Defective binding of macrophages to bone in rodent osteomalacia and vitamin D deficiency. In vitro evidence for a cellular defect and altered saccharides in the bone matrix.

In the osteomalacic as well as normal skeleton, few osteoclasts are associated with osteoid-covered bone surfaces. The reason for this particular cellular deficit is not clear, but may relate to the inability of osteoclasts and/or osteoclast precursors (monocyte-macrophages) to attach to immature, unmineralized bone matrix, a step apparently essential for normal resorptive activity and osteoclast differentiation. In this study, we have examined cell-bone binding using macrophages (M phi) and bone isolated from vitamin D-deficient rats and hypophosphatemic, osteomalacic mice and from their normal counterparts. The data show that M phi-bone attachment is greatly reduced (P less than 0.001) in both vitamin D deficiency and hypophosphatemia, but that the mechanisms responsible for this reduction are apparently different in the two disorders. In hypophosphatemia, the reduction in binding appears solely attributable to the absence or inaccessibility of bone matrix oligosaccharides or glycoproteins essential to the attachment process. In vitamin D deficiency, on the other hand, not only is the bone matrix defective as a binding substrate, but the M phi, per se, is limited in its capacity to attach to normal, vitamin D-deficient, and hypophosphatemic bone.

Saccharides mediate the attachment of rat macrophages to bone in vitro.

Macrophages (M phi) are multipotential cells capable of giving rise to osteoclasts and of resorbing bone. Since both of these processes are ultimately dependent upon the attachment of cells to a mineralized bone surface, we have examined in this study the mechanism by which such attachment is achieved. The data show that elicited rat peritoneal M phi bind to bone in a temperature-dependent and -saturable manner with half-maximal attachment occurring within 10 min at 37 degrees C and reaching a plateau by approximately 60 min. The kinetics of binding are essentially the same whether devitalized bone particles or viable calvaria are used as a substrate. The attachment of M phi to bone is inhibited by some sugars (e.g., N-acetyl-galactosamine, thiogalactoside, beta-lactose), fetuin and asialofetuin, and by pretreating the bone with periodate. Binding is also significantly reduced when M phi are preincubated with tunicamycin and swainsonine at nontoxic concentrations sufficient to inhibit or alter glycosylation. On the other hand, exposing the cells to neuraminidase increases the capacity of M phi to bind to bone. Collectively, our observations indicate that the attachment of M phi to bone is a highly regulated process and is mediated, at least in part, by saccharides located on both the cell and the bone surface.

Human alveolar macrophages produce a fibroblast-like collagenase and collagenase inhibitor.

Human macrophages have been implicated in connective tissue remodeling; however, little is known about their direct effects upon collagen degradation. We now report that human alveolar macrophages in culture produced both a collagenase and a collagenase inhibitor. The collagenase was secreted in latent form and could be activated by exposure to trypsin. Collagenase production could be increased three- to fourfold by incubating the cells with lipopolysaccharide, but synthesis was largely unaffected by exposure to phorbol myristate acetate. By several criteria, macrophage collagenase was the same as the collagenase secreted by human skin fibroblasts: (a) they were antigenically indistinguishable in double immunodiffusion; (b) both degraded type III collagen preferentially to type I, had little activity against type II collagen, and none against types IV and V, and (c) their affinity for susceptible collagens was equivalent, Michaelis constant = 1-2 microM. Collagenase inhibitory activity was also present in the macrophage-conditioned medium, and was accounted for by an antigen that showed immunologic and functional identity with the collagenase inhibitor secreted by human skin fibroblasts. The amount of inhibitor released by unstimulated cells, approximately 100 ng/10(6) cells per 24 h, was substantially augmented by both phorbol and lipopolysaccharide, although considerable variability in response to these agents was observed between macrophage populations derived from different subjects. As negligible quantities of collagenase or collagenase inhibitor were detectable intracellularly, it appeared that both proteins were secreted rapidly after synthesis. Thus, human macrophages have the capacity to modulate collagen degradation directly by production of collagenase and collagenase inhibitor.

Glucocorticoids modulate macrophage surface oligosaccharides and their bone binding activity.

