Development of a novel, nonimmunogenic, soluble human TNF receptor type I (sTNFR-I) construct in the baboon.

Pharmacokinetics and immunogenicity of six different recombinant human soluble p55 tumor necrosis factor (TNF) receptor I (sTNFR-I) constructs were evaluated in juvenile baboons. The constructs included either an sTNFR-I IgG1 immunoadhesin (p55 sTNFR-I Fc) or five different sTNFR-I constructs covalently linked to polyethylene glycol. The constructs were administered intravenously three times, and pharmacokinetics and immunogenicity were examined over 63 days. All of the constructs were immunogenic, with the exception of a 2.6-domain monomeric sTNFR-I. To evaluate whether the nonimmunogenic 2.6-domain monomeric construct could protect baboons against TNF-alpha-induced mortality, baboons were pretreated with 1, 5, or 10 mg/kg body wt and were compared with baboons receiving either placebo or 1 mg/kg body wt of the dimeric 4.0-domain sTNFR-I construct (n = 3 each) before lethal Escherichia coli bacteremia. The monomeric construct protected baboons and neutralized TNF bioactivity, although greater quantities were required compared with the dimeric 4.0-domain sTNFR-I construct. We conclude that E. coli-recombinant-derived human sTNFR-I constructs can be generated with minimal immunogenicity on repeated administration and still protect against the consequences of exaggerated TNF-alpha production.

Regulation of osteoclastogenesis and RANK expression by TGF-beta1.

Transforming growth factor-beta (TGF-beta) has been shown to both inhibit and to stimulate bone resorption and osteoclastogenesis. This may be due, in part, to differential effects on bone marrow stromal cells that support osteoclastogenesis vs. direct effects on osteoclastic precursor cells. In the present study, we used the murine monocytic cell line, RAW 264.7, to define direct effects of TGF-beta on pre-osteoclastic cells. In the presence of macrophage-colony stimulating factor (M-CSF) (20 ng/ml) and receptor activator of NF-kappaB ligand (RANK-L) (50 ng/ml), TGF-beta1 (0.01-5 ng/ml) dose-dependently stimulated (by up to 120-fold) osteoclast formation (assessed by the presence of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells and expression of calcitonin and vitronectin receptors). In addition, TGF-beta1 also increased steady state RANK mRNA levels in a time- (by up to 3.5-fold at 48 h) and dose-dependent manner (by up to 2.2-fold at 10 ng/ml). TGF-beta1 induction of RANK mRNA levels was present both in undifferentiated RAW cells as well as in cells that had been induced to differentiate into osteoclasts by a 7-day treatment with M-CSF and RANK-L. Using a fluorescence-labeled RANK-L probe, we also demonstrated by flow cytometry that TGF-beta1 resulted in a significant increase in the percentage of RANK+ RAW cells (P < 0.05), as well as an increase in the fluorescence intensity per cell (P < 0.05), the latter consistent with an increase in RANK protein expression per cell. These data thus indicate that TGF-beta directly stimulates osteoclastic differentiation, and this is accompanied by increased RANK mRNA and protein expression.

Characterization of osteoclast precursors in human blood.

