Osteoclast formation elicited by interleukin-33 stimulation is dependent upon the type of osteoclast progenitor

Molecular and Cellular Endocrinology 399 (2015) 259–266

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Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e

Osteoclast formation elicited by interleukin-33 stimulation is dependent upon the type of osteoclast progenitor Damien G. Eeles a,b,1, Jason M. Hodge c,d,1, Preetinder P. Singh a, Johannes A. Schuijers b, Brian L. Grills b, Matthew T. Gillespie a,e, Damian E. Myers f,g,2, Julian M.W. Quinn a,e,*,2 a

MIMR-PHI Institute of Medical Research, Clayton, VIC, Australia Department of Human Biosciences, La Trobe University, Bundoora, VIC, Australia c Barwon Biomedical Research, Department of Medicine, The Geelong Hospital, Geelong, VIC, Australia d School of Medicine, Deakin University, Geelong, VIC, Australia e Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia f Department of Orthopaedics, St Vincent’s Hospital, Fitzroy, VIC, Australia g Department of Surgery, St Vincent’s Hospital, Fitzroy, VIC, Australia b

A R T I C L E

I N F O

Article history: Received 17 June 2014 Received in revised form 3 October 2014 Accepted 14 October 2014 Available online 18 October 2014 Keywords: Osteoclast Th2 cytokine Bone resorption Interleukin

A B S T R A C T

Osteoclasts are bone resorbing multinucleated cells (MNCs) derived from macrophage progenitors. IL33 has been reported to drive osteoclastogenesis independently of receptor activator of NFκB ligand (RANKL) but this remains controversial as later studies did not confirm this. We found IL-33 clearly elicited functional dentine-resorbing osteoclast formation from human adult monocytes. However, monocytes from only 3 of 12 donors responded this way, while all responded to RANKL. Human cord blood-derived progenitors and murine bone marrow macrophages lacked an osteoclastogenic response to IL-33. In RAW264.7 cells, IL-33 elicited NFκB and p38 responses but not NFATc1 signals (suggesting poor osteoclastogenic responses) and formed only mononuclear tartrate-resistant acid phosphatase positive (TRAP+) cells. Since TGFβ boosts osteoclastogenesis in RAW264.7 cells we employed an IL-33/TGFβ co-treatment, which resulted in small numbers of MNCs expressing key osteoclast markers TRAP and calcitonin receptors. Thus, IL-33 possesses weak osteoclastogenic activity suggesting pathological significance and, perhaps, explaining previous conflicting reports. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Osteoclasts are multinucleated haematopoietically-derived cells that possess the unique ability to excavate resorption pits in bone, a process central to both normal adult bone remodelling and pathological bone destruction (Martin et al., 2006; Raggatt and Partridge, 2010). The formation and activity of osteoclasts is normally depen-

Abbreviations: BMM, bone marrow-derived macrophages; CBMC, cord blood mononuclear cells; CTR, calcitonin receptor; FBS, foetal bovine serum; GM-CFU, granulocyte-macrophage-colony forming unit; IL-33, interleukin-33; LPS, lipopolysaccharides; M-CSF, macrophage-colony stimulating factor; MEM, alpha minimal essential medium; MES, morpholino-ethanesulphonic acid buffer; MNCs, multinucleated cells; OPG, osteoprotegerin; PBMC, peripheral blood mononuclear cells; PBS, phosphate buffered saline; PVDF, polyvinylidene difluoride; RANKL, receptor activator of NFκB ligand; TGFβ, transforming growth factor beta; TNF, tumour necrosis factor; TRAP, tartrate-resistant acid phosphatase. * Corresponding author. Current address: The Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW 2010, Australia. Tel.: +612 9295 8280; fax: +612 9295 8110. E-mail address: [email protected] (J.M.W. Quinn). 1 These authors contributed equally to this work. 2 Joint senior authors. http://dx.doi.org/10.1016/j.mce.2014.10.014 0303-7207/© 2014 Elsevier Ireland Ltd. All rights reserved.

dent upon the tumour necrosis factor (TNF)-related protein receptor activator of NFκB ligand (RANKL) (Yasuda et al., 1998). The central importance of RANKL is demonstrated by the lack of osteoclasts in mice lacking either RANKL or RANK (RANKL receptor), both of which result in osteopetrosis (Dougall et al., 1999). Consistent with this, RANKL blockade reduces osteoclast numbers and bone loss both clinically (using humanised anti-RANKL antibody Denosumab) and in numerous animal models (Sinningen et al., 2012), suppressing both normal physiological bone resorption and pathological osteolysis caused by tumour-induced inflammation and other disorders. Cultured cell populations that contain immature macrophage progenitors (such as human adult and cord blood monocytes and mouse bone marrow cells) can form large numbers of functional osteoclasts when treated with recombinant RANKL together with proliferation factor macrophage-colony stimulating factor (MCSF) (Hodge et al., 2004; Quinn et al., 1998, 2002). Physiologically, membrane-bound and soluble RANKL protein is produced by mesenchymally-derived cells in the bone microenvironment, particularly osteoblasts and osteocytes, in a manner regulated by osteolytic factors and hormones such as parathyroid hormone and prostaglandins. Such pro-osteoclastic stimuli also typically cause a reduction in osteoblast expression of osteoprotegerin (OPG),

