Identification of osteoclast-specific monoclonal antibodies.

Studies on the origin, identification, and characterization of osteoclasts have been difficult. This is in part due to a lack of definitive osteoclast markers and the similarity of these cells in form and function to cells of the mononuclear phagocyte system. To solve this problem, we inoculated isolated chick osteoclasts into mice to generate osteoclast-specific monoclonal antibodies. Supernatants from growth-positive hybridomas were screened by indirect immunofluorescent methods against cultured osteoclasts, monocyte-derived multinucleated giant cells, cultured monocytes, fibroblasts, and limb mesenchyme. Select hybridomas were cloned to produce 375 clones, which were analyzed as described above. Antibody from select clones was also reacted with paraffin sections of bone. In addition, two clones have been analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Antibody binding from an osteoclast-specific clone and a clone reactive with osteoclasts, giant cells, and cultured monocytes (as determined by immunohistochemical assay) was confirmed by antibody-binding and titration curves quantitated by ELISA. The above studies demonstrate that osteoclast specific antigens exist, and that osteoclasts, giant cells, and cultured monocytes share common determinants not found on other cells screened.

Characterization of the purified Chlamydomonas minus agglutinin.

Chlamydomonas flagellar sexual agglutinins are responsible for the adhesion of opposite mating-type (plus and minus) gametes during the first stages of mating. Purification and partial characterization of the plus agglutinin was previously reported (Adair, W. S., C. J. Hwang, and U. W. Goodenough, 1983, Cell, 33:183-193). Here we characterize the purified minus molecule. We show it to be a high molecular weight, hydroxyproline-rich glycoprotein that migrates in the 3% stacking region of an SDS-polyacrylamide gel and is absent from two nonagglutinating minus mutants. Plus and minus agglutinins are remarkably similar, although nonidentical, in amino acid composition, molecular morphology, and reactivity in vivo and in vitro with monoclonal antibodies raised against the plus agglutinin. Moreover, the adhesiveness of both plus and minus agglutinins, when coupled to agarose beads, is abolished by thermolysin, trypsin, periodate, alkaline borohydride, reducing agents, or heat, but unaffected by exo- or endoglycosidases. The minus agglutinin, however, migrates just ahead of the plus molecule on SDS PAGE, is excluded from an anion-exchange (Mono Q) column, elutes earlier during hydrophobic interaction (Bio-gel TSK Phenyl 5PW) chromatography, and is sensitive to chymotrypsin digestion (unlike the plus agglutinin); therefore, it differs from the plus agglutinin in apparent molecular weight, net charge, relative hydrophobicity and proteolytic susceptibility. Nevertheless, our results generally demonstrate a high degree of homology between these complementary cell-cell recognition/adhesion molecules, which suggests that they are specified by genes that have a common evolutionary origin.

Structure of the Chlamydomonas agglutinin and related flagellar surface proteins in vitro and in situ.

Using the quick-freeze, deep-etch technique, we compare the structure of the cane-shaped plus and minus sexual agglutinin molecules purified from gametes of Chlamydomonas reinhardi. We also describe the structure of three additional gamete-specific fibrillar molecules, called short canes, loops, and crescents, which are structurally related to the agglutinins. Four non-agglutinating mutant strains are found to produce the three latter fibrils but not canes, supporting our identification of the cane-shaped molecule as the agglutinin. The heads of the plus and minus canes are shown to differ in morphology. Moreover, two treatments that inactivate the plus agglutinin in vitro--thermolysin digestion and disulfide reduction/alkylation--bring about detectable structural changes only in the head domain of the cane, suggesting that the head may play an indispensible role in affecting gametic recognition/adhesion. We also present quick-freeze, deep-etch images of the flagellar surfaces of gametic, vegetative, and mutant cells of Chlamydomonas reinhardi. The gametic flagella are shown to carry the canes, short canes, loops, and crescents present in in vitro preparations. The cane and crescent proteins self-associate on the flagellar surface into stout fibers of uniform caliber, and they align along the longitudinal axis of the flagellum. The short canes and loops co-purify with flagella but, in the presence of mica, dissociate so that they lie to the sides of the flagella. The agglutinin canes of both mating types are oriented with their hooks at the membrane surface and their heads directed outward, where they are positioned to participate in the initial events of sexual agglutination.

