. Wani, National Cen- obtained from R&D Systems. Human soluble RANKL was obtained from ter for Cell Science, University of Pune Campus, Pune-411 007, India. E-mail ad- Insight Biotechnology. Polyclonal anti-NF-␬B p65 and FITC-conjugated dress: [email protected] anti-rabbit Abs were purchased from Santa Cruz Biotechnology. Curcumin 3 Abbreviations used in this paper: MNC, multinuclear cell; CTR, calcitonin receptor; and FITC phalloidin were obtained from Sigma-Aldrich. BALB/c and IL-4 tm2Nnt RANK, receptor activator of NF-␬B; RANKL, RANK ligand; TRAP, tartrate-resis- knockout (BALB/c-Il4 ) mice 5–8 wk old were obtained from the tant acid phosphatase; S, sense; AS, antisense. Experimental Animal Facility of the National Center for Cell Science

(Pune, India). The institutional ethics committee approved the use of ani- TAGGGTTCAGGGGG-3Ј; GAPDH, S, 5Ј- TCGGTGTGAACGGATTT mals and human blood for experiments. Slices of devitalized bovine cor- GGC-3Ј, AS, 5Ј- CATGTAGGCCATGAGGTCCACCAC-3Ј. ␤-Actin and tical bone were prepared as described previously (22). All cultures were GAPDH were used as internal controls. incubated in ␣MEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 ␮g/ml streptomycin (all from Assessment of actin ring formation Sigma-Aldrich). All cultures were maintained at 37°C in a humidified at- mosphere of 5% CO in air. Formation of F-actin was examined as described previously (11). Briefly, 2 mature osteoclasts prepared on bone slices were incubated for1hat37°C Preparation of mouse mature osteoclasts in ␣MEM ϩ 10% FBS and then incubated further with M-CSF and RANKL with or without IL-4 (20 ng/ml) for 6 h. After incubation, the bone To prepare mature osteoclasts, we first isolated stromal and lymphocyte- slices were fixed for 5 min in 10% formalin and permeabilized with 0.1% free, M-CSF-dependent osteoclast precursors from bone marrow as de- Triton X-100 for 5 min. Bone slices were then incubated in 1 ␮g/ml FITC- scribed previously (23). The osteoclast precursors were added to 96-well conjugated phalloidin (Sigma-Aldrich) for 45 min at 37°C, washed thor- 5 plates (5 ϫ 10 cells/well) containing Thermonax plastic coverslips (In- oughly, and mounted onto glass slides in antifade mounting medium vitrogen Life Technologies) and bone slices, and incubated with M-CSF (Sigma-Aldrich). Actin rings were visualized using a Zeiss LSM 510 con- (30 ng/ml) and RANKL (30 ng/ml). Cells were fed on day 2, and after 4 focal microscope equipped with argon and helium lasers (Zeiss). The num- days, formation of mature multinucleated osteoclasts (more than three nu- ber of complete, disrupted, and less intense actin rings per bone slice was clei) was confirmed by TRAP staining. TRAP-positive MNCs were de- counted by a blinded observer. The intensity of the F-actin was represented pleted of mononuclear cells by incubating the cultures for 5 min in 20 mM graphically using the Zeiss LSM 510 software. EDTA in Ca2ϩ- and Mgϩ-free PBS. The cultures were then washed thoroughly with ␣MEM. This procedure removes the majority of mononuclear Osteoclast migration assay cells, while leaving MNCs adherent. In this study, we called these MNCs as mature osteoclasts. The mature osteoclasts were further incubated for Osteoclast migration assay was performed using the Transwell migration Downloaded from 48 h with M-CSF (30 ng/ml) and RANKL (30 ng/ml) for survival and full chambers (Corning). Mature osteoclasts prepared as above were seeded in activation and were treated with or without different concentrations of IL-4. the upper chamber with M-CSF (5 ng/ml) with or without IL-4 (10 and 30 In all additional experiments, M-CSF and RANKL were used at 30 ng/ml ng/ml), and RANKL was added to the lower chamber. Cells were allowed except where indicated. Cells on coverslips were stained for TRAP, and the to migrate through a polycarbonate filter for 8 h. Nonmigrated cells in the bone slices were assessed for bone resorption by reflected light microscopy upper chamber were removed with a cotton swab. Migrated cells on lower or scanning electron microscopy. side of insert were fixed, stained for TRAP, and counted.