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the secreted decoy receptor of RANKL, and thereby further increases the levels of functional RANKL protein in bone (Fuller et al., 1998). These actions are well established to underlie the osteoclastogenic effects of such hormones. In addition, T and B cells also produce RANKL upon their cellular activation, and it is likely that this contributes significantly to pathological bone loss (Horwood et al., 1999; Onal et al., 2012). Despite the undoubted importance of RANKL, a number of reports have emerged that other TNF-related proteins (including TNF itself) have some ability to stimulate osteoclast formation in vitro and in vivo (Edwards et al., 2006; Hemingway et al., 2011, 2013; Quinn et al., 2001; Yao et al., 2009). Expression of some of these factors in bone lesions suggests they may contribute to pathological osteolysis (Edwards et al., 2006; Hemingway et al., 2011). One notable recent report by Mun and colleagues (Mun et al., 2010) described IL-33 as a RANKL-independent osteoclastogenic factor. IL-33 is a 30 kD secreted pro-inflammatory protein factor and is a member of the IL-1 family rather than the TNF family. IL-33 and RANKL nevertheless elicit similar intracellular pathway signals in macrophages, although unlike RANKL (or other TNF-family members) IL-33 participates in Th2-mediated inflammatory processes (Hayakawa et al., 2007) and stimulates osteoblast maturation (Keller et al., 2012; Saleh et al., 2011) both of which may indirectly reduce osteoclast formation. The findings described by Mun et al. (2010) suggested that IL33 may directly drive osteoclast formation in cultured adult human monocytes, and that this action was not dependent on any RANKL production (or OPG sensitivity) in the cultures. They also found that IL-33 treatment of monocytic cells elicited the activity of key osteoclast transcription factors, notably the osteoclastogenesis controlling transcription factor NFATc1, making IL-33-driven osteoclast formation plausible. However, two other reports employing similar monocyte cultures did not replicate this result, finding no osteoclast formation at all with IL-33 treatment (Schulze et al., 2011; Zaiss et al., 2011). In addition, in several murine osteoclast culture systems IL-33 actually inhibits RANKL-dependent osteoclast formation (Saleh et al., 2011; Schulze et al., 2011), which makes any pro-osteoclastic effects of IL-33 surprising to observe. It should also be noted that proof of presence of osteoclasts in human monocyte cultures is harder to demonstrate than the proof of their absence as there are several markers, notably tartrate-resistant acid phosphatase (TRAP), that are expressed by all osteoclasts but also by some activated macrophages (Elleder, 1986; Heymann et al., 1998). Thus, lack of TRAP expression conclusively demonstrates a lack of osteoclasts, while the presence of TRAP in human monocyte cultures does not prove osteoclasts are present without corroborating evidence of pit excavations on bone or dentine surfaces (i.e., conclusive evidence of osteoclast function) or cell expression of calcitonin receptors (CTR). Thus, in the light of these conflicting observations by different groups we re-examined the ability of IL-33 to stimulate the formation of functional osteoclasts, since IL-33 is produced by bone cells and T cells and is abundant in inflammatory lesions. Our studies found that functional osteoclast formation does indeed occur in response to IL-33 treatment but that this stimulus is weak (relative to the strong and consistent effects of RANKL) and highly variable in its effects, with some types of osteoclast progenitor-containing populations appearing unresponsive. These data indicate the basis for the recent contradictory reports on this subject, and suggests that IL-33 should not be ignored as a potential osteolytic factor. 2. Materials and methods Cells were cultured in alpha minimal essential medium (Life Technologies, Inc., Gaithersburg, MD) with 10% foetal bovine serum (CSL Biosciences, Parkville, Australia) and penicillin 50 U/ ml; streptomycin 50 μg/ml and 2 mM L-glutamine (MEM/FBS).

