Differential effects of interleukin-6 receptor activation on intracellular signaling and bone resorption by isolated rat osteoclasts.

The effects of the related cytokines interleukin-6 (IL-6), leukemia inhibitory factor (LIF) and oncostatin-M on bone resorption and cytosolic Ca(2+) signaling were compared in isolated rat osteoclasts. In the traditional disaggregated osteoclast (pit) assay, IL-6 and LIF, but not oncostatin-M, conserved the bone resorption otherwise inhibited by high extracellular [Ca(2+)] (15 mM). It produced a paradoxical, concentration-dependent stimulation of resorption by elevated extracellular Ca(2+). In the micro-isolated single osteoclast resorption assay, IL-6, high [Ca(2+)] or IL-6 plus high [Ca(2+)] all increased pit formation. In contrast, the IL-6 receptor (IL-6R)-specific agonist antibody MT-18 inhibited bone resorption in a concentration-dependent manner (1:500 to 1:500 000). MT-18 triggered cytosolic Ca(2+) signals in fura 2-loaded osteoclasts within approximately 10 min of application. Each cytosolic Ca(2+) transient began with a peak deflection that persisted in Ca(2+)-free, EGTA-containing extracellular medium, consistent with a release of intracellularly stored Ca(2+). This was followed by a sustained elevation of cytosolic [Ca(2+)] that was abolished in Ca(2+)-free medium, as expected from an entry of extracellular Ca(2+), and by the Ca(2+) channel antagonist Ni(2+). The inclusion of either IL-6 or soluble human (sh) IL-6R specifically reversed both the above effects of MT-18, confirming that both effects were specific for the IL-6R. The findings suggest that IL-6R activation by IL-6 stimulates osteoclastic bone resorption either by reversing the inhibitory effect of high extracellular Ca(2+) in stromal-containing systems or itself stimulating bone resorption along with Ca(2+) by micro-isolated osteoclasts. In contrast, activation of the IL-6R by an agonist antibody produces an inhibition of bone resorption and an associated triggering of the cytosolic Ca(2+) signals previously associated with regulation of bone resorptive function in other situations.

Expression of functional RANK on mature rat and human osteoclasts.

Although the important roles of RANK/RANKL in osteoclastogenesis have been established, their roles in the regulation of mature osteoclasts remain uncertain. Microisolation has been used to obtain pure populations of rat and human osteoclasts for RT-PCR analysis. RANK and calcitonin receptor mRNA was detected in all the samples whereas OPG and ALP mRNA was not present in any. RANKL mRNA was detected in two of eight rat and one of four human samples. Treatment of osteoclasts with soluble RANKL resulted in translocation of NF-kappaB to the nucleus and elevation of cytosolic and nuclear calcium levels. We have shown that RANK is highly expressed in mature osteoclasts and that its stimulation by RANKL results in activation of NF-kappaB and calcium signalling.

Role of the osteoclast at the bone-implant interface.

A thorough understanding of the processes of healing, repair, and remodeling of bone is critical for the establishment and maintenance of osseointegration of dental implants. In this regard, much attention has been paid to the anabolic aspects of bone remodeling, including the cell biology of the osteoblast and the various cytokines and growth factors which regulate these processes. In contrast, there is little information on the bone-resorptive activity that occurs around implants during osseointegration, and of the role of osteoclasts, macrophages, and stromal cells in those catabolic processes associated with bone remodeling. This paper reviews osteoclast cell biology, the interaction of osteoclasts and biomaterials, and the information available on osteoclasts and dental implants, and poses some questions for future research.

Mode of action of interleukin-6 on mature osteoclasts. Novel interactions with extracellular Ca2+ sensing in the regulation of osteoclastic bone resorption.