Preparation of human mature osteoclasts Immunofluorescence http://www.jimmunol.org/ Human mature osteoclasts were generated using mononuclear cells from Mature osteoclasts prepared in an eight-well glass Lab-Tek chamber slide blood of healthy adult donors. PBMCs were obtained by density gradient (Nunc) were washed with PBS, incubated in the presence of M-CSF and centrifugation using Ficoll-Hypaque. The cells were resuspended (5 ϫ 106 IL-4 (20 ng/ml) for2hat37°C, and then stimulated with RANKL as cells/ml) in ␣MEM containing 15% FBS. The cell suspension (100 ␮l) was indicated. The cells were washed, fixed, permeabilized, and blocked with added to 96-well plates containing bone slices. After 1 h, bone slices were 5% BSA for 20 min (all steps were performed at 4°C). Cells were treated washed thoroughly and lymphocyte-free adherent cells were incubated for with primary Ab against p65 NF-␬B subunit for 30 min, washed, and 12 days with M-CSF (15 ng/ml) and RANKL (30 ng/ml). At 12 days, treated with FITC-labeled secondary Ab for 20 min. Cells were washed mature osteoclasts were depleted of mononuclear cells as described above thoroughly and assessed for the nuclear translocation of p65 using Zeiss and further treated for 3 days with M-CSF (15 ng/ml) and RANKL (30 LSM 510 confocal microscope. The number of mature osteoclasts showing ␬ ng/ml) without or with increasing concentrations of IL-4. The bone slices nuclear translocation of p65 NF- B was scored. by guest on September 26, 2021 were assessed for bone resorption by reflected light microscopy. Measurement of intracellular Ca2ϩ changes Assessment of bone resorption Intracellular Ca2ϩ changes in mature osteoclasts were assessed using the After incubation, bone slices were immersed in 4% sodium hypochlorite Ca2ϩ indicator Fluo-4 AM (Molecular Probes) as described (13). Mature for 15 min to remove the cells, and were washed thoroughly. After drying, osteoclasts cultured in 35-mm petri plates were loaded with 2 ␮M Fluo-4 bone slices were either mounted onto stubs or glass slides and sputter- AM for 30 min at 37°C in ␣MEM containing 0.1% FBS, and subsequently coated with gold. Bone slices on glass slides were examined by reflected washed thoroughly with fresh medium. Petri plates were mounted on the light microscopy, and bone resorption was quantified using an eyepiece stage of the Zeiss LSM 510 Axiovert microscope (Zeiss), and cells were graticule. Bone slices on stubs were examined by scanning electron mi- maintained at room temperature in the physiological buffer containing 130

croscope (XL-30; Philips), and the pit size was measured using microscope mM NaCl, 5 mM KCl, 10 mM glucose, 1 mM MgCl2, 1 mM CaCl2, and software. 20 mM HEPES, pH 7.4. RANKL with or without IL-4 were added in the cultures, and images were acquired for 30 min at 30-s intervals. Intracel- TRAP cytochemistry lular changes in Ca2ϩ were analyzed using the Zeiss LSM 510 software, Mature osteoclast formation was evaluated by quantification of TRAP- and mean values of fluorescent intensities of the sequential images were positive MNCs as described previously (24). After incubation, cells on plotted. coverslips were washed in PBS, fixed in 10% formalin for 10 min, and 2ϩ stained for acid phosphatase in the presence of 0.05 M sodium tartrate Measurement of blood ionized Ca in mice (Sigma-Aldrich). The substrate used was napthol AS-BI phosphate (Sigma- Adult BALB/c and IL-4 knockout mice were used for the in vivo mea- Aldrich). TRAP-positive and TRAP-negative MNCs were counted by light surement of blood ionized Ca2ϩ levels. Mice were injected i.v. with microscopy. RANKL without or with IL-4 in Ca2ϩ- and Mgϩ-free PBS carrier, or PBS RNA isolation and RT-PCR alone as control. After 1 h, retro-orbital blood was collected from anes- thetized mice and levels of whole blood ionized Ca2ϩ were measured using ϩ Mature osteoclasts as described above were prepared in tissue culture flask. a Chiron Diagnostics Rapidlab 865 Ca2 /pH analyzer (Chiron Diagnos- Expression of TRAP, calcitonin receptor (CTR), RANK, IL-4R␣, ␤-actin, and tics). Mean values (ϮSEM) from a minimum of five mice per group were GAPDH mRNAs was assessed by RT-PCR. RNA was isolated using the evaluated statistically. TRIzol reagent (Invitrogen Life Technologies) and used for cDNA synthesis (cDNA synthesis kit; Invitrogen Life Technologies). The cDNA was amplified Statistical analysis of data using PCR for 35 cycles. Each cycle consisted of 30 s of denaturation at 94°C The data is presented as mean Ϯ SEM. Statistical differences between the and 30 s of annealing and 30 s of extension at 72°C. The sequences of sense mean values of control and experimental groups were analyzed using t test. (S) and antisense (AS) primers used were TRAP, S, 5Ј-GGATTCATGGGT GGTGCTG-3Ј, AS, 5Ј-TGGCTAACAATGGTCGCAAG-3Ј; CTR, S, 5Ј-CT GGTTGAGGTTGTGCCC-3Ј, AS, 5Ј-CTCGTGGGTTTGCCTCATC-3Ј; Results RANK, S, 5Ј-ACACCTGGAATGAAGAAGATAAATG-3Ј, AS, 5Ј-AGC IL-4 inhibits bone resorption by mature osteoclasts CACTACTACCACAGAGATGAAG-3Ј; IL-4R␣,S,5Ј-GCTCCAGA CAACCTCACACTCC-3Ј, AS, 5Ј-TCACAGATTTTCATTACTTGGG-3Ј; Mature osteoclasts were prepared from osteoclast precursors as ␤-actin, S, 5Ј-GTGGGCCGCTCTAGGCACCA-3Ј, AS, 5Ј-TGGCCT described in Materials and Methods and purified (Ͼ95% pure) by The Journal of Immunology 919

