The quest for a biomarker of circulating osteoclast precursors

Osteoclast precursors arise from the CD14+ CD16- population in controls but details about cell surface marker expression and functional characteristics of these cells is unknown, particularly in patients with inflammatory arthritis. In a recent issue of Arthritis, Research and Therapy, Lari and colleagues found that osteoclasts developed from a proliferative CD14+ CD16- subset in healthy controls. These cells took on the morphology of osteoclasts, expressed mRNA for osteoclast-related genes and excavated pits on bone wafers. These findings provide new insights into monocyte diversity and provide evidence that osteoclast precursors arise from a small proliferating monocyte population in controls. Additional studies are needed in patients with inflammatory arthritis     

HUVEC co-culture and haematopoietic growth factors modulate human proliferative monocyte activity

Monocytes and macrophages are often claimed to have limited potential for proliferation in vivo and in vitro although a human monocyte subset with increased potential to proliferate in culture, termed the proliferative monocyte (PM), has previously been identified. The response of the putatively less mature PM to conditions conducive to haematopoietic stem cell culture was determined. Co-culture of monocytes on a HUVEC monolayer induced up to four cell divisions in a 9 day period. The PM response to haematopoietic growth factors (Flt3L, SCF, IL-6, IL-3 and M-CSF) was determined. M-CSF induced the greatest proliferative response in PM; IL-3 and Flt3L reduced basal and M-CSF-induced proliferation. The inhibition of M-CSFR kinase activity by GW2580 indicated that the ligand(s) for this receptor was a potent inducer of proliferation of this subset; inhibitors of intracellular signalling pathways also reduced PM proliferation.     

Reciprocal osteoblastic and osteoclastic modulation in co-cultured MG63 osteosarcoma cells and human osteoclast precursors

Osteosarcoma is usually associated with a disturbed bone metabolism. The aim of this work was to characterize the reciprocal interactions between MG63 osteosarcoma cells and osteoclasts, in a co-culture system. Co-cultures were characterized throughout 21 days for the osteoclastogenic response and the expression of osteoblastic markers. Monocultures of MG63 cells and peripheral blood mononuclear cell (PBMC) and co-cultures of PBMC + human bone marrow cells (hBMC) were also performed. Compared to PBMC cultures, co-cultures yielded significantly increased gene expression of osteoclast-related markers, tartarate-acid resistant phosphatase (TRAP) activity, TRAP-positive multinucleated cells, cells with actin rings and vitronectin receptors (VNR) and calcitonin receptors (CTR) and calcium phosphate resorbing ability. Results showed that the development of functional osteoclasts required a very low number of MG63 cells, suggesting a high osteoclastogenic-triggering capacity of this cell line. Subjacent mechanisms involved the pathways MEK and NF-kB, although with a lower relevance than that observed on PBMC monocultures or co-cultures of hBMC + PBMC; PGE2 production also had a contribution. Compared to MG63 cell monocultures, the co-culture expressed lower levels of COL1 and ALP, and higher levels of BMP-2, suggesting that PBMC also modulated the osteoblastic behavior. While M-CSF appeared to be involved in the osteoclastogenic response on the MG63 + PBMC co-cultures, RANKL does not seem to be a key player in the process. On the other hand, sphingosine-1-phosphate production might contribute to the modulation of the osteoblastic behavior. Results suggest that the reciprocal modulation between osteosarcoma and osteoclastic cells might contribute to the disturbed bone metabolism associated with bone tumors.     

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.     