We describe a physiologically significant mechanism through which interleukin-6 (IL-6) and a rising ambient Ca2+ interact to regulate osteoclastic bone resorption. VOXEL-based confocal microscopy of nonpermeabilized osteoclasts incubated with anti- IL-6 receptor antibodies revealed intense, strictly peripheral plasma membrane fluorescence. IL-6 receptor expression in single osteoclasts was confirmed by in situ reverse transcriptase PCR histochemistry. IL-6 (5 ng/l to 10 microg/l), but not IL-11 (10 and 100 microg/l), reversed the inhibition of osteoclastic bone resorption induced by high extracellular Ca2+ (15 mM). The IL-6 effect was abrogated by excess soluble IL-6 receptor (500 microg/l). Additionally, IL-6 (5 pg/l to 10 microg/l) inhibited cytosolic Ca2+ signals triggered by high Ca2+ or Ni2+. In separate experiments, osteoclasts incubated in 10 mM Ca2+ or on bone released more IL-6 than those in 1.25 mM Ca2+. Furthermore, IL-6 mRNA histostaining was more intense in osteoclasts in 10 or 20 mM Ca2+ than cells in 1.25 mM Ca2+. Similarly, IL-6 receptor mRNA histostaining was increased in osteoclasts incubated in 5 or 10 mM Ca2+. Thus, while high Ca2+ enhances IL-6 secretion, the released IL-6 attenuates Ca2+ sensing and reverses inhibition of resorption by Ca2+. Such an autocrine-paracrine loop may sustain osteoclastic activity in the face of an inhibitory Ca2+ level generated locally during resorption.

Contortrostatin, a homodimeric snake venom disintegrin, is a potent inhibitor of osteoclast attachment.

Disintegrins are small disulfide-rich proteins containing an Arg-Gly-Asp (RGD) sequence near their carboxyl terminus. These polypeptides inhibit binding of adhesion molecules to their receptors (integrins) on the surface of cells. Osteoclasts express integrins, heterodimeric cell surface adhesion receptors, that have been shown to be involved in interactions with the extracellular matrix (ECM), including attachment to bone and bone resorption. It has recently been shown that disintegrins effectively inhibit attachment of osteoclasts to components of the ECM and also disrupt osteoclast-mediated bone resorption. Here we characterize the effects of contortrostatin (CTS), a novel homodimeric snake venom disintegrin, on osteoclast attachment. Plastic dishes coated with CTS were able to support osteoclast attachment with a high affinity (EC50,CTS = 86 +/- 6.7 nM) similar to that of vitronectin (VTN; EC50,VTN = 80 +/- 20 nM). Further, CTS was observed to inhibit completely osteoclast attachment to fetal bovine serum (FBS; IC50,FBS = 0.36 +/- 0.04 nM) and VTN (IC50,VTN = 0.85 +/- 0.13 nM). We used monoclonal antibodies directed against the beta1 (monoclonal antibody [MAb] CD29) and beta3 (MAb F11) integrin subunits to explore the mechanism of osteoclast attachment to immobilized CTS. Only MAb F11 inhibited attachment to immobilized CTS (IC50 = 0.41 +/- 0.12 microg/ml), suggesting that binding to CTS is mediated in part by a beta3 integrin, presumably the alpha(v)beta3 VTN receptor. In further support of an integrin-mediated mechanism, binding of osteoclasts to CTS is inhibited by the RGD peptide, GRGDSP. CTS was also more potent (IC50,FBS = 0.36 +/- 0.04 nM) at inhibiting osteoclast attachment to FBS-coated wells than the monomeric snake venom disintegrin echistatin (IC50,FBS = 8.9 +/- 1.5 nM) or VTN (IC50FBS = 97.5 +/- 25.5 nM). Taken together, these data suggest that the snake venom disintegrin CTS is a potent inhibitor of beta3 integrin-mediated osteoclast attachment, presumably involving the VTN receptor (an alpha[v]beta3 integrin). Further studies of the mechanism of CTS-osteoclast interactions may aid in the design of peptide mimetics to act as antiresorptive agents for the treatment of osteoporosis and other skeletal pathology.

Plasminogen activator system in osteoclasts.

To determine which genes of the plasminogen activator (PA) system were expressed in osteoclasts, RNA extracted from microisolated mouse osteoclasts was used as template for reverse transcribed polymerase chain reaction (RT-PCR) with gene-specific primer pairs. Using this approach, the expression of RNAs for tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, protease nexin, and urokinase receptor isoform 1 (uPAR1) were detected in mouse osteoclasts. The expression of uPAR RNA in osteoclasts was confirmed by in situ hybridization with a uPAR1 probe. RNA encoding the uPAR isoform 2 was not detected in mouse osteoclasts, but a novel unspliced uPAR RNA variant was detected in these cells. The novel uPAR variant and uPAR1 RNA were also detected in mouse calvarial osteoblasts, kidney, muscle, and the mouse macrophage cell line J774A.1 by RT-PCR. The presence of RNAs for most of the components of the PA system in osteoclasts suggests that it may have a functional role in this cell type.