Estrogen's bone-protective effects may involve differential IL-1 receptor regulation in human osteoclast-like cells.

Declining estrogen levels during the first postmenopausal decade lead to rapid bone loss and increased fracture risk that can be reversed by estrogen replacement therapy. The bone-protective effects of estrogen may involve suppression of inflammatory cytokines that promote osteoclastogenesis and bone resorption, such as IL-1, TNF-alpha, and IL-6. We investigated whether estrogen modulates IL-1 actions on human osteoclasts (OCs) and other bone cell types. Isolated human OCs and primary bone marrow-derived OC-like cells expressed both the signaling (IL-1RI) and decoy (IL-1RII) IL-1 receptors, whereas only IL-1RI was detected in osteoblasts. IL-1RII/IL-1RI mRNA ratios and release of soluble IL-1RII (sIL-1RII) were lower in OC-like cells derived from women in the late postmenopausal period compared with younger women, but were unrelated to male donor age, suggesting that estrogen might play a role in regulating IL-1 receptor levels in vivo. Estrogen directly reduced in vitro OC-like cell IL-1RI mRNA levels while increasing IL-1RII mRNA levels and sIL-1RII release. These estrogenic events were associated with inhibited IL-1-mediated cytokine (IL-8) mRNA induction and cell survival, i.e., increased apoptosis. In contrast, estrogen did not alter IL-1R levels or IL-1 responsiveness in primary human osteoblasts or bone marrow stromal cells. We conclude that one novel mechanism by which estrogen exerts bone-protective effects may include a selective modulation of IL-1R isoform levels in OC or OC-like cells, thereby reducing their IL-1 responsiveness and cell survival. Conversely, this restraint on IL-1 actions may be lost as estrogen levels decline in aging women, contributing to an enhanced OC-mediated postmenopausal bone loss.

Osteoclast-specific monoclonal antibodies coupled to magnetic beads provide a rapid and efficient method of purifying avian osteoclasts.

Osteoclasts are the major cell type responsible for normal and pathologic bone resorption. Obtaining highly purified populations of these multinucleated cells has been problematic, although such populations would greatly facilitate investigations of osteoclast regulation and activity. A new immunomagnetic protocol has been devised to surmount these difficulties, employing avian osteoclast-directed monoclonal antibodies (designated 121F, 35L, and 75B) surface coupled to uniformly small, magnetic polystyrene beads covalently conjugated with sheep antimouse IgG. Presentation of these antiosteoclast antibody-coated beads to mixed cell preparations derived from marrow-depleted, collagenase- and/or trypsin-treated chick tibiae and wing bones, followed by magnetic separation and washing, results in efficient and selective binding of osteoclasts to the immunomagnetic beads within minutes. The specific nature of this bead-cell interaction is further demonstrated by the progressive decline in antiosteoclast antibody-coated bead binding to osteoclasts by uncoated beads or beads coated with an irrelevant antibody. Under optimal conditions, these isolations typically yield more than a 100-fold enrichment and greater than a 90% purification of osteoclasts from subpopulations of either predominantly nonviable or viable osteoclasts. Although scanning electron microscopy reveals that immunomagnetically purified and cultured osteoclasts internalize large numbers of the antibody-coated beads, such cells appear unimpaired in their ability to attach to tissue culture plastic or devitalized cortical bone slices and to produce resorption pits characteristic for osteoclasts. Additional studies to ascertain the most effective method for removal (desorption) of antibody-coated beads from magnetically isolated osteoclasts demonstrate that moderate physical agitation is at present the most effective protocol to dislodge antibody-coated beads from the cell surface while maintaining osteoclast viability and function. This immunomagnetic technique therefore provides a gentle method for the isolation of highly purified populations of osteoclasts from heterogeneous bone cell populations in a rapid, efficient, and selective manner.

Decreased nitric oxide levels stimulate osteoclastogenesis and bone resorption both in vitro and in vivo on the chick chorioallantoic membrane in association with neoangiogenesis.

