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Editorial: Cytokines, Estrogen, and Postmenopausal Osteoporosis—The Second Decade

Washington University School of Medicine and Barnes-Jewish Hospital St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Roberto Pacifici, M.D., Division of Bone and Mineral Diseases, Washington University School of Medicine, Barnes-Jewish Hospital, North Campus, 216 S. Kingshighway, St. Louis, Missouri 63110. E-mail: Pacifici{at}imgate.wustl.edu

	Introduction

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Postmenopausal osteoporosis is a heterogeneous disorder characterized by a progressive loss of bone tissue that begins after natural or surgical menopause and leads to fracture within 15–20 yr from the cessation of the ovarian function. Although suboptimal skeletal development ("low peak bone mass") and age-related bone loss may be contributing factors, a hormone-dependent increase in bone resorption and accelerated loss of bone mass in the first 5 or 10 yr after the menopause appears to be the main pathogenetic factor.

During the past decade, considerable evidence has accumulated that suggests that estrogen prevents bone loss by blocking the production of proinflammatory cytokines by bone marrow and bone cells (1, 2). The main consequence of increased cytokine production in the bone microenvironment is an expansion of the osteoclastic pool due to increased osteoclast (OC) formation and elongation of their life-span (3). In addition, enhanced cytokine production results in increased activity of mature OCs and in increased osteoblastic activity. The latter compensates, in part, the consequences of increased bone formation on bone mass.

Cytokines exert their regulatory effects on bone turnover by stimulating both the secretory and proliferative activities of mature cells. However, they also condition the differentiation of immature cells leading to the emergence of new phenotypes that favor osteoclastogenesis. Examples of this phenomenon are the differentiation of stromal cells precursors in "high osteoclastogenic" stromal cells, as a result of the increased bone marrow levels of interleukin (IL)-1 and tumor necrosis factor (TNF) induced by estrogen deficiency (4), and the differentiation of cells of the monocytic lineage into osteoclasts in response to factors produced by stromal cells such as IL-6, IL-11 (2), and macrophage colony-stimulating factor (M-CSF) (5).

Among the cytokines now known to be regulated by estrogen are IL-1, IL-6, and TNF (1, 2). IL-1 and TNF are the most powerful locally produced stimulators of bone resorption and are well recognized inhibitors of bone formation. These cytokines promote bone resorption in vitro and cause bone loss and hypercalcemia when infused in vivo. IL-1 and TNF activate mature osteoclasts indirectly via a primary effect on osteoblasts and inhibit osteoclast apoptosis. In addition, they markedly enhance osteoclast formation by stimulating the proliferation of osteoclast precursor both directly and by enhancing the pro-osteoclastogenic activity of stromal cells. IL-1 and TNF are also powerful inducers of other cytokines that regulate the differentiation of osteoclast precursor cells into mature osteoclasts, such as IL-6, M-CSF, and granulocyte M-CSF. Therefore, with respect to osteoclastogenesis, IL-1 and TNF should be regarded as "upstream" cytokines necessary for inducing the secretion of "downstream" factors that stimulate hematopoietic osteoclast precursors. This cascade mechanism ensures that small changes in IL-1 and TNF levels results in large changes in osteoclast production. A specific competitive inhibitor of IL-1, known as IL-1ra, has also been identified (6). IL-1ra, which has a 26% amino acid sequence homology with IL-1ß, binds to cells expressing primarily the 87-kD type I IL-1 receptor with nearly the same affinity as IL-1 and competes with either IL-1 or IL-1ß on these cells without detectable IL-1 agonist effects. The type I IL-1 receptor is expressed in T cells, tissue macrophages, endothelial cells, and bone cells. IL-1ra also binds, although with a lower affinity, to the type II IL-1 receptor, which is expressed mainly in blood neutrophils and B cells. Because the binding of 5 molecules of IL-1 per cell is sufficient to induce a full biological response, a 50% IL-1 inhibition in bone cells requires amounts of IL-1ra up to 100 times in excess of the amounts of IL-1 or IL-1ß present.