Osteoclast precursors (OCPs) circulate in the mononuclear fraction of peripheral blood (PB), but their abundance and surface characteristics are unknown. Previous studies suggest that the receptor activator for NF-kappaB (RANK) on cytokine-treated OCPs in mouse bone marrow interacts with osteoprotegerin ligand (OPGL/TRANCE/RANKL/ODF) to initiate osteoclast differentiation. Hence, we used a fluorescent form of human OPGL (Hu-OPGL-F) to identify possible RANK-expressing OCPs in untreated peripheral blood mononuclear cells (PBMCs) using fluorescence-activated cell sorting analysis. Monocytes [CD14-phycoerythrin (PE) antibody (Ab) positive (+) cells, 10-15% of PBMCs] all (98-100%) co-labelled with Hu-OPGL-F (n > 18). T lymphocytes (CD3-PE Ab+ cells, 66% of PBMCs) did not bind Hu-OPGL-F; however, B cells (CD19-PE Ab+ cells, 9% of PBMCs) were also positive for Hu-OPGL-F. All Hu-OPGL-F+ monocytes also co-labelled with CD33, CD61, CD11b, CD38, CD45 and CD54 Abs, but not CD34 or CD56 Abs. Hu-OPGL-F binding was dose dependent and competed with excess Hu-OPGL. When Hu-OPGL-F+, CD14-PE Ab+, CD33-PE Ab+, Hu-OPGL-F+/CD14-PE Ab+ or Hu-OPGL-F+/CD33-PE Ab+ cells were cultured with OPGL (20 ng/ml) and colony-stimulating factor (CSF)-1 (25 ng/ml), OC-like cells readily developed. Thus, all freshly isolated monocytes demonstrate displaceable Hu-OPGL-F binding, suggesting the presence of RANK on OCPs in PB; also, OCPs within a purified PB monocyte population form osteoclast-like cells in the complete absence of other cell types in OPGL and CSF-1 containing medium.

The osteoclast differentiation factor osteoprotegerin-ligand is essential for mammary gland development.

Osteoprotegerin-ligand (OPGL) is a key osteoclast differentiation/activation factor essential for bone remodeling. We report that mice lacking OPGL or its receptor RANK fail to form lobulo-alveolar mammary structures during pregnancy, resulting in death of newborns. Transplantation and OPGL-rescue experiments in opgl-/- and rank-/- pregnant females showed that OPGL acts directly on RANK-expressing mammary epithelial cells. The effects of OPGL are autonomous to epithelial cells. The mammary gland defect in female opgl-/- mice is characterized by enhanced apoptosis and failures in proliferation and PKB activation in lobulo-alveolar buds that can be reversed by recombinant OPGL treatment. These data provide a novel paradigm in mammary gland development and an evolutionary rationale for hormonal regulation and gender bias of osteoporosis in females.

Osteoprotegerin reverses osteoporosis by inhibiting endosteal osteoclasts and prevents vascular calcification by blocking a process resembling osteoclastogenesis.

High systemic levels of osteoprotegerin (OPG) in OPG transgenic mice cause osteopetrosis with normal tooth eruption and bone elongation and inhibit the development and activity of endosteal, but not periosteal, osteoclasts. We demonstrate that both intravenous injection of recombinant OPG protein and transgenic overexpression of OPG in OPG(-/-) mice effectively rescue the osteoporotic bone phenotype observed in OPG-deficient mice. However, intravenous injection of recombinant OPG over a 4-wk period could not reverse the arterial calcification observed in OPG(-/-) mice. In contrast, transgenic OPG delivered from mid-gestation through adulthood does prevent the formation of arterial calcification in OPG(-/-) mice. Although OPG is normally expressed in arteries, OPG ligand (OPGL) and receptor activator of NF-kappaB (RANK) are not detected in the arterial walls of wild-type adult mice. Interestingly, OPGL and RANK transcripts are detected in the calcified arteries of OPG(-/-) mice. Furthermore, RANK transcript expression coincides with the presence of multinuclear osteoclast-like cells. These findings indicate that the OPG/OPGL/RANK signaling pathway may play an important role in both pathological and physiological calcification processes. Such findings may also explain the observed high clinical incidence of vascular calcification in the osteoporotic patient population.

Osteoprotegerin ligand modulates murine osteoclast survival in vitro and in vivo.

