Lactosylceramide is essential for the osteoclastogenesis mediated by macrophage-colony-stimulating factor and receptor activator of nuclear factor-kappa B ligand.

Glycosphingolipids and their metabolites play important roles in a variety of biological processes. Several signal molecules are localized in a glycolipid-enriched microdomain on the cell surface, and their signals are regulated by the glycolipid composition. However, the function of glycolipids in osteoclastogenesis has not been clearly understood. We found that D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a glucosylceramide synthase inhibitor, completely inhibits the osteoclast formation induced by macrophage-colony-stimulating factor and receptor activator of nuclear factor-kappa B ligand (RANKL) in a dose-dependent manner. Expression of RANK, the receptor of RANKL, induced by macrophage colony-stimulating factor, was reduced markedly in D-PDMP-treated cells. d-PDMP also inhibited the phosphorylation of the inhibitor of nuclear factor-kappa B and extracellular signal-regulated kinase 1/2 induced by RANKL. In several experiments with the addition of glycolipids to D-PDMP-treated purified bone marrow cells, lactosylceramide (LacCer) strongly affected the differentiation into tartrate-resistant acid phosphatase mononucleated cells, but not positive multinucleated cells. GM3 and GM1 also recovered, but less effectively compared with LacCer. Moreover, exogenous LacCer recovered the reduced expression of RANK and the phosphorylation of inhibitor of NF-kappa B and extracellular signal-regulated kinase 1/2 after stimulation by RANKL at the same level of cells without D-PDMP treatment. Our data suggest that glycosphingolipids, especially LacCer, are necessary for the initiation step of RANKL-induced osteoclastogenesis.

Force-induced osteoclast apoptosis in vivo is accompanied by elevation in transforming growth factor beta and osteoprotegerin expression.

The mechanism controlling the disappearance of osteoclasts from bone surfaces after bone resorption in vivo is largely unknown. This is because there is no suitable experimental system to trace the final fate of osteoclasts. Here, we used an experimental model of tooth movement in rats to show that preexisting osteoclasts disappeared from the bone surface through apoptosis during a force-induced rapid shift from bone resorption to formation. On the distal alveolar bone surface of the maxillary molar in growing rats, many mature osteoclasts were present. When light tensional force was applied to the bone surface through an orthodontic appliance, these preexisting osteoclasts gradually disappeared. One day after the application of force, about 24% of the osteoclasts exhibited apoptotic morphology and the proportion of apoptotic cells was increased to 41% by day 2, then decreased afterward. These changes were undetectable on the control distal alveolar bone surface, which is free from tensional force. As shown by in situ hybridization, a marked increase in transforming growth factor beta1 (TGF-beta1) and osteoprotegerin (OPG) messenger RNA (mRNA) was observed in the stretched cells on the tensioned distal bone surface, simultaneously with the loss of osteoclasts. Both of these factors are known to have a negative effect on osteoclast recruitment and survival. As early as 2 days after force application, some of these stretched cells were identified as cuboidal osteoblasts showing intense signals for both factors. Our data suggest there may be a sequential link in tensional force applied on the bone lining cells, up-regulation of TGF-beta1/OPG, and disappearance of osteoclasts.

A common downstream signaling activity of osteoclast survival factors that prevent nitric oxide-promoted osteoclast apoptosis.

Treatment with NO-releaser NOC18 significantly promoted apoptosis in murine osteoclast-like cells, with a transient increase in caspase-3-like protease activity. In contrast, the apoptosis was protected against by caspase inhibitors, most efficiently with the broadly acting caspase specific inhibitor z-Asp-CH2-DCB, indicating involvement of multiple caspases in progression of the apoptosis. Among osteoclast survival factors examined, calcitonin completely protected against morphologically defined-apoptosis and the increase of caspase-3-like protease activity. The effect of calcitonin was mimicked by treatment of cells with (Bu)2cAMP and forskolin, and abolished by protein kinase-A inhibitor H-89. Independently from the PKA activation, colony stimulating factor-1, interleukin-1beta and the receptor activator of NF-kappaB ligand also protected against the apoptosis but were less effective than calcitonin. All survival factors investigated inhibited conversion of procaspases-3 and -9 to their mature forms in the cells. Thus, downstream antiapoptotic signaling activity from each factor overlapped in inhibition of caspases. However, how this was attained seemed to be different from each other. Typically, only colony stimulating factor-1 up-regulated expression of endogenous caspase inhibitor protein, X-linked inhibitor of apoptosis (XIAP), in the osteoclast-like cells.

Fluorescence microscopic demonstration of cathepsin K activity as the major lysosomal cysteine proteinase in osteoclasts.

The enzyme activity of lysosomal cysteine proteinases in vital rabbit osteoclasts and mouse osteoclast-like cells was visualized with Z-Leu-Arg-4-methoxy-beta-naphthylamide (Z-LR-MNA) as the enzyme substrate. The MNA liberated by proteolysis forms a fluorescent insoluble Schiff-base product in the presence of 5-nitrosalicylaldehyde. Many small fluorescent particles, endproducts of the Z-LR-MNA hydrolysis, were observed in proximity to the bone surface underneath the actively resorbing osteoclasts, as well as in the cytoplasm. The Z-LR-MNA hydrolase activity was markedly diminished by bafilomycin A1 and chloroquine treatment. Moreover, the activity was completely inhibited by cysteine proteinase inhibitors such as leupeptin and E-64d, but not by other classes of proteinase inhibitors. About 60% of the hydrolase activity in mouse osteoclast-like cells was immunoabsorbed by anti-cathepsin K antibody-coupled Sepharose CL-4B beads, and about 10% of the activity was absorbed with the anti-cathepsin L antibody-coupled beads. Thus, the majority of the Z-LR-MNA hydrolase activity in osteoclasts was derived from cathepsin K. In contrast, using the same substrate in the assay, no detectable cathepsin K activity was observed in mouse peritoneal macrophages. The abundant cathepsin K activity in osteoclasts would therefore indicate a significant role of this enzyme in bone matrix degradation.

[Management of personnel exposure data with a personal computer].

This paper describes a software package that manages dosimeter inventory and exposure data with a personal computer. The computer, connected with the access control system, deals with both records of the film badge (FB) supply to workers and the ordering of new FB for a company, processes exposure data and produces reports. The software not only ensures accurate reports but also significantly reduces the tedious labour involved in managing the control system.

Inhibition of in vitro neutrophil responses to chemotactic factors by piroxicam.

The effect of piroxicam on polymorphonuclear neutrophils (PMN) functions induced by several stimuli was evaluated in vitro. Preincubation of rabbit or human PMN with piroxicam inhibited the cellular responses elicited by N-formyl-methionyl-leucyl-phenylalanine (FMLP) such as superoxide anion (O2-) generation, granule enzyme release and chemotaxis. The effectiveness of piroxicam on each response was superior to those of indomethacin and ibuprofen. Also when either concanavalin A, zymosan-treated serum or ionophore A23187 was used as stimuli, piroxicam inhibited O2- generation