Recombinant GST-RANKL158-316 (RANKL) was obtained from the Oriental Yeast Company Ltd. (Tokyo, Japan). Human transforming growth factor-β1 (TGFβ) and other recombinant proteins were obtained from R&D Systems (Minneapolis, MN) except recombinant mouse and human IL-33, both obtained from Axxora, LLC (San Diego, CA). For tartrate resistant acid phosphatase (TRAP) histochemical staining, fast red violet LB Salt, naphthol AS-MX phosphate and dimethylformamaide were purchased from Sigma-Aldrich (St Louis, MO). Sperm whale dentine slices (4 × 4 × 0.1 mm) were prepared as previously described (Saleh et al., 2011) by use of a Buehler Isomet low speed diamond wafering saw (Buehler, Lake Bluff, IL). Antibodies specific for NFATc1, NFκBp65, phospho-NFκBp65, p38 and phospho-p38 were obtained from Abcam (Cambridge, MA), the antibody for β-actin from Sigma-Aldrich (Castle Hill, Australia). 2.1. Sources of human and mouse cells for experimentation Collection and use of adult human blood was conducted according to the guidelines and approval of The University of Melbourne (Melbourne, Australia) and the Human Ethics Committee of St Vincent’s Hospital, Melbourne, Australia. Blood was obtained from either 50 ml donations of whole blood obtained by vein puncture of healthy human volunteers or from buffy coat preparations obtained from the Australian Red Cross blood service (Melbourne, Australia under St Vincent’s Hospital Research Governance Human Ethics Committee approval; HREC-A 041/11. Human umbilical cord blood was obtained from healthy donors under a protocol approved by Barwon Health Research and Ethics Advisory Committee (Geelong, Australia). C57BL/6J mice were obtained from Monash Animal Services (Clayton, Australia) and mice were maintained at Monash Medical Centre Animal facility (Clayton, Australia) with procedures approved by Monash Medical Centre Animal Ethics Committee B, Clayton, Australia. 2.2. Osteoclast formation cultures Peripheral blood mononuclear cells (PBMC) were prepared by discontinuous gradient sedimentation; blood diluted in phosphate buffered saline (PBS) was gently layered over Ficoll/Paque Plus (GE Healthcare, Uppsala, Sweden) and centrifuged at 383 g for 15 min and the PBMC-rich interface layer removed and rinsed by centrifugation and resuspended in PBS. PBMCs were then used in osteoclast differentiation assays as previously described (Quinn et al., 1998). Briefly, cells were resuspended in MEM/FBS and added to 6 mm diameter culture wells containing 4 mm diameter dentine slices. After allowing cells to settle and attach for 1 h, non-adherent cells such as lymphocytes were removed by vigorous rinsing and dentine slices placed in fresh well in 200 μl MEM/FBS containing M-CSF (25 ng/ ml), M-CSF + RANKL (100 ng/ml) or M-CSF + human IL-33 (20 ng/ ml). Cultures were maintained at 37 °C in 5% CO2 gassed incubator for 2 weeks, with a complete change of medium and mediators every 3 days. Cord blood mononuclear cells (CBMC) were prepared from whole cord blood Ficoll-Hypaque discontinuous gradient centrifugation as above, viably frozen and stored in liquid nitrogen. Thawed CBMC aliquots (5 × 107 cells) were expanded to generate granulocytemacrophage-colony forming unit (GM-CFU) progenitors by incubation in semi-solid media containing growth factors for 10 days as previously described (Hodge et al., 2004). These expanded progenitors (4 × 104/well) were seeded into 6 mm diameter tissue culture wells containing dentine slices as a cell substrate and cultured in 200 μl MEM/FBS containing M-CSF + RANKL, M-CSF + IL-33 and M-CSF + RANKL + IL-33; culture in M-CSF alone results only in