FIGURE 1. Effect of IL-4 on bone resorption by mature osteoclasts. Ma- ture osteoclasts on bone slices were incubated with M-CSF and RANKL in the absence or the presence of increasing concentrations of IL-4. A, Percent bone surface resorbed, mean Ϯ SEM of six cultures per vari- -p Ͻ 0.01 vs control. B, Re ,ء ;able sorption pits (magniﬁcation, ϫ250). C, Pit size (square micrometers) measured by software in scanning elec- -p Ͻ 0.01 vs con ,ء .tron microscope trol. D, Mature osteoclasts were incubated for 48 h with M-CSF and RANKL in the presence or the absence of IL-4, and M-CSF, RANKL, IL-4, and increasing concentrations of anti-IL-4 Ab. Similar results were Downloaded from obtained in three independent experiments. E, Expression of IL-4R␣ mRNA on mature osteoclasts by RT- PCR. Mature osteoclasts were incubated for 0, 24, and 48 h in the presence of M-CSF and RANKL. NC, Nonloading control. The relative in- http://www.jimmunol.org/ tensity of IL-4R␣ and ␤-actin was analyzed by densitometry.

removing mononuclear cells using EDTA treatment. To examine gest that IL-4 inhibits bone resorption by direct action on activated the effect of IL-4 on bone resorption, purified mature osteoclasts mature osteoclasts. on bone slices were further incubated for 48 h with M-CSF (30 IL-4 effects depend upon binding to and signaling through a by guest on September 26, 2021 ng/ml) and RANKL (30 ng/ml) with or without different concen- receptor complex consisting of the IL-4R␣ chain and the common trations of IL-4. As shown in Fig. 1A, IL-4 inhibited the RANKL- ␥-chain (25). Receptors of IL-4 are expressed on a wide range of induced bone resorption in a dose-dependent manner. IL-4 (20 cells including hemopoietic cells (15). To examine whether mature ng/ml) also significantly decreased the size of an individual re- osteoclasts express IL-4R, we incubated mature osteoclasts with sorption pit (Fig. 1, B and C). The inhibitory effect of IL-4 on bone M-CSF and RANKL and assessed for IL-4R␣ mRNA expression. resorption was confirmed by anti-mouse IL-4 Ab. As shown in Fig. As shown in Fig. 1E, mature osteoclasts induced by RANKL on 1D, simultaneous addition of anti-IL-4 Ab neutralized the inhibi- day 4 (0 h) expressed IL-4R, and cells further incubated for 24 and tory effect of IL-4 in a dose-dependent manner. These results sug- 48 h also showed strong expression of IL-4R.

FIGURE 2. Effect of IL-4 on TRAP expression in mature osteoclasts. Mature osteoclasts were incubated with M-CSF and RANKL in the absence or the presence of various concentrations of IL-4. After 48 h, the number of TRAP-positive (A) and TRAP-negative (B) MNCs was scored. Results are expressed as the mean Ϯ ,ء .SEM of six cultures per variable .p Ͻ 0.05 vs control ,ءء p Ͻ 0.01 and Results were reproducible in three independent experiments. C, TRAP staining of MNCs (magniﬁcation, ϫ20). 920 IL-4 INHIBITS FUNCTION OF MATURE OSTEOCLASTS

