Osteomyelitis commonly causes bone destruction and is most frequently due to infection by Staphylococcus aureus. S. aureus is known to secrete a number of surface-associated proteins which are extremely potent stimulators of bone resorption in the mouse calvarial assay system. The precise cellular and humoral mechanisms whereby this stimulatory effect is mediated, in particular whether osteoclast formation or activity is directly promoted by these factors, have not been determined by this study. Surface-associated material (SAM)(0.001ug/ml)obtained from 24 hour cultures of S. aureus was added to cultures of mouse and human osteoclast precursors (RAW 264.7 cells and human peripheral blood mononuclear cells respectively). These cultures were incubated in the presence and absence of receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony stimulating factor (M-CSF). It was found that independent of RANKL, SAM was capable of inducing osteoclast formation in cultures of RAW cells and human monocytes. This was evidenced by the generation of tartrate-resistant acid phosphatase-positive multinucleated cells, which formed lacunar resorption pits when these cells were cultured on dentine slices. In cultures where M-CSF, RANKL and SAM were added, osteoclast formation was increased, but did not exceed the osteoclast formation in cultures with M-CSF and RANKL. These findings indicate that S. aureus produces a soluble factor which can promote osteoclast formation. Identification of this factor may help to develop therapeutic strategies for treating bone destruction due to Staphylococcal osteomyelitis.

Introduction: Osseous metastases from melanoma are relatively common (7% of cases), and occur most often in the axial skeleton. Bone destruction in skeletal metastases of solid tumours is due to stimulation of osteoclast formation and bone resorption. Osteoclasts are formed by the fusion of marrow-derived mononuclear phagocyte precursors which express RANK (receptor activator of nuclear factor κB) which interacts with RANKL expressed by osteoblasts/bone stromal cells in the presence of macrophage colony-stimulating factor (M-CSF). Osteoclast formation by a RANKL-independent, tumour necrosis factor α (TNFα)-induced mechanism has also been reported. Tumour-associated macrophages (TAMs) are present in both primary and secondary tumours and TAMs are known to be capable of osteoclast differentiation. Our aim in this study was to determine the role of TAMs and the humoral mechanisms of osteolysis associated with melanoma metastases.

Materials and Method: In this study we isolated TAMs from extraskeletal primary melanomas and lymph node metastases. TAMs were cultured for up to 21 days in the presence and absence of M-CSF and RANKL or TNF. In a separate experiment, conditioned medium was extracted from the melanoma cell line, SK-Mel-29, and cultured with human peripheral blood mononuclear cells in the presence of M-CSF.

Results: TAM-osteoclast differentiation, as evidenced by the expression of tartrate-resistant acid phosphatase, vitronectin receptor and lacunar resorption pit formation, occurred by both RANKL-dependent and RANKL-independent mechanisms. Osteoclast formation induced by RANKL-independent mechanism was not abolished by the addition of osteoprotegerin or RANK:Fc, decoy receptors for RANK. Conditioned medium from SK-Mel-29 could support osteoclast differentiation in the absence of RANKL. This effect was not abolished by antibodies to RANKL, TNFα, TGFβ, IL-8 or gp130.

Discussion: These results indicate that melanoma TAMs are capable of differentiation into osteoclasts and that both RANKL-dependent and RANKL-independent (TNFα) mechanisms are involved. Melanoma tumour cells also secrete a soluble factor that supports osteoclastogenesis.

Conclusion: Inhibitors of osteoclast formation targeting TAM-osteoclast differentiation and osteoclast activity and identification of the osteoclastogenic factor produced by melanoma cells may have a therapeutic potential in controlling tumour osteolysis.