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proliferating immature macrophages and does not result in osteoclast formation. All cultures were maintained for 2 weeks at 37 °C in 5% CO2 gassed incubator with twice weekly replacement of mediators in half volume of media. RAW264.7 cells, a bi-potential macrophage/pre-osteoclast cell line, were cultured (104 cells/well) in 200 μl MEM/FBS in 6 mm diameter wells (Life Technologies) with RANKL (50 ng/ml), murine IL33 (20 ng/ml), IL-33 + TGFβ) or as indicated, for 7 days, with complete change of medium and mediators at day 3. Bone marrow-derived macrophages (BMM) were prepared as previously from freshly obtained mouse bone marrow as previously described (Saleh et al., 2011). 2.3. TRAP histochemical staining of osteoclast cultures Cells were fixed in 4% phosphate buffered formaldehyde, permeabilised by 30 s rinse in acetone: methanol (1:1) mixture followed by incubation with TRAP substrate solution as previously described (Martin et al., 2006). Cells were rinsed in water after 10 min and histochemical staining determined and analysed by light microscopy; TRAP positive cells with >2 nuclei counted as multinucleated cells (MNCs). 2.4. Dentine resorption analysis Cells cultured on dentine were removed by sonication in 2:1 chloroform: methanol mixture. Dentine resorption pits were visualised by applying xylene-free black ink (from a standard laboratory marker pen) to the surface of each dentine slice, then removing the residual ink from unresorbed areas by wiping on absorbent paper. Resorption was assessed by light microscopy and where indicated quantification was performed using microcomputer image analysis software (MCID-Imaging Research Inc., ON, Canada). In some cultures where pit formation was observed the dentine slices were further examined by field emission scanning electron microscopy. Briefly, ink on the dentine slices was removed by ethanol washes which were then dried and sputter coated with platinum (Polaron sputter coater, Quorum Technologies, East Sussex, UK) then examined and photographed under 2 kV accelerating voltage conditions in a JEOL JSM 6340F scanning electron microscope (SEMTech Solutions, North Billerica, MA). 2.5. Western blot analysis of RANKL and IL-33-dependent signals in RAW264.7 cells To analysed NFκB and p38 signals RAW264.7 cells were cultured in MEM/FBS in the presence of 50 ng/ml RANKL or 20 ng/ml murine IL-33 over a 60 min time course. For NFATc1 levels, cells were cultured for 24 h in the presence of 50 ng/ml RANKL, 20 ng/ml IL33, 5 ng/ml TGFβ or 20 ng/ml IL-33 + 5 ng/ml TGFβ. Cells were lysed in modified RIPA buffer [50 mmol/l Tris/HCl (pH 7.4), 1% Nonidet P40, 0.25% sodium deoxycholate and 150 mmol/l NaCl] containing phosphatase and protease inhibitors. Lysate proteins were separated by gel electrophoresis on 10 well 4–12% Bis-Tris gels (Life Technologies) under reducing conditions in morpholino-ethanesulphonic acid buffer (MES; Life Technologies) according to manufacturer’s instructions. Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes by wet transfer in Tris-Glycine transfer buffer at 90 v for 2 h. Following transfer, membranes were soaked for 30 min in TBS containing 3% skimmed milk (Diploma Skim Milk powder, Fonterra, Australia) before incubation in the following primary antibodies overnight at 4 °C: rabbit α-mouse phosphop65, 1:1000 (Abcam); rabbit α-mouse p65, 1:1000 (Abcam); rabbit α-mouse phospho-p38, 1:1000 (Abcam); rabbit α-mouse p38, 1:1000 (Abcam); rabbit α-mouse NFATc1, 1:1000 (Abcam) or α-mouse β-actin, 1:10000 (Sigma-Aldrich). Following this, membranes were

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then incubated in HRP-conjugated secondary antibodies for 1 h at room temperature and visualised by chemiluminescence using the Chemi-Doc MP system (Bio-Rad, Philadelphia, PA) or by traditional film methods as indicated. 2.6. Statistical analyses Statistical analysis was conducted using ANOVA/Tukey’s post hoc test, or Mann–Whitney U-test where indicated, using Graphpad Prism® software (La Jolla, CA). 3. Results 3.1. IL-33 stimulates osteoclast formation from PBMCs independently of RANKL, but not in all monocyte isolates PBMCs extracted from blood taken from healthy, adult donors cultured on dentine in the presence of M-CSF (30 ng/ml) and RANKL (50 ng/ml) for 14 days resulted in the formation of numerous TRAP+ MNCs (Fig. 1A, Table 1), consistent with osteoclast formation seen in previous studies (Quinn et al., 1998). In the presence of M-CSF alone TRAP+ MNCs were not observed, although large numbers of TRAP+ mononuclear cells were typically present. PBMCs cultured in the presence of M-CSF and IL-33 (20 ng/ml) also resulted in TRAP+ MNC formation (Fig. 1A). Since large numbers of mononuclear TRAP+ cells were present, TRAP+ MNCs formed in the presence of IL-33 cannot be confirmed as osteoclasts as they may be fused TRAP+ macrophage polykaryons; macrophage activation and Th2 cytokines can cause such fusion. Thus, to confirm that bona fide osteoclasts were formed in the latter cultures, we removed the cells and inspected the dentine by light microscopy for evidence of resorption pits. Cultures stimulated with M-CSF alone showed no evidence of dentine resorption pit formation at all, while the M-CSF plus RANKL treatment gave rise to resorption pits in all experiments, in which the dentine surface was heavily resorbed (Fig. 1B). Dentine slices cultured with monocytes treated with M-CSF and IL-33 where TRAP+ MNCs were observed showed evidence of pit excavation; this indicated that IL-33 was inducing functional osteoclast formation in these cultures. To put the presence of resorption pits in IL-33 treated cultures beyond doubt, dentine slices were examined by scanning electron microscopy (Fig. 1C). This clearly showed unambiguous and conclusive evidence of pit excavation. However, not all PBMC preparations responded in this way to IL-33 treatment,

Table 1 Evidence of functional osteoclast formation from PBMCs extracted from peripheral blood from 12 individuals. Donor