Recruitment of osteoclast precursors by purified bone matrix constituents

The osteoclast, the multinucleated giant cell of bone, is derived from circulating blood cells, most likely monocytes. Evidence has accrued that is consistent with the hypothesis that the recruitment of monocytes for osteoclast development occurs by chemotaxis. In the present study, we have examined the chemotactic response of human peripheral blood monocytes and related polymorphonuclear leucocytes to three constituents of bone matrix: peptides from Type I collagen, alpha 2-HS glycoprotein, and osteocalcin (bone gla protein). The latter two substances are among the major noncollagenous proteins of bone and are uniquely associated with calcified connective tissue. In chemotaxis assays using modified Boyden chambers, Type I collagen peptides, alpha 2HS glycoprotein, and osteocalcin evoke a dose-dependent chemotactic response in human monocytes. No chemotaxis is observed on PMNs despite their ontogenetic relationship to monocytes and their documented sensitivity to a broad range of other chemical substances. Our observations are consistent with the view that osteoclast precursors (monocytes) are mobilized by chemotaxis, and suggest that the chemoattractants responsible for this activity are derived from the bone matrix or, in the case of collagen and osteocalcin; directly from the osteoblasts which produce them.     

Gradient fluid shear stress regulates migration of osteoclast precursors

Cell migration is highly sensitive to fluid shear stress (FSS) in blood flow or interstitial fluid flow. However, whether the FSS gradient can regulate the migration of cells remains unclear. In this work, we constructed a parallel-plate flow chamber with different FSS gradients and verified the gradient flow field by particle image velocimetry measurements and finite element analyses. We then investigated the effect of FSS magnitudes and gradients on the migration of osteoclast precursor RAW264.7 cells. Results showed that the cells sensed the FSS gradient and migrated toward the low-FSS region. This FSS gradient-induced migration tended to occur in low-FSS magnitudes and high gradients, e.g., the migration angle relative to flow direction was approximately 90° for 0.1 Pa FSS and 0.2 Pa mm^?1 FSS gradient. When chemically inhibiting the calcium signaling pathways of the mechanosensitive cation channel, endoplasmic reticulum, phospholipase C, and extracellular calcium, the cell migration toward the low-FSS region was significantly reduced. This study may provide insights into the mechanism of the recruitment of osteoclast precursors at the site of bone resorption and of mechanical stimulation-induced bone remodeling.     

The subpopulation of brain coated vesicles that carries synaptic vesicle proteins contains two unique polypeptides

Coated vesicles have been purified in the past on the basis of their remarkably homogeneous structure, not their function. We have succeeded in isolating two subpopulations of bovine brain coated vesicles that carry specific "cargoes," in this case two synaptic vesicle membrane polypeptides (Mr = 95,000 and 65,000). Monoclonal antibodies that recognize cytoplasmic domains of these polypeptides can penetrate the clathrin coat and recognize them on the outer surface of the coated vesicle membrane. An immunoadsorption technique could therefore be used to fractionate coated vesicles on the basis of their membrane composition. The subpopulations have the normal complement of conventional coated vesicle proteins. Exclusive, however, to the subpopulations that carry synaptic vesicle polypeptides are two new coated vesicle polypeptides (Mr = 38,000 and 29,000).     

Direct inhibition of human RANK+ osteoclast precursors identifies a homeostatic function of IL-1beta

IL-1β is a key mediator of bone resorption in inflammatory settings, such as rheumatoid arthritis (RA). IL-1β promotes osteoclastogenesis by inducing RANKL expression on stromal cells and synergizing with RANKL to promote later stages of osteoclast differentiation. Because IL-1Rs share a cytosolic Toll-IL-1R domain and common intracellular signaling molecules with TLRs that can directly inhibit early steps of human osteoclast differentiation, we tested whether IL-1β also has suppressive properties on osteoclastogenesis in primary human peripheral blood monocytes and RA synovial macrophages. Early addition of IL-1β, prior to or together with RANKL, strongly inhibited human osteoclastogenesis as assessed by generation of TRAP(+) multinucleated cells. IL-1β acted directly on human osteoclast precursors (OCPs) to strongly suppress expression of RANK, of the costimulatory triggering receptor expressed on myeloid cells 2 receptor, and of the B cell linker adaptor important for transmitting RANK-induced signals. Thus, IL-1β rendered early-stage human OCPs refractory to RANK stimulation. Similar inhibitory effects of IL-1β were observed using RA synovial macrophages. One mechanism of RANK inhibition was IL-1β-induced proteolytic shedding of the M-CSF receptor c-Fms that is required for RANK expression. These results identify a homeostatic function of IL-1β in suppressing early OCPs that contrasts with its well-established role in promoting later stages of osteoclast differentiation. Thus, the rate of IL-1-driven bone destruction in inflammatory diseases, such as RA, can be restrained by its direct inhibitory effects on early OCPs to limit the extent of inflammatory osteolysis.     

