Mechanisms of TNF-α– and RANKL-mediated osteoclastogenesis and bone resorption in psoriatic arthritis (original) (raw)
In psoriatic arthritis (PsA), bone erosions can be extensive, resulting in joint deformity and disability. These erosions differ markedly from the periarticular osteopenia and pericapsular bone loss commonly observed in rheumatoid joints (34). While these radiographic features suggest a different mechanism of bone loss in PsA, understanding of the basis of this difference has been impeded because the events that lead to psoriatic bone resorption have not been well defined. To elucidate this process, we sought to clarify how OCPs and the regulatory molecules RANK, RANKL, and OPG may orchestrate osteolysis in PsA.
Our results demonstrate that osteoclasts are prominently situated at the bone-pannus junction and in cutting cones traversing the subchondral bone in the psoriatic joint. In addition, OCPs are markedly increased in the circulation of PsA patients, most strikingly in those with bone erosions on plain radiographs. These cells express the surface markers CD11b, CD14, CD51/CD61, and RANK. A pivotal role for TNF-α in promoting OCP formation is supported by the observations that blocking TNF-α in vivo markedly suppressed the number of circulating OCPs and that cultured PsA PBMCs spontaneously release high quantities of biologically active TNF-α. Immunohistochemical studies delineated the presence of RANK-positive cells in synovium and adjacent to blood vessels in subchondral bone. Furthermore, synovial lining cells stained strongly for RANKL, while OPG expression was confined to the endothelium. These data suggest that OCPs enter a synovial microenvironment characterized by a high ratio of RANKL to OPG expression, facilitating osteoclastogenesis and bone resorption.
To our knowledge, this is the first study demonstrating the presence of increased numbers of circulating OCPs in patients with inflammatory arthritis. The initial impetus for the concept of an expanded pool of OCPs arose from studies on patients with Paget disease and multiple myeloma. Examination of bone marrow cultures from Paget patients revealed an increase in the number of committed OCPs compared with that in healthy controls (12). Similarly, PBMCs cultured from patients with multiple myeloma and bone lesions, but not those without bony involvement or healthy controls, gave rise to osteoclasts that resorbed bone in vitro when cultured in the presence of a murine stromal line (11). Faust et al. extended these observations by showing that osteoclasts can develop from unstimulated PBMCs derived from healthy controls when grown at high density; however, the number of osteoclasts was not quantified, and they demonstrated weak bone resorption properties (35). In pilot studies, we noted that numerous osteoclasts were present in unstimulated wells of PBMCs cultured from PsA patients, even when the cultures were seeded at low density. Thus, we modified our experimental protocol, analyzing OCP frequency at low density in the absence of exogenous factors such as RANKL and MCSF. Using this approach, osteoclasts were identified by positive TRAP staining and multinuclearity. These cells were shown to be functional by their ability to form pits on bone wafers. Compared with healthy controls, PsA patients had markedly more OCPs, and these cells resorbed significantly greater quantities of bone. It should be noted that the difference in resorption area between control and PsA patients was less than the difference in OCP frequency between the two groups. Potential explanations include the inability to measure the depth of resorption pits, species differences (human PBMCs on bovine bone), and the fact that there is no literature demonstrating a correlation between the number of OCPs and in vitro measures of bone loss. The additional finding that the increase in OCP frequency correlated with clinical erosions indicates that the size of the precursor pool may be a dependent factor that contributes directly to bone resorption in PsA.
Studies in mice demonstrated that systemic TNF-α directly increases OCP frequency and that this elevation is reversible by anti-TNF therapy (10). Here we show that this is also true in PsA, as increased OCP frequency declined significantly in five of five patients treated with anti-TNF therapy, which paralleled clinical improvement. Moreover, PsA PBMCs spontaneously released high levels of TNF-α in vitro. TNF-α secreted by these cells promoted osteoclastogenesis that was blocked with anti–TNF-α antibodies. Blocking RANKL with OPG also substantially decreased the number of OCPs that arose from PsA PBMCs. Thus, our results imply that TNF-α triggers a systemic increase in the number of circulating OCPs and suggest that this may be a critical event in the modulation of psoriatic bone resorption. While these data strongly support the concept that TNF-α released by PsA PBMCs promotes an increased OCP frequency, they do not establish these cells as the principal source of the TNF-α. Further studies designed to specifically address this question are required.
Of particular relevance to the observations outlined above are data demonstrating that TNF-α is a pivotal cytokine in PsA. TNF has been isolated from psoriatic synovial fluid, and psoriatic synovial explants release elevated levels of TNF-α, which were highest in patients with erosive arthritis (19, 20). Also, psoriatic synovial lining cells express TNF-α protein (21). Perhaps the most convincing evidence stems from clinical trials showing that TNF blockade dramatically ameliorates psoriatic joint pain and swelling; this evidence led to the Food and Drug Administration’s first approval of a drug, etanercept, for treatment of PsA (36, 37). Lastly, in a recent report, TNF inhibition improved clinical parameters of arthritis and reversed abnormal MRI bone and soft tissue signals in spondyloarthropathy patients with active joint and entheseal inflammation (38).
Although the precise phenotype of the precursor cell was not directly addressed in these experiments, we did find that PBMCs express CD11b, CD14, CD51/CD61, and RANK, established markers of mononuclear OCPs (14). It has been shown that approximately 2% of PBMCs can be stimulated to give rise to osteoclasts in vitro (39, 40). Interestingly, CD14+ monocytes can also differentiate into dendritic cells and macrophages (9, 41). Presumably, events in the bone marrow, circulation, and possibly the synovium determine the fate of a particular monocyte. Indeed, following exposure to RANKL and MCSF, a subpopulation of monocytes rapidly loses the CD14 marker and acquires an osteoclast phenotype (42), underscoring the critical importance of the RANK signaling pathway in osteoclastogenesis.