High nitric oxide (NO) levels inhibit osteoclast (OC)-mediated bone resorption in vivo and in vitro, and nitrate donors protect against estrogen-deficient bone loss in postmenopausal women. Conversely, decreased NO production potentiates OC bone resorption in vitro and is associated with in vivo bone loss in rats and humans. Previously, we reported that bone sections from rats administered aminoguanidine (AG), a selective inhibitor of NO production via inducible NO synthase, exhibited both increased OC resorptive activity as well as greater numbers of OC. Here, we investigated further whether AG promoted osteoclastogenesis, in addition to stimulating mature OC function, using a modified in vivo chick chorioallantoic membrane (CAM) system and an in vitro chick bone marrow OC-like cell developmental model. AG, focally administered in small agarose plugs placed directly adjacent to a bone chip implanted on the CAM, dose-dependently elicited neoangiogenesis while stimulating the number, size, and bone pit resorptive activity of individual OC ectopically formed in vivo. In addition to enhancing OC precursor recruitment via neoangiogenesis, AG also exerted other vascular-independent effects on osteoclastogenesis. Thus, AG promoted the in vitro fusion and formation from bone marrow precursor cells of larger OC-like cells that contained more nuclei per cell and exhibited multiple OC differentiation markers. AG stimulated development was inversely correlated with declining medium nitrite levels. In contrast, three different NO donors each dose-dependently inhibited in vitro OC-like cell development while raising medium nitrite levels. Therefore, NO sensitively regulates OC-mediated bone resorption through affecting OC recruitment (angiogenesis), formation (fusion and differentiation), and bone resorptive activity in vitro and in vivo. Possibly, the stimulation of neoangiogenesis and OC-mediated bone remodeling via AG or other pro-angiogenic agents may find clinical applications in reconstructive surgery, fracture repair, or the treatment of avascular necrosis.

Correlation of an osteoclast antigen and ruffled border on giant cells formed in response to resorbable substrates.

The osteoclast is the specialized multinucleated cell primarily responsible for the degradation of the organic and inorganic components of bone matrix. The functional and developmental relationship between osteoclasts and foreign body giant cells is unclear. The osteoclast plasma membrane ruffled border juxtaposed to the bone surface is a unique morphologic characteristic of active osteoclasts. In the studies reported here giant cell formation was induced in response to a variety of materials implanted onto the richly vascularized chick chorioallantoic membrane. Light and electron microscopic techniques were used to examine the morphologic characteristics of the giant cells. In addition, immunohistochemical methods were used to demonstrate the appearance of a 150 kD cell surface antigen on chicken osteoclasts recognized by monoclonal antibody 121F. Giant cells that formed in response to mineralized bone particles exhibited ruffled borders and stained positively with the 121F antibody. Many giant cells that formed in response to hydroxyapatite possessed ruffled borders similar to but not as extensive as those observed on giant cells formed on bone. Immunohistochemical localization of the 121F antigen on these cells suggested that the antigen was present, but staining intensity was reduced compared to that of bone-associated giant cells. The formation of mineral matrix complexes by the adsorption to hydroxyapatite of bone extract or osteocalcin enhanced ruffled borders and the presence of the 121F antigen on elicited giant cells. In contrast, giant cells that formed on non-resorbable materials, such as Sepharose beads, mica, and methacrylate, lacked ruffled borders and were negative for the 121F antigen. It appears that expression of the 121F osteoclast antigen correlates with the appearance and extent of ruffled membranes on giant cells. Furthermore, it appears that giant cell ruffled membrane development and the presence of the 121F osteoclast antigen are related to giant cell formation in response to resorbable materials that are subject to extracellular dissolution. Expression of this antigen may be indicative of the developmental and/or functional state of giant cells (osteoclasts) that form on resorbable substrates. In addition, components of the bone matrix, including osteocalcin, in association with bone mineral, lead to elevated levels of this osteoclast antigen.

Isolation of avian osteoclasts: improved techniques to preferentially purify viable cells.

