CIA develops more readily in IFN-γR KO mice than in wild-type mice: arthritic lesions appear about 2 weeks earlier and symptoms are more severe. The myelopoietic burst that accompanies the local disease manifestations and that is most evident in the spleen also occurs earlier and is more pronounced. The cell population generated by this myelopoietic burst consists mainly of CD11b+
doughnut-like cells, namely immature macrophages and neutrophils [3
]. Total numbers of other investigated splenocyte subpopulations, namely CD4+
T cells and B cells, remain unchanged. The parallelism between systemic myelopoiesis and local lesion development has led us to postulate that the CD11b+
cells have a crucial role in CIA by invading the joint tissues and by differentiating into osteoclasts. We have already provided evidence that invasion of periarticular tissues by myeloid cells does take place and is an important element in the pathogenesis [34
]. The experiments described in the present paper were conducted to provide evidence for the potential of the CD11b+
cells to differentiate into osteoclasts.
We showed that osteoclasts, identifiable by their multinucleated appearance, by their localisation close to the calcified bone material and by TRAP staining, resided in the CIA lesions of IFN-γR KO mice at the time (day 27) when macroscopic joint involvement was maximal. At that time, lesions were not yet present in wild-type mice, and osteoclasts could not yet be seen in their joint tissues. At a later time, when lesions eventually developed in these mice, osteoclasts also became visible, although their numbers were smaller. Thus, intra-articular osteoclast formation was accelerated and more pronounced in IFN-γR KO mice, in concordance with the earlier and more prolific myelopoietic burst. Immunocytochemical staining of joint sections revealed osteoclasts to be positive for CD11b+, supporting the hypothesis that mature osteoclasts in the inflamed joints tissues might be derived from extramedullar CD11b+ myelopoiesis.
Evidence for CD11b+ splenocytes being able to differentiate into osteoclasts was obtained by observations both in vivo and in vitro. Osteoclastogenesis induced by RANKL as well as by TNF-α could be demonstrated in splenocyte cultures. It was more pronounced if these cells were derived from IFN-γR KO rather than from wild-type mice in both CIA and naive conditions. IFN-γ levels were present in RANKL- and TNF-α-induced splenocyte cultures derived from immunised as well as from naive mice. Spleens of naive IFN-γR KO and wild-type mice did not significantly differ in their proportions of the splenocyte subpopulations (CD11b+ cells, T cells and B cells). This suggests that IFN-γ, aside from causing a delay in the myelopoietic response to the CII/CFA immunisation, also inhibits differentiation of immature myeloid cells into osteoclast precursors. Pit-forming assays failed to reveal any difference between osteoclasts from IFN-γR KO and wild-type mice, indicating that endogenous IFN-γ, while inhibiting differentiation of osteoclasts, does not affect their activation.
RANKL, when used in optimal doses, seemed to be a more potent osteoclast differentiating stimulus than TNF-α. Moreover, stimulation with RANKL, but not with TNF-α, revealed a facilitating effect of CII/CFA immunisation on differentiation into osteoclasts. The different RANKL sensitivities of splenocytes from wild-type versus IFN-γR KO mice and from CII/CFA-immunised versus naive ones led us to investigate whether RANKL production could also vary between these groups of mice and whether the receptor and the signalling system for RANKL (RANK and TRAF6) could be differently tuned.
Production of RANKL and TNF-α by anti-CD3-stimulated splenocyte cultures was higher if these cells were derived from IFN-γR KO (rather than wild-type) mice and from CII/CFA-immunised (rather than naive) mice, suggesting that augmented osteoclastogenesis in the immunised IFN-γR KO mice might be due in part to an increased production of RANKL and TNF-α. We found expression of RANK in the 10-fold expanded CD11b+ splenocyte population of immunised IFN-γR KO mice, whereas CD11b- cells were RANK-negative. Furthermore, the intracellular concentration of TRAF6, a RANK adapter protein, seemed to be strongly decreased in the splenocyte cultures derived from 21-day CII/CFA-immunised IFN-γR KO mice, in comparison with levels in splenocytes taken at the same time point from nonimmunised or wild-type mice. This indicates that the status of the RANKL signalling system in 21-day immunised IFN-γR KO mice is profoundly different from that in the nonimmunised mice or the wild-type controls.
Because our in vitro data prove that spleen cells are able to differentiate into osteoclasts, and that anti-CD3-stimulated splenocyte cultures can produce RANKL and TNF-α, we investigated whether osteoclast formation can occur in vivo in the spleen. No osteoclasts were detected by TRAP staining of freshly isolated splenocytes of diseased mice. Quantitative reverse transcriptase PCR revealed that mRNA of the calcitonin receptor was absent from splenocytes but present in cells residing in the inflamed synovium. These data prove that no osteoclast differentiation takes place within the spleen.