Effect of IL-4 on TRAP, CTR, and RANK mRNA expression by mature osteoclasts To examine the effect of IL-4 on expression of osteoclast-speciﬁc genes TRAP and CTR, mature osteoclasts were incubated with M-CSF and RANKL in the absence or the presence of IL-4 (20 ng/ml), and the mRNA expression was analyzed. RANKL-activated mature osteoclasts showed strong expression of TRAP and CTR genes at 24 and 48 h, and it was down-regulated by IL-4 (Fig. 3). RANKL-RANK interaction is required for activation of mature osteoclasts (10, 11, 13); therefore, effect of IL-4 on RANK mRNA expression was examined. IL-4 showed no effect on RANK expression, suggesting the inhibitory effect of IL-4 on bone resorption is not mediated by blockade in RANK expression in mature FIGURE 3. Effect of IL-4 on TRAP, CTR, and RANK mRNAs expres- osteoclasts. sion. Mature osteoclasts were incubated with M-CSF and RANKL in the absence or the presence of IL-4 (20 ng/ml) for 24 and 48 h. Lane 1, Non- loading control; lanes 2 and 4, M-CSF and RANKL for 24 and 48 h, IL-4 inhibits actin ring formation and osteoclast migration respectively; lanes 3 and 5, M-CSF, RANKL, and IL-4 for 24 and 48 h, Downloaded from respectively. The relative intensity of genes was analyzed by densitometry. induced by RANKL Similar results were obtained in two independent experiments. Activated status of the mature osteoclasts is indicated by formation of distinct polymerized actin rings (26, 27). RANKL induces actin IL-4 inhibits TRAP expression in mature osteoclasts ring formation and motility of mature osteoclasts (10, 11). There- fore, we determined whether IL-4 inhibits actin ring formation and

To investigate the mechanism of IL-4 action on activated mature osteoclast migration induced by RANKL. Mature osteoclasts were http://www.jimmunol.org/ osteoclasts, we first examined whether IL-4 inhibits bone resorp- incubated for 6 h with M-CSF and RANKL with or without IL-4 tion by inducing the apoptosis in mature osteoclasts. Osteoclasts (20 ng/ml). As shown in Fig. 4, A and C, RANKL rapidly induced were incubated for 48 h with M-CSF and RANKL in the absence the formation of complete and well-defined actin rings in mature or presence of various concentrations of IL-4. No apoptotic osteoclasts. In the presence of IL-4, two changes were seen in the changes such as chromosome condensation, nuclear fragmentation structure of actin rings. IL-4 disrupted the formation of RANKL- were seen in the presence of IL-4 (data not shown). We then examined the effect of IL-4 on TRAP expression. As shown in Fig. induced actin rings (Fig. 4B), and also significantly decreased the 2A, IL-4 dose-dependently decreased TRAP expression, and the intensity of actin rings (Fig. 4D). Fig. 4, E and F, is the graphical majority of MNCs were TRAP-negative and increased with in- representation of the intensity of actin rings shown in Fig. 4, C and by guest on September 26, 2021 creasing concentrations of IL-4 (Fig. 2B). These TRAP-negative D, respectively. IL-4 significantly decreased the number of well- MNCs did not express CTR (data not shown). Fig. 2C shows the defined actin rings, and the majority of actin rings were either effect of IL-4 on TRAP expression in mature osteoclasts. The disrupted or less intense (Fig. 4G). Using Transwell migration as- TRAP-negative MNCs in the presence of IL-4 were fused to form say, we found that IL-4 (20 ng/ml) significantly inhibited RANKL- giant cells with accumulation of large vacuoles. These results sug- induced migration of majority of osteoclasts (Fig. 4H). These re- gest that IL-4 inhibits expression of TRAP in mature osteoclasts sults suggest that IL-4 inhibits bone resorption by disruption of without affecting the multinuclearity of cells. actin rings and prevention of osteoclast migration.

FIGURE 4. Effect of IL-4 on actin ring formation and migration of mature osteoclasts. Mature osteoclasts on bone slices were treated for 6 h with M-CSF and RANKL without or with IL-4 (20 ng/ml) and were stained for F-actin. A and B show the structure and C and D show the intensity of actin rings (magniﬁcation, ϫ360). E and F are the graphical presentation of C and D, respectively. G, Number of complete, disrupted, and less intense actin rings in mature osteoclasts. Results are from six cultures per variable in three independent experiments. H, Osteoclast migration assay was performed using the Transwell migration chambers. Mature osteoclasts incubated with or without IL-4 were allowed to migrate toward RANKL (30 ng/ml) for 8 h. Migrated osteoclasts were stained for TRAP and counted. Results p Ͻ 0.01 vs ,ء .are from two independent experiments control. The Journal of Immunology 921