Among the many different methods that have been used to obtain and study isolated osteoclasts from a variety of species, the egg-laying hen maintained on a low-calcium diet has proven to be one of the richest sources of relatively large numbers of osteoclasts. However, recent reports and our own observations indicate that only a very small proportion of the osteoclasts harvested by such methods are viable. The difficulty in obtaining large numbers of viable osteoclasts has restricted studies of osteoclast function and regulation, and so new isolation methods were sought. This report describes an osteoclast isolation procedure designed to substantially enrich for large numbers of viable authentic osteoclasts. Size and cell density differences between osteoclasts and contaminating mononuclear cells have been exploited in developing the methods for osteoclast enrichment. Sequential nonenzymatic and enzymatic procedures, followed by cell density separations, have yielded three populations of osteoclasts derived from chick hatchlings maintained on a low-calcium diet. A corresponding decrease in bone-associated osteoclasts during the sequential isolation scheme has been monitored using an osteoclast-directed monoclonal antibody, 121F. The first two populations contain 40% osteoclasts, which are predominantly (greater than 99%) nonviable, but the third population contains 8-fold more viable osteoclasts, effectively increasing the proportion of viable osteoclasts more than 25-fold in comparison with the first two populations. The osteoclast-like nature of the isolated viable population 3 cells was established by demonstrating ruffled border formation, possession of the 121F monoclonal antibody-reactive osteoclast antigen, bone particle resorption activity, and resorption pit formation on cortical bone slices revealed by transmission and scanning electron microscopy.

Osteoclast 121F antigen expression during osteoblast conditioned medium induction of osteoclast-like cells in vitro: relationship to calcitonin responsiveness, tartrate resistant acid phosphatase levels, and bone resorptive activity.

Osteoclast differentiation from hematopoietic precursors into multinucleated cells uniquely capable of removing the organic and inorganic components of bone matrix occurs in multistep process, during which osteoclasts acquire the specialized characteristics necessary for bone resorptive activity and physiological regulation. Among those traits is a novel plasma membrane glycoprotein, reactive with the anti-osteoclast monoclonal antibody 121F, which is expressed during the course of osteoclast differentiation, shares structural and functional homologies with Mn2+/Fe2+ superoxide dismutase, and has been hypothesized to protect the osteoclast from the damaging effects of superoxide radicals generated during active bone resorption. We have reported previously that the expression of this membrane antigen is induced on multinucleated giant cells when the prefusion marrow mononuclear cells are cultured in conditioned medium from avian calvaria. The studies reported here were designed to investigate the relationship between expression of the 121F antibody-reactive osteoclast membrane antigen and tartrate resistant acid phosphatase levels, bone resorptive activity, calcitonin responsiveness, and ultrastructural features of avian bone marrow-derived multinucleated giant cells formed either in the presence or absence of diffusible osteoblast secreted factors. Parallel analyses of in vivo formed osteoclasts isolated from the same animals were performed for direct comparisons. In this report we demonstrate: (1) that the 121F monoclonal antibody-reactive osteoclast membrane antigen is stably induced in giant cells by soluble osteoblast-derived factors in a species nonrestricted but concentration- and temporal-dependent manner; (2) that osteoblast-mediated antigen induction is reflected in both increased numbers of cells and elevated expression of individual cells that are reactive with the 121F antibody, as determined by ELISA and histomorphometry; (3) that osteoblast conditioned medium, in addition to inducing this antigen in bone marrow cells, also elevates other defining osteoclast characteristics in these avian giant cells including their TRAP activity, cell retraction from the bone surface in response to calcitonin, bone resorptive function, and expression of a series of additional osteoclast antigenic markers; and (4) that secreted osteoblast products alone do not raise the levels of these traits for in vitro formed marrow giant cells to the extent associated with in vivo formed osteoclasts. Therefore, osteoblast soluble factors alone appear unable to promote the full differentiation of bone marrow cells in vitro into mature bone-resorbing osteoclasts.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of avian osteoclast bone resorption by monoclonal antibody 121F: a mechanism involving the osteoclast free radical system.

