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

National Center for Cell Science, Pune, India

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
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 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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Osteoclasts, the multinuclear cells (MNCs)³ responsible for bone resorption, play a crucial role in bone remodeling. The bone loss in many important skeletal disorders such as osteoporosis, rheumatoid arthritis, hypercalcemia of malignancy, and bone metastases occurs mainly because of increased osteoclast activity (1). A major breakthrough in understanding the regulation of osteoclastogenesis occurred after the discovery of novel molecules such as receptor activator of NF-B (RANK), RANK ligand (RANKL), and osteoprotegerin (2, 3, 4, 5, 6, 7). RANKL, in the presence of M-CSF, mediates osteoclastogenesis through binding to its receptor RANK on osteoclast precursors (5, 7). Transgenic and gene knockout studies in mice established the absolute dependency of osteoclast differentiation and activation on the expression of RANKL and RANK (7, 8). The distinct signaling pathways such as NF-B, JNK, p38, ERK, and Src pathways mediated by protein kinases are activated by RANKL during osteoclastogenesis and bone resorption (9).

RANKL also plays an important role in survival and activation of mature osteoclasts and rapidly induces actin ring formation (10, 11, 12). In vivo studies have shown that RANKL increases blood ionized Ca²⁺ levels within 1 h suggesting its direct effect on preexisting mature osteoclasts (11). Stimulation of RANK on mature osteoclasts by RANKL results in activation of transcriptional factor NF-B and Ca²⁺ signaling (13, 14). The study of molecular mechanisms by which cytokines secreted by T cells or other immune cells regulate bone-resorbing activity of mature osteoclasts had been limited because of difficulties in obtaining a sufficient number of mature osteoclasts. Induction of mature osteoclasts by RANKL in mouse and human osteoclast precursors is an excellent in vitro model to investigate the novel mechanisms of cytokines that regulate bone resorption.

IL-4 is a 19-kDa pleiotropic type I cytokine secreted by activated T_H2 lymphocytes, mast cells, eosinophils, and basophils (15). IL-4, an important immune cytokine that regulates function of lymphocytes and macrophages, also regulates osteoclastogenesis and bone resorption (16, 17). Recent work has clarified the role and molecular mechanisms by which IL-4 inhibits RANKL-induced osteoclast differentiation in osteoclast precursors (18, 19, 20, 21). However, the mechanism of IL-4 action on mature osteoclasts and its function is not fully delineated. Moreno et al. (21) have reported the inhibitory effect of IL-4 on mouse mature osteoclast function and showed that the effect requires STAT6. In this study, we prepared a large number of highly purified mature osteoclasts induced by RANKL using both mice and human osteoclast precursors, and provide further advances that clarify in detail the mechanisms of IL-4 action on bone-resorbing mature osteoclasts. We show here that IL-4 acts directly on mature osteoclasts and significantly inhibits bone resorption and tartrate-resistant acid phosphatase (TRAP) expression. IL-4 inhibits bone resorption by disruption of RANKL-induced actin ring formation in mouse and as well as human mature osteoclasts. Furthermore, IL-4 prevents RANKL-induced nuclear translocation of p65 NF-B subunit, and intracellular Ca²⁺ changes in mature osteoclasts. In addition, RANKL-induced hypercalcemia in vivo is attenuated by IL-4 and accentuated by IL-4 deficiency. In conclusion, IL-4 acts directly on mature osteoclasts and inhibits bone resorption by inhibiting NF-B and Ca²⁺ signaling.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Chemicals and animals

Mouse and human rIL-4, anti-mouse IL-4 Ab, and human M-CSF were obtained from R&D Systems. Human soluble RANKL was obtained from Insight Biotechnology. Polyclonal anti-NF-B p65 and FITC-conjugated anti-rabbit Abs were purchased from Santa Cruz Biotechnology. Curcumin and FITC phalloidin were obtained from Sigma-Aldrich. BALB/c and IL-4 knockout (BALB/c-Il4^tm2Nnt) mice 5–8 wk old were obtained from the Experimental Animal Facility of the National Center for Cell Science (Pune, India). The institutional ethics committee approved the use of animals and human blood for experiments. Slices of devitalized bovine cortical bone were prepared as described previously (22). All cultures were incubated in MEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (all from Sigma-Aldrich). All cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂ in air.