Osteoclast molecular phenotyping by random cDNA sequencing.

The osteoclast is a cell type that is highly specialized for its bone resorption function. In order to decipher the numerous biochemical functions of osteoclasts, a description of the gene expression profile of osteoclasts would be beneficial. We have sought to identify genes that are highly expressed in osteoclasts by partially sequencing 194 randomly chosen cDNA clones from a representative rabbit osteoclast cDNA library. Comparison to nucleic acid and protein sequence databases indicates that 135 of these cDNAs are identical to or homologous to known mammalian genes. Reverse transcription-polymerase chain reaction (RT-PCR) assays with microisolated osteoclasts were used to verify the osteoclast expression of some of these genes. Fifty-nine cDNAs, including two abundantly expressed species, have no significant similarity to the sequence databases and likely represent novel genes. The most abundant of the osteoclast expressed genes encode cofilin and the vacuolar H(+)-ATPase 16 kd subunit. Each were represented at a frequency of 4.1% of the clones in the library (95% confidence interval = 2.4-6.6%). The high expression of these gene products is consistent with the high motility of osteoclasts and their very active hydrogen ion secretion. Other abundantly expressed sequences include beta-actin (95% C.I. = 2.0-6.0%), creatine kinase B (95% C.I. = 1.2-4.9%), c-fms and ribosomal protein L18 (95% C.I. = 0.8-4.3%), and cathepsin-OC2, cyclophilin, delta-aminolevulinate synthetase, 16S mitochondrial rRNA, and two novel gene sequences (95% C.I. = 0.5-3.6%).

Murine osteoclasts and spleen cell polykaryons are distinguished by mRNA phenotyping.

To probe osteoclast gene expression, we combined the techniques of cell microisolation and RT-PCR to develop a novel and sensitive method for the isolation and mRNA phenotyping of small numbers of authentic osteoclasts and spleen cell polykaryons. Using this method we report (1) direct evidence for the presence of calcitonin receptor mRNA in osteoclasts, (2) confirmation of the recent finding of osteopontin mRNA in osteoclasts, and (3) demonstration that the specific expression of mRNA for tartrate-resistant acid phosphatase, carbonic anhydrase II, calcitonin receptor, and osteopontin enable one to distinguish the osteoclast from the morphologically similar and developmentally related spleen cell polykaryon. We also show that mRNA associated with the osteoblast phenotype, such as alkaline phosphatase, osteocalcin, and type I collagen, are absent in osteoclasts. This is the first report in which such an approach has been used successfully to distinguish the mRNA expression pattern of an authentic osteoclast from a macrophage polykaryon, and as such it should provide an important new tool for evaluating the results of various cell culture model systems designed to examine the origin and ontogeny of osteoclasts. Our results also indicate that these procedures can be used as an alternative to in situ hybridization methods for the cell-specific localization of specific mRNA in a mixed cell preparation and for colocalization of multiple mRNA species to a single cell type.

The effects of 1,25-dihydroxyvitamin D3 on osteoclast formation in fetal mouse metatarsal organ cultures.