Bone morphogenic protein 2 directly enhances differentiation of murine osteoclast precursors

Previous studies found that bone morphogenic proteins (BMPs) support osteoclast formation, but it is not clear whether this is a direct effect on osteoclasts or mediated indirectly through osteoblasts. We have shown that a mouse deficient for the BMP antagonist Twisted gastrulation suggested a direct positive role for BMPs on osteoclastogenesis. In this report, we further determine the significance of BMP signaling on osteoclast formation in vitro. We find that BMP2 synergizes with suboptimal levels of receptor activator of NF‐κB ligand (RANKL) to enhance in vitro differentiation of osteoclast‐like cells. The enhancement by BMP2 is not a result of changes in the rate of proliferation or survival of the bone marrow‐derived cultures, but is accompanied by an increase in expression of genes involved in osteoclast differentiation and fusion. Treatment with BMP2 did not significantly alter expression of RANKL or OPG in our osteoclast cultures, suggesting that the enhancement of osteoclastogenesis is not mediated indirectly through osteoblasts or stromal cells. Consistent with this, we detected phosphorylated SMAD1,5,8 (p‐SMAD) in the nuclei of mononuclear and multinucleated cells in osteoclast cultures. Levels of p‐SMAD, BMP2, and BMP receptors increased during differentiation. RNAi suppression of Type II BMP receptor inhibited RANKL‐stimulated formation of multinuclear TRAP‐positive cells. The BMP antagonist noggin inhibited RANKL‐mediated osteoclast differentiation when added prior to day 3, while addition of noggin on day 3 or later failed to inhibit their differentiation. Taken together, these data indicate that osteoclasts express BMP2 and BMP receptors, and that autocrine BMP signaling directly promotes the differentiation of osteoclasts‐like cells. J. Cell. Biochem. 109: 672–682, 2010. © 2009 Wiley‐Liss, Inc.     

Migration and differentiation of osteoclast precursors under gradient fluid shear stress

Biomechanics and Modeling in Mechanobiology - The skeleton can adapt to mechanical loading through bone remodeling, and osteoclasts close to microdamages are believed to initiate bone resorption....     

Membrane associated events during proliferative inhibition of granulocyte precursors

A new way of assessing the significance of intracellular signals that may regulate cellular proliferation, would be to analyze possible 'second messengers' when proliferation is slowed down, rather than stimulated. Therefore, we examined proliferating mononuclear blood cells from leukaemic patients which had been exposed to an inhibitory ox leucocyte extract. The extract decreased 3H-thymidine incorporation in leukaemic cells in short-term cultures. The inhibition was not cell-line specific, but was nevertheless non-toxic and not due to endotoxin. The K+ flux into the leukaemic cells was assessed with 86Rb+, a K+ analogue. An inverse relationship was found between 86Rb+ uptake and 3H-thymidine incorporation. The increased 86Rb+ influx was probably due to leakage or exchange mechanisms other than the Na+/K+ membrane pump, as suggested by ouabain inhibition experiments. However, the long lag time (greater than 45 min) between addition of inhibitor and a marked increase in 86Rb+ uptake does not support a role for the K+ flux as an early mediator of the inhibitory signal.     