An inhibitory effect of IFN-γ on osteoclast formation via cross-talk with the RANK/RANKL system has been described in another in vivo
model of bone degradation involving the injection of lipopolysaccharide into calvarial bone in mice, and, in vitro
, in bone marrow macrophage (BMM) cultures exposed to RANKL [35
]. Intriguingly, inhibition of osteoclast formation by IFN-γ in the BMM cultures was accompanied by decreased TRAF6 levels and by increased TRAF6 turnover. In the BMM model, IFN-γ induced degradation of TRAF6 was found to require RANK/RANKL signalling and a functional proteasome. These observations are in contrast to our findings in ex vivo
lysed splenocytes (Fig. ) and in splenocyte cultures, in which decreased TRAF6 levels occurred in association with increased osteoclastogenesis and with an absence of IFN-γ signalling in the IFN-γR KO-derived cells. The lower TRAF6 levels were not caused by decreased transcription because no differences in TRAF6 mRNA were found between IFN-γR KO and wild-type mice (data not shown). In concordance with our TRAF6 results, recent findings by Huang and colleagues [36
] have shown that early exposure to IFN-γ renders osteoclast precursors resistant to the effects of RANKL and that this effect is not associated with degradation of TRAF6.
TRAF6 is a ubiquitin ligase, becoming activated by ubiquitination [37
]. It has been shown that the TRAF6 protein is ubiquitinated in response to RANKL and IL-1. It has recently also been shown that ubiquitination of TRAF6 does not necessarily lead to degradation but can be followed by de-ubiquitination [38
]. The ubiquitinated form of TRAF6 is not necessarily detectable with anti-TRAF6 antibody because modification with ubiquitin might alter its native epitopes. Hence, the presence of non-ubiquitinated TRAF6 can be indicative of the absence of TRAF6-activating stimuli, whereas decreasing the concentration of the non-ubiquitinated form can be indicative of a high activation state of TRAF6. It is therefore possible that the absence of non-ubiquitinated TRAF6 from the IFN-γR KO cells of our model is due to a high activation status of the RANK/RANKL system, requiring TRAF6 activation and thus resulting in a high turnover between ubiquitinated and non-ubiquitinated TRAF6.
As regards the role of TRAF6 during osteoclastogenesis, it is also important to keep in mind that, at least in vitro
, RANK has been shown to associate with TRAFs 1, 2, 3, 5 and 6. Consequently, the possibility cannot be excluded that signalling through one of these occurs in the IFN-γR KO splenocytes. Moreover, TRAF6-deficient mice reportedly do produce osteoclasts within their bone, and the numbers of TRAP+
cells per square millimetre of tissue area are comparable in wild-type and knock-out mice [39
]. The phenotype is nonetheless osteopetrotic owing to the inactivity of these osteoclasts. This shows that TRAF6 is not indispensable for the formation of osteoclasts but is vital for their activation. In this respect, the absence of TRAF6 should not in the first place influence the number of osteoclasts developing from splenocytes, but rather their activity. Although osteoclasts in TRAF6-deficient mice are inactive, and the splenocytes of our IFN-γR KO mice show low levels of TRAF6, the osteoclasts derived from the splenocytes of both the IFN-γR KO mice and the wild-type mice were indeed active, as proved with the pit-forming assay. This supports our first hypothesis that the lowered levels of non-ubiquitinated TRAF6 in the IFN-γR KO mice, as detected by Western blotting, are indicative of a high activity of the RANK/RANKL signalling.
Not only is the RANK/RANKL system operational in the CII/CFA-immunised IFN-γR KO mice; so also is the IL-1β system, as was evident from comparatively low levels of pro-IL-1β and pro-caspase-1 but a high level of mature caspase-1, indicating the active conversion of pro-IL-1β into secreted active IL-1β. In this respect, Guedez and colleagues [40
] found that genetic ablation of IFN-γ upregulates IL-1β and enables the elicitation of CIA in the nonsusceptible C57BL/6 mouse strain. Moreover, treatment of IFN-γ KO mice with anti-IL-1β antibody reduced the incidence and severity of arthritis, indicating that in the absence of IFN-γ, IL-1β is important in the pathogenesis of CIA [40
]. Important in this respect is the known osteoclast-activating property of IL-1β [41
Together, these data indicate that, in CII/CFA-immunised mice, all the conditions are fulfilled for the expanded CD11b+
myeloid splenocytes to differentiate into osteoclasts. Our study thereby provides a link between the increased expansion of CD11b+
cells in the IFN-γR KO mouse spleens, the higher capacity of IFN-γR KO splenocytes to produce key mediators in the osteoclast-differentiating process and the higher susceptibility of the IFN-γR KO mice to CIA. Moreover, control by endogenous IFN-γ over CD11b+
myelopoiesis and osteoclastogenesis might also account for the recently reported observation that IL-10-deficient mice have an increased susceptibility to CIA in association with decreased production of IFN-γ [42
]. Support for the importance of extramedullar CD11b+
myelopoiesis during the development of CIA comes from a recent study on spontaneously occurring arthritis in TNF-α-transgenic mice [43
]. Higher numbers of CD11b+
osteoclast precursors were recorded in the blood and spleen of the transgenic mice. The increased numbers were correlated with the appearance of TNF-α in the circulation and with the initiation of joint inflammation. TNF-α blockade with the TNF-α antagonist etanercept did not affect enhanced RANKL-induced osteoclast formation in vitro
, suggesting that TNF-α-stimulated osteoclasto-genesis in vivo
was indeed due to the generation of larger numbers of osteoclast precursors rather than to accelerated differentiation beyond the precursor stage.
Our present and previous studies [3
] stress the predominant role of innate immunity in the pathogenesis of CIA. Innate immunity, triggered by the mycobacterial cell wall components in CFA, might be the primary motor of the disease by stimulating myelopoiesis, causing migration and regulating osteoclast differentiation. The role of specific immunity directed at CII might consist of restricting the inflammatory response to the specific location of the joints.