Osteoprotegerin ligand (OPGL) targets osteoclast precursors and osteoclasts to enhance differentiation and activation, however, little is known about OPGL effects on osteoclast survival. In vitro, the combination of OPGL + colony-stimulating factor-1 (CSF-1) is required for optimal osteoclast survival. Ultrastructurally, apoptotic changes were observed in detached cells and culture lysates exhibited elevated caspase 3 activity, particularly in cultures lacking CSF-1. DEVD-FMK (caspase 3 inhibitor) partially protected cells when combined with OPGL, but not when used alone or in combination with CSF-1. CSF-1 maintained NF-kappaB activation and increased the expression of bcl-2 and bcl-X(L) mRNA, but had no effect on JNK activation. In contrast, OPGL enhanced both NF-kappaB and JNK kinase activation and increased the expression of c-src, but not bcl-2 and bcl-X(L) mRNA. These data suggest that aspects of both OPGL's and CSF-1's signaling/survival pathways are required for optimal osteoclast survival. In mice, a single dose of OPG, the OPGL decoy receptor, led to a >90% loss of osteoclasts because of apoptosis within 48 hours of exposure without impacting osteoclast precursor cells. Therefore, OPGL is essential, but not sufficient, for osteoclast survival and endogenous CSF-1 levels are insufficient to maintain osteoclast viability in the absence of OPGL.

TACI is a TRAF-interacting receptor for TALL-1, a tumor necrosis factor family member involved in B cell regulation.

We and others recently reported tumor necrosis factor (TNF) and apoptosis ligand-related leukocyte-expressed ligand 1 (TALL-1) as a novel member of the TNF ligand family that is functionally involved in B cell proliferation. Transgenic mice overexpressing TALL-1 have severe B cell hyperplasia and lupus-like autoimmune disease. Here, we describe expression cloning of a cell surface receptor for TALL-1 from a human Burkitt's lymphoma RAJI cell library. The cloned receptor is identical to the previously reported TNF receptor (TNFR) homologue transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI). Murine TACI was subsequently isolated from the mouse B lymphoma A20 cells. Human and murine TACI share 54% identity overall. Human TACI exhibits high binding affinities to both human and murine TALL-1. Soluble TACI extracellular domain protein specifically blocks TALL-1-mediated B cell proliferation without affecting CD40- or lipopolysaccharide-mediated B cell proliferation in vitro. In addition, when injected into mice, soluble TACI inhibits antibody production to both T cell-dependent and -independent antigens. By yeast two-hybrid screening of a B cell library with TACI intracellular domain, we identified that, like many other TNFR family members, TACI intracellular domain interacts with TNFR-associated factor (TRAF)2, 5, and 6. Correspondingly, TACI activation in a B cell line results in nuclear factor kappaB and c-Jun NH(2)-terminal kinase activation. The identification and characterization of the receptor for TALL-1 provides useful information for the development of a treatment for B cell-mediated autoimmune diseases such as systemic lupus erythematosus.

RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism.

We have generated RANK (receptor activator of NF-kappaB) nullizygous mice to determine the molecular genetic interactions between osteoprotegerin, osteoprotegerin ligand, and RANK during bone resorption and remodeling processes. RANK(-/-) mice lack osteoclasts and have a profound defect in bone resorption and remodeling and in the development of the cartilaginous growth plates of endochondral bone. The osteopetrosis observed in these mice can be reversed by transplantation of bone marrow from rag1(-/-) (recombinase activating gene 1) mice, indicating that RANK(-/-) mice have an intrinsic defect in osteoclast function. Calciotropic hormones and proresorptive cytokines that are known to induce bone resorption in mice and human were administered to RANK(-/-) mice without inducing hypercalcemia, although tumor necrosis factor alpha treatment leads to the rare appearance of osteoclast-like cells near the site of injection. Osteoclastogenesis can be initiated in RANK(-/-) mice by transfer of the RANK cDNA back into hematopoietic precursors, suggesting a means to critically evaluate RANK structural features required for bone resorption. Together these data indicate that RANK is the intrinsic cell surface determinant that mediates osteoprotegerin ligand effects on bone resorption and remodeling as well as the physiological and pathological effects of calciotropic hormones and proresorptive cytokines.

Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice.