The Ly-6Chigh monocyte subpopulation transports Listeria monocytogenes into the brain during systemic infection of mice

Mononuclear phagocytes can be used by intracellular pathogens to disseminate throughout the host. In the bloodstream these cells are generically referred to as monocytes. However, blood monocytes are a heterogeneous population, and the exact identity of the leukocyte(s) relevant for microbial spreading is not known. Experiments reported in this study used Listeria monocytogenes-infected mice to establish the phenotype of parasitized blood leukocytes and to test their role in systemic dissemination of intracellular bacteria. More than 90% of the blood leukocytes that were associated with bacteria were CD11b(+) mononuclear cells. Analysis of newly described monocyte subsets showed that most infected cells belonged to the Ly-6C(high) monocyte subset and that Ly-6C(high) and Ly-6C(neg-low) monocytes harbored similar numbers of bacteria per cell. Interestingly, systemic infection with wild-type or DeltaactA mutants of L. monocytogenes, both of which escape from phagosomes and replicate intracellularly, caused expansion of the Ly-6C(high) subset. In contrast, this was not evident after infection with Deltahly mutants, which neither escape phagosomes nor replicate intracellularly. Importantly, when CD11b(+) leukocytes were isolated from the brains of lethally infected mice, 88% of these cells were identified as Ly-6C(high) monocytes. Kinetic analysis showed a significant influx of Ly-6C(high) monocytes into the brain 2 days after systemic infection. This coincided with both bacterial invasion and up-regulation of brain macrophage chemoattractant protein-1 gene expression. These data indicate that the Ly-6C(high) monocyte subset transports L. monocytogenes into the brain and establish their role as Trojan horses in vivo.     

The proteoglycan chondroitin sulfate is present in a subpopulation of cultured astrocytes and in their precursors

We have used an antibody raised against the bovine nasal cartilage proteoglycan chondroitin sulfate (CS) digested with chondroitinase ABC (anti-CS serum) to stain cerebellar glial cells maintained in culture. In cultures grown in the presence of serum, the antibody stained a subclass of GFAP+ astrocytes which we have previously shown to selectively bind the monoclonal antibodies A2B5 and LB1. Also the direct bipotential precursors of these cells, capable of differentiating into GFAP+ astrocytes or into Gal-C+, O1+ oligodendrocytes depending on the culture conditions, were stained, but stopped to produce CS when they differentiated into oligodendrocytes.     

Interaction of Fas ligand and Fas expressed on osteoclast precursors increases osteoclastogenesis

We incidentally found that osteoclast precursors and mature osteoclasts express Fas ligand (FasL) as well as Fas, which was confirmed by flow cytometry, immunofluorescent staining, and RT-PCR. The aim of this study was to determine the role of FasL in differentiation and cell death of osteoclasts. To study the role of FasL in osteoclastogenesis, neutralizing anti-FasL mAb or rFasL was added during receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis using bone marrow-derived macrophages. Neutralization of endogenous FasL by anti-FasL mAb decreased osteoclastogenesis, whereas rFasL enhanced osteoclast differentiation in a dose-dependent manner. In addition, rFasL up-regulated the secretion of osteoclastogenic cytokines, such as IL-1beta and TNF-alpha, and the activation of NF-kappaB. Functional blocking of IL-1beta and TNF-alpha using IL-1 receptor antagonist and soluble TNFR confirmed that those cytokines mediated the effect of FasL on osteoclastogenesis. The osteoclast precursors were relatively resistant to rFasL-induced apoptosis especially before RANKL treatment, resulting in minimal cell loss by rFasL treatment during osteoclastogenesis. Although rFasL increased the cell death of mature osteoclasts, growth factor withdrawal induced much more cell death. However, anti-FasL mAb did not affect the survival of mature osteoclasts, suggesting that the endogenous FasL does not have a role in the apoptosis of osteoclasts. Finally, in contrast to the effect on apoptosis, rFasL-assisted osteoclastogenesis was not mediated by caspases. In conclusion, FasL has a novel function in bone homeostasis by enhancing the differentiation of osteoclasts, which was not considered previously.     