FIGURE 5. Effect of IL-4 on RANKL-induced nuclear translocation of p65 NF-␬B subunit, and intracellular Ca2ϩ in mature osteoclasts. A, Mature osteoclasts were preincubated in the presence of M-CSF with or without IL-4 (20 ng/ml) and stimulated with RANKL. Cells were analyzed for nuclear translocation of p65. B, The number of MNCs showing p65 nuclear transloca- -p Ͻ 0.01 vs control. C, Mature os ,ء .tion was scored teoclasts were loaded with 2 ␮M Fluo-4 AM and stimulated with RANKL with or without IL-4 (20 ng/ml). Intracellular Ca2ϩ changes were analyzed by acquiring images in time-dependent manner, and mean values (n ϭ 8) of ﬂuorescent intensities of the sequential images were plotted. Downloaded from http://www.jimmunol.org/ IL-4 prevents RANKL-induced nuclear translocation of p65 NF- also prevents RANKL-induced intracellular Ca2ϩ changes. We ␬B subunit and intracellular Ca2ϩ changes found that IL-4, in a time-dependent manner, prevented the tran- 2ϩ To further address the molecular mechanism by which IL-4 inhib- sient increase in both cytoplasmic and nuclear Ca induced by its bone resorption, we examined the effect of IL-4 on NF-␬B. RANKL (Fig. 5C). These results suggest that decrease in intracel- 2ϩ ␬ RANKL is a strong activator of NF-␬B that plays a functional role lular Ca by IL-4 is associated with decrease in NF- B in bone resorption, and NF-␬B knockout mice are osteopetrotic activation. because of defective osteoclast formation (28–30). In our studies using NF-␬B inhibitors, we also conﬁrmed the functional role of

Effect of IL-4 on RANKL-induced hypercalcemia in mice by guest on September 26, 2021 NF-␬B in bone resorption. When mature osteoclasts were preincubated for 4 h with curcumin, a strong inhibitor of NF-␬B (31), RANKL has been shown to activate preexisting mature osteoclasts RANKL does not induce bone resorption (data not shown). This and stimulate hypercalcemia in mice (11). Also, hypercalcemia in effect of curcumin at low concentration was without inducing the many bone metastases and adult T cell leukemia occur due to apoptosis of mature osteoclasts (data not shown). To examine the increase in RANKL secretion (32). Because IL-4 has previously effect of IL-4 on NF-␬B, mature osteoclasts were incubated with been shown to inhibit parathyroid hormone-related protein-in- M-CSF with or without IL-4, and stimulated with RANKL. duced hypercalcemia in mice (33, 34), we examined the in vivo RANKL stimulated nuclear translocation of the p65 NF-␬B sub- effect of IL-4 on RANKL-induced hypercalcemia. Adult male unit within 15 min and decreased its cytoplasmic level (Fig. 5A, mice were injected with RANKL in the absence or the presence of upper panel). Interestingly, IL-4 totally prevents the nuclear trans- different concentrations of IL-4, and levels of ionized Ca2ϩ were location of p65 with accumulation of this protein in the cytoplasm examined. As shown in Fig. 6A, RANKL rapidly stimulated hy- (Fig. 5A, lower panel). The number of MNCs showing nuclear percalcemia in dose-dependent manner by increasing blood ion- translocation of p65 was decreased signiﬁcantly in the presence of ized Ca2ϩ levels. IL-4 signiﬁcantly decreased RANKL-stimulated IL-4 (Fig. 5B). Activation of NF-␬B and its nuclear translocation ionized Ca2ϩ level in 1 h (Fig. 6B). These results suggest that IL-4 in mature osteoclasts by RANKL is associated with increase in acts on preexisting mature osteoclasts and decreases ionized Ca2ϩ intracellular Ca2ϩ (13, 14). Therefore, we examined whether IL-4 levels in blood.

FIGURE 6. Effect of IL-4 on RANKL-induced hypercalcemia in mice. Adult male mice were injected i.v. with RANKL or PBS as a carrier control (A), and RANKL (0.05 mg/kg) with or without different concentrations of IL-4 (B). After 1 h, blood samples were collected and levels of whole blood ionized Ca2ϩ were measured. Mean values (ϮSEM) from a minimum of p Ͻ ,ء .ﬁve mice per group were evaluated statistically .p Ͻ 0.05 vs RANKL alone ,ءء ;vs PBS alone 0.05 922 IL-4 INHIBITS FUNCTION OF MATURE OSTEOCLASTS

FIGURE 7. Sensitivity of IL-4 knockout mice to RANKL action. A, IL-4 knockout and control male mice were injected i.v. with RANKL (0.05 mg/kg) or PBS as a carrier control. After 1 h, levels of whole blood ionized Ca2ϩ were measured. Mean values (ϮSEM) from a minimum of ﬁve mice per group were evaluated statis- p Ͻ ,ءء ;p Ͻ 0.05 vs PBS alone from control ,ء .tically 0.05 vs PBS alone from IL-4 knockout. B, Osteoclast precursors from IL-4 knockout and control mice were incubated with M-CSF and different concentrations of RANKL. TRAP-positive mature osteoclasts were counted after 5 days. C, percent bone resorption was .p Ͻ 0.05 vs control mice ,ء .counted after 8 days Downloaded from