We have previously described a model system, using 15-day fetal mouse metatarsals cultured in serumless medium, in which osteoclasts and their precursors develop from in situ progenitors in a manner which is similar, both temporally and spatially, to that which occurs in vivo. In this report we evaluate the role of the osteotropic hormone 1,25-dihydroxyvitamin D3 (1,25-D3) on osteoclast formation in this model system by characterizing its effects on proliferation, differentiation, and fusion of cells of the osteoclast lineage. Morphologic evaluation was used to enumerate osteoclast precursors, mono- and multinucleate osteoclasts, and osteoclast nuclei in serial paraffin sections. Dose response data reveal a significant stimulation of osteoclast formation by 1,25-D3 in the range of 0.6 nM to 40 nM, and kinetic analyses suggest that these effects are on proliferation of osteoclast progenitors as well as on differentiation of precursors to form osteoclasts. A single 48 h exposure between day 4 and 6 of culture is necessary and sufficient to induce maximal osteoclast formation, while continuous exposure beyond this "critical period" inhibits multinucleate osteoclast formation. Simultaneous treatment with indomethacin inhibits the effects of 1,25-D3, while treatment with PGE2 stimulates osteoclast formation without significantly increasing precursor numbers, or inhibiting multinucleate osteoclast formation. These results suggest that the effect of 1,25-D3 to induce differentiation of precursors to form mono- and multinucleate osteoclasts is mediated by endogenous prostaglandin synthesis. On the other hand, the inhibition of polykaryon formation observed with continuous 1,25-D3 treatment, does not appear to be a prostaglandin mediated phenomena.(ABSTRACT TRUNCATED AT 250 WORDS)

Mammalian osteoclasts express a transient potassium channel with properties of Kv1.3.

Previous studies have revealed that expression of K+ channels in osteoclasts correlates with cell morphology and is influenced by interaction with the extracellular matrix. In this study, we investigated the electrophysiological properties of an outwardly rectifying K+ channel in rat and mouse osteoclasts using patch-clamp techniques. Cell-attached patch recordings revealed a channel of approximately 14 pS conductance that opened upon depolarization, and had a reversal potential close to that predicted for a K+ channel. Channel activity was transient; inactivation of ensemble currents, like that of whole-cell currents, occurred as a single exponential process. Both single-channel and macroscopic currents exhibited use-dependent inactivation in response to repetitive depolarizations. Two scorpion toxins, margatoxin and charybdotoxin, blocked this transient K+ channel, with half-maximal inhibition at 200 pM and 5 nM, respectively. In contrast, dendrotoxin (500 nM) had little effect. In summary, the outwardly rectifying K+ channel in osteoclasts resembles the Shaker-related K+ channel, Kv1.3. When membrane potential was recorded in whole-cell configuration, charybdotoxin (50 nM) caused a depolarization of 5 to 10 mV from resting levels of -50 mV or more positive; therefore this K+ channel contributes to the membrane potential of osteoclasts under some conditions. To investigate the molecular nature of osteoclast K+ channels, we performed RT-PCR on osteoclast RNA using primers for Kv1.3 and the inward rectifier, IRK1. mRNA encoded by Kv1.3 and IRK1 was detected and message identity confirmed by restriction enzyme digestion and sequence analysis. We conclude that osteoclasts exhibit, in addition to the previously described inward rectifier, an outwardly rectifying K+ conductance with properties of the Kv1.3. channel.

Calcitonin receptor expression and its regulation by 1 alpha-25-dihydroxyvitamin D3 during de novo osteoclast formation in organ cultures of fetal mouse metatarsals.

Organ cultures of 15-day embryonic mouse metatarsals cultured in serumless, chemically-defined medium were used to investigate the influence of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) on calcitonin receptor (CTR) expression in osteoclasts formed from in situ progenitors. CTR expression was demonstrated in tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts and their precursors. The expression of TRAP preceded that of CTR and was first observed in cells identified as osteoclast precursors. Not all TRAP+ precursors were CTR+ and from 40 to 60% of newly formed osteoclasts lacked CTR. Treatment with 1,25(OH)2D3 increased precursor numbers without affecting CTR expression, whereas both the numbers and the percentage of newly formed osteoclasts expressing CTR was increased by 1,25(OH)2D3. Grain count analysis revealed that this increased percentage of newly formed CTR+ osteoclasts were cells with low and medium levels of CTR expression. This apparent down-regulation of CTR expression in newly formed osteoclasts may help to explain the 'escape' of osteoclastic bone resorption from the inhibitory effects of calcitonin.

Skeletal development and formation of osteoclast-like cells from in situ progenitors in fetal mouse metatarsals cultured in chemically defined medium.