The protocol for the isolation and cryopreservation of osteoclast precursors from mouse bone marrow and spleen

Osteoclasts are responsible for physiological bone remodeling as well as pathological bone destruction in osteoporosis, periodontitis and rheumatoid arthritis, and thus represent a pharmacological target for drug development. We aimed to characterize and compare the cytokine-induced osteoclastogenesis of bone marrow and spleen precursors. Established protocols used to generate osteoclasts from bone marrow were modified to examine osteoclastogenesis of the spleen cells of healthy mice. Osteoclast formation was successfully induced from spleen precursors using receptor activator of nuclear factor κB ligand (50 ng/ml) and macrophage colony stimulating factor (50 ng/ml). Compared to bone marrow cultures, differentiation from spleen required a longer cultivation time (9 days for spleen, as compared to 5 days for marrow cultures) and a higher plating density of non-adherent cells (75,000/cm² for spleen, as compared to 50,000/cm² for bone marrow). Osteoclasts generated from spleen precursors expressed osteoclast marker genes calcitonin receptor, cathepsin K and matrix metalloproteinase 9 and were capable of resorbing hydroxyapatite. The differentiation capacity of spleen and bone marrow precursors was comparable for BALB/c, C57BL/6 and FVB mice. We also developed and tested a cryopreservation protocol for the osteoclast precursors. While 70–80 % of cells were lost during the first week of freezing, during the subsequent 5 weeks the losses were within 2–5 % per week. Osteoclastogenesis from the recovered bone marrow precursors was successful up to 5 weeks after freezing. Spleen precursors retained their osteoclastogenic capacity for 1 week after freezing, but not thereafter. The described protocol is useful for the studies of genetically modified animals as well as for screening new osteoclast-targeting therapeutics.     

Cloning and characterization of osteoclast precursors from the raw264.7 cell line

Summary Osteoclasts are bone-resorbing cells that differentiate from macrophage precursors in response to receptor activator of NF-κB ligand (RANKL). In vitro models of osteoclast differentiation are principally based on primary cell cultures, which are poorly suited to molecular and transgene studies because of the limitations associated with the use of primary macrophage. RAW264.7 is a transfectable macrophage cell line with the capacity to form osteoclast-like cells. In the present study, we have identified osteoclast precursors among clones of RAW264.7 cells. RAW264.7 cell were cloned by limiting dilution and induced to osteoclast differentiation by treatment with recombinant RANKL. Individual RAW264.7 cell clones formed tartrate resistant acid phosphatase (TRAP)-positive multinuclear cells to various degrees with RANKL treatment. All clones tested expressed the RANKL receptor RANK. Each of the clones expressed the osteoclast marker genes TRAP and cathepsin-K mRNA with RANKL treatment. However, we noted that only select clones were able to form large, well-spread, TRAP-positive multinuclear cells. Clones capable of forming large TRAP-positive multinuclear cells also expressed β₃ integrin and calcitonin receptor mRNAs and were capable of resorbing a mineralized matrix. All clones tested activated NF-κB with RANKL treatment. cDNA expression profiling of osteoclast precursor RAW264.7 cell clones demonstrates appropriate expression of a large number of genes before and after osteoclastic differentiation. These osteoclast precursor RAW264.7 cell clones provide a valuable model for dissecting the cellular and molecular regulation of osteoclast differentiation and activation.     

Soluble Sema4D cleaved from osteoclast precursors by TACE suppresses osteoblastogenesis

Bone remodelling is mediated by orchestrated communication between osteoclasts and osteoblasts which, in part, is regulated by coupling and anti-coupling factors. Amongst formally known anti-coupling factors, Semaphorin 4D (Sema4D), produced by osteoclasts, plays a key role in downmodulating osteoblastogenesis. Sema4D is produced in both membrane-bound and soluble forms; however, the mechanism responsible for producing sSema4D from osteoclasts is unknown. Sema4D, TACE and MT1-MMP are all expressed on the surface of RANKL-primed osteoclast precursors. However, only Sema4D and TACE were colocalized, not Sema4D and MT1-MMP. When TACE and MT1-MMP were either chemically inhibited or suppressed by siRNA, TACE was found to be more engaged in shedding Sema4D. Anti-TACE-mAb inhibited sSema4D release from osteoclast precursors by ~90%. Supernatant collected from osteoclast precursors (OC-sup) suppressed osteoblastogenesis from MC3T3-E1 cells, as measured by alkaline phosphatase activity, but OC-sup harvested from the osteoclast precursors treated with anti-TACE-mAb restored osteoblastogenesis activity in a manner that compensates for diminished sSema4D. Finally, systemic administration of anti-TACE-mAb downregulated the generation of sSema4D in the mouse model of critical-sized bone defect, whereas local injection of recombinant sSema4D to anti-TACE-mAb-treated defect upregulated local osteoblastogenesis. Therefore, a novel pathway is proposed whereby TACE-mediated shedding of Sema4D expressed on the osteoclast precursors generates functionally active sSema4D to suppress osteoblastogenesis.     