Preparation of mouse mature osteoclasts

To prepare mature osteoclasts, we first isolated stromal and lymphocyte-free, M-CSF-dependent osteoclast precursors from bone marrow as described previously (23). The osteoclast precursors were added to 96-well plates (5 x 10⁵ cells/well) containing Thermonax plastic coverslips (Invitrogen Life Technologies) and bone slices, and incubated with M-CSF (30 ng/ml) and RANKL (30 ng/ml). Cells were fed on day 2, and after 4 days, formation of mature multinucleated osteoclasts (more than three nuclei) was confirmed by TRAP staining. TRAP-positive MNCs were depleted of mononuclear cells by incubating the cultures for 5 min in 20 mM EDTA in Ca²⁺- and Mg⁺-free PBS. The cultures were then washed thoroughly with MEM. This procedure removes the majority of mononuclear cells, while leaving MNCs adherent. In this study, we called these MNCs as mature osteoclasts. The mature osteoclasts were further incubated for 48 h with M-CSF (30 ng/ml) and RANKL (30 ng/ml) for survival and full activation and were treated with or without different concentrations of IL-4. In all additional experiments, M-CSF and RANKL were used at 30 ng/ml except where indicated. Cells on coverslips were stained for TRAP, and the bone slices were assessed for bone resorption by reflected light microscopy or scanning electron microscopy.

Preparation of human mature osteoclasts

Human mature osteoclasts were generated using mononuclear cells from blood of healthy adult donors. PBMCs were obtained by density gradient centrifugation using Ficoll-Hypaque. The cells were resuspended (5 x 10⁶ cells/ml) in MEM containing 15% FBS. The cell suspension (100 µl) was added to 96-well plates containing bone slices. After 1 h, bone slices were washed thoroughly and lymphocyte-free adherent cells were incubated for 12 days with M-CSF (15 ng/ml) and RANKL (30 ng/ml). At 12 days, mature osteoclasts were depleted of mononuclear cells as described above and further treated for 3 days with M-CSF (15 ng/ml) and RANKL (30 ng/ml) without or with increasing concentrations of IL-4. The bone slices were assessed for bone resorption by reflected light microscopy.

Assessment of bone resorption

After incubation, bone slices were immersed in 4% sodium hypochlorite for 15 min to remove the cells, and were washed thoroughly. After drying, bone slices were either mounted onto stubs or glass slides and sputter-coated with gold. Bone slices on glass slides were examined by reflected light microscopy, and bone resorption was quantified using an eyepiece graticule. Bone slices on stubs were examined by scanning electron microscope (XL-30; Philips), and the pit size was measured using microscope software.

TRAP cytochemistry

Mature osteoclast formation was evaluated by quantification of TRAP-positive MNCs as described previously (24). After incubation, cells on coverslips were washed in PBS, fixed in 10% formalin for 10 min, and stained for acid phosphatase in the presence of 0.05 M sodium tartrate (Sigma-Aldrich). The substrate used was napthol AS-BI phosphate (Sigma-Aldrich). TRAP-positive and TRAP-negative MNCs were counted by light microscopy.

RNA isolation and RT-PCR

Mature osteoclasts as described above were prepared in tissue culture flask. Expression of TRAP, calcitonin receptor (CTR), RANK, IL-4R, -actin, and GAPDH mRNAs was assessed by RT-PCR. RNA was isolated using the TRIzol reagent (Invitrogen Life Technologies) and used for cDNA synthesis (cDNA synthesis kit; Invitrogen Life Technologies). The cDNA was amplified using PCR for 35 cycles. Each cycle consisted of 30 s of denaturation at 94°C and 30 s of annealing and 30 s of extension at 72°C. The sequences of sense (S) and antisense (AS) primers used were TRAP, S, 5'-GGATTCATGGGTGGTGCTG-3', AS, 5'-TGGCTAACAATGGTCGCAAG-3'; CTR, S, 5'-CTGGTTGAGGTTGTGCCC-3', AS, 5'-CTCGTGGGTTTGCCTCATC-3'; RANK, S, 5'-ACACCTGGAATGAAGAAGATAAATG-3', AS, 5'-AGCCACTACTACCACAGAGATGAAG-3'; IL-4R, S, 5'-GCTCCAGACAACCTCACACTCC-3', AS, 5'-TCACAGATTTTCATTACTTGGG-3'; -actin, S, 5'-GTGGGCCGCTCTAGGCACCA-3', AS, 5'-TGGCCTTAGGGTTCAGGGGG-3'; GAPDH, S, 5'- TCGGTGTGAACGGATTTGGC-3', AS, 5'- CATGTAGGCCATGAGGTCCACCAC-3'. -Actin and GAPDH were used as internal controls.

