Increased osteoclastic activity in acute Charcot’s osteoarthopathy: the role of receptor activator of nuclear factor-kappaB ligand

This study shows that monocytes from patients with acute Charcot’s osteoarthropathy cultured in the presence of M-CSF alone were capable of differentiating into mature osteoclasts that exhibited increased resorption compared with diabetic and healthy control participants. Furthermore, osteoclasts generated after the addition of sRANKL were functionally more aggressive, exhibiting a considerable increase in the extent of resorbing activity in patients with acute Charcot’s osteoarthropathy. This resorption was partially blocked by the addition of excess concentrations of OPG, a soluble receptor decoy for RANKL. This suggests that the increased osteoclastic activity in patients with acute Charcot’s osteoarthropathy is mediated through both a RANKL-dependent and a RANKL-independent pathway.

Cultures from the patients with Charcot’s osteoarthropathy showed increased osteoclast formation and resorption when cultured with M-CSF alone. Although M-CSF is an essential factor for proliferation, differentiation and survival of the monocyte-macrophage lineage [9, 10], it is not an osteoclastogenic factor and it is unusual to detect osteoclast formation and resorption in cultures with M-CSF, as was seen in the diabetic and healthy controls. This observation suggests that in acute Charcot’s osteoarthropathy there may be increased levels of other circulating pro-inflammatory factors such as TNF-α [11, 12], IL-6 [13], IL-8 [14] and LIGHT (homologous to lymphotoxins exhibiting inducible expression and competing with herpes simplex virus glycoprotein D for herpes virus entry mediator [HVEM], a receptor expressed by T lymphocytes) [15], which have been previously shown to stimulate osteoclastogenesis independently of RANK/RANKL mechanisms. The concentrations of these circulating factors in diabetic and healthy control participants may not be sufficient to induce the formation and differentiation of active osteoclasts in the presence of M-CSF alone.

After the addition of sRANKL to M-CSF cultures, the newly formed osteoclasts exhibited markedly increased resorption in the patients with Charcot’s osteoarthropathy, although the number of osteoclasts did not significantly increase in these patients compared with cultures with M-CSF alone. These observations may not be unique to Charcot’s osteoarthropathy, and indeed similar observations have been reported in other conditions associated with increased bone resorption, such as rheumatoid arthritis where the addition of sRANKL resulted in a significant increase in lacunar resorption, but did not lead to a significant increase in the number of TRAcP-positive cells [16]. Overall, the observed extensive resorption in acute Charcot patients, in the presence of M-CSF and sRANKL, as compared with the diabetic or healthy control groups, may suggest that the osteoclast precursors circulating in acute Charcot patients are in a higher activated state and as such are more primed to becoming osteoclasts (mediated through RANKL) than those in the control groups.

In order to ascertain that RANKL was a major osteoclastic activator in patients with Charcot’s osteoarthropathy, excess concentrations of OPG, the soluble receptor decoy to RANKL, were added to the cultures with M-CSF and RANKL. The rationale for this approach was that if osteoclastogenesis is mediated solely through RANK–RANKL interaction, addition of excess concentrations of OPG (as had been previously determined to be sufficient to block osteoclastogenesis through RANKL [15]) would completely abolish the process of osteoclast differentiation and activation. In the current study, although osteoclast formation and resorption in the diabetic and healthy control groups was completely blocked by the addition of OPG, the latter did not achieve total inhibition of osteoclast formation and resorption in patients with acute Charcot’s osteoarthropathy. These results suggest that although RANKL-dependent pathways do play a significant role in the osteoclastic activity of Charcot’s osteoarthropathy, an alternative pathway (other than RANK/RANKL) may also be involved. Osteoclastogenic mediators other than RANKL that have been reported to stimulate osteoclast differentiation independently of the RANKL pathway include TNF-α [11, 12], IL-6 [13], IL-8 [14] and LIGHT [15]. In acute Charcot’s osteoarthropathy, it is possible that one or a combination of these factors may have initiated the circulating osteoclast precursors to be in a more ‘primed’ condition, a situation which as such could explain the observed resorption in Charcot monocyte cultures supplemented with M-CSF alone, without the exogenous addition of any osteoclastogenic mediators.

The osteolysis of Charcot’s osteoarthropathy may be explained by our observation that osteoclast precursors from Charcot patients develop into mature osteoclasts that exhibit increased resorptive activity, especially in response to RANKL, unlike the increased resorption in response to bacterial infection, which is not mediated by RANKL [17]. Increased expression of RANKL has been previously demonstrated in pathological osteolysis associated with the development of various bone diseases [18] and a similar mechanism may contribute to osteolysis of Charcot’s osteoarthropathy [5]. Furthermore, patients with Charcot’s osteoarthropathy have severe neuropathy, which itself can also lead to increased expression of RANKL as a result of the loss of nerve-derived peptides known to antagonise its effect such as calcitonin gene-related peptide [5]. In addition to the RANKL-dependent pathway, our results suggest that a RANKL-independent pathway, mediated by pro-inflammatory cytokines, may also be important. Indeed, Charcot’s foot is characterised by excessive inflammation and proinflammatory cytokines have been implicated in its pathogenesis [19]. In support of this, a recent immunohistochemical analysis of bone samples isolated from Charcot’s osteoarthropathy patients showed excessive osteoclastic activity in a microenvironment enriched with mediators of bone resorption (IL-1, IL-6 and TNF-α) [20]. Thus a RANKL-independent pathway, which is also known to play a role in other osteolytic disorders such as rheumatoid arthritis [21] and aseptic loosening [22], could contribute also to the pathogenesis of the Charcot’s osteoarthropathy.

This study has indicated, for the first time that the RANKL-dependent pathway is important in the pathogenesis of Charcot’s osteoarthropathy, thereby raising the possibility of the use of RANKL inhibition in the management of Charcot’s foot. However, our observations also suggest that a RANKL-independent pathway may play a role, but further investigation is required to fully clarify the mechanism involved. If confirmed, specific pharmacological agents that counteract the RANKL-independent pathway, such as anti-TNF strategies, may be useful in the treatment of Charcot’s osteoarthropathy. Whatever the relative importance of either pathway, this in vitro technique of generating human osteoclasts from PBMCs may allow specific characterisation of osteoclastic activity in each patient and could, in the future, lead to individually tailored anti-osteoclastic treatment for the patient with acute Charcot’s osteoarthropathy.