TALL-1/Blys/BAFF is a member of the tumor necrosis factor (TNF) ligand superfamily that is functionally involved in B cell proliferation. Here, we describe B cell hyperplasia and autoimmune lupus-like changes in transgenic mice expressing TALL-1 under the control of a beta-actin promoter. The TALL-1 transgenic mice showed severe enlargement of spleen, lymph nodes, and Peyer's patches because of an increased number of B220+ cells. The transgenic mice also had hypergammaglobulinemia contributed by elevations of serum IgM, IgG, IgA, and IgE. In addition, a phenotype similar to autoimmune lupus-like disease was also seen in TALL-1 transgenic mice, characterized by the presence of autoantibodies to nuclear antigens and immune complex deposits in the kidney. Prolonged survival and hyperactivity of transgenic B cells may contribute to the autoimmune lupus-like phenotype in these animals. Our studies further confirm TALL-1 as a stimulator of B cells that affect Ig production. Thus, TALL-1 may be a primary mediator in B cell-associated autoimmune diseases.

The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption.

Although multiple hormones and cytokines regulate various aspects of osteoclast formation, the final two effectors are osteoprotegerin ligand (OPG-L)/osteoclast differentiation factor (ODF), a recently cloned member of the tumor necrosis factor superfamily, and macrophage colony-stimulating factor. OPG-L/ODF is produced by osteoblast lineage cells and exerts its biological effects through binding to its receptor, osteoclast differentiation and activation receptor (ODAR)/receptor activator of NF-kappa B (RANK), on osteoclast lineage cells, in either a soluble or a membrane-bound form, the latter of which requires cell-to-cell contact. Binding results in rapid differentiation of osteoclast precursors in bone marrow to mature osteoclasts and, at higher concentrations, in increased functional activity and reduced apoptosis of mature osteoclasts. The biological activity of OPG-L/ODF is neutralized by binding to osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF), a member of the TNF-receptor superfamily that also is secreted by osteoblast lineage cells. The biological importance of this system is underscored by the induction in mice of severe osteoporosis by targeted ablation of OPG/OCIF and by the induction of osteopetrosis by targeted ablation of OPG-L/ODF or overexpression of OPG/OCIF. Thus, osteoclast formation may be determined principally by the relative ratio of OPG-L/ODF to OPG/OCIF in the bone marrow microenvironment, and alterations in this ratio may be a major cause of bone loss in many metabolic disorders, including estrogen deficiency and glucocorticoid excess. That changes in but two downstream cytokines mediate the effects of large numbers of upstream hormones and cytokines suggests a regulatory mechanism for osteoclastogenesis of great efficiency and elegance.

APRIL and TALL-I and receptors BCMA and TACI: system for regulating humoral immunity.

We report that the tumor neurosis factor homolog APRIL (a proliferation-inducing ligand) stimulates in vitro proliferation of primary B and T cells and increases spleen weight due to accumulation of B cells in vivo. APRIL functions via binding to BCMA (B cell maturation antigen) and TACI (transmembrane activator and CAML-interactor) and competes with TALL-I (also called BLyS or BAFF) for receptor binding. Soluble BCMA and TACI specifically prevent binding of APRIL and block APRIL-stimulated proliferation of primary B cells. BCMA-Fc also inhibits production of antibodies against keyhole limpet hemocyanin and Pneumovax in mice, indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI are required for generation of humoral immunity. Thus, APRIL-TALL-I and BCMA-TACI form a two ligands-two receptors pathway involved in stimulation of B and T cell function.

Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand.

Bone remodelling and bone loss are controlled by a balance between the tumour necrosis factor family molecule osteoprotegerin ligand (OPGL) and its decoy receptor osteoprotegerin (OPG). In addition, OPGL regulates lymph node organogenesis, lymphocyte development and interactions between T cells and dendritic cells in the immune system. The OPGL receptor, RANK, is expressed on chondrocytes, osteoclast precursors and mature osteoclasts. OPGL expression in T cells is induced by antigen receptor engagement, which suggests that activated T cells may influence bone metabolism through OPGL and RANK. Here we report that activated T cells can directly trigger osteoclastogenesis through OPGL. Systemic activation of T cells in vivo leads to an OPGL-mediated increase in osteoclastogenesis and bone loss. In a T-cell-dependent model of rat adjuvant arthritis characterized by severe joint inflammation, bone and cartilage destruction and crippling, blocking of OPGL through osteoprotegerin treatment at the onset of disease prevents bone and cartilage destruction but not inflammation. These results show that both systemic and local T-cell activation can lead to OPGL production and subsequent bone loss, and they provide a novel paradigm for T cells as regulators of bone physiology.