Osteoclasts generate high levels of superoxide anions during bone resorption that contribute to the degradative process, although excessive levels of this free radical may be damaging. One mechanism for their removal is via superoxide dismutase (SOD), a protective superoxide scavenging enzyme. We have previously described a novel developmentally regulated 150 kDa plasma membrane glycoprotein of avian osteoclasts which is reactive with the osteoclast-specific monoclonal antibody (Mab) 121F and is related immunologically, biochemically, and in protein sequence to mitochondrial Mn2+/Fe2+ SOD. We hypothesized that this unusual osteoclast surface component may be involved in protection against superoxides generated during active bone resorption. Increasing concentrations of monovalent Fab fragments prepared from Mab 121F, but not those from another antiosteoclast Mab designated 29C, markedly inhibited both bone particle and bone pit resorption by avian osteoclasts, while reducing tartrate-resistant acid phosphatase activity and causing the morphological contraction of osteoclasts on bone. Thus, the SOD-related membrane antigen may be essential for osteoclast bone resorption. Osteoclast superoxide production, monitored kinetically by cytochrome c reduction and histochemically by nitroblue tetrazolium reduction staining, was significantly greater in the presence of 121F, but not 29C, Fab treatment. Furthermore, the release of another free radical known as nitric oxide, which is produced by osteoclasts, can scavenge superoxides, and acts to potently inhibit osteoclast bone resorption, was dose-dependently increased by 121F Fab in resorbing osteoclast cultures. Therefore, Mab 121F binding may block the potential protective function of the osteoclast plasma membrane SOD-related glycoprotein, leading to a rapid elevation of superoxide levels and a subsequent rise in osteoclast nitric oxide release, feedback messages which may be sensed by the osteoclast as signals to cease active bone resorption.

Charged beads: generation of bone and giant cells.

Based on reports of electrically induced bone formation and findings that some materials used to promote bone ingrowth are stimulatory in bead form, the osteogenic potential of beads with different surface charges was examined. In this preliminary study, three types of Sephadex beads were injected into chick femora: type I, DEAE beads, positively charged; type II, CM beads, negatively charged; type III, G-25, uncharged. Beads were injected into the femoral midshaft, and after 3 days, 4 days, and 1 week, birds were sacrificed and femora were processed for histology. Type I beads: at 3 days, were surrounded by multinucleated giant cells; by 4 days, patches of bead-associated new bone were present along with giant cells; after 1 week, occasional bead-associated multinucleated cells were seen, but now most beads were surrounded by new intramedullary bone, forming an extensive bead-bone lattice. With bead types II and III, bead-associated new bone was seen at 3 days and 4 days only when beads lodged near the endosteum or in the metaphysis. At 7 days, no bone was seen with either of these two bead types. The response to the type I beads may be likened to a remodeling phenomenon with large numbers of giant cells at 3 days, new bone and giant cells at 4 days, and evidence only of bone formation at 7 days.

Influence of osteoclasts and osteoclast-like cells on osteoblast alkaline phosphatase activity and collagen synthesis.

Osteoblasts have been shown to modulate osteoclast activity, but the reverse process has not been investigated. In the current study conditioned medium (CM) was collected from osteoclasts and osteoclast-like cells and its effects on osteoblast alkaline phosphatase (ALPase) activity and collagen synthesis ([3H]proline hydroxylation) were determined. In primary chick osteoblasts, cultured chick embryo frontal bones, and UMR-106-01 cells, collagen synthesis and ALPase activity, but not [3H]thymidine incorporation, were inhibited by CM from chick marrow-derived giant cells, which possess some of the phenotypic characteristics of osteoclasts. However, collagen synthesis in chick embryo fibroblasts was not affected by giant cell CM. CM collected from cultures of chicken osteoclasts and human osteoclastoma cells and marrow-derived giant cells inhibited collagen synthesis in UMR-106-01 cells, but the effects of ALPase activity varied with the cell type. In contrast, mononuclear cell and fibroblast CM did not alter collagen synthesis. Initial characterization studies demonstrate that the inhibitor is a heat-labile factor with a molecular weight greater than 3500. In summary, authentic osteoclasts, tumor osteoclast-like cells, and chicken and human multinucleated giant cells produce a soluble factor that alters osteoblast collagen synthesis, suggesting that osteoclasts play a role in the modulation of osteoblast activity.