Assessment of actin ring formation

Formation of F-actin was examined as described previously (11). Briefly, mature osteoclasts prepared on bone slices were incubated for 1 h at 37°C in MEM + 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 slices were fixed for 5 min in 10% formalin and permeabilized with 0.1% Triton X-100 for 5 min. Bone slices were then incubated in 1 µg/ml FITC-conjugated phalloidin (Sigma-Aldrich) for 45 min at 37°C, washed thoroughly, and mounted onto glass slides in antifade mounting medium (Sigma-Aldrich). Actin rings were visualized using a Zeiss LSM 510 confocal microscope equipped with argon and helium lasers (Zeiss). The number of complete, disrupted, and less intense actin rings per bone slice was counted by a blinded observer. The intensity of the F-actin was represented graphically using the Zeiss LSM 510 software.

Osteoclast migration assay

Osteoclast migration assay was performed using the Transwell migration chambers (Corning). Mature osteoclasts prepared as above were seeded in the upper chamber with M-CSF (5 ng/ml) with or without IL-4 (10 and 30 ng/ml), and RANKL was added to the lower chamber. Cells were allowed to migrate through a polycarbonate filter for 8 h. Nonmigrated cells in the upper chamber were removed with a cotton swab. Migrated cells on lower side of insert were fixed, stained for TRAP, and counted.

Immunofluorescence

Mature osteoclasts prepared in an eight-well glass Lab-Tek chamber slide (Nunc) were washed with PBS, incubated in the presence of M-CSF and IL-4 (20 ng/ml) for 2 h at 37°C, and then stimulated with RANKL as indicated. The cells were washed, fixed, permeabilized, and blocked with 5% BSA for 20 min (all steps were performed at 4°C). Cells were treated with primary Ab against p65 NF-B subunit for 30 min, washed, and treated with FITC-labeled secondary Ab for 20 min. Cells were washed thoroughly and assessed for the nuclear translocation of p65 using Zeiss LSM 510 confocal microscope. The number of mature osteoclasts showing nuclear translocation of p65 NF-B was scored.

Measurement of intracellular Ca²⁺ changes

Intracellular Ca²⁺ changes in mature osteoclasts were assessed using the Ca²⁺ indicator Fluo-4 AM (Molecular Probes) as described (13). Mature osteoclasts cultured in 35-mm petri plates were loaded with 2 µM Fluo-4 AM for 30 min at 37°C in MEM containing 0.1% FBS, and subsequently washed thoroughly with fresh medium. Petri plates were mounted on the stage of the Zeiss LSM 510 Axiovert microscope (Zeiss), and cells were maintained at room temperature in the physiological buffer containing 130 mM NaCl, 5 mM KCl, 10 mM glucose, 1 mM MgCl₂, 1 mM CaCl₂, and 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. Intracellular changes in Ca²⁺ were analyzed using the Zeiss LSM 510 software, and mean values of fluorescent intensities of the sequential images were plotted.

Measurement of blood ionized Ca²⁺ in mice

Adult BALB/c and IL-4 knockout mice were used for the in vivo measurement of blood ionized Ca²⁺ levels. Mice were injected i.v. with RANKL without or with IL-4 in Ca²⁺- and Mg⁺-free PBS carrier, or PBS alone as control. After 1 h, retro-orbital blood was collected from anesthetized mice and levels of whole blood ionized Ca²⁺ were measured using a Chiron Diagnostics Rapidlab 865 Ca²⁺/pH analyzer (Chiron Diagnostics). Mean values (±SEM) from a minimum of five mice per group were evaluated statistically.