Number of blood samples assayed

Control (M-CSF) treatment

RANKL + M-CSF treatment

IL-33 + M-CSF treatment

#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12

6 8 1 2 2 1 1 1 2 1 1 1

0 0 0 0 0 0 0 0 0 0 0 0

6 8 1 2 2 1 1 1 2 1 1 1

2 2 0 0 0 0 0 0 0 0 1 0

Five individuals (donors 1, 2, 4, 5 and 9) supplied blood samples more than once. PBMCs from these blood samples were cultured on dentine for 21 days with M-CSF alone (Control), M-CSF + RANKL treatment or M-CSF + IL-33 treatment. The cultures were then assayed for the presence of active osteoclasts by stripping the cells from the dentine surface to determine the presence of resorption pits. The table shows the number of blood samples that yielded at least one dentine slice with evidence of resorption pits.

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Fig. 1. Evidence for in vitro functional osteoclast formation from IL-33 treated human PBMCs. (A) TRAP positive cells evident in PBMCs cultured for 14 days in on dentine slices in the presence of M-CSF alone, RANKL (50 ng/ml) plus M-CSF, or IL-33 (20 ng/ml) plus M-CSF as indicated. Outlines of deep resorption pits are seen around or near many TRAP+ MNCs. (B) Staining of resorption pits formed on dentine slices from PBMCs cultured in the same conditions as (A), but for 21 days. Cultured cells were removed prior to dentine staining. (C), Scanning electron microscope images of resorption pits formed in PBMC cultures cultured on dentine as in (B). Black bars = 100 μm.

monocytes from only three donors showing resorption pits out of 12 tested; resorption in these three cases was associated with TRAP+ MNC formation (Table 1). Furthermore, in the case of donors 1 and 2 which were tested repeatedly, IL-33 only resulted in resorption pits in 2 out of 6 and 2 out of 8 experiments, respectively. In contrast, RANKL treatment always resulted in resorption in monocyte cultures (Table 1). 3.2. IL-33 does not stimulate osteoclast formation from cord bloodderived monocytes Previously, we have reported that CFU-GM derived from cord blood monocytes rapidly form active bone resorbing osteoclasts with RANKL plus M-CSF treatment (Hodge et al., 2004).

In a small previous study we investigated whether such cells also respond to IL-33 and found little conclusive evidence for IL-33-driven osteoclast formation (Saleh et al., 2011). Given that in adult monocytes there was a great deal of variability in IL-33 response we repeated this study with cells from five different cord blood donors. None resulted in any resorption pits in dentine and IL-33 itself did not affect any osteoclastic actions of RANKL (Fig. 2A, B). 3.3. IL-33 weakly stimulates TRAP+ MNC formation in RAW264.7 cultures, but only with TGFβ co-treatment Having determined that IL-33 could drive osteoclast formation from human PBMCs, we examined IL-33 actions on mouse primary

Fig. 2. Expanded cord blood progenitors do not form functional osteoclasts in response to IL-33. (A) TRAP+ multinucleated cell (MNC) numbers on dentine slices after 14 days with M-CSF alone, in combination with 50 ng/ml RANKL or the indicated concentration of IL-33. (B) Resorption area on the dentine surface analysed after 14 days of culture in cultures treated as in (A). Data shown as mean ± SEM, 3 independent replicate experiments. ANOVA, Tukey’s post hoc test, **p < 0.01 relative to positive controls.

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Fig. 3. IL-33 induces mononuclear TRAP+ cell formation in RAW264.7 cells. (A) RAW264.7 cells were cultured for 7 days with no treatment, 50 ng/ml RANKL or 20 ng/ml IL-33 and histochemically stained for TRAP. TRAP+ MNC and mononuclear cells indicated by black arrows. (B) The number of TRAP+ mononuclear cells formed in RAW264.7 cells; RANKL treatment (data not shown) gave a mean of 592 ± 72.7 TRAP+ cells/well; n = 4. Data plot shows mean ± SEM. Mann–Whitney U test, ***p < 0.001.