IL-6 is a potent osteoclastogenic factor that exerts its effects via a cell surface receptor that consists of a ligand binding chain (IL-6R) and a signal transducing chain known as gp130. When bound to soluble IL-6R, IL-6 stimulates the early stages of osteoclastogenesis in human and murine cultures, presumably by forming a complex with gp130 expressed on either stromal cell or osteoblasts (7). IL-6 increases in vitro bone resorption in systems rich in osteoclast precursors, such as the mouse fetal metacarpal assay (8), whereas it has no effect in organ cultures where more mature cells predominate, such as murine fetal radii (9). This suggests that IL-6 is more potent in increasing the formation of osteoclasts from hemopoietic precursors than in activating mature osteoclasts.

Over the past decades, numerous reports have been published demonstrating that natural or surgical menopause increases blood, bone marrow, and monocytic levels of IL-1, IL-6, TNF, and the related factors IL-1ra and IL-6R (1, 2). In vitro studies have also documented the ability of estrogen treatment to suppress the production of these three cytokines by bone and bone marrow cells. In spite of these observations, controversy persists concerning the specific contribution of each of these factors to postmenopausal bone loss.

In this issue of Endocrinology, Lorenzo et al. (10) report that mice insensitive to IL-1 due the absent expression of type I IL-1 receptor (IL-IRI) are protected against the bone loss induced by ovx, thus demonstrating that IL-1 is an essential mediator of the effects of estrogen deficiency in bone. The findings of Lorenzo et al. (10) are particularly convincing because IL-1RI deficient mice have both normal bone mass and growth rates. These data exclude the possibility that resistance to ovx-induced bone loss may results from an abnormal skeleton development induced by the IL-1RI deficiency. Lorenzo et al. (10) also confirmed their findings in a second strain of IL-1RI deficient mice characterized by a distinct genetic backgrounds. These is an important precaution that mitigates the concern that the results of the study may be explained by a genetic anomaly. Ovx-induced bone loss results, in fact, from the impact of estrogen deficiency on a normally developed skeleton. Conversely, in conventional knock-out mice, the muted gene may alter both the development and the involution of the skeleton. Thus, knock-out mice may be characterized by bone modeling and remodeling defects that ensue during fetal development and lead to the formation of an abnormal mature skeleton.

A critical finding of the study of Lorenzo et al. is that the lack of IL-1RI does not alter bone mass in sham-operated mice, thus demonstrating that IL-1 is not essential for maintaining normal bone remodeling in estrogen replete mice. However, in response to estrogen withdrawal, IL-1 induces bone loss either directly or by inducing the secretion of "downstream factors" that stimulate osteoclastogenesis and bone loss.

IL-1 dependent bone loss may result from either increased production of the cytokine or increased responsiveness of target cells to IL-1. The study of Lorenzo et al. (10) did not address this question, as bone marrow levels of IL-1 were not measured. However, published data provide evidence that estrogen deficiency increases the cell response to IL-1 either by modulating the expression of IL-1 receptors on IL-1 target cells, or by decreasing antagonistic molecules that prevent the binding of IL-1 to its signaling receptor. Previous studies have, in fact, shown that ovx increases IL-1 bioactivity (as measured by bioassay) but not IL-1ß levels (as measured by ELISA or IRMA). IL-1 bioactivity reflects the relative amounts of biologically active IL-1 and IL-1 antagonists present in the test sample. Consequently, IL-1 bioassays provide a reliable estimate of target cell response to IL-1. In contrast, ELISAs and IRMAs, provide a measurement of the net amount of IL-1 but do not provide information on the amount of biologically active IL-1, which binds to the signaling type I IL-1 receptor. The binding of IL-1 to the type I receptor is, in fact, antagonized by IL-1ra, soluble type I (sIL-1RI) and type II IL-1 receptor (sIL-1RII) (11, 12), anti IL-1a autoantibodies and IL-1ß binding proteins. Moreover, whereas sIL-1RI antagonizes the effects of IL-1ra (12), sIL-1 RII binds IL-1ß but does not bind IL-1ra (11, 12). Thus, sIL-1 RII can compete with cell-associated receptors for IL-1ß and potentiate the inhibitory action of IL-1ra. Because, at least in humans, estrogen regulates the monocytic production of IL-1ra, it is possible that ovx may result in increased IL-1 bioactivity without changes in IL-1 levels.

Recent studies have also shown that estrogen increases the expression of the decoy type II, IL-1 receptor in bone marrow cells and osteoclasts (13). Thus upregulation of cell responsiveness to IL-1 via down-regulation of IL-1RII is also likely to be a key mechanism by which estrogen deficiency induces bone loss.

