Osteoclasts are multinucleated cells responsible for bone resorption and are derived from hematopoietic precursor cells that circulate in the blood 
. It is currently thought that two critical factors supplied by osteoblasts, M-CSF and RANKL, are essential for the differentiation and maturation of osteoclast precursors 
. Although M-CSF defective mice (op/op) show an osteopetrotic phenotype, it can spontaneously reverse with age, suggesting that alternative osteoclastic pathways do exist 
. Hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and Flt3 ligand have all been shown to support osteoclast formation in the absence of M-CSF 
. Furthermore mice deficient in either RANKL or its receptor RANK also show an osteopetrotic phenotype that is caused by the complete lack of osteoclast in their bones 
. Although no osteoclasts can be identified in the bone of RANKL or RANK deficient mice, this may not be simply because of the complete failure of osteoclastogenesis. Indeed, RANKL is a survival factor for differentiated osteoclasts 
and it is plausible that impaired osteoclast differentiation superimposed on a shortened lifespan might also explain the observed phenotype in RANKL or RANK deficient mice 
. As such, alternative RANKL-independent pathways (e.g. LIGHT, TGFβ and TNF-α) have been reported to support osteoclastogenesis in the presence of excess osteoprotegerin, an inhibitor of RANKL-RANK interactions 
. The emergence of the osteoimmunology field has demonstrated that activated T cells directly modulate osteoclastogenesis and bone resorption 
, and that T cell products, such as IL-17, TWEAK, GM-CSF and IFN-γ, can regulate osteoclast formation 
. The present study sought to determine the role of IL-32, a newly described cytokine presenting characteristic of pro-inflammatory cytokine and involved in a variety of inflammatory disorders 
in osteoclast differentiation and activation. We have demonstrated that multinucleated cells formed in response to IL-32 expressed several specific markers of osteoclast such as activation of the NF-κB and MAP kinase pathways, expression of TRAcP and VNR, up-regulation of NFATc1, OSCAR and Cathepsin K which were also observed in RANKL-treated cultures. However, IL-32-treated multinucleated cells were unable to induce bone resorption in vitro
. One explanation for the lack of bone resorption could be attributed to the failure of the multinucleated cells generated in response to IL-32 to express F-actin ring which ultimately is required for anchorage of the cells prior to bone resorption.
An important step in the process of osteoclast differentiation is the induction of NFATc1 above a critical threshold 
. This is consistent with the findings that overexpression of NFATc1 is sufficient to induce osteoclast differentiation 
. In the present study, IL-32 was capable of up-regulating the expression of NFATc1 compared to M-CSF-treated cultures or even M-CSF/RANKL-treated cultures. Moreover, in response to IL-32, these cells expressed high levels of OSCAR and Cathepsin K, two markers that are specific for osteoclasts 
. According to the different markers expressed by the cells in response to IL-32, it is reasonable to conclude that these multinucleated cells are likely to be “immature osteoclasts”. Recently, Kim et al. 
have reported that TNF-α is capable of inducing multinucleation of osteoclast precursors and expression of osteoclast phenotypic markers (TRAcP, F-actin) in RANK-deficient cells, but was unable to induce evidence of lacunar bone resorption. In the present study, although PBMCs released pro-osteoclastic mediators (TNF-α, IL-6, LIGHT, MIP-1α and VEGF), the lack of lacunar resorption in IL-32-treated cultures could be explained by the fact that IL-32 stimulated the release of known osteoclastic inhibitors, i.e. IL-4 and IFN-γ. Our results herein indeed indicated that this is the case as we demonstrated a significant increase in the release of IL-4 and IFN-γ from IL-32-stimulated PBMCs as compared to unstimulated cells.
Tumour necrosis factor receptor-associated factor 6 (TRAF6) has been reported to be important for osteoclast activation, i.e. lacunar bone resorption 
. The complex role of IFN-γ in osteoclastogenesis has been previously addressed by Takayanagi et al. 
