IL-10 is an anti-inflammatory cytokine that suppresses both immunoproliferative and inflammatory responses. It is produced primarily from T cells and activated macrophages. Animal studies implicate IL-10 in the development and progression of arthritis and chronic colitis. IL-10 has a critical role in the in vivo regulation of pro-inflammatory cytokine levels. It functions in a negative feedback loop, in which it suppresses the release of inflammatory cytokines and dampens the acute inflammatory response. Clinical studies and IL-10-deficient mice back up the theoretical importance of this work, as discussed in the introduction. IL-10 has been known for some time to have a potent inhibitory effect on osteoclastogenesis . It prevents differentiation of osteoclast progenitors to preosteoclasts, however its mechanism of action is still unclear.
We have studied the expression of eleven osteoclast signalling genes and have found that one of the ways in which IL-10 directly inhibits osteoclastogenesis is by disrupting NFATc1 mRNA expression and nuclear translocation. NFATc1 is crucial for osteoclastogenesis, and so its downregulation may be expected to have major downstream effects. We also provide evidence that this disruption to NFATc1 may be mediated through the effect of IL-10 on Ca2+ mobilisation. In addition, it was found that only NFATc1 isoform 1 transcribed from promoter P1 was downregulated by IL-10.
Osteoclast formation was not abolished in the presence of IL-10 and similarly NFATc1 levels did not revert to basal level in the IL-10 treated cells. Several pathways contribute to induction of NFATc1 levels and it may well be the case that IL-10 does not inhibit all of them. The initial expression of NFATc1 is dependent on both the TRAF6 and c-Fos pathways, which are unaffected by IL-10. RANKL stimulation also results in the induction of Ca2+ oscillations which activate NFATc1 via a calcineurin-dependent mechanism, targeting it to the nucleus . For the generation of osteoclasts it has been demonstrated that the high NFATc1 levels generated by autoregulation are necessary for cell lineage commitment [reviewed in ]. Therefore the reduction in levels, rather than complete abrogation, brought about by IL-10 will be sufficient to cause the potent inhibition of osteoclastogenesis shown in these studies.
There are two variants of NFATc1 in Mus musculus but only one of these, isoform1, was downregulated by IL-10. It is not known why the shorter isoform 2 is not similarly modified by IL-10. This may in part be related to the relative inability of RANKL to induce the expression of isoform 2 in RAW cells, or it could be that the conformation of the DNA is such that IL-10-induced transcription factors only have access to the P1 promoter. Linked in with this, it is likely that the finding that NFATc1 expression was not completely abolished is due to a combination of residual expression from the P1 promoter and the inability of IL-10 induced factors to suppress basal expression from the P2 promoter. Alternatively, it could also be that IL-10 acts only on a subset of the various pathways that contribute to NFATc1 upregulation.
The activity of NFATc1 is not only dependent on its level of expression, but also its location within the cell. It is active only when in the nucleus, as would be expected of a transcription factor. This nuclear transport relies upon active calcineurin which triggers dephosphorylation of NFATc1. Therefore we used antibodies to establish the intracellular distribution of NFATc1. The fact that very little staining, and in particular nuclear staining, was observed in the IL-10 sample strengthens our evidence that the inhibitory effect of IL-10 on osteoclastogenesis can be explained at least in part by its effect on NFATc1 expression and activity.
PKC plays a critical role in T cell receptor-induced NFAT activation . Deficiency of PKC primarily abrogates NFAT transactivation. This NFAT transactivation defect appears to be secondary to reduced inositol 1,4,5-triphosphate generation and intracellular Ca2+ mobilisation. Therefore, we set out to test whether IL-10 is disrupting NFATc1 activity through its effect on Ca2+ mobilisation. We found that PMA inhibits the suppressive effect of IL-10 on osteoclast formation, as determined by TRAP staining and NFATc1 expression. PMA is an activator of PKC, and so if PMA is overcoming the suppressive effects of IL-10, knowing how PMA acts it would appear that IL-10 is disrupting NFATc1 activity through its effect on Ca2+ mobilisation. Therefore the effect of IL-10 could involve PKC as PMA reverses the suppressive action of IL-10.
The mechanism by which IL-10 interferes with osteoclast formation appears to be different from that used by other inhibitors, namely IFN-γ, IL-4, and TRAIL. IL-4 also downregulates NFATc1 but it acts through suppression of c-Fos , we did not find any evidence for c-Fos being downregulated although it remains possible that it could be inactivated by other means. IFN-γ causes rapid degradation of TRAF6 protein; we found no difference in TRAF6 mRNA levels but did not look at protein levels. Likewise, TRAIL interferes with the p38 pathway and there was no downregulation of p38 mRNA in IL-10 treated cultures.
It is valuable to pinpoint the effect of IL-10 on osteoclast signalling pathways since evidence is accumulating, particularly from studies of IL-10-deficient mice, to illustrate the importance of this cytokine in bone homeostasis. We have demonstrated that one of the ways by which IL-10 directly inhibits osteoclast formation is to suppress NFATc1 expression and activation. In vivo, IL-10 may be expected to have an even more pronounced effect due to its indirect action of suppressing RANKL expression  which would further reduce NFATc1 activation through TRAF6. This is the first report indicating that one of the ways in which IL-10 inhibits osteoclastogenesis is by suppressing NFATc1 activity.