Mature osteoclasts in IL-4 knockout mice are sensitive to (16, 17). These studies suggested that IL-4 may target both oste-

RANKL action oclast precursors and mature osteoclasts. Recently, IL-4 has been http://www.jimmunol.org/ To further elucidate the role of IL-4, we checked the in vivo sen- shown to act directly on osteoclast precursors and inhibit oste- sitivity of mature osteoclasts in IL-4 knockout mice. Control and oclastogenesis through inhibition of RANKL signaling pathways IL-4 knockout mice were injected with RANKL (0.05 mg/kg) or (18–21). In this study, we investigated the mechanism by which PBS as carrier, and levels of blood ionized Ca2ϩ were measured. IL-4 inhibits bone-resorbing activity of mature osteoclasts. Puri- There was no difference in levels of ionized Ca2ϩ in wild-type and fication of mature osteoclasts induced by RANKL has enabled us knockout mice when injected with PBS. To our surprise, there was to fully delineate the mechanism of IL-4 action. significant increase in RANKL-induced ionized Ca2ϩ levels in Although RANKL alone is sufficient for activation of isolated IL-4 knockout compared with control mice (Fig. 7A). These results rat mature osteoclasts (11), we found that mature osteoclasts re- show the sensitivity of mature osteoclasts to RANKL in the ab- quire both M-CSF and RANKL for survival and full activation. by guest on September 26, 2021 sence of IL-4. To further check whether the sensitivity of IL-4 IL-4 inhibited bone resorption, and anti-IL-4 Ab neutralized its knockout mice to RANKL reflect in vitro formation of mature effect, suggesting the direct action of IL-4 on mature osteoclasts. osteoclasts and bone resorption, we compared the effect of differ- IL-4 also decreased the individual pit size, suggesting that each ent concentrations of RANKL on mature osteoclast formation and osteoclast is defective in terms of resorptive activity. Receptors for bone resorption. At low concentrations of RANKL (10 and 20 IL-4 have been found on a various cell types of both hemopoietic ng/ml) there was 2-fold increase in osteoclast formation and bone and nonhemopoietic lineages (15). In the present study, we provide resorption in knockout mice (Fig. 7, B and C) vs wild-type mice. the evidence of presence of IL-4R on mature osteoclasts by RT- However, at a high concentration of RANKL (30 ng/ml), there was PCR. The expression of IL-4R has previously been noted on hu- no significant difference. These results suggest that osteoclast pre- man mature osteoclasts from giant cell tumor of bone (35). IL-13, cursors from IL-4 knockout are also sensitive to a low concentra- another T cell-derived cytokine, which shares numerous biologic tion of RANKL. properties with IL-4 (15), showed no effect on bone resorption by mature osteoclasts (data not shown). These divergent results may IL-4 inhibits bone resorption and actin ring formation by human be partially due to the sharing and differential expression of IL-4 mature osteoclasts and IL-13 receptor components on various cell types (36, 37). The role of IL-4 on bone resorption by human mature osteoclasts No apoptotic changes were seen in mature osteoclasts in the is not known. Therefore, we finally examined whether IL-4 inhib- presence of IL-4. We found that IL-4 markedly inhibited TRAP its activity of human mature osteoclasts. RANKL, in the presence expression in mature osteoclasts without affecting its multinucle- of M-CSF, induced formation of mature osteoclasts at 12 days with arity. The enzyme TRAP is strongly expressed in actively bone- few resorption pits. These mature osteoclasts were further acti- resorbing mature osteoclasts, and TRAP-deficient mice have been vated with M-CSF (15 ng/ml) and/or RANKL for 3 days without shown to exhibit osteopetrotic phenotype with normal differenti- or with different concentrations of IL-4. IL-4 inhibited bone re- ation of osteoclasts that are dysfunctional in vitro, suggesting a sorption by human mature osteoclasts in a dose-dependent manner role of TRAP in the bone resorption process (38–40). Transgenic (Fig. 8A). We also observed that IL-4 inhibits formation of actin mice overexpressing the TRAP gene showed mild osteoporosis, rings induced by RANKL (Fig. 8, B and C). Consistent with mouse with decreased trabecular bone density (41). Overexpression of mature osteoclasts, IL-4 decreased the intensity and disrupted the IL-4 in transgenic mice showed normal numbers of osteoclasts; formation of actin rings in human mature osteoclasts (Fig. 8, D–F). however, their function was altered by the decrease in TRAP expression (42). Thus, our results suggest that IL-4 inhibits bone Discussion resorption predominantly by decrease in TRAP expression and not In previous studies using complex in vitro and in vivo models, IL-4 by apoptosis of mature osteoclasts. Increased number of vacuoles has been shown to inhibit osteoclastogenesis and bone resorption in the presence of IL-4 is consistent with those of Suter et al. (39) The Journal of Immunology 923