BMM and RAW264.7 cells; the latter, unlike BMM and human monocytes, do not require M-CSF to proliferate and survive so this was not added to RAW264.7 cultures. As previously described (Saleh et al., 2011), we could find no TRAP staining in IL-33-treated BMM cultures, although BMMs were highly responsive to RANKL (data not shown). As expected, in RAW264.7 cell cultures, RANKL stimulation caused formation of many TRAP+ MNCs after 7 days incubation (Fig. 3A), but IL-33 treatment alone did not. However, in the latter cultures we did note a small and variable number of TRAP+ mononuclear cells (Fig. 3A, B). In RAW264.7 and BMM cultures we commonly observe TRAP+ mononuclear cell formation (but few if any TRAP+ MNCs) with TNFα stimulation, a weak osteoclastogenic stimulus that can be boosted by co-treatment with TGFβ (Quinn et al., 2001). Therefore, we investigated the effects of IL-33 plus TGFβ (5 ng/ml) co-treatment of RAW264.7 cells. This stimulus did indeed result in the consistent formation of small numbers of TRAP+ MNCs (Fig. 4A, B). This suggested that there was some induction of osteoclastogenesis occurring in these cultures, however, evidence of TRAP expression was not entirely conclusive, even though (unlike long term cultured human monocytes) no TRAP activity was seen in untreated controls. Since osteoclasts formed from RANKL-treated RAW264.7 cells form very few resorption pits in vitro, given the small number of TRAP+ MNCS formed with IL-33 + TGFβ treatment we used a different approach to confirm osteoclast formation, namely their CTR expression, determined by immunohistochemistry. CTR immunostaining was indeed noted on MNCs in RANKL-treated and IL-33 + TGFβ-treated but not in untreated RAW264.7 cultures (Fig. 4C). No TRAP+ MNCs were observed in any BMM cultures treated with IL-33 + TGFβ (data not shown), indicating that no osteoclastogenesis was elicited with this stimulus. 3.4. IL-33 stimulates phosphorylation of p65 and p38, but not NFATc1 RANKL action via its cognate receptor RANK induces NFκB signalling in RAW264.7 cells by eliciting phosphorylation of NFκB p65 subunit. Increased NFκB signal is required for osteoclast forma-

tion and, indeed, both RANKL treatment and IL-33 treatment of RAW264.7 cultures increased p65 phosphorylation levels (Fig. 5A). A second RANKL-elicited signal essential for osteoclast formation is phosphorylation of p38 MAPK; as with NFκB signalling, p38 phosphorylation was induced in RAW264.7 cells by RANKL treatment and with IL-33 treatment (Fig. 5B). NFATc1 is a transcription factor whose cellular and nuclear levels are enhanced by NFκB activity and is required for osteoclast formation. NFATc1 levels in RAW264.7 cells were increased with RANKL treatment but this was not observable with IL-33 treatment, either alone or in combination with TGFβ (Fig. 5C). This failure to properly induce NFATc1 levels would explain the extremely weak osteoclastogenic action of IL-33 in these cells. 4. Discussion While the role of RANKL is central to osteoclast formation, the intracellular signals this factor elicits upon binding its receptor, RANK, are similar to those elicited by other TNF family members, IL-1 related proteins and lipopolysaccharides (LPS). This raises the possibility that these factors might also drive osteoclastogenesis in the absence of RANKL. There have indeed been several reports of factors that elicit osteoclastogenic effects but, with the exception of TNF, they have not generally found wide acceptance as significant factors in controlling bone resorption. In some cases, the claims are based on observing extremely weak effects on osteoclast formation that have proven difficult to replicate. In other cases, the claims are based upon data resulting from the inappropriate or questionable use of osteoclast-associated markers to show that the experimental protocols have generated osteoclasts. Here, we have successfully replicated some of the controversial observations made by Mun et al. (2010), confirming that human monocytes do indeed have the capacity (at least in long term culture) to respond to IL-33 by forming bone fide osteoclasts. While in this previous work IL-33 was observed to cause monocyte formation of MNCs expressing TRAP, two subsequent studies failed to confirm this (Schulze et al., 2011; Zaiss et al., 2011). Since TRAP is a lysosomal enzyme expressed by all osteoclasts and is probably required for efficient bone resorption (van de Wijngaert and Burger, 1986), lack of detectable TRAP expression is conclusive evidence that osteoclasts are not present. In

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Fig. 4. TGFβ boosts the weak osteoclastogenic action of IL-33 in RAW264.7 cells. (A) RAW264.7 cells were cultured for 7 days with no treatment, 50 ng/ml RANKL, 20 ng/ml IL-33 or 5 ng/ml TGFβ + 20 ng/ml IL-33, then histochemically stained to detect TRAP activity. Scale bars = 100 μm. (B) Quantification of TRAP+ MNC formation in these cultures; n = 4, mean ± SEM shown. ANOVA, Tukey’s post hoc test, ***p < 0.001 relative to negative controls. (C) RAW264.7 cells cultured as in (A), above, but immunostained using anti-CTR antibody or (as immunostain controls) using rabbit immunoglobulin, IgG, where indicated; scale bars = 100 μm.

