A novel T cell cytokine, secreted osteoclastogenic factor of activated T cells, induces osteoclast formation in a RANKL-independent manner.

OBJECTIVE: Chronic T cell activation is central to the etiology of rheumatoid arthritis (RA), an inflammatory autoimmune disease that leads to severe focal bone erosions and generalized systemic osteoporosis. Previous studies have shown novel cytokine-like activities in medium containing activated T cells, characterized by potent induction of the osteoblastic production of interleukin-6 (IL-6), an inflammatory cytokine and stimulator of osteoclastogenesis, as well as induction of an activity that directly stimulates osteoclast formation in a manner independent of the key osteoclastogenic cytokine RANKL. This study was undertaken to identify the factors secreted by T cells that are responsible for these activities. METHODS: Human T cells were activated using anti-human CD3 and anti-human CD28 antibodies for 72 hours in AIM V serum-free medium to obtain T cell-conditioned medium, followed by concentration and fractionation of the medium by fast-protein liquid chromatography. Biologically active fractions were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major bands were analyzed by mass spectrometry, and a major candidate protein was identified. This novel cytokine was cloned, and its expression was analyzed using recombinant DNA technologies. RESULTS: A single novel cytokine that could induce both osteoblastic IL-6 production and functional osteoclast formation in the absence of osteoblasts or RANKL and that was insensitive to the effects of the RANKL inhibitor osteoprotegerin was identified in the activated T cell-conditioned medium; this cytokine was designated secreted osteoclastogenic factor of activated T cells (SOFAT). Further analysis of SOFAT revealed that it was derived from an unusual messenger RNA splice variant coded by the threonine synthase-like 2 gene homolog, which is a conserved gene remnant coding for threonine synthase, an enzyme that functions only in microorganisms and plants. CONCLUSION: SOFAT may act to exacerbate inflammation and/or bone turnover under inflammatory conditions such as RA or periodontitis and in conditions of estrogen deficiency.

The mechanism of phosphorus as a cardiovascular risk factor in CKD.

Hyperphosphatemia and vascular calcification have emerged as cardiovascular risk factors among those with chronic kidney disease. This study examined the mechanism by which phosphorous stimulates vascular calcification, as well as how controlling hyperphosphatemia affects established calcification. In primary cultures of vascular smooth muscle cells derived from atherosclerotic human aortas, activation of osteoblastic events, including increased expression of bone morphogenetic protein 2 (BMP-2) and the transcription factor RUNX2, which normally play roles in skeletal morphogenesis, was observed. These changes, however, did not lead to matrix mineralization until the phosphorus concentration of the media was increased; phosphorus stimulated expression of osterix, a second critical osteoblast transcription factor. Knockdown of osterix with small interference RNA (siRNA) or antagonism of BMP-2 with noggin prevented matrix mineralization in vitro. Similarly, vascular BMP-2 and RUNX2 were upregulated in atherosclerotic mice, but significant mineralization occurred only after the induction of renal dysfunction, which led to hyperphosphatemia and increased aortic expression of osterix. Administration of oral phosphate binders or intraperitoneal BMP-7 decreased expression of osterix and aortic mineralization. It is concluded that, in chronic kidney disease, hyperphosphatemia stimulates an osteoblastic transcriptional program in the vasculature, which is mediated by osterix activation in cells of the vascular tunica media and neointima.

The role of noggin in human mesenchymal stem cell differentiation.