Ca2+ or phorbol ester but not inflammatory stimuli elevate inducible nitric oxide synthase messenger ribonucleic acid and nitric oxide (NO) release in avian osteoclasts: autocrine NO mediates Ca2+-inhibited bone resorption.

Osteoclast bone resorption is essential for normal calcium homeostasis and is therefore tightly controlled by calciotropic hormones and local modulatory cytokines and factors. Among these is nitric oxide (NO), a multifunctional free radical that potently inhibits osteoclast bone resorption in vitro and in vivo. Previous findings led us to propose that NO might serve as an autocrine, as well as paracrine, regulator of osteoclast function. This premise was investigated using isolated bone-resorptive avian osteoclasts and focusing on the inducible isoform of NO synthase (iNOS) responsible for inflammatory stimulated high-level NO synthesis in other cells. Avian osteoclasts expressed both iNOS messenger RNA (mRNA) and protein. However, inflammatory cytokines that induce iNOS mRNA, protein, and NO in other cells did not do so in avian osteoclasts, consistent with the known role of inflammatory stimuli in promoting osteoclast resorption and localized bone loss. In searching for potential modulators of osteoclast iNOS, protein kinase C activation [by phorbol 12-myristate 13-acetate (PMA)] and intracellular Ca2+ rises (A23187) were each found to elevate osteoclast iNOS mRNA and protein levels, while increasing NO release and reducing osteoclast bone resorption. The iNOS selective inhibitor aminoguanidine suppressed stimulated osteoclast NO production elicited by either signal, but reversed only the resorption inhibition due to raised Ca2+. Thus, whereas additional inhibitory signals are presumably coproduced in osteoclasts treated with PMA, osteoclast iNOS-derived NO may act as an autocrine signal to mediate Ca2+-inhibited bone resorption. These findings document for the first time an iNOS whose mRNA levels are regulated by Ca2+ or PMA, but not inflammatory stimuli, and the autocrine production of NO as a Ca2+ sensing signal to suppress osteoclast bone resorption. The unusual regulation of osteoclast iNOS makes it a potentially attractive target for designing novel therapeutic agents to alleviate excessive bone loss.

Estrogen modulation of avian osteoclast lysosomal gene expression.

We have previously demonstrated that avian osteoclasts contain high levels of 17 beta-estradiol (17 beta E2) receptors and respond to 17 beta E2 treatment with a dose-dependent decrease in in vitro resorption of [3H] proline-labeled bone particles. To more accurately assess the influence of 17 beta E2 on osteoclastic activity, the specificity of estrogen modulation of resorption levels was determined using a quantitative pit resorption assay. Treatment with 17 beta E2 significantly decreased the number of osteoclast resorption pits formed compared with that after either vehicle or 17 alpha E2 treatment. Cotreatment with 17 beta E2 and hydroxytamoxifen (a complete 17 beta E2 antagonist in birds) abrogated the influence of 17 beta E2 on resorption activity. To elucidate the mechanism by which 17 beta E2 inhibits osteoclast activity, the effects of 17 beta E2 on the steady state mRNA levels of two avian osteoclast lysosomal proteins, lysozyme and a lysosomal membrane protein (LEP-100), were examined. Using highly purified avian osteoclasts, 17 beta E2 was shown to decrease lysosomal protein mRNA levels in a dose-dependent manner within 8 h of treatment in a process that required de novo protein synthesis. This response was specific for 17 beta E2, since the inactive stereoisomer 17 alpha E2 had no effect. Furthermore, coincubation of 17 beta E2 with hydroxytamoxifen eliminated the 17 beta E2 influence. After removal of 10(-8) M 17 beta E2, lysosomal gene mRNA levels returned to near-normal levels within 24 h. This is consistent with the previously reported ability of avian osteoclast-mediated resorption activity to recover from 17 beta E2 treatment. Lysozyme protein levels similarly decreased after 17 beta E2 treatment. These data suggest that avian osteoclasts are target cells for 17 beta E2 in vitro, that osteoclast activity in vivo is likely to be modulated by circulating levels of 17 beta E2, and that the 17 beta E2 inhibition of osteoclast resorption activity may be mediated at least in part via regulation of osteoclast lysosomal gene expression.