The discovery of RANK, RANKL, and OPG as the final effector molecules ultimately regulating osteoclastogenesis and bone resorption has provided a fundamental insight into the mechanisms of osteolysis in metabolic bone diseases (8, 43, 44). Definitive proof in support of this paradigm has also been provided in animal models of inflammatory arthritis (10, 39–41). In RA, investigators found that RANKL mRNA is expressed by T lymphocytes and synoviocytes isolated from lining membranes (26, 45, 46). It has also been demonstrated that fibroblast-like synoviocytes can induce osteoclastogenesis when cocultured with PBMCs (47). In our immunohistochemistry experiments, we found that PsA synovial lining cells stained intensely for RANKL, a finding not observed in OA synovial tissues. The RANKL appeared to be relatively unopposed by OPG, since staining for this molecule was restricted to the endothelium. The likely targets for this synovial cell RANKL are the perivascular RANK-positive mononuclear cells in the synovium and subchondral bone. The finding of RANK-positive mononuclear cells in the synovium, confirmed by elevated RANK mRNA expression in at least some of our PsA patients, was in line with previous studies that detected TRAP-positive cells in RA synovium and report that osteoclasts can be generated from RA synovium and bone (33, 48, 49). We observed a gradient of RANK staining by mononuclear cells that increased in intensity from the perivascular region in the subsynovium to the erosion front, where synoviocytes and osteoclasts exhibited the strongest RANK expression. We speculate that this gradient is directed by the elevated RANKL and TNF-α expressed by PsA synoviocytes. Ultimately, RANKL stimulation of these precursor cells could result in the genesis of RANK-positive multinucleated osteoclasts that directly erode the bone matrix. Support for the critical role of RANKL is provided by our experiments indicating that OPG significantly blocked osteoclast formation in PsA PBMCs.
A central question that remains to be addressed regards the specificity of these findings to PsA. As previously discussed, osteoclasts have been detected in rheumatoid synovium (48, 50). Furthermore, while osteoclast numbers in PsA tissues were considerably greater than in OA samples, they were not significantly different from those in RA samples. Clearly, larger sample sizes are required to determine whether the number of osteoclasts differs in RA and PsA; but, assuming that it does not, what mechanisms could account for the aggressive bone resorption observed in many PsA patients? First, the number of circulating OCPs may be higher in PsA, resulting in a more sustained assault on bone. Second, the ratio of RANKL to OPG may be significantly greater in patients with destructive PsA, or, alternatively, levels of antiosteoclastogenic factors such as IFN-γ, IL-12, or GM-CSF could be higher in the rheumatoid joint. Third, the striking increased vascularity and vessel tortuosity characteristic of PsA but not RA (51) may facilitate enhanced recruitment and entry of OCPs into the joint. Finally, other pro-osteoclastogenic factors such as IL-1 may be present in greater quantities in PsA joints, providing an additional osteoclast activation signal. In support of this latter mechanism is the observation that IL-1 was markedly elevated in psoriatic but not rheumatoid synovial explants obtained from patients with erosive joint disease (19).
Taken together with the established literature, the results of this study lead us to propose a mechanism for the destructive pathology observed in many psoriatic joints (Figure 12). In this model, TNF-α increases the number of circulating OCPs in PsA patients. In the case of “outside-in” erosion, OCPs enter a highly vascular psoriatic synovial membrane containing tortuous blood vessels and adhere to activated endothelial cells that have been stimulated by proinflammatory cytokines (52). Exposure to TNF-α could induce the expression of fibronectin and vitronectin receptors on endothelial cells, as described by McGowan et al., facilitating OCP binding and tissue migration (53). Simultaneously, the high level of OPG expressed by the endothelial cells would suppress osteoclastogenesis, permitting smaller undifferentiated OCPs to migrate through the dense pannus and target bone at a significant distance from the vessel. Upon arrival at the bone-pannus junction, OCPs bind RANKL on the surface of synoviocytes and, in the presence of TNF-α and MCSF, undergo osteoclastogenesis and erode bone. In the case of “outside-in” resorption, OCPs enter the subchondral environment in vessels that are in immediate proximity to bone. Following translocation through the endothelium, it is conceivable that OCPs are exposed to TNF-α–induced RANKL on the surface of osteoblasts and stromal cells (52, 54), resulting in the generation of osteoclasts that line cutting cones devoid of synovial tissue. In this scenario, mature osteoclasts mount a bidirectional assault, resorbing bone matrix in the subchondral bone and at the pannus-bone interface. Thus, there are two critical steps in the osteolytic pathway mediated by TNF-α: increase in the frequency of circulating OCPs, and upregulation of RANKL expression in the joint. In this model, patients with generalized inflammatory disease (Crohn disease, psoriasis) may have an expansion of CD14+CD11b+ cells that differentiate into dendritic cells or macrophages, but not osteoclasts. In view of the reported findings, antagonism of TNF-α may prove to be an effective strategy for inhibiting bone destruction in PsA.
Schematic model of osteolysis in the psoriatic joint. Extensive erosions observed in the PsA joint are mediated by a bidirectional attack on bone. We propose that circulating OCPs enter the synovium and are induced to become osteoclasts by RANKL expressed by synoviocytes (outside-in). In parallel, OCPs traverse endothelial cells in the subchondral bone and undergo osteoclastogenesis following RANKL stimulation from osteoblasts and stromal cells (inside-out).