The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts.

Osteoprotegerin (OPG) and OPG-ligand (OPGL) potently inhibit and stimulate, respectively, osteoclast differentiation (Simonet, W.S., D.L. Lacey, C.R. Dunstan, M. Kelley, M.-S. Chang, R. Luethy, H.Q. Nguyen, S. Wooden, L. Bennett, T. Boone, et al. 1997. Cell. 89:309-319; Lacey, D.L., E. Timms, H.-L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, et al. 1998. Cell. 93: 165-176), but their effects on mature osteoclasts are not well understood. Using primary cultures of rat osteoclasts on bone slices, we find that OPGL causes approximately sevenfold increase in total bone surface erosion. By scanning electron microscopy, OPGL-treated osteoclasts generate more clusters of lacunae on bone suggesting that multiple, spatially associated cycles of resorption have occurred. However, the size of individual resorption events are unchanged by OPGL treatment. Mechanistically, OPGL binds specifically to mature OCs and rapidly (within 30 min) induces actin ring formation; a marked cytoskeletal rearrangement that necessarily precedes bone resorption. Furthermore, we show that antibodies raised against the OPGL receptor, RANK, also induce actin ring formation. OPGL-treated mice exhibit increases in blood ionized Ca++ within 1 h after injections, consistent with immediate OC activation in vivo. Finally, we find that OPG blocks OPGL's effects on both actin ring formation and bone resorption. Together, these findings indicate that, in addition to their effects on OC precursors, OPGL and OPG have profound and direct effects on mature OCs and indicate that the OC receptor, RANK, mediates OPGL's effects.

Osteoprotegerin ligand: a common link between osteoclastogenesis, lymph node formation and lymphocyte development.

The TNF-family molecule osteoprotegerin ligand (OPGL; also known as TRANCE, RANKL or ODF) has been identified as the osteoclast differentiation factor and a regulator of T cell-dendritic cell interactions in the immune system. Surprisingly, the same molecule was identified as a crucial factor in early lymphocyte development and lymph node organogenesis. We will discuss the role of OPGL in bone remodelling and the immune system.

TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling.

Bone resorption and remodeling is an intricately controlled, physiological process that requires the function of osteoclasts. The processes governing both the differentiation and activation of osteoclasts involve signals induced by osteoprotegerin ligand (OPGL), a member of tumor necrosis factor (TNF) superfamily, and its cognate receptor RANK. The molecular mechanisms of the intracellular signal transduction remain to be elucidated. Here we report that mice deficient in TNF receptor-associated factor 6 (TRAF6) are osteopetrotic with defects in bone remodeling and tooth eruption due to impaired osteoclast function. Using in vitro assays, we demonstrate that TRAF6 is crucial not only in IL-1 and CD40 signaling but also, surprisingly, in LPS signaling. Furthermore, like TRAF2 and TRAF3, TRAF6 is essential for perinatal and postnatal survival. These findings establish unexpectedly diverse and critical roles for TRAF6 in perinatal and postnatal survival, bone metabolism, LPS, and cytokine signaling.

Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.

A receptor that mediates osteoprotegerin ligand (OPGL)-induced osteoclast differentiation and activation has been identified via genomic analysis of a primary osteoclast precursor cell cDNA library and is identical to the tumor necrosis factor receptor (TNFR) family member RANK. The RANK mRNA was highly expressed by isolated bone marrow-derived osteoclast progenitors and by mature osteoclasts in vivo. Recombinant OPGL binds specifically to RANK expressed by transfected cell lines and purified osteoclast progenitors. Transgenic mice expressing a soluble RANK-Fc fusion protein have severe osteopetrosis because of a reduction in osteoclasts, similar to OPG transgenic mice. Recombinant RANK-Fc binds with high affinity to OPGL in vitro and blocks osteoclast differentiation and activation in vitro and in vivo. Furthermore, polyclonal Ab against the RANK extracellular domain promotes osteoclastogenesis in bone marrow cultures suggesting that RANK activation mediates the effects of OPGL on the osteoclast pathway. These data indicate that OPGL-induced osteoclastogenesis is directly mediated through RANK on osteoclast precursor cells.