An in vitro model system is described, using metatarsal explants from 15-day mouse embryos (E15) cultured in serumless chemically defined medium, to study fetal skeletal development with particular emphasis on de novo osteoclast formation. The normal pattern of growth and differentiation observed in vitro, assessed by ultrastructure and morphometry, demonstrate a permissive local environment which replicates physiologic temporal and spatial relationships which exist in vivo. The population of committed osteoclast progenitors present in E15 metatarsals form osteoclasts and precursors which have cytochemical and ultrastructural features, as well as kinetics of formation, that are similar to that which occurs in vivo. The responsiveness of osteoclast formation to the effects of added 1,25(OH)2D3 illustrates that controlled manipulation enables one to exploit the system for investigating the role of cytokines, growth factors and osteotropic hormones in skeletal development and osteoclast ontogeny.

Bone remodeling in W/Wv mast cell deficient mice.

Strong experimental evidence exists for a relationship between mast cells and bone disease, but the role of mast cells in the regulation of bone remodeling is unknown. In order to address this question, mast cell deficient mice (W/Wv) were paired with their mast cell sufficient (+/+) littermates and evaluated for differences in response to an induced cycle of bone remodeling. This was achieved using a tooth egression protocol, in which a synchronous cycle of bone remodeling was induced in the mandibular buccal alveolar periosteum by extraction of the opposing dentition. Quantitative histomorphometric changes during the activation, resorption, reversal, and formation phases of bone remodeling were documented using standard techniques. Most cell deficient mutants exhibited the following defects in response to an induced cycle of bone remodeling: (a) the onset of the remodeling cycle was delayed by a prolonged activation phase, (b) the duration and extent of the active formation phase was decreased, and (c) the amount of new bone matrix synthesized was diminished while mineralization rates were found to be normal. These results suggest that mast cells and their mediators provide a paracrine mechanism which influences the recruitment of osteoclast and osteoblast progenitors and their participation in bone remodeling. Nonetheless, since bone remodeling occurs in mast cell deficient mice, albeit less efficiently, this mechanism is most likely one of several redundant mechanisms that provide for adequate skeletal homeostasis.

Mouse major histocompatibility complex (H-2) and fetal lung development: implications for human pulmonary maturation.

Using C57/10Sn (B10, H-2b) and B10.A/SgSn (B10.A,H-2a) congenic mice, we measured 1) the level of endogenous pulmonary corticosterone during mouse development; 2) the degree of lung morphological maturation on gestation day 17, with or without corticosteroid treatment; and 3) the maternal influence on normal lung development and fetal response to corticosteroids. The results of our study indicate that there was a progressive increase in the level of endogenous hormone with time in fetal B10 (H-2b) and B10.A (H-2a) mice; throughout mid- to late gestation, the detectable amount of hormone was almost identical in lungs of both strains. Evaluating the degree of lung maturation by morphometry, B10.A mouse lungs were found to be less mature than B10 mouse lungs. Following corticosteroid treatment on day 12 of gestation, H-2a lungs were equal to or more mature than H-2b lungs. We also compared heterozygous mouse lungs from reciprocal crosses (B10.B10.A, b/a and B10.A.B10, a/b). Mice with a maternally derived H-2a haplotype had less mature lungs than those with a maternally derived H-2b haplotype, suggesting a maternal effect. When exogenous hormone was administered, all heterozygous mouse lungs increased in maturity regardless of the origin of the H-2a haplotype. The treated a/b or b/a lungs were more mature than homozygous b/b and less mature than homozygous a/a lungs. We conclude that progressive lung maturation is associated with a gene(s) at or near the H-2 complex, as is the ability to respond to corticosteroids.

In vitro bone resorption activity produced by a hypercalcemic adenocarcinoma tumor line (CAC-8) in nude mice.

Tumor extracts and conditioned tissue culture media from a canine adenocarcinoma tumor line (CAC-8) propagated in nude mice significantly increased in vitro bone resorption in neonatal mouse calvaria as measured by release of previously incorporated 45Ca. In vitro bone resorption activity was induced in a dose-dependent manner, was not suppressible by indomethacin, and was heat- and acid-stable. Gel exclusion chromatography demonstrated peak bone resorbing activity at a relative molecular mass of approximately 28,000. The parathyroid hormone (PTH) antagonist (8,18norleucine, 34tyrosine) bovine PTH (3-34) amide did not inhibit CAC 8-stimulated or (1-34) bPTH-induced bone resorption. There was an increased number of tartrate-resistant, acid phosphatase-positive cells in calvariae exposed to CAC-8 extract. Ultrastructural evaluation of calvaria revealed hypertrophy and maturation of osteoclasts in calvaria exposed to CAC-8 extract. The maturation effects included close contact to bone surfaces and the presence of clear zones and ruffled borders in osteoclasts. Similar structures were observed infrequently in osteoclasts of control calvaria. These data demonstrate that the tumor line (CAC-8) contained activity capable of stimulating in vitro bone resorption by increasing osteoclast numbers and the activity of existing osteoclasts.