Statistical analysis of data

The data is presented as mean ± SEM. Statistical differences between the mean values of control and experimental groups were analyzed using t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
IL-4 inhibits bone resorption by mature osteoclasts

Mature osteoclasts were prepared from osteoclast precursors as described in Materials and Methods and purified (>95% pure) by removing mononuclear cells using EDTA treatment. To examine the effect of IL-4 on bone resorption, purified mature osteoclasts on bone slices were further incubated for 48 h with M-CSF (30 ng/ml) and RANKL (30 ng/ml) with or without different concentrations of IL-4. As shown in Fig. 1A, IL-4 inhibited the RANKL-induced bone resorption in a dose-dependent manner. IL-4 (20 ng/ml) also significantly decreased the size of an individual resorption pit (Fig. 1, B and C). The inhibitory effect of IL-4 on bone resorption was confirmed by anti-mouse IL-4 Ab. As shown in Fig. 1D, simultaneous addition of anti-IL-4 Ab neutralized the inhibitory effect of IL-4 in a dose-dependent manner. These results suggest that IL-4 inhibits bone resorption by direct action on activated mature osteoclasts.

View larger version (40K): [in this window] [in a new window]   FIGURE 1. Effect of IL-4 on bone resorption by mature osteoclasts. Mature 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 ± SEM of six cultures per variable; *, p < 0.01 vs control. B, Resorption pits (magnification, x250). C, Pit size (square micrometers) measured by software in scanning electron microscope. *, p < 0.01 vs control. 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 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 intensity of IL-4R and -actin was analyzed by densitometry.

IL-4 inhibits TRAP expression in mature osteoclasts

To investigate the mechanism of IL-4 action on activated mature osteoclasts, we first examined whether IL-4 inhibits bone resorption by inducing the apoptosis in mature osteoclasts. Osteoclasts were incubated for 48 h with M-CSF and RANKL in the absence or presence of various concentrations of IL-4. No apoptotic changes such as chromosome condensation, nuclear fragmentation 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. 2A, IL-4 dose-dependently decreased TRAP expression, and the majority of MNCs were TRAP-negative and increased with increasing concentrations of IL-4 (Fig. 2B). These TRAP-negative MNCs did not express CTR (data not shown). Fig. 2C shows the effect of IL-4 on TRAP expression in mature osteoclasts. The TRAP-negative MNCs in the presence of IL-4 were fused to form giant cells with accumulation of large vacuoles. These results suggest that IL-4 inhibits expression of TRAP in mature osteoclasts without affecting the multinuclearity of cells.

View larger version (27K): [in this window] [in a new window]   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 ± SEM of six cultures per variable. *, p < 0.01 and **, p < 0.05 vs control. Results were reproducible in three independent experiments. C, TRAP staining of MNCs (magnification, x20).

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 osteoclasts.

View larger version (58K): [in this window] [in a new window]   FIGURE 3. Effect of IL-4 on TRAP, CTR, and RANK mRNAs expression. 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, Nonloading control; lanes 2 and 4, M-CSF and RANKL for 24 and 48 h, respectively; lanes 3 and 5, M-CSF, RANKL, and IL-4 for 24 and 48 h, respectively. The relative intensity of genes was analyzed by densitometry. 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 ring formation and motility of mature osteoclasts (10, 11). Therefore, we determined whether IL-4 inhibits actin ring formation and osteoclast migration induced by RANKL. Mature osteoclasts were incubated for 6 h with M-CSF and RANKL with or without IL-4 (20 ng/ml). As shown in Fig. 4, A and C, RANKL rapidly induced the formation of complete and well-defined actin rings in mature osteoclasts. In the presence of IL-4, two changes were seen in the structure of actin rings. IL-4 disrupted the formation of RANKL-induced actin rings (Fig. 4B), and also significantly decreased the intensity of actin rings (Fig. 4D). Fig. 4, E and F, is the graphical representation of the intensity of actin rings shown in Fig. 4, C and D, respectively. IL-4 significantly decreased the number of well-defined actin rings, and the majority of actin rings were either disrupted or less intense (Fig. 4G). Using Transwell migration assay, we found that IL-4 (20 ng/ml) significantly inhibited RANKL-induced migration of majority of osteoclasts (Fig. 4H). These results suggest that IL-4 inhibits bone resorption by disruption of actin rings and prevention of osteoclast migration.

View larger version (36K): [in this window] [in a new window]   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, x360). 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 are from two independent experiments. *, p < 0.01 vs control.