Human osteoclasts and osteoclast-like cells synthesize and release high basal and inflammatory stimulated levels of the potent chemokine interleukin-8.

Chemokines, including interleukin-8 (IL-8), function as key mediators in diverse inflammatory disorders via promoting the recruitment, proliferation, and activation of vascular and immune cells. IL-8 levels are elevated in inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, osteomyelitis, and periodontal disease, that also exhibit progressive bone loss. Therefore, it is possible that IL-8 contributes to the osteopenia associated with these pathological conditions. Although macrophages, neutrophils, and endothelial cells are considered the primary sources of inflammation-induced IL-8 increases, we report here for the first time that human bone marrow-derived osteoclast-like cells (hOCL) as well as authentic bone-resorbing human osteoclasts (hOC) isolated from osteoporotic femoral heads express messenger RNA (mRNA) for IL-8 and secrete high levels of IL-8 during culture. Basal IL-8 release by cultured hOC or hOCL was orders of magnitude greater than the release of the proinflammatory cytokines IL-1beta, IL-6, and tumor necrosis factor-alpha. At a cellular level, in situ hybridization analysis revealed that IL-8 mRNA was expressed in resorbing hOC of rheumatoid arthritic pannus and was substantially greater than that expressed in hOC of noninflammatory giant cell tumor of bone tissue. Therefore, the potential inflammation-mediated induction of IL-8 was directly assessed using cultured hOCL. IL-8 release was stimulated by proinflammatory signals (IL-1alpha, tumor necrosis factor-alpha, lipopolysaccharide, or phorbol 12-myristate 13-acetate), unaffected by various other osteotropic modulators (transforming growth factor-beta1 and -beta3, IL-6, 17beta-estradiol, or calcitonin) and was decreased by interferon-gamma, vitamin D3, and the antiinflammatory glucocorticoid dexamethasone. Changes in IL-8 secretion were paralleled by corresponding changes in IL-8 mRNA steady state levels. We conclude that hOC and hOCL synthesize and secrete high constitutive and inflammation-stimulated levels of the chemokine IL-8. Consequently, hOC-derived IL-8 could act as an important regulatory signal for bone, vascular, and immune cell recruitment and activation during normal and pathological bone remodeling.

Osteoclast recruitment in response to human bone matrix is age related.

The effect of bone matrix age on the recruitment and differentiation of osteoclast precursors was studied using the chick chorioallantoic membrane (CAM) implant system. Devitalized mineralized bone particles (75-250 microns) were prepared from human femoral cortical bone obtained postmortem from 8 men (age range: 18-72 years). The particles were implanted onto the CAM and 8 days later implants were harvested and processed for light microscopic, morphometric or immunohistochemical analysis. Histomorphometric analysis was performed on samples representing each donor age. The analysis was grouped into three categories consisting of bone from young adults (18-20 years), adults (34-53 years) and aged individuals (67 years and older). Total osteoclast number, osteoclast number per bone particle, cell area, cell size, number of nuclei per cell profile, nucleocytoplasmic ratio, and the presence of a distinctive osteoclast antigen defined by monoclonal anti-body 121F were determined. Bone matrix from older individuals, and therefore the oldest age group (67 years and older), elicited significantly fewer multinucleated cells when compared to bone matrix from younger donors. The number of nuclei per cell profile was highest in the adult population (34-53 years), and there was a continuous increase in cell area with aging. As a consequence, the nucleocytoplasmic ratio decreased from the youngest to the oldest age group. These findings indicate that, relative to factors that affect the recruitment and differentiation of osteoclast precursor cells, bone matrix of older individuals is changed in quality and/or quantity compared to bone matrix from younger individuals. It is hypothesized that this decline in osteoclast formation in response to older bone matrix may contribute to the impaired bone remodeling associated with aging.

