Estrogen deficiency increases osteoclastogenesis up-regulating T cells activity: A key mechanism in osteoporosis
Introduction
Post-menopausal osteoporosis is a systemic skeletal disorder characterised by reduced bone mineral density (BMD), micro architectural deterioration of bone tissue resulting in fragility and susceptibility to fractures [1] and uncoupling of osteoblast-mediated bone formation and osteoclast (OC)-mediated bone resorption.
Post-menopausal osteoporosis stems from the cessation of ovarian function at menopause and from genetic and non genetic factors which heighten and prolong the rapid phase of bone loss characteristic of the early post-menopausal period. The anti resorptive activity of estrogen is a result of multiple genomic and non genomic effects on bone marrow and bone cells, which leads to decreased OC formation, increased OC apoptosis and decreased capacity of mature OCs to reabsorb bone. Although it is now recognized that stimulation of bone resorption in response to estrogen deficiency is mainly due to cytokine-driven increases in OC formation, the responsible factors are not completely understood. OC formation occurs when monocytes are co-stimulated by the essential osteoclastogenic factors RANKL and M-CSF [2], [3], [4], but additional inflammatory cytokines are responsible for the up-regulation of OC formation observed in estrogen deficiency. One of the cytokines responsible for the augmented osteoclastogenesis in states of estrogen deficiency is TNFα, a factor which enhances OC formation by up-regulating stromal cell production of RANKL and M-CSF, and by augmenting the responsiveness of OC precursors to RANKL [5], [6]. Furthermore, TNF directly induces marrow precursor differentiation into OCs in the absence of RANKL, although according to some studies, TNF is not osteoclastogenic in cells not previously primed by RANKL [7]. However, the source of this cytokine, which is relevant to post-menopausal bone loss in humans, remains to be determined. Studies in mice suggest that activated T cells are the most relevant source of TNF in conditions of estrogen deficiency [8], [9]. In support of this hypothesis there are reports demonstrating that T cell deficient nude mice are protected against post-ovariectomy bone loss [10], [11]. Furthermore, data show that adoptive transfer of wild type T cells restores the capacity of ovariectomy to induce bone loss, while transfer of T cells from TNF null mice does not [8], [9], [10], [12]. Other studies argued against a pivotal role of T cells in bone loss induced by ovariectomy in mice models [13], [14], [15]. In particular, Lee et al [13] suggested that nude mice lose trabecular bone as well as wild type after ovariectomy and that T cells may have important effects on the cortical rather than on the trabecular compartment.
Little information is available regarding the role of T cells in human bone loss. Studies show a key role of T cell produced TNF in rheumatoid arthritis [16], multiple myeloma [17], [18] and bone metastasis [5], [19]. Other reports show that estrogen deficiency increases the production of TNF and RANKL by bone marrow cells, including T cells, and that their increase correlate with indices of bone resorption [20], [21], [22].
This cross-sectional investigation was designed to determine how estrogen deficiency affects the production of osteoclastogenic cytokines by peripheral blood mononuclear cells (PBMC), OC formation and bone resorption.
We report that estrogen deficiency increases the ability of PBMC to form OCs in vitro and that this ability is due both to increased output of OC precursors from bone marrow and to an increased production of TNFα. Furthermore, we demonstrate that T cells are more active in women affected by post-menopausal osteoporosis as respect to post-menopausal healthy women and to pre-menopausal healthy controls. We suggest T cell cytokine production as the primary driver of osteoclastogenesis in post-menopausal osteoporosis.
Section snippets
Experimental subjects and markers of bone turnover
The study was approved by the human study review board of the Azienda Sanitaria Ospedaliera San Giovanni Battista in Torino. The study population included twenty-five post-menopausal women with osteoporosis, 23 healthy post-menopausal and 10 healthy pre-menopausal women. All study subjects had levels of 25-OH vitamin D, bone alkaline phosphatase (BAP) activity and routine blood tests within normal limits. Subjects with secondary forms of osteoporosis or taking drugs active on bone turnover such
Osteoclasts formation is higher in women affected by post-menopausal osteoporosis
Unstimulated PBMC cultures from osteoporotic women produced more OCs than those from controls, whereas this difference disappeared when the cultures were stimulated with M-CSF and RANKL. Furthermore, the OCs formed in patients are more active than those formed in controls in unstimulated condition as demonstrated by the higher percentage of hydroxyapatite resorbed (Fig. 1). PBMC from pre-menopausal controls do not form OCs nor reabsorb hydroxyapatite in unstimulated condition by contrast with
Discussion
Although estrogen deficiency is known to induce bone loss through various mechanisms, to our knowledge, this report is the first to demonstrate the contribution of T cells to cytokine-driven osteoclastogenesis in post-menopausal osteoporosis. We found menopause to increase the number of OC precursors in the PBMC and the ability of T cells to produce pro-osteoclastogenic cytokines. Estrogen deficiency leads to an increase in OC formation more relevant in patients affected by osteoporosis.
Acknowledgments
This work was supported by a grant from the Ministry for Education, the Universities and Research (MIUR) and a grant from the Fondazione Internazionale Ricerche Medicina Sperimentale (FIRMS) Compagnia San Paolo. S.Z.M. Brianza and P. D'Amelio were supported by a fellowship of MIUR (COFIN 2003).
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These two authors contributed equally to the present paper.