IFN-γ has complex and apparently contradictory actions on osteoclastogenesis and bone resorption, depending on the system and model employed. In this study, we have examined the balance between the direct anti-osteoclastogenic effects of IFN-γ and the indirect proresorptive activities of IFN-γ in vivo, using 3 different murine models of pathological bone turnover. Our data demonstrates that in experimental models of postmenopausal osteoporosis, bacteria-induced infection, and inflammation, the net balance between the direct antiresorptive activities of IFN-γ and its proresorptive effects is biased in favor of osteoclastogenesis and bone resorption. Our data confirms previous studies demonstrating direct anti-osteoclastogenic activities of IFN-γ on the process of osteoclast differentiation in vitro. However, we further demonstrate that in vivo, the net effect of IFN-γ is to potently stimulate osteoclastogenesis, a consequence of its capacity to upregulate antigen presentation. This process leads to T cell activation and proliferation, with a consequent upregulation in the production of the key osteoclastogenic cytokines RANKL and TNF-α. In vivo, this indirect pro-osteoclastogenic activity overcomes the direct suppressive activities of IFN-γ on osteoclast precursors, leading to a net bone loss.
It is reported that preexposure of osteoclast precursors to RANKL renders them resistant to the inhibitory effect of IFN-γ (9
). It is thus likely that in vivo, as RANKL concentrations begin to rise — a consequence of IFN-γ–driven antigen-dependent T cell activation — the direct anti-osteoclastogenic activity of IFN-γ may become progressively muted. Furthermore, as the dominant source of IFN-γ is activated T cells, a potent amplificatory loop is likely established that further drives up and sustains antigen presentation and T cell activation, thus perpetuating an inflammatory and proresorptive environment.
Our experimental disease models highlight the central role of T cells in the process of IFN-γ–driven bone resorption. This is underscored by experiments showing that T cell–deficient nude mice fail to undergo bone loss following rIFN-γ administration. IFN-γ–induced bone loss was reversed by T cell reconstitution and is consistent with the fact that IFN-γ has been shown to stimulate bone resorption only in humans or experimental models possessing a normal T cell lineage (14
), while the vast majority of previous studies concluding that IFN-γ is a bone-sparing cytokine made use of the nude mouse model and/or T cell–depleted in vitro cultures (12
). Interestingly, Takayanagi et al. reported that IFN-γ represses bone resorption in a T cell–replete model, but the only in vivo data presented in the report were derived from calvariae from prepubertal mice (7
). Thus, the significance of these observations in mature bone and long bones remains unknown. Our studies failed to identify a significant repressive effect of IFN-γ on bone resorption in nude mice, an unexpected finding in view of the absence of the T cell–driven proresorptive activity. However, it is conceivable that a longer IFN-γ treatment period may have allowed for the direct inhibitory effect of IFN-γ to predominate. Alternatively, it is possible that a relatively higher concentration of IFN-γ might be required to decrease bone resorption in this strain. This hypothesis is suggested by the presence of an increased number of osteoclasts and a higher rate of bone resorption in nude mice than WT controls (43
Of additional interest was the finding of a lower baseline bone density in IFN-γ–/– mice than in WT mice. This was not unexpected, since the indirect proresorptive activity of IFN-γ overrides its direct anti-osteoclastogenic activity only in conditions of increased T cell activation. In contrast, in unstimulated conditions, IFN-γ exerts only its direct anti-osteoclastogenic activity. As a result, bone density is lower in adult IFN-γ–/– than congenic WT mice, because of the long-term impact of the absence of the direct anti-resorptive activity of IFN-γ.