RANKL. Thus, in our study, decreased TRAP expression, struc- tural disturbances in actin rings, and inhibition of osteoclast migration by IL-4 contributed largely to the reduced bone resorption and pit size by mature osteoclasts. We also observed that IL-4 inhibits the formation of actin rings in2hinisolated osteoclasts of 2- to 5-day-old mice (data not shown). Expression of RANK on mature osteoclasts provides evidence that it is required for signaling in activated osteoclasts (13). Also RANK-dependent signaling is essential for osteoclast cytoskeleton organization and resorption (44). Because IL-4 does not inhibit RANK expression we investigated the molecular mechanisms of IL-4 action by examining its effect on NF-␬B activation. NF-␬B activation is essential for the osteoclast differentiation, and its role has been implicated in bone resorption (29, 30). In our study, IL-4 inhibited the nuclear translocation of NF-␬B induced by RANKL. Also, increase of intracellular Ca2ϩ in mature osteoclasts in re- sponse to RANKL was prevented by IL-4. RANKL-induced Ca2ϩ signaling is more prominent in mature osteoclasts than in precur- ϩ sors, and elevation of intracellular Ca2 regulates NF-␬B nuclear Downloaded from translocation in mature osteoclasts (13, 14). Bizzari et al. (45) have reported increase in intracellular Ca2ϩ in mature osteoclasts by IL-4. In their study, effect of IL-4 was examined on cytoplasmic calcium level only for 10 min. However, in our study, we studied both cytoplasmic and nuclear calcium levels up to 30 min. Fur-

thermore, IL-4 inhibits RANKL-induced hypercalcemia in vivo in http://www.jimmunol.org/ 1 h suggesting its direct inhibitory action on preexisting active mature osteoclasts. Hypercalcemia is one of the most frequent and serious complications experienced by patients with adult T cell leukemia leading to accumulation of osteoclasts and marked increase in bone resorption (32). As reported previously (33, 34), IL-4 is a strong inhibitor of hypercalcemia, and in our studies we show that IL-4 knockout mice are more sensitive to RANKL action in inducing hypercalcemia. Also IL-4 knockout mice showed more sensitivity to RANKL for in vitro osteoclast formation and by guest on September 26, 2021 bone resorption. Binding of IL-4 to its receptor recruits the members of the Janus tyrosine kinase family that activates STAT6 (15). Recently, FIGURE 8. Effect of IL-4 on bone resorption and actin ring formation Moreno et al. (21) has shown that IL-4 inhibits bone resorption by human mature osteoclasts. A, Mature osteoclasts on bone slices were through STAT6-dependent mechanism. However, we provide fur- incubated further for 3 days with M-CSF (15 ng/ml) and/or RANKL (30 ther advances that IL-4 inhibition of NF-␬B activation and Ca2ϩ ng/ml) without or with increasing concentrations of IL-4, and bone resorp- signaling may be central to the mechanism of action of IL-4. It is tion was counted. Results are expressed as a mean Ϯ SEM of six cultures per variable obtained in two independent experiments. B and C, Mature possible that IL-4 inhibits activity of mature osteoclasts through ␬ osteoclasts prepared on bone slices were incubated for 6 h with M-CSF and inhibition of NF- B pathway in STAT6-dependent manner. Our RANKL without or with IL-4 (20 ng/ml). Bone slices were fixed and in- observation of IL-4 inhibition of RANKL induced intracellular ϩ cubated in 1 ␮g/ml FITC-conjugated phalloidin, and actin rings were vi- Ca2 changes besides inhibition of NF-␬B is novel; however, the ϩ sualized (magnification, ϫ360). D and E are the graphical presentation of mechanism by which IL-4 acts on intracellular Ca2 is yet to be B and C, respectively. F, Number of complete, disrupted and less intense determined. Our results suggest that IL-4-induced disruption of actin rings in mature osteoclasts. Results are from six cultures per variable actin ring is associated with the decrease of intracellular Ca2ϩ. Ͻ ء in three experiments. , p 0.01 vs control. This is consistent with the recent report that depletion of intracellular Ca2ϩ leads to the disruption of the F-actin in smooth muscle cells (46). We also provide in vivo validation of in vitro data that in which TRAP-deficient osteoclasts showed the accumulation of RANKL-induced hypercalcemia is attenuated by IL-4 and accen- vacuoles. IL-4 has been known to act directly on macrophages and tuated by IL-4 deficiency. In conclusion, our results suggest that induces their fusion to form foreign body giant cells (43). In our IL-4 acts directly on mature osteoclasts and inhibits bone resorp- studies also IL-4 induced the fusion of TRAP-negative MNCs. tion through inhibition of NF-␬B activation and Ca2ϩ signaling There is an excellent correlation between actin ring formation probably by IL-4R-mediated mechanism. and bone resorption (26, 27). Our results demonstrate that IL-4- In vivo, we found a normal number of osteoclasts and an unal- treated mouse and human mature osteoclasts showed reduced in- tered level of basal ionized calcium levels in IL-4 knockout mice, tensity and disruption of actin ring structures. The disrupted actin suggesting that endogenous IL-4 has no physiological significance rings and diffuse cytoplasmic staining observed in the presence of in bone metabolism. Increased evidence has revealed that IL-4 IL-4 reflects the improper assembly of osteoclast cytoskeleton. The inhibits bone resorption not only through inhibition of osteoclast disassembly of actin rings would not allow the formation of the formation, but also through suppression of bone resorption by ma- tight-sealing zone, resulting in the formation of the leaky zone. ture osteoclasts (Ref. 21 and our results). It is also reported that IL-4 also inhibited the migration of osteoclasts induced by IL-4 prevents bone and cartilage destruction in collagen-induced 924 IL-4 INHIBITS FUNCTION OF MATURE OSTEOCLASTS