. They have shown that IFN-γ induces rapid degradation of TRAF6, which results in strong inhibition of the RANKL-induced activation. We therefore speculated that the inhibitory effects of IL-32 alone or in combination with soluble RANKL could partly be attributed to TRAF-6 degradation. However, surprisingly, we found that TRAF6 is not degraded but it is overexpressed in response to IL-32 treatment compared to RANKL. Recently, Gao et al. have shown that IFN-γ exhibits a “direct” anti-resorptive effect by blunting osteoclast differentiation. However, IFN-γ can act “indirectly” as a pro-resorptive factor by stimulating T-cells to express RANKL and TNF-α 
. In the present study, we used PBMCs as a source of osteoclast precursors and although cells were washed thoroughly to eliminate non-adherent cells (mainly B- and T-cells), it is plausible that some T-cells could have been present in the culture and contributed to osteoclastogenesis. This hypothesis is also reinforced by the evidence that the addition of excess OPG to the IL-32-treated cultures led to a marked decrease in the number and size of newly-formed multinucleated cells. Although we were unable to detect any soluble RANKL in the supernatant of IL-32-treated cultures, it is conceivable to suggest that effects of IL-32 could have been partially mediated through a RANKL-dependent mechanism. We have previously observed that IL-32 increase the expression of membrane-bound RANKL in T-cell cultures (unpublished data) and it is likely that a few numbers of RANKL-expressing T-cells may have been present in the PBMC cultures. As IL-32 is known to be produced by PBMCs in response to IFN-γ 
, the effects of IFN-γ in combination with IL-32 on T-cells could have contributed to the inhibitory effects of OPG observed herein.
The downstream signalling of RANK/RANKL interactions has been extensively studied in the last decade. It has been shown that the binding of RANKL to its receptor activated NF-κB, MAP kinase and Akt pathways. However, downstream pathways involved in response to IL-32 in osteoclasts are not fully elucidated. In our study, we found that PBMCs treatments with M-CSF/IL-32 or M-CSF/RANKL dramatically increased the activation of NF-κB and JNK pathways compared to M-CSF-treated cultures. However, the activation of the Akt pathways appeared more complicated. M-CSF or M-CSF/IL-32 treatments were capable of strongly activating Akt pathways compared to M-CSF/RANKL. These results appeared controversial with the consensus that RANKL activates Akt 
. However, most of the studies showing Akt activation in response to RANKL have been done in in vitro
models of bone marrow co-cultures or using murine cell line, RAW264.7 cells. In these studies, treatment of serum-starved osteoclast precursors with soluble RANKL resulted in a significant activation of the Akt pathway as compared to cultures in the absence of RANKL. This is in conflict with the present findings whereby after exposure of M-CSF–treated PBMCs to sRANKL, Akt activation was down-regulated compared with M-CSF alone. Interestingly, M-CSF/IL-32 treatment of PBMCs resulted in a similar level of Akt activation compared to M-CSF treatment. It has also been extensively shown that M-CSF, via its receptor c-fms, is a strong activator of PI-3 Kinase which in turn activates Akt 
. In the present study, we demonstrated that M-CSF/IL-32 treatment exhibited levels of Akt activation similar to M-CSF treatment and it is questionable whether Akt activation in response to IL-32 or RANKL results in the same downstream effectors ().
Schematic representation of downstream pathways activated by RANKL or IL-32 treatment.
We have also evidence of a marked increase in the phosphorylation of ERK1/2 in M-CSF/IL-32 compared to M-CSF/RANKL treated cultures. It is well known that activated ERK1/2 translocates to the nucleus and activates its target to promote the expression of specific genes (reviewed in 
). It is plausible to suggest that the downstream targets of activated ERK1/2 in M-CSF/IL-32 are different to those known for M-CSF/RANKL (). This is also reinforced by the morphological differences (e.g. differences in cell size and number of nuclei, lack of F-actin ring) observed in the multinucleated cells generated in presence of IL-32 or RANKL.
Our current findings suggest that IL-32 was capable of inducing osteoclast differentiation in a manner partially independent of the RANK/RANKL pathway. However, although IL-32 could increase the release of pro-inflammatory mediators known to positively influence osteoclastogenesis, it was unable to induce the activation of these newly-formed multinucleated cells into bone-resorbing osteoclasts and had a direct inhibitory effect on osteoclast activation in vitro. It is worth noting that IL-32 has direct effects on other cell types such as epithelial cells, T-cells, natural killer cells and monocytes. Although the present study only addressed its direct role on osteoclast precursors, IL-32 could also indirectly modulate osteoclastogenesis in vivo. The complex role of IL-32 in the patho-physiology of bone disorder therefore requires further clarification prior to the development of any potential therapeutic approach.