Inhibition of bone resorption in vitro by compound 48/80.

The mechanisms that control cycles of bone formation and bone resorption are not well understood. In this report we provide evidence that compound 48/80 is a potent inhibitor of bone resorption in vitro. Resorption was assessed by the release of calcium-45 from pre-labelled newborn mouse calvaria that were treated with compound 48/80 and/or parathyroid hormone (PTH) in organ culture. Our results demonstrate that co-incubation of calvaria with PTH plus compound 48/80 (concentrations 1-10 mcg/ml) produces a marked reduction of calcium-45 release compared to PTH alone. Furthermore, pre-incubation of calvaria with compound 48/80, for as little as three hours, inhibits resorption by subsequent treatment with PTH alone. Measurement of lactate dehydrogenase (LDH) released into the culture medium indicated that treatment with compound 48/80, at the doses and time periods studied, was not cytotoxic. This novel effect of compound 48/80 may provide a useful tool for studying the cellular mechanisms involved in the bone resorption process.

Multiwell chamber chemotaxis assays: improved experimental design and data analysis.

The chemotaxis assay using the Boyden transfilter technique has become widely used in recent years for assessing migratory responses of a wide variety of cell types. In the study reported here we examined the migratory responses of mouse peritoneal macrophages using a multiwell chamber. The experiments were designed to analyze the components of variance in the assay method, to optimize the experimental design, and to develop objective statistical criteria for choosing among experiments with disparate results. Cell counts were obtained with the aid of an image analyzer coupled to a light microscope. Microcomputer software was developed to drive the image analyzer, collect data and conduct statistical analyses. Nested analysis of variance (ANOVA, either 2- or 3-level) was employed to partition the components of variance and F-tests were used to determine their significance. Significant sources of experimental error were identified both within and among wells and were attributed mostly to variability in the chamber/filter assembly and counting procedure. Statistical analyses demonstrated that there was significant variation among assays conducted in different multiwell chambers on the same day, among assays where the same agent was tested on different days in the same chamber, and among replicate counts of the same assay. The following recommendations were made: use ANOVA to distinguish differences due to biological effects from those due to experimental error, design experiments so that all relevant comparisons are included in the same chamber and the same assay, avoid pooling data from different assays unless ANOVA treatment variances are comparable, and when replicate assays yield disparate results choose the assay with the lowest percentage of variation due to experimental error.

Bone-derived macrophage chemotactic factors: methods of extraction and further characterization.

Mononuclear phagocytes have been implicated as important cellular elements in the process of bone resorption. We have postulated that the recruitment and migration of mononuclear phagocytes to bone occurs via a mechanism(s) in which bone-derived chemotactic factors (BDCF) are released from foci undergoing resorption. In the experiments presented here we have used newborn mouse calvaria and examined a variety of extraction protocols, both dissociative and non-dissociative, as means of obtaining stable and reproducible chemotactic activity for mouse peritoneal macrophages. Chemotaxis and chemokinesis were assessed using a multi-well chamber modification of the Boyden transfilter method. Further, we have attempted to purify the BDCF by both molecular sieve and anion exchange chromatography. Our results indicated that non-dissociative extraction with 0.5 M EDTA in the presence of 1% DMSO yielded the most potent and reproducible chemotactic activity. The results of molecular sieve and anion exchange chromatography suggested that there were several BDCF activities in these preparations and that their molecular weights were probably in the range of from 14,000-67,000 daltons. Anion exchange chromatography also demonstrated the presence of a fraction, eluted with 2 M NaCl, with high chemotactic activity and minimal protein concentration. These observations confirmed the suggestion that there are several macrophage chemotactic factors in bone which have as yet to be identified, and suggest methods for pursuing their isolation.