The circumstantial evidence that indicates that glucocorticoids (GC) may stimulate osteoclastic resorption in vivo has recently found support in observations that demonstrate that these compounds effectively increase the activity of isolated resorptive cells (osteoclasts, macrophage polykaryons, and elicited macrophages [MO] ) in vitro. Data are presented here that indicate that this stimulation by GC is due to an enhancement of the initial stage of the resorption process, the attachment of cells to bone, and that this is caused by alterations of cell surface oligosaccharides. Specifically, dexamethasone and cortisol enhance by 80% the attachment of MO to bone surfaces in a dose dependent manner but do not alter or reduce the binding of these cells to other surfaces (plastic, collagen, and hydroxyapatite crystals). The effect of GC on cell-bone attachment is blocked by the glycosylation inhibitor, tunicamycin, and the glycosylation modifier, swainsonine; this demonstrates that asparagine-linked oligosaccharides are involved in the stimulatory process. Flow cytometric analysis of GC-treated cells using a panel of fluoresceinated lectins confirms this by indicating a selective, enhanced exposure of plasma membrane-associated N-acetylglucosamine and N-acetylgalactosamine residues, sugars we have previously shown to be pivotal in MO-bone binding. Finally, progesterone, a known GC antagonist, blocks GC-stimulated resorption, macrophage-bone binding, and membrane oligosaccharide modification, presumably by competing for the GC receptor. Progesterone alone alters none of these processes. Thus, GC stimulates the resorptive activity of macrophages by enhancing the initial events in the degradative process (cell-bone binding) and does so, apparently, via receptor-mediator alteration of cell surface glycoproteins.

Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties.

This study examines the osteoblastic properties of the established human osteosarcoma cell line Saos-2. Saos-2 cells inoculated into diffusion chambers, which were implanted i.p. into nude mice, produced mineralized matrix in 4 of 6 chambers at 8 weeks. In 5 of 6 chambers there was a strong positive alkaline phosphatase reaction. In culture the alkaline phosphatase levels increased with time and cell density, reaching very high levels at confluence: 4-7 mumol/mg protein/min. The cells show a sensitive adenylate cyclase response to parathyroid hormone, 50% effective dose = 2.8 nM, which increases with cell density and is further raised by dexamethasone treatment. They also exhibit typical binding of 1-25-dihydroxyvitamin D3 to 3.2S receptor protein with an apparent Kd of 0.21 nM; the numbers of sites per cell were 3,300 at 50,000 cells/cm2 and 1,800 at 280,000 cells/cm2. The presence of osteonectin was visualized with a monoclonal antibody which revealed a reticular pattern on the cell surface. Osteonectin was also detected in the medium by Western blots, migrating at around Mr 40,000 in nonreduced gels and Mr 44,000 in reduced gels. The Saos-2 cells thus possess several osteoblastic features and could be useful as a permanent line of human osteoblast-like cells and as a source of bone-related molecules.

Developmental regulation of proenkephalin gene expression in osteoblasts.

Proenkephalin (PENK), a classically defined opioid gene, was originally thought to be expressed almost exclusively in the mature nervous and neuroendocrine systems. In the last few years, it was demonstrated, however, that high levels of PENK messenger RNA and PENK-derived peptides are expressed in embryonic mesenchymal tissues during differentiation into mature tissues and organs. Shortly after birth, as development progresses, PENK expression drops in those tissues to undetectable levels. Very little is known about the molecular mechanisms regulating this transient expression. To investigate those mechanisms, we used primary cell cultures of calvaria-derived osteoblasts. These cultures express PENK and exhibit a normal pattern of osteoblastic differentiation. In the present study we demonstrate that 1) a reciprocal interrelationship exists between PENK expression and osteoblastic differentiation in vivo, ex vivo, and in vitro; namely, PENK expression is down-regulated upon cellular differentiation; 2) PENK promoter usage and messenger RNA splicing function similarly in osteoblasts and in neural cells; 3) osteoblastic PENK expression is modulated by bone-targeting hormones; and 4) this down-regulation is inhibited by the serine/threonine kinase inhibitor H-8. The link between osteoblastic differentiation and down-regulation of PENK expression together with our preliminary findings indicating the existence of an osteoblastic opioid receptor suggest that opioids act in an autocrine/paracrine mechanism on undifferentiated osteoblasts and play a significant role in bone development.

Arachidonic acid metabolism by a vitamin D3-differentiated human leukemic cell line.