Fig. 5. IL-33 induces NFκB and p38 signalling but not NFATc1 levels in RAW264.7 cells: Western blot analyses. (A) Increased levels of phosphorylated forms of NFκB p65 and p38 after treatment with 50 ng/ml RANKL and with 20 ng/ml IL-33, as indicated. (B) RANKL treatments strongly elevates NFATc1 protein levels in RAW264.7 cells after 24 h treatment while IL-33, TGFβ and IL-33 + TGFβ fails to cause detectable increases in NFATc1 levels. ‘pp65’ = phosphorylated NFκB p65, ‘pp38’ = phosphop38.

contrast, since TRAP can be produced by activated macrophages (which can fuse) the contrary is not true: the presence of TRAPexpressing MNCs is not itself conclusive evidence for the formation of osteoclasts (Hattersley and Chambers, 1989). Other features employed by Mun et al. (2010), including DC-STAMP and removal of artificial calcified substrate also, like TRAP expression, do not prove osteoclast formation. Only two features have been found to reliably distinguish osteoclasts from macrophages: CTR expression and resorption pit formation on solid bone or dentine surfaces. The ability to make such resorption pits is the defining characteristic of osteoclasts, a complex function which involves coordinated substrate adhesion, ruffled border formation and acid/enzyme secretion. Macrophages cannot make resorption pits although they can phagocytose and break down small particulate bone matter. In the studies that we present here we have confirmed not only that TRAP+ MNCs form in human monocyte cultures in response to IL-33 but that these cells could excavate intact dentine, demonstrating formation of true and functional osteoclasts in these IL-33-treated cultures beyond reasonable doubt. In these resorption experiments presented in Fig. 1 and Table 1 it was a very striking observation that we observed osteoclastic effects of IL-33 only in a small minority of our human monocyte cultures, indicating that monocytes from different sources vary widely in IL-33 responsiveness. This may reflect some attribute of the donor or of the monocyte adaptation to culture which may differ between individual donors yet to be defined. It may be the case that the more immature and highly proliferative osteoclast progenitors that are present in the monocyte populations are responsive to RANKL but are unresponsive to the pro-osteoclastic effects of IL33. If this is the case, it may explain the lack of osteoclast formation that we observed in our cord blood-derived CFU-GM cells since these principally contain highly proliferative progenitors which resemble those found in bone marrow; like bone marrow cells, they

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differentiate far more rapidly and consistently in response to RANKL treatment than adult monocytes, which require 2 weeks or more of stimulus and proliferate only little. The osteoclast differentiation profile of CFU-GM cells thus resemble mouse BMM and RAW264.7 cells in many ways; however, one exception is that TGFβ does not enhance osteoclast differentiation human CFU-GM cultures (data not shown) and hence could not be used to enhance IL33 actions. A link between progenitor maturity and osteoclastogenic responsiveness to IL-33 is hard to gauge in such populations by current methods, but it is interesting to note that at least in mouse populations it is the more mature macrophage populations that responded to IL-33-treatment with production of osteoclast inhibitory factors such as IL-10 and GM-CSF (Liew, 2012; Saleh et al., 2011). Thus, the very populations responding in a pro-osteoclastic manner to IL-33 are the populations producing factors that limit or completely ablate such osteoclast formation. This is also a feature seen in RANKL treatment, i.e., RANKL induces in progenitors the secretion of factors that oppose its own actions, e.g., interferon β (Takayanagi et al., 2002); however RANKL exerts a very powerful effect on immature progenitors such as CFU-GM and strongly stimulates NFATc1 levels in responsive cells. In addition to extrinsic factors that regulate osteoclastogenesis, powerful effects of RANKL and TNF-regulated intracellular factors that inhibit osteoclast formation have recently emerged. For example, it is notable that TNF treatment causes osteoclast formation in a number of in vitro experimental systems; however in vivo TNF injections in RANK null mice (i.e., mice unresponsive to RANKL) do not induce osteoclast formation unless the mice also lack NFκBp52 production (Yao et al., 2009). Other RANKL-induced negative regulators include IRF8, MafB, Blimp1 and RBP-J (Kim et al., 2007; Zhao et al., 2009, 2012). By implication, such regulators may reduce the effects of factors that would otherwise drive osteoclast formation. This suggests that the responsiveness of osteoclast progenitors to weak stimulators such as IL-33 or TNFSF-14/LIGHT (Edwards et al., 2006; Hemingway et al., 2011) may vary greatly according to the levels of cell intrinsic factors. Osteoclast research has long been hampered by a lack of good, highly specific markers to identify osteoclasts and distinguish them from other related cell types. Resorption pit formation requires live cells adhesion to a prepared substrate for at least 12 h and preferably much longer. However, this does not allow us to determine osteoclast number directly as individual osteoclasts frequently make large clusters of pits; also, difficulties in preparing or obtaining properly ground bone or dentine substrate has understandably led to the common use of artificial substrates, even though these can be broken down by macrophages. Some osteoclasts can have low activity, including those derived from RAW264.7 cells, which leads to inconclusive results. CTR is highly specific in the myelomonocytic lineage for osteoclasts, although osteocytes as well as some cell types not found in bone can express CTR (Gooi et al., 2014; Nicholson et al., 1986). This has led us previously to investigate the use of antiCTR antibodies to identify murine osteoclasts in culture (Quinn et al., 1999) (peptides derived from human CTR seem to be poorly immunogenic in rabbits); we therefore have employed this approach with IL-33 + TGFβ-treated RAW264.7 cells. CTR immunostaining confirmed the conclusions of the TRAP histochemical staining, that very low numbers of osteoclasts do form in such cultures. Indeed, RAW264.7 cells responded strongly to IL-33 with activation of NFκB (p65 phosphorylation) and p38, essential early response mediators for RANKL on osteoclast progenitors, consistent with an osteoclastogenic response. However, consistent with the observation of very low levels of osteoclast formation induced by IL-33 was the lack of any observable NFATc1 induction by IL-33 or IL-33 + TGFβ treatment. Indeed, the levels of NFATc1 protein were lower with IL33 treatment than that of untreated mice (Fig. 5B) while in cells treated by RANKL NFATc1 levels were clearly raised. This is consis-