IL-4 prevents RANKL-induced nuclear translocation of p65 NF-B subunit and intracellular Ca²⁺ changes

To further address the molecular mechanism by which IL-4 inhibits bone resorption, we examined the effect of IL-4 on NF-B. RANKL is a strong activator of NF-B that plays a functional role in bone resorption, and NF-B knockout mice are osteopetrotic because of defective osteoclast formation (28, 29, 30). In our studies using NF-B inhibitors, we also confirmed the functional role of NF-B in bone resorption. When mature osteoclasts were preincubated for 4 h with curcumin, a strong inhibitor of NF-B (31), RANKL does not induce bone resorption (data not shown). This effect of curcumin at low concentration was without inducing the apoptosis of mature osteoclasts (data not shown). To examine the effect of IL-4 on NF-B, mature osteoclasts were incubated with M-CSF with or without IL-4, and stimulated with RANKL. RANKL stimulated nuclear translocation of the p65 NF-B subunit within 15 min and decreased its cytoplasmic level (Fig. 5A, upper panel). Interestingly, IL-4 totally prevents the nuclear translocation of p65 with accumulation of this protein in the cytoplasm (Fig. 5A, lower panel). The number of MNCs showing nuclear translocation of p65 was decreased significantly in the presence of IL-4 (Fig. 5B). Activation of NF-B and its nuclear translocation in mature osteoclasts by RANKL is associated with increase in intracellular Ca²⁺ (13, 14). Therefore, we examined whether IL-4 also prevents RANKL-induced intracellular Ca²⁺ changes. We found that IL-4, in a time-dependent manner, prevented the transient increase in both cytoplasmic and nuclear Ca²⁺ induced by RANKL (Fig. 5C). These results suggest that decrease in intracellular Ca²⁺ by IL-4 is associated with decrease in NF-B activation.

View larger version (70K): [in this window] [in a new window]   FIGURE 5. Effect of IL-4 on RANKL-induced nuclear translocation of p65 NF-B subunit, and intracellular Ca2+ 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 translocation was scored. *, p < 0.01 vs control. C, Mature osteoclasts were loaded with 2 µM Fluo-4 AM and stimulated with RANKL with or without IL-4 (20 ng/ml). Intracellular Ca2+ changes were analyzed by acquiring images in time-dependent manner, and mean values (n = 8) of fluorescent intensities of the sequential images were plotted.

RANKL has been shown to activate preexisting mature osteoclasts and stimulate hypercalcemia in mice (11). Also, hypercalcemia in many bone metastases and adult T cell leukemia occur due to increase in RANKL secretion (32). Because IL-4 has previously been shown to inhibit parathyroid hormone-related protein-induced hypercalcemia in mice (33, 34), we examined the in vivo effect of IL-4 on RANKL-induced hypercalcemia. Adult male mice were injected with RANKL in the absence or the presence of different concentrations of IL-4, and levels of ionized Ca²⁺ were examined. As shown in Fig. 6A, RANKL rapidly stimulated hypercalcemia in dose-dependent manner by increasing blood ionized Ca²⁺ levels. IL-4 significantly decreased RANKL-stimulated ionized Ca²⁺ level in 1 h (Fig. 6B). These results suggest that IL-4 acts on preexisting mature osteoclasts and decreases ionized Ca²⁺ levels in blood.

View larger version (14K): [in this window] [in a new window]   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 Ca2+ were measured. Mean values (±SEM) from a minimum of five mice per group were evaluated statistically. *, p < 0.05 vs PBS alone; **, p < 0.05 vs RANKL alone.

To further elucidate the role of IL-4, we checked the in vivo sensitivity of mature osteoclasts in IL-4 knockout mice. Control and IL-4 knockout mice were injected with RANKL (0.05 mg/kg) or PBS as carrier, and levels of blood ionized Ca²⁺ were measured. There was no difference in levels of ionized Ca²⁺ in wild-type and knockout mice when injected with PBS. To our surprise, there was significant increase in RANKL-induced ionized Ca²⁺ levels in IL-4 knockout compared with control mice (Fig. 7A). These results show the sensitivity of mature osteoclasts to RANKL in the absence of IL-4. To further check whether the sensitivity of IL-4 knockout mice to RANKL reflect in vitro formation of mature osteoclasts and bone resorption, we compared the effect of different concentrations of RANKL on mature osteoclast formation and bone resorption. At low concentrations of RANKL (10 and 20 ng/ml) there was 2-fold increase in osteoclast formation and bone resorption in knockout mice (Fig. 7, B and C) vs wild-type mice. However, at a high concentration of RANKL (30 ng/ml), there was no significant difference. These results suggest that osteoclast precursors from IL-4 knockout are also sensitive to a low concentration of RANKL.