HL-60 is a human promyelocytic cell line that differentiates along the granulocytic pathway when incubated with dimethylsulfoxide and along the monocytic pathway when incubated with 1,25-(OH)2D3. We compared arachidonic acid metabolism in undifferentiated, DMSO-differentiated, and 1,25-(OH)2D3-differentiated cells. DMSO- and 1,25-(OH)2D3-differentiated cells metabolized exogenous arachidonic acid to both cyclo-oxygenase products (predominantly thromboxane B2 and prostaglandin E2) and 5-lipoxygenase products, including leukotriene B4. Undifferentiated cells produce these metabolites in much smaller amounts. DMSO-differentiated cells released a large percentage of phospholipid-bound arachidonic acid in response to stimulation with the ionophore A23187, zymosan, or formylmethionylleucylphenylalanine (FMLP). DMSO-differentiated cells stimulated with A23187 converted released arachidonate to LTB4 and TxB2. In contrast, 1,25-(OH)2D3-differentiated cells released a smaller percentage of phospholipid-bound arachidonate in response to stimuli, and undifferentiated cells released none at all. The three cell types (undifferentiated, DMSO-differentiated, and 1,25-(OH)2D3-differentiated) were homogenized and the 10,000 X g supernatant incubated with [14C]arachidonic acid. The supernatants from the homogenates of the DMSO- and 1,25-(OH)2D3-differentiated cells metabolized [14C]arachidonic acid to cyclooxygenase and lipoxygenase products, but the supernatant from the homogenate of undifferentiated cells did not. These data indicate that differentiation of HL-60 cells with DMSO or 1,25-(OH)2D3 induces cyclooxygenase and 5-lipoxygenase and induces mechanisms for the release of arachidonate from phospholipids by soluble and particulate stimuli.

Reversibility of vitamin D-induced human leukemia cell-line maturation.

HL-60 cells are induced to differentiate along a monocytic pathway by the active metabolites of vitamin D3, e.g. 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. All such differentiated cells share a number of features in common but are heterogeneous in their ability to adhere to solid substrates and to resorb devitalized bone matrix. Here, we show that, in addition, as compared to the nonadherent, adherent cells are smaller, less likely to be in the S phase, more enriched in the human monocyte-specific cell surface antigen, 63D3, and contain less cmyc messenger RNA (mRNA). In addition, we document that removal of the hormone leads to dedifferentiation. For these susceptible mononuclear cells, removal of 1,25-(OH)2D3 results in a reversion to a more myeloblastic phenotype, renewed cell proliferation, and the rapid appearance of elevated levels of cmyc mRNA. Finally, we report that the cells that do not revert upon 1,25-(OH)2D3 removal are those that became multinucleated during treatment.

Neurotensin modulates human neutrophil locomotion and phagocytic capability.

Neurotensin (NT) was found to induce oriented locomotion and augment the phagocytic capability of human blood neutrophils over 10(-11) - 10(-7) M. The tridecapeptide also causes Ca2+ extrusion from neutrophils, very likely as a result of intracellular Ca2+ mobilization. It is suggested that the NT-mediated functional modulation of neutrophils correlates with the capacity of NT to affect the intracellular compartmentalization of Ca2+. Peripheral NT-elicited phenomena such as vasodilation, enhanced vascular compartmentalization of Ca2+. Peripheral NT-elicited phenomena such as vasodilation, enhanced vascular permeability, mast cell degranulation and the enhancement of directional migration and phagocytosis of neutrophils described here, classify NT as a typical mediator of inflammation.

Modulation of the expression of CD4 on HL-60 cells by exposure to 1,25-dihydroxyvitamin D3.

The CD4 molecule functions as a receptor for the binding and infectivity of the human immunodeficiency virus (HIV). It is of interest, therefore, to develop procedures for its down-regulation. In the present study, the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the expression of cell surface antigens of the HL-60 promyelocytic leukemia cell line was analyzed. Exposure of HL-60 cells to 1,25(OH)2D3 resulted in down-regulation of CD4 as assessed by their staining with the Leu-3a monoclonal antibody (MoAb). This treatment increased the staining of HL-60 cells with the monocyte-specific 63D3 MoAb. In contrast to the rapid elimination of cell surface CD4 by exposure of HL-60 to phorbol myristate acetate (PMA), the maximal reduction of CD4 by 1,25(OH)2D3 was attained within 48 h after the beginning of the exposure.

Enhancement of phagocytosis by neurotensin, a newly found biological activity of the neuropeptide.