Combined measurements of cortical and trabecular bone by DXA revealed that ovx caused a significant bone loss in WT but not in IFN-
mice. These data are consistent with a previous report from our laboratory showing that IFN-
mice fail to undergo bone loss following ovx (2
). Selective measurements of trabecular bone by μCT showed that silencing of IFN-γ affords significant protection against the bone loss induced by ovx, although it did not completely prevent bone loss. These findings may suggest a more relevant role of IFN-γ in the loss of cortical bone than in the loss of trabecular bone following ovx. Although the finding that silencing of IFN-γ protects, in part, against ovx-induced bone loss does not establish a cause-effect relationship, the data strongly suggest that under in vivo conditions of estrogen deficiency, IFN-γ possesses pro-osteoclastogenic and bone-wasting effects that prevail over the direct anti-osteoclastogenic activity that is dominant in vitro. The results of the current investigation are consistent with an earlier report demonstrating that ovx expands the size of the T cell pool via an IL-7–dependent increase in both the peripheral expansion of T cells and the output of recently produced naive T cells from the thymus (45
). Attesting to the relevance of these phenomena, ovx-induced bone loss is decreased by thymectomy and completely prevented by IL-7 neutralization (45
). IL-7 stimulates the production of IFN-γ, while TGF-β, a cytokine directly regulated by estrogen, blocks the production of both IL-7 and IFN-γ. It is thus likely that the increase in the levels of both IL-7 and IFN-γ characteristic of ovx mice results from the blunted production of TGF-β induced by ovx. Because IL-7 and IFN-γ are under reciprocal control, silencing of either factor affords significant protection against ovx-induced bone loss. The finding that IFN-γ is implicated in the mechanism of ovx-induced bone loss provides further support for our hypothesis that an increased production of osteoclastogenic cytokines by activated T cells plays a pivotal role in the bone loss that follows estrogen withdrawal (1
). Evidence in favor of this hypothesis includes the reported failure of ovx to induce trabecular and cortical bone loss in nude mice (33
). While an independent confirmation was provided by a preliminary report by Watanabe et al. (46
), a more recent study by Lee et al. (44
) showed that nude mice lose trabecular bone after ovx, although they are protected against the loss of cortical bone. The partially negative outcome of this study is most likely explained by experimental design differences. Our μCT analysis of the effects of ovx in nude mice was conducted in mature, 16-week-old animals (37
). In the same investigation, prospective in vivo measurements of BMD were obtained by DXA (37
). Lee et al. (44
) conducted their μCT analysis in 5- to 6-week-old mice, an age when in most strains ovx blunts trabecular bone formation (38
). Accordingly, they found ovx to decrease osteoblast surface per bone surface (ObS/BS) (a static histomorphometric index of bone formation) in nude mice and to increase it in WT controls (44
). They also found ovx not to increase osteoclast surface per bone surface (OcS/BS) and the number of osteoclasts per unit of bone perimeter (NOcBpm) (2 sensitive indices of bone resorption) in both WT and nude mice (44
). These findings raise the possibility that the main effect of ovx in very young T cell–deficient mice is that of blunting bone formation rather than increasing bone resorption, leading ovx to cause an arrest in skeletal growth, rather than bone loss. Prospective in vivo measurements of bone density and analysis of dynamic histomorphometric indices of bone formation in mature mice will be required to reconcile our studies with those of Lee et al.
The mechanisms of LPS-induced bone loss are poorly understood. Our data demonstrate that LPS treatment led to a significant increase in antigen presentation and T cell activation in WT mice, while IFN-
mice displayed low basal antigen presentation and failed to undergo a significant upregulation in antigen presentation following LPS administration. T cell activation was also blunted in IFN-
mice. Nonetheless, LPS elicited similar trabecular bone loss in IFN-
and WT mice, although LPS-induced osteoclastic bone resorption and BMD decrements were modestly, but significantly, reduced in the IFN-
mice. These data suggest that the direct and indirect effects of IFN-γ on trabecular bone mass cancel each other out, thus resulting in an apparent similar trabecular bone–wasting effects of LPS in IFN-
and WT mice. The data further suggest that LPS mediates bone resorption by both IFN-γ–dependent and IFN-γ–independent pathways, although the independent pathway appears to be dominant. This interpretation is consistent with studies reporting that LPS induces osteoclast formation by multiple mechanisms, including direct stimulation of RANKL expression in osteoblasts (47
); production of TNF-α, IL-1, and IL-6 by macrophages; and stimulation of osteoclast survival (48
). Our data suggest that these proresorptive effects are further amplified by IFN-γ–mediated pathways.
TGF-β is a potent antiinflammatory cytokine that prevents T cell activation via 2 distinct mechanisms, direct signaling in T cells and inhibition of antigen presentation secondary to blunted T cell production of IFN-γ (49
). The loss of TGF-β–mediated repression of T cell activation results in inflammation, autoimmunity (35
), and increased T cell production of IFN-γ, TNF, and RANKL (37
). IFN-γ further stimulates T cell production of osteoclastogenic cytokines by promoting antigen presentation and antigen-dependent T cell activation. In our model whereby T cell activation is potentiated through selective silencing of TGF-β signaling in T cells, silencing of IFN-γ was observed to completely suppress antigen presentation. By contrast, T cell activation and T cell production of TNF-α and RANKL were only 50% lower in mice lacking both TGF-β signaling and IFN-γ production compared with those lacking TGF-β signaling only, leading to a 50% reduction in bone loss. These data suggest that IFN-γ–driven antigen-dependent T cell activation is responsible for approximately 50% of T cell TNF-α and RANKL production in this model. The remaining 50% of TNF and RANKL production is driven by the blockade of the direct repressive effects of TGF-β on T cell activation (50
In conclusion, our data reveal that IFN-γ has complex effects that explain the apparent conflicting reports regarding the effects of this cytokine in bone. In common conditions such as estrogen deficiency, infection, and inflammation, inhibition of IFN-γ signaling may represent a novel target for the concomitant prevention of both inflammation and bone loss.