The findings of Lorenzo et al. are not in contrast with a previous report indicating that IL-1ß deficient mice are not protected against ovx-induced bone loss (6). Simultaneous suppression of both the IL-1 and IL-1ß genes is, in fact, required for blocking ovx-induced bone loss, as demonstrated by the finding that the functional block of both species of IL-1 by infusion of IL-1ra duplicates the bone-sparing effects of estrogen (14).

The study of Lorenzo et al. confirm and expand the evidence supporting the hypothesis that IL-1 is an essential mediator of the effects of estrogen deficiency in bone. However, two other cytokines, TNF and IL-6, are known to be regulated by estrogen and are suspected to play a causal role in inducing bone loss in conditions of estrogen deficiency. The published evidence supporting the notion that TNF (either alone or in cooperation with IL-1) is a key mediator of the effects of estrogen deficiency in bone is particularly compelling. Treatment of ovx mice with TNF binding protein (TNFbp), a potent inhibitor of TNF, completely prevents the bone loss and the increase in OC formation and bone resorption induced by ovx, whereas no effects are observed in sham-operated mice (15). Moreover, a recent elegant study by Ammann et al. has demonstrated that transgenic mice insensitive to TNF due to the overexpression of soluble TNF receptor, are also protected against ovx-induced bone loss (16). Finally, an orally active inhibitor of IL-1 and TNF production has also been shown to completely prevent bone loss in ovx rats (17). Although the finding that functional block of either IL-1 or TNF is sufficient to prevent ovx-induced bone loss may appear to be difficult to reconcile, it should be emphasized that in most biological system IL-1 and TNF have potent synergistic effects. Thus, the functional block of one of these two cytokines elicits biological effects identical to those induced by the block of both IL-1 and TNF. The long-term stimulation of bone resorption that follows ovx is sustained primarily by an expansion of the osteoclastic pool. Because OC formation is synergistically stimulated by IL-1 and TNF (18), it is not surprising that long-term inhibition of either IL-1 or TNF results in complete prevention of ovx-induced bone loss. Apparently in conflict with this interpretation, is a study in the rat demonstrating that in the first 2 weeks after ovx bone loss is prevented, in part, by either IL-1ra or TNFbp. However, simultaneous treatment with IL-1ra + TNFbp is required to completely prevent ovx-induced bone loss (19). Because the acute stimulation of bone resorption that occurs in the first 2 weeks after ovx is sustained primarily by the increased activity of preexisting, mature OCs, the data suggest that IL-1 and TNF have weak additive effects on mature OCs. Thus simultaneous inhibition of both cytokines is required to restore normal bone resorption.

Whereas studies with transgenic mice and inhibitors of IL-1 and TNF have consistently demonstrated that IL-1 and TNF are key inducers of bone loss in ovx animals, investigations aimed at assessing the contribution of IL-6 to ovx-induced bone loss have yielded conflicting results. In favor of a causal role for IL-6 in ovx-induced bone loss is the report of Poli et al. (20) indicating that IL-6 knock out mice are protected against the loss of trabecular bone induced by ovx. Against a significant pathogenetic role of IL-6 are studies demonstrating that osteoporosis is not a feature of transgenic mice overexpressing IL-6 (21). Studies have also been conducted by injecting an antibody neutralizing IL-6 in ovx mice. Neutralizing IL-6 prevents the increase in OC formation induced by estrogen deficiency (15, 22) but does prevent ovx-induced bone loss and does not decrease in vivo bone resorption (15). These findings confirm that IL-6 contributes to the expansion of the osteoclastic pool induced by ovx. However, this cytokine does not appear to be the dominant factor in inducing bone loss in estrogen deficient mice.

In summary, there is now substantial evidence supporting the hypothesis that a network of estrogen-regulated cytokines is responsible for the changes in bone turnover and the loss of bone induced by estrogen deficiency. It is likely that during the current decade the development of orally active, tissue selective cytokine inhibitors will lead to new strategies for the prevention and treatment of postmenopausal osteoporosis.

Received March 26, 1998.

	References

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This Article Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pacifici, R. Search for Related Content PubMed PubMed Citation Articles by Pacifici, R. Pubmed/NCBI databases Substance via MeSH Medline Plus Health Information Osteoporosis