Rheumatoid arthritis (RA) is a chronic inflammatory disease with a complex etiology. Juxtaarticular bone loss occurring around the inflamed joints and generalized systemic bone loss are common features of RA (for review, see refs.1–3). One of the main characteristics of RA is a dense lymphoid cell infiltration into the synovial membrane. Activated T cells are now considered to be potent modulators of bone turnover and are a key source of osteoclastogenic cytokines under inflammatory conditions such as RA (4, 5) and periodontitis (6, 7) and in estrogen deficiency (8–11). We recently reported that activated T cells secrete cytokines that potently stimulate the differentiation of human bone marrow stromal cells into osteoblasts (12, 13) and also secrete an unknown factor that is capable of stimulating the production of interleukin-6 (IL-6) by osteoblasts (14). IL-6 is an osteoclastogenic factor that has been implicated in the bone destruction associated with estrogen deficiency in humans (15–17) and in mice (18, 19) and in the inflammation and osteoporosis associated with RA (14, 16, 20).
In addition, activated T cells have long been known to stimulate osteoclast formation (21–24). T cell–derived production of tumor necrosis factor α (TNFα) has been reported to play a critical role in ovariectomy-induced bone loss in mice (25), and T cell–derived RANKL is reported to be relevant in animal models of RA (26). We and other investigators (27–30) have reported that activated T cells stimulate osteoclastogenesis in vitro by secretion of RANKL. Interestingly, our observations prompted the further controversial finding that activated T cells also significantly induce osteoclast formation by a mechanism that is independent of RANKL, since saturating concentrations of the RANKL inhibitor osteoprotegerin (OPG) failed to neutralize more than 30% of the observed osteoclast formation induced by activated T cells (28).
In the present study, with the use of sequential biochemical purification, mass spectrometry, and recombinant DNA technologies, we identified and analyzed the expression of a novel activated human T cell–secreted cytokine, herein referred to as secreted osteoclastogenic factor of activated T cells (SOFAT). This single cytokine was observed to elicit RANKL- and osteoblast-independent osteoclast formation in an OPG-insensitive manner and also to stimulate IL-6 production by osteoblasts.
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Threonine synthase is a pyridoxal-5′-phosphate–dependent enzyme that synthesizes L-threonine in plants and microorganisms. However, L-threonine is an essential amino acid, since it is not biosynthetically generated in insects, birds, and mammals. Interestingly, 2 homologs of threonine synthase, THNSL1 and THNSL2, have nonetheless been conserved throughout evolution and persist in rodents and mammals, including humans (33). The high degree of sequence conservation of threonine synthase genes through evolution is surprising, given the apparent lack of L-threonine synthesis in higher organisms. The full-length THNSL2 protein has been reported to bind pyridoxal-5′-phosphate and O-phospho-homoserine, degrading them to alpha-ketobutyrate, phosphate, and ammonia, and degrading O-phospho-threonine to alpha-ketobutyrate, suggesting that the protein exhibits catabolic phospholyase activity (33). Interestingly, SOFAT does not contain the entire pyridoxal-5′-phosphate binding domain, suggesting that unlike threonine synthase, SOFAT is not pyroxidal-5′-phosphate dependent.
Our results suggest that a secreted fragment coded by the threonine synthase homolog may play a critical, previously unrecognized function in the immune/ skeletal interface. Furthermore, the enhanced expression of this cytokine may drive inflammation and bone destruction in pathologic conditions that are characterized by exuberant T cell activation, such as in RA.
We have previously reported that activated T cells produce factors capable of inducing osteoblastic IL-6 production (14) as well as factors that stimulate osteoclastogenesis, including RANKL (27, 28). However the role of RANKL in inflammatory bone destruction is not clear. RANKL has been implicated in systemic bone destruction in animal models of RA (26). However, patients with RA have elevated levels of serum OPG (40), and the OPG:RANKL ratio tends to be high in the synovium. Furthermore, there is often no correlation between the levels of synovial RANKL and disease severity (41). It is thus possible that SOFAT is a critical osteoclastogenic factor in RA.
TNFα has also been found to induce osteoclasts synergistically with RANKL (42) as well as to act independent of RANKL (43), although these latter effects are controversial. Although SOFAT was not found to require TNFα to induce osteoclast formation, the presence of TNFα potently amplified osteoclastogenesis. We speculate that the high levels of TNFα present in RA may act to potently magnify SOFAT-induced osteoclastogenesis and bone loss.
In T cells, the RANKL gene is regulated by a calcineurin-dependent signaling pathway (44). In contrast, SOFAT is secreted by activated T cells via a calcineurin-independent pathway, as demonstrated by findings indicating that CSA treatment of activated T cells failed to block production of this cytokine (14). Taken together, these observations suggest that SOFAT production by T cells is stimulated by an intracellular pathway different from that utilized by RANKL, although the specific pathways involved remain to be elucidated.
The role of IL-6 in bone resorption has been primarily associated with postmenopausal osteoporosis, but IL-6 may also play a role in the bone loss seen in RA and other inflammatory arthritides (20). Our results demonstrated that SOFAT is unable to stimulate, or augment, osteoclast formation by directly inducing IL-6 secretion by osteoclast precursors. However, IL-6 has been reported to stimulate RANKL production by synovial fibroblasts derived from patients with RA and to mediate TNFα-induced RANKL production in this system (45). IL-6 also functions to induce the proliferation of mononuclear osteoclast precursor cells (46). The demonstration that SOFAT is a potent inducer of IL-6 by osteoblasts suggests that SOFAT could play a significant role in the local inflammatory response, and also could exacerbate bone destruction in RA indirectly through multiple IL-6–mediated events. Interestingly, results of a new study suggest that IL-6 modulates production of T cell–derived cytokines in antigen-induced arthritis and drives inflammation-induced osteoclastogenesis (47). These findings suggest the potential existence of a feedback mechanism between activated T cells and osteoblasts, and SOFAT could play a critical role in the coupling of bone cells to the adaptive immune response, thus perpetuating inflammation and osteoclastic bone resorption.
SOFAT may represent the first in a potential family of novel cytokines possessing biologic activities in the absence of classic cytokine-like motifs. The pathophysiologic functions and mechanisms of action of SOFAT remain to be elucidated.