arthritis (47). Our results suggest the pathological signiﬁcance of 16. Watanabe, K., Y. Tanaka, I. Morimoto, K. Yahata, K. Zeki, T. Fujihira, IL-4 in the bone. IL-4 inhibition of NF-␬B activation in mature U. Yamashita, and S. Eto. 1990. Interleukin-4 as a potent inhibitor of bone resorption. Biochem. Biophys. Res. Commun. 172: 1035–1041. osteoclasts and its in vivo rapid action to decrease acute hypercal- 17. Riancho, J. A., M. T. Zarrabeitia, and J. Gonzalez-Macias. 1993. Interleukin-4 cemia induced by RANKL increases its therapeutic potential in modulates osteoclast differentiation and inhibits the formation of resorption pits skeletal disorders such as osteoporosis, rheumatoid arthritis, and in mouse osteoclast cultures. Biochem. Biophys. Res. Commun. 196: 678–685. hypercalcemia of malignancy cases, when delivered as a recom- 18. Abu-Amer, Y. 2001. IL-4 abrogates osteoclastogenesis through STAT6-depen- ␬ binant cytokine or in combination with other drugs in gene ther- dent inhibition of NF- B. J. Clin. Invest. 107: 1375–1385. 19. Wei, S., M. W. Wang, S. L. Teitelbaum, and F. P. Ross. 2002. Interleukin-4 apy. Our results also strengthened the potent inhibitory nature of reversibly inhibits osteoclastogenesis via inhibition of NF-␬B and mitogen-acti- IL-4 by showing its inhibitory effects on bone resorption by human vated protein kinase signaling. J. Biol. Chem. 277: 6622–6630. mature osteoclasts. 20. Mirosavljevic, D., J. M. Quinn, J. Elliott, N. J. Horwood, T. J. Martin, and M. T. Gillespie. 2003. T-cells mediate an inhibitory effect of interleukin-4 on osteoclastogenesis. J. Bone Miner. Res. 18: 984–993. Acknowledgments 21. Moreno, J. L., M. Kaczmarek, A. D. Keegan, and M. Tondravi. 2003. IL-4 suppresses osteoclast development and mature osteoclast function by a STAT6-de- We extend our sincere thanks to Dr. G. C. Mishra, Director, National pendent mechanism: irreversible inhibition of the differentiation program acti- Center for Cell Science, for encouragement and support. We thank vated by RANKL. Blood 102: 1078–1086. S. D. Yogesha for critically reading the manuscript, Satish Pote for tech- 22. Wani, M. R., K. Fuller, N. S. Kim, Y. Choi, and T. Chambers. 1999. Prosta- nical assistance, and Ashwini Atre for confocal microscopy. We also thank glandin E2 cooperates with TRANCE in osteoclast induction from hemopoietic Dr. Cecilia Dayaraj from National Institute of Virology (Pune, India) for precursors: synergistic activation of differentiation, cell spreading, and fusion. Endocrinology 140: 1927–1935. help in confocal microscopy.

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