Specific binding of neurotensin (NT) to mouse peritoneal thioglycollate-elicited macrophages and macrophages differentiated in vitro from bone marrow cells was demonstrated and characterized. NT binding to these phagocytes modulated their phagocytic capacity in a biphasic manner. At concentrations of 10(-14) to 10(-9) M NT, a dose-dependent augmentation of phagocytosis (up to 2-fold) was observed. Further increases in the concentration of NT resulted in a gradual decrease of the augmented response until the basal phagocytic activity (in the absence of NT) was reached. Three partial sequences of NT, NT (8-13), NT (6-13) and NT (1-10), were also effective in augmenting the phagocytic response of thioglycollate elicited macrophages, but the maximal effect was attained at about 10(-7) M and stayed at that level up to a concentration of 10(-5) M. The activity of the three NT partial sequences was comparable to that of substance P and tuftsin. Scatchard analysis of (3H)NT binding to macrophages suggested the existence of two populations of binding sites, a major population of relatively low affinity binding sites and a small population of high affinity binding sites. NT (8-13), NT (6-13), substance P and tuftsin competed with (3H)NT binding to the low affinity sites with a comparable KI to that of NT. NT (1-10) did not compete for the binding at the low affinity sites. It is suggested that NT binding to the high affinity sites leads to enhancement of phagocytosis, whereas its binding to the low affinity sites leads to inhibition of the augmented response. However, the low affinity sites are the sites of interaction of NT (8-13), NT (6-13), substance P and tuftsin with the phagocytes and their saturation with the peptides leads to augmentation of phagocytosis.

TNF-alpha expression is transcriptionally regulated by RANK ligand.

Tumor necrosis factor (TNF)-alpha is known for its osteoclastogenic and resorptive activities. Induction of osteoclastogenesis by receptor activator of NF-kappaB ligand (RANKL) is accompanied by increased TNF-alpha expression. In the present study we investigated the mechanism by which RANKL induces expression of TNF-alpha in osteoclast precursors. The macrophage-like cell-line, RAW 264.7 was used as a model for osteoclast precursors. To examine if RANKL-mediated increase in TNF-alpha expression involves increased stability of its transcript, RAW264.7 cells were treated with or without RANKL, and then a transcription inhibitor was added. At different time points, TNF-alpha and L32 mRNA levels were examined. TNF-alpha mRNA stability was not altered by RANKL. We next measured directly the transcription rate of TNF-alpha by a run-on assay and found that RANKL increases TNF-alpha transcription rate by 2.9-fold in RAW264.7 cells. We further characterized this transcriptional induction of TNF-alpha by RANKL. Gel shift assays using nuclear extracts derived from RANKL-treated RAW264.7 cells show increased specific NF-kappaB binding activity on the murine TNF-alpha promoter. Gliotoxin, known for its ability to inhibit NF-kappaB activation blocked RANKL-induced TNF-alpha expression. We finally used 1,260 bp of the murine TNF-alpha promoter fused to luciferase, as well as four mutants of this promoter carrying mutations in each of the four NF-kappaB sites to stably transfect RAW 264.7 cells. Reporter activity was increased in response to RANKL in wild type promoter transfected cells, whereas treatment of the mutants' transfected cells did not elicit reporter activity. In conclusion, RANKL induces TNF-alpha expression via a transcriptional mechanism, depending on the NF-kappaB sites in the TNF promoter.

Impaired bone marrow-derived macrophage differentiation in vitamin D deficiency.

The calcium-regulating hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is also recognized as an immunomodulator. In vitro addition of the hormone to bone marrow-derived macrophages (BMDMs) results in a decreased proliferation and an increased differentiation. In the present work we compare the in vitro differentiation of BMDMs derived from vitamin D-depleted and -repleted mice (-D and +D BMDMs, respectively). -D BMDMs proliferate in vitro slower than +D BMDMs. Addition of the hormone to BMDM cultures inhibited the rate of their proliferation, which was more pronounced in low-density cultures. The ability of mononuclear phagocytes to produce reactive oxygen metabolites is an important element in the microbicidal functions of these cells. We found that -D BMDMs produce less H2O2 than +D BMDMs, which was corrected by the in vitro addition of 1,25(OH)2D3. Analyses of various macrophage-specific surface antigens revealed a reduction in their expression on -D BMDMs. In vitro addition of 1,25(OH)2D3 to BMDM cultures increased the expression of these antigens. The activity of the lysosomal enzyme acid phosphatase was similarly affected by vitamin D deficiency and by the in vitro addition of the hormone. Thus, vitamin D deficiency is associated with impaired maturation of BMDMs suggesting that the hormone is a natural modulator of macrophage maturation.