Osteoprotegerin and osteoprotegerin ligand effects on osteoclast formation from human peripheral blood mononuclear cell precursors.

Osteoprotegerin (OPG) and its ligand (OPGL) negatively and positively regulate osteoclastogenesis in the mouse. OPG inhibits osteoclastogenesis by sequestering its ligand, OPGL, the osteoclast differentiation and activation factor. This study demonstrates the effects of soluble muOPGL and huOPG on the developing human osteoclast phenotype, on bone slices, using peripheral blood mononuclear cells (PBMCs), cultured for 2 weeks, without stromal cells. OPGL (2-50 ng/ml), in combination with CSF-1, hydrocortisone (HC), and 1,25(OH)2D3, increases the size of osteoclast-like cells on bone, as defined by the acquisition of osteoclast markers: vitronectin receptor (VR), tartrate-resistant acid phosphatase (TRAP), multinuclearity, and bone resorption. By 14 days, with 20 ng/ml OPGL, the largest cells/10x field have achieved an average diameter of 163+/-38 microm, but only approximately 10-20 microm in its absence and the number of osteoclast-like cells/mm2 bone surface is about 128. By scanning electron microscopy, OPGL-treated (20-ng/ml) cultures contain small osteoclast-like cells on bone with ruffled "apical" surfaces by day 7; by day 15, large osteoclast-like cells are spread over resorption lacunae. At 15 ng/ml OPGL, about 37% of the bone slice area is covered by resorption lacunae. OPG (5-250 ng/ml) antagonizes the effects of OPGL on the morphology of the osteoclast-like cells that form, as well as bone erosion. For cells grown on plastic, Cathepsin K mRNA levels, which are barely detectable at plating, are elevated 7-fold, by 5 days, in the presence, not the absence, of OPGL (20 ng/ml) + CSF-1 (25 ng/ml). Similar findings are observed in experiments performed in the absence of HC and 1,25(OH)2D3, indicating that HC and 1,25(OH)2D3 are not needed for OPGL-induced osteoclast differentiation. In conclusion, this study confirms a pivotal role for OPGL and OPG in the modulation of human osteoclast differentiation and function, suggesting a use for OPG for treating osteoclast-mediated bone disease in humans.

OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis.

The tumour-necrosis-factor-family molecule osteoprotegerin ligand (OPGL; also known as TRANCE, RANKL and ODF) has been identified as a potential osteoclast differentiation factor and regulator of interactions between T cells and dendritic cells in vitro. Mice with a disrupted opgl gene show severe osteopetrosis and a defect in tooth eruption, and completely lack osteoclasts as a result of an inability of osteoblasts to support osteoclastogenesis. Although dendritic cells appear normal, opgl-deficient mice exhibit defects in early differentiation of T and B lymphocytes. Surprisingly, opgl-deficient mice lack all lymph nodes but have normal splenic structure and Peyer's patches. Thus OPGL is a new regulator of lymph-node organogenesis and lymphocyte development and is an essential osteoclast differentiation factor in vivo.

osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification.

Osteoprotegerin (OPG) is a secreted protein that inhibits osteoclast formation. In this study the physiological role of OPG is investigated by generating OPG-deficient mice. Adolescent and adult OPG-/- mice exhibit a decrease in total bone density characterized by severe trabecular and cortical bone porosity, marked thinning of the parietal bones of the skull, and a high incidence of fractures. These findings demonstrate that OPG is a critical regulator of postnatal bone mass. Unexpectedly, OPG-deficient mice also exhibit medial calcification of the aorta and renal arteries, suggesting that regulation of OPG, its signaling pathway, or its ligand(s) may play a role in the long observed association between osteoporosis and vascular calcification.