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tent with the induction of intrinsic inhibitors discussed above, or with IL-33 induction of macrophage-derived inhibitors we have previously noted (Saleh et al., 2011) since these can all reduce NFATc1 levels. A small drop in NFATc1 levels is thus explainable but is surprising to observe with a stimulus that actually triggers some degree of osteoclast differentiation. This may suggest that NFATc1 levels at or below levels seen in untreated cells are nevertheless permissive for low levels of osteoclast formation if other signalling pathway components (such as NFκB) are stimulated strongly enough. Although we found some osteoclastogenic effects of IL-33 on RAW264.7 cells (Fig. 3) we found none at all with primary BMM. Both types are immature proliferative cells that do not display an anti-osteoclastic (inhibitory) response to IL-33 treatment which we have previously argued is due to their immature nature (Saleh et al., 2011). Thus, it is not clear why they should differ in their proosteoclastic responses to IL-33, albeit that the response of RAW264.7 cells is extremely small. These cell types do differ substantially in other ways for example: BMM are primary cells dependent on M-CSF for proliferation while RAW264.7 cells are an immortalised leukemic cell line that is M-CSF independent. They also differ in their osteoclastic responses to Th2 cytokines such as IL-4 (Mirosavljevic et al., 2003) which, although not directly relevant in this context, does underline important differences in their hormonal responses that may be reflected in our observations. In summary, our data point to a pro-osteoclastic action of IL33, perhaps similar to that seen with TNF and TNFSF-14/LIGHT actions. The bone phenotype of IL-33 null mice has not been reported but it seems unlikely that IL-33 actions are required for physiological bone resorption, given that ST2 (IL-33 receptor) null mice do not lack osteoclasts. The indirect inhibitory effects of IL33 may also offset any osteoclastogenesis it causes. However, IL33 may play a role in pathological bone loss, as it is abundant in inflammatory rheumatoid arthritis conditions that are associated with bone loss (Liew, 2012). IL-33 may act in concert with other stimulators of osteoclast formation (such as TGFβ or TNFSF-14/ LIGHT) or may perhaps drive osteoclastogenesis in progenitor cells that have low levels of intrinsic inhibitors. We would argue that although RANKL is undoubtedly the key driver of osteoclast formation the influence of other, ostensibly weaker, local stimuli should certainly not be discounted as they may emerge as significant in specific pathological circumstances. Acknowledgements The work was supported by NHMRC (Australia) Project Grant (611805) to J. Quinn and M.T. Gillespie, and by the Victorian State Government’s Operational Infrastructure Support Program. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.mce.2014.10.014. References Dougall, W.C., Glaccum, M., Charrier, K., Rohrbach, K., Brasel, K., De Smedt, T., et al., 1999. RANK is essential for osteoclast and lymph node development. Genes Dev. 13 (18), 2412–2424. Edwards, J.R., Sun, S.G., Locklin, R., Shipman, C.M., Adamopoulos, I.E., Athanasou, N.A., et al., 2006. LIGHT (TNFSF14), a novel mediator of bone resorption, is elevated in rheumatoid arthritis. Arthritis Rheum. 54 (5), 1451–1462. Elleder, M., 1986. Enzyme patterns in human endocytotic multinucleate giant cells – a histochemical study. Acta Histochem. 79 (1), 1–10. Fuller, K., Wong, B., Fox, S., Choi, Y., Chambers, T.J., 1998. TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts. J. Exp. Med. 188 (5), 997–1001. Gooi, J.H., Chia, L.Y., Walsh, N.C., Karsdal, M.A., Quinn, J.M., Martin, T.J., et al., 2014. Decline in calcitonin receptor expression in osteocytes with age. J. Endocrinol. 221 (2), 181–191.

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