Functional characterization of human thymocytes: a limiting dilution analysis of precursors with proliferative and cytolytic activities

In this report we have analyzed the pool size of human thymocytes capable of proliferating or mediating cytolytic activity. Furthermore, the relationship between these functional capabilities and cell surface antigen expression was studied. Graded numbers of human thymocytes were plated under limiting dilution conditions with irradiated human spleen cells (as a source of feeder cells) and phytohemagglutinin (PHA) in the presence of a saturating concentration of interleukin 2. Cell proliferation, which was evaluated after 20 days of culture, was usually compared with the proliferation of peripheral blood T cell populations cultured under identical conditions. Although essentially all peripheral blood T cells were clonogenic, only 3 to 8% of thymocytes proliferated. Of proliferating microcultures, 48 to 86% showed cytolytic activity in a PHA-dependent assay, whereas 26 to 83% killed the NK-sensitive target cell K-562 in the absence of added lectin. Similar frequency analysis of functional precursors was performed on thymocyte subsets selected according to their expression of T3, T6, T4, and T8 antigens. All precursors of proliferating cells (PTL-P) were found in the T3+ subset. From the comparison of the percentages of total thymocytes capable of proliferation and the proportion of cells brightly stained with anti-T3 antibody, it was evident that only a fraction of T3+ cells was clonogenic. Although the large majority of PTL-P was found in the T6- subpopulation, a small fraction of functional precursors was detected in the T6+ subset. When thymocytes were fractionated according to T4 or T8 antigen expression, it was found that 80 to 90% of the recovered PTL-P were confined to the T4+ fraction, whereas only 20 to 28% of the recovered PTL-P were present in the T8+ subset. Analysis of the precursors of cytolytic T lymphocytes (CTL-P) in thymocyte populations fractionated according to T4 or T8 antigen expression showed that 70 to 90% of the recovered CTL-P were found in the T4+ fraction and 17 to 30% were in the T8+ subset. Because approximately 80% of proliferating T4+ thymocytes had CTL activity (as compared with less than 5% in peripheral blood T4+ lymphocytes), it appears that the subset distribution of thymic CTL-P differs from that of peripheral blood T cells.     

1,25-Dihydroxyvitamin D(3) and prostaglandin E(2) act directly on circulating human osteoclast precursors.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and prostaglandin E(2) (PGE(2)) are known to influence osteoclast formation indirectly through their effects on osteoblasts. To determine whether 1, 25(OH)(2)D(3) and PGE(2) also have a direct effect on circulating osteoclast precursors, these factors were added to long-term cultures of human peripheral blood mononuclear cells (PBMCs) in the presence of osteoprotegerin ligand and macrophage colony-stimulating factor (M-CSF) (+/-dexamethasone). The number of TRAP(+) and VNR(+) multinucleated cells and the area of lacunar resorption were decreased when 1,25(OH)(2)D(3) alone was added. A marked increase in resorption pit formation was noted when the combination of 1, 25(OH)(2)D(3) and dexamethasone was added to PBMC cultures. Dose-dependent inhibition of osteoclast formation and lacunar resorption was seen when PGE(2) was added to PBMC cultures in both the presence and the absence of dexamethasone. Thus, 1,25(OH)(2)D(3) and PGE(2) not only influence osteoclast formation in the presence of bone stromal cells but also act directly on circulating osteoclast precursors to influence osteoclast differentiation.