Noggin is a secreted protein that inhibits the binding of bone morphogenetic proteins (BMPs) to their cognate receptor. Its role in human mesenchymal stem cell differentiation has not been well studied. Here, we studied the effect of noggin on human mesenchymal stem cell differentiation induced by inflammatory cytokines (activated T-cell conditioned medium (ACTTCM) or the combination of four T-cell cytokines, TNF-alpha, TGF-beta, IFN-gamma, and IL-17 (TTII)), BMPs, or dexamthasone (DEX). HMSC treated with TTII alone rapidly induced alkaline phosphatase (AlkP) activity. Inclusion of noggin resulted in an additive effect. Noggin acted additively with DEX to induce a significantly higher level of AlkP induction than either noggin or DEX alone. Noggin was examined for its ability to inhibit mineralization in long-term cultures of HMSC stimulated with BMP-2, BMP-6, BMP-7, DEX, or TTII. Surprisingly, noggin alone induced mineralization while it did not inhibit mineralization induced by TTII or BMP-2, BMP-6, or BMP-7. Interestingly, when HMSC were treated with both noggin and DEX they acted synergistically to induce mineralization nearly 3-fold over DEX alone and 30-fold over noggin alone. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that T-cell cytokines induced noggin, Runx2, BMP-2, and osteocalcin gene expression, while noggin alone induced BMP-2 and osteocalcin gene expression, but not Runx2, although it increased the expression of ActRII, a receptor for BMP-2. These results suggest that in HMSC, the anabolic action of inflammation on bone formation occurs through the induction of noggin, which then induces BMP-2 receptor and BMP-2 leading to the activation of the differentiation process.

T-cell cytokine induction of BMP-2 regulates human mesenchymal stromal cell differentiation and mineralization.

How T-cells, attracted to local sites of inflammation in arthritides, affect heterotopic ossification is presently unknown. Here, we tested the hypothesis that T-cell cytokines play a role in the differentiation of human mesenchymal stromal cells (HMSC) into the osteoblast phenotype by inducing autologous BMP-2, providing a possible mechanism for heterotopic ossification. HMSC from multiple donor bones were treated with either activated T-cell conditioned medium (ACTTCM) or physiological concentrations of the major inflammatory cytokines, TNF-alpha, TGF-beta, IFN-gamma, and IL-17 (TTII), individually or in combinations. ACTTCM induced BMP-2 protein in a time-dependent manner over a 48 h period and alkaline phosphatase (AlkP) within 7 days. In combination, TTII, like ACTTCM, induced AlkP and synergistically induced BMP-2 protein. Either individually, or in combinations of up to three, the T-cell cytokines failed to induce BMP-2 above control levels while a combination of all four cytokines synergistically induced BMP-2 10-fold as assessed by ELISA. TTII induced mineralized matrix as effectively as dexamethasone. Inhibition of p38 MAPK completely inhibited TTII-induced BMP-2 production and matrix mineralization. Real time RT-PCR analysis demonstrated a striking early (within 4 h) increase in BMP-2 gene expression by TTII, which was suppressed by p38 MAP kinase inhibition. In localized chronic inflammatory diseases, T-cell cytokines released at localized sites of inflammation may be the driving force for differentiation of local mesenchymal stromal cells into the osteoblast phenotype thereby playing a significant role in the heterotopic ossification observed in these diseases.

IL-13 regulates vascular cell adhesion molecule-1 expression in human osteoblasts.

Activated T cells (Act T) produce multiple cytokines that affect osteoblast function as well as osteoclastogenesis. One of these cytokines, IL-13, is a multifunctional cytokine elaborated by Act T that regulates vascular cellular adhesion molecule (VCAM)-1 expression in endothelial cells. VCAM-1 has also been implicated in osteoclast formation by myeloma cells. We therefore studied whether IL-13 regulates VCAM-1 in human osteoblastic cells since these cells express RANKL, the major osteoclastogenic factor and osteoclast precursors are found adjacent to osteoblasts. Human T cells were activated in the absence or presence of Cyclosporin A (CsA), an inhibitor of the production of most activated T cell cytokines. Conditioned media were assayed for IL-13 by ELISA. Act T produced IL-13 and, unlike other T cell cytokines, this was elevated 3-fold by CsA. Exposure of human osteoblasts (hOB) to doses of recombinant human IL-13 (rhIL-13, 0-10 ng/ml) resulted in an increase of VCAM-1 mRNA (up to 5-fold) within 4 h with a maximum stimulation at 1 ng/ml. CsA had no effect on basal hOB VCAM-1 mRNA expression. Examination of VCAM-1 on the cell surface of hOB, by immunocytochemistry, revealed increasing levels of surface expression of the protein within 16 h after stimulation with doses of rhIL-13 (0.1-10 ng/ml) which were reflective of the mRNAs. IL-6 production was also stimulated in a dose dependent manner with a maximum of 2.5-fold with 1 ng/ml rhIL-13 within 16 h. Since both VCAM-1 and IL-6 showed similar responses to IL-13, IL-6 was examined for its ability to induce VCAM-1. Immunocytochemistry demonstrated no effect of IL-6 on VCAM-1 expression. These data demonstrate that during pathological processes associated with T cell activation, such as rheumatoid arthritis or possibly post-menopausal osteoporosis, T cells may play a pivotal role in osteoclast precursor adhesion to osteoblasts as a first step prior to RANKL signaling.