Bone cell function, regulation, and communication: a role for nitric oxide.

A large array of factors serve as vital communication links between cells and the characterization, regulation, and mechanisms of action of such factors are topics of intense research efforts. Most intercellular messenger molecules which have been described over the years are represented by proteins, small peptides, amino acids or their derivatives, ions, lipid metabolites, or steroids. However, a small uncharged free radical, nitric oxide, has recently garnered much attention as a potent multifunctional signal molecule with widespread actions within and between diverse tissues. Biochemical, molecular, and regulatory studies of the family of enzymes responsible for nitric oxide synthesis, nitric oxide synthases, have established that there are at least three distinct isoforms of this enzyme which are differentially expressed and regulated in various cells or tissues. Modulation of these isoenzyme levels or activities by diverse signals is mediated via transcriptional, translational, and/or post-translational mechanisms, and consequently, alterations in such control may influence normal or pathological processes. Nitric oxide appears to exert pronounced effects on skeletal physiology and its production by various bone cells, elicited target cell responses, modulation by other signalling molecules (e.g., cytokines, hormones, fatty acid derivatives), and chemical interactions with other free radicals (e.g., superoxide anions, hydroxyl radicals) may form one important facet of the many complicated communication pathways controlling bone cell physiology and remodeling. Further cell and molecular studies are needed to address the precise roles that nitric oxide plays in bone development and in the formation and degradation of bone during ordinary bone metabolism. In addition, alterations in the regulation and action of the bone nitric oxide system as a function of certain bone disorders may be manifested by perturbations in bone integrity or mineral homeostasis. In this article, we review the current evidence implicating nitric oxide as an important messenger molecule in bone intercellular communication, speculate on potential roles for this radical in bone biology, and discuss possible future directions for advanced research into the function of nitric oxide in skeletal physiology.

The possible differentiation of osteogenic elements in vitro from chick limb mesodermal cells. I. Morphological evidence.

In the developing chick limb bud, myogenic, fibrogenic, chondrogenic, and osteogenic tissues are derived from embryonic mesenchyme. Previous reports show that when stage 24 limb mesenchymal cells are cultured in vitro, chondrocytes, fibrocytes, and myocytes can be identified on the basis of morphological and biochemical parameters. The studies reported here clearly demonstrate that, in similar cultures, crystalline calcium phosphate material is deposited in the chondrocytic and fibrocytic matrices. Such crystalline material is not observed before Day 10 of culture life. Subsequent to Day 10 of culture, first amorphous, then crystalline calcium phosphate is observed. On the basis of light and electron microscopic analysis, it appears that the in vitro calcification phenomenon closely resembles the morphological and temporal sequence of osteogenesis observed in vivo.

Growth behavior and lineage of isolated and cultured cells derived from giant cell granuloma of the mandible.

A central giant cell granuloma of the mandible was fractionated into its mononuclear and multinuclear cellular constituents. The cells were subsequently grown in tissue culture. Sections from the original lesion and the cultured cells were analysed histochemically and immunocytochemically. Acid phosphatase, non-specific esterase, Lysozyme and alpha-1-antitrypsin were employed as markers for cells of histiocytic origin and Factor VIII-related antigen served as an endothelial cell marker. The mononuclear cells were of 2 types; a spindle-shaped cell and a round macrophage-like cell. The giant cells and the macrophage-like cells had a limited life span in culture and survived for up to 2 and 5 weeks respectively. However, the spindle-shaped cells continued to proliferate with a doubling time of 48 h. The giant cells and the macrophage-like cells were identical in their staining characteristics and showed positive staining for all the histiocytic markers tested. In contrast, the spindle-shaped cells were negative for those markers. None of the 3 cell types stained positively for Factor VIII-related antigen. These findings suggest that the giant cells in giant cell granuloma of the jaw are reactive, fully differentiated end-cells that are probably derived from stromal macrophages. The histogenesis of the spindle-shaped cell is not yet known. It is also shown in this study that the histochemical and in vitro growth characteristics of the cells of central giant cell granuloma of the mandible are analogous to those of giant cell tumors of long bones.