View larger version (19K): [in this window] [in a new window]   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 Ca2+ were measured. Mean values (±SEM) from a minimum of five mice per group were evaluated statistically. *, p < 0.05 vs PBS alone from control; **, p < 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 counted after 8 days. *, p < 0.05 vs control mice.

The role of IL-4 on bone resorption by human mature osteoclasts is not known. Therefore, we finally examined whether IL-4 inhibits activity of human mature osteoclasts. RANKL, in the presence of M-CSF, induced formation of mature osteoclasts at 12 days with few resorption pits. These mature osteoclasts were further activated with M-CSF (15 ng/ml) and/or RANKL for 3 days without or with different concentrations of IL-4. IL-4 inhibited bone resorption by human mature osteoclasts in a dose-dependent manner (Fig. 8A). We also observed that IL-4 inhibits formation of actin rings induced by RANKL (Fig. 8, B and C). Consistent with mouse mature osteoclasts, IL-4 decreased the intensity and disrupted the formation of actin rings in human mature osteoclasts (Fig. 8, D–F).

View larger version (23K): [in this window] [in a new window]   FIGURE 8. Effect of IL-4 on bone resorption and actin ring formation by human mature osteoclasts. A, Mature osteoclasts on bone slices were incubated further for 3 days with M-CSF (15 ng/ml) and/or RANKL (30 ng/ml) without or with increasing concentrations of IL-4, and bone resorption was counted. Results are expressed as a mean ± SEM of six cultures per variable obtained in two independent experiments. B and C, Mature osteoclasts prepared on bone slices were incubated for 6 h with M-CSF and RANKL without or with IL-4 (20 ng/ml). Bone slices were fixed and incubated in 1 µg/ml FITC-conjugated phalloidin, and actin rings were visualized (magnification, x360). D and E are the graphical presentation of B and C, respectively. F, Number of complete, disrupted and less intense actin rings in mature osteoclasts. Results are from six cultures per variable in three experiments. *, p < 0.01 vs control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Although RANKL alone is sufficient for activation of isolated rat mature osteoclasts (11), we found that mature osteoclasts require both M-CSF and RANKL for survival and full activation. IL-4 inhibited bone resorption, and anti-IL-4 Ab neutralized its effect, suggesting the direct action of IL-4 on mature osteoclasts. IL-4 also decreased the individual pit size, suggesting that each osteoclast is defective in terms of resorptive activity. Receptors for IL-4 have been found on a various cell types of both hemopoietic and nonhemopoietic lineages (15). In the present study, we provide the evidence of presence of IL-4R on mature osteoclasts by RT-PCR. The expression of IL-4R has previously been noted on human mature osteoclasts from giant cell tumor of bone (35). IL-13, another T cell-derived cytokine, which shares numerous biologic properties with IL-4 (15), showed no effect on bone resorption by mature osteoclasts (data not shown). These divergent results may be partially due to the sharing and differential expression of IL-4 and IL-13 receptor components on various cell types (36, 37).

No apoptotic changes were seen in mature osteoclasts in the presence of IL-4. We found that IL-4 markedly inhibited TRAP expression in mature osteoclasts without affecting its multinuclearity. The enzyme TRAP is strongly expressed in actively bone-resorbing mature osteoclasts, and TRAP-deficient mice have been shown to exhibit osteopetrotic phenotype with normal differentiation of osteoclasts that are dysfunctional in vitro, suggesting a role of TRAP in the bone resorption process (38, 39, 40). Transgenic mice overexpressing the TRAP gene showed mild osteoporosis, with decreased trabecular bone density (41). Overexpression of IL-4 in transgenic mice showed normal numbers of osteoclasts; however, their function was altered by the decrease in TRAP expression (42). Thus, our results suggest that IL-4 inhibits bone resorption predominantly by decrease in TRAP expression and not by apoptosis of mature osteoclasts. Increased number of vacuoles in the presence of IL-4 is consistent with those of Suter et al. (39) in which TRAP-deficient osteoclasts showed the accumulation of vacuoles. IL-4 has been known to act directly on macrophages and induces their fusion to form foreign body giant cells (43). In our studies also IL-4 induced the fusion of TRAP-negative MNCs.

There is an excellent correlation between actin ring formation and bone resorption (26, 27). Our results demonstrate that IL-4-treated mouse and human mature osteoclasts showed reduced intensity and disruption of actin ring structures. The disrupted actin rings and diffuse cytoplasmic staining observed in the presence of IL-4 reflects the improper assembly of osteoclast cytoskeleton. The disassembly of actin rings would not allow the formation of the tight-sealing zone, resulting in the formation of the leaky zone. IL-4 also inhibited the migration of osteoclasts induced by RANKL. Thus, in our study, decreased TRAP expression, structural 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 in 2 h in isolated 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 Ca²⁺ in mature osteoclasts in response to RANKL was prevented by IL-4. RANKL-induced Ca²⁺ signaling is more prominent in mature osteoclasts than in precursors, and elevation of intracellular Ca²⁺ regulates NF-B nuclear translocation in mature osteoclasts (13, 14). Bizzari et al. (45) have reported increase in intracellular Ca²⁺ 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. Furthermore, IL-4 inhibits RANKL-induced hypercalcemia in vivo in 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 bone resorption.

Binding of IL-4 to its receptor recruits the members of the Janus tyrosine kinase family that activates STAT6 (15). Recently, Moreno et al. (21) has shown that IL-4 inhibits bone resorption through STAT6-dependent mechanism. However, we provide further advances that IL-4 inhibition of NF-B activation and Ca²⁺ signaling may be central to the mechanism of action of IL-4. It is possible that IL-4 inhibits activity of mature osteoclasts through inhibition of NF-B pathway in STAT6-dependent manner. Our observation of IL-4 inhibition of RANKL induced intracellular Ca²⁺ changes besides inhibition of NF-B is novel; however, the mechanism by which IL-4 acts on intracellular Ca²⁺ is yet to be determined. Our results suggest that IL-4-induced disruption of actin ring is associated with the decrease of intracellular Ca²⁺. This is consistent with the recent report that depletion of intracellular Ca²⁺ leads to the disruption of the F-actin in smooth muscle cells (46). We also provide in vivo validation of in vitro data that RANKL-induced hypercalcemia is attenuated by IL-4 and accentuated by IL-4 deficiency. In conclusion, our results suggest that IL-4 acts directly on mature osteoclasts and inhibits bone resorption through inhibition of NF-B activation and Ca²⁺ signaling probably by IL-4R-mediated mechanism.

In vivo, we found a normal number of osteoclasts and an unaltered level of basal ionized calcium levels in IL-4 knockout mice, suggesting that endogenous IL-4 has no physiological significance in bone metabolism. Increased evidence has revealed that IL-4 inhibits bone resorption not only through inhibition of osteoclast formation, but also through suppression of bone resorption by mature osteoclasts (Ref. 21 and our results). It is also reported that IL-4 prevents bone and cartilage destruction in collagen-induced arthritis (47). Our results suggest the pathological significance of IL-4 in the bone. IL-4 inhibition of NF-B activation in mature osteoclasts and its in vivo rapid action to decrease acute hypercalcemia induced by RANKL increases its therapeutic potential in skeletal disorders such as osteoporosis, rheumatoid arthritis, and hypercalcemia of malignancy cases, when delivered as a recombinant cytokine or in combination with other drugs in gene therapy. Our results also strengthened the potent inhibitory nature of IL-4 by showing its inhibitory effects on bone resorption by human mature osteoclasts.

	Acknowledgments

 
We extend our sincere thanks to Dr. G. C. Mishra, Director, National Center for Cell Science, for encouragement and support. We thank S. D. Yogesha for critically reading the manuscript, Satish Pote for technical assistance, and Ashwini Atre for confocal microscopy. We also thank Dr. Cecilia Dayaraj from National Institute of Virology (Pune, India) for help in confocal microscopy.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Department of Biotechnology, Government of India. L.S.M. is the recipient of Senior Research Fellowship from Department of Biotechnology (India). S.M.K. is the recipient of Senior Research Fellowship from the Council for Scientific and Industrial Research (New Delhi, India).

² Address correspondence and reprint requests to Dr. Mohan R. Wani, National Center for Cell Science, University of Pune Campus, Pune-411 007, India. E-mail address: mohanwani{at}nccs.res.in

³ Abbreviations used in this paper: MNC, multinuclear cell; CTR, calcitonin receptor; RANK, receptor activator of NF-B; RANKL, RANK ligand; TRAP, tartrate-resistant acid phosphatase; S, sense; AS, antisense.

Received for publication February 18, 2005. Accepted for publication May 10, 2005.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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