T cells regulate the expression of matrix metalloproteinase in human osteoblasts via a dual mitogen-activated protein kinase mechanism.

OBJECTIVE: To investigate the role of T cell induction of matrix metalloproteinase 13 (MMP-13) production by human osteoblasts in order to better understand the process of bone loss in rheumatoid arthritis (RA). METHODS: Activated T cell-conditioned medium (ACTTCM) was used to mimic the physiologic conditions of inflammation. MMP-13 production by human osteoblasts was assessed using a specific enzyme-linked immunosorbent assay. Specific inhibitors of the p38 mitogen-activated protein (MAP) kinase and the extracellular signal-regulated kinase 1/2 (ERK-1/2) MAP kinase signaling pathways were used to assess their roles in T cell-mediated MMP-13 production. Finally, recombinant cytokines representative of the major components in ACTTCM were assessed for their ability to induce MMP-13. RESULTS: ACTTCM powerfully induced MMP-13 in human osteoblasts. Inhibition of p38 activity abolished, while inhibition of ERK-1/2 activity enhanced, MMP-13 production. We next investigated physiologic levels of the T cell cytokines tumor necrosis factor alpha (TNFalpha), transforming growth factor beta (TGFbeta), interferon-gamma (IFNgamma), and interleukin-17 (IL-17) for their roles in MMP-13 induction. Although individual cytokines had no significant effect, the combination of TNFalpha, TGFbeta, IFNgamma, and IL-17 resulted in a dramatic p38-dependent induction of MMP-13 identical to that produced by ACTTCM. CONCLUSION: These studies demonstrate for the first time that human osteoblasts produce MMP-13. The results also show that under conditions of chronic inflammation, multiple T cell cytokines synergize to induce high levels of MMP-13 via a mechanism that is dependent on activated p38 MAP kinase and is suppressed by activated ERK-1/2. Selective inhibition of p38 activity may offer a target for pharmacologic inhibition of bone loss in RA.

Inflammatory T cells rapidly induce differentiation of human bone marrow stromal cells into mature osteoblasts.

Activated T cells secrete multiple osteoclastogenic cytokines which play a major role in the bone destruction associated with rheumatoid arthritis. While the role of T cells in osteoclastogenesis has received much attention recently, the effect of T cells on osteoblast formation and activity is poorly defined. In this study, we investigated the hypothesis that in chronic inflammation activated T cells contribute to enhanced bone turnover by promoting osteoblastic differentiation. We show that T cells produce soluble factors that induce alkaline phosphatase activity in bone marrow stromal cells and elevated expression of mRNA for Runx2 and osteocalcin. This data indicate that T cell derived factors have the capacity to stimulate the differentiation of bone marrow stromal cells into the osteoblast phenotype. RANKL mRNA was undetectable under any conditions in highly purified bone marrow stromal cells. In contrast, RANKL was constitutively expressed in primary osteoblasts and only moderately up-regulated by activated T cell conditioned medium. Interestingly, both bone marrow stromal cells and osteoblasts expressed mRNA for RANK, which was strongly up-regulated in both cell types by activated T cell conditioned medium. Although, mRNA for the RANKL decoy receptor, osteoprotegerin, was also up-regulated by activated T cell conditioned medium, it's inhibitory effects may be mitigated by a simultaneous rise in the osteoprotegerin competitor TNF-related apoptosis-inducing ligand. Based on our data we propose that during chronic inflammation, T cells regulate bone loss by a dual mechanism involving both direct stimulation of osteoclastogenesis, by production of osteoclastogenic cytokines, and indirectly by induction of osteoblast differentiation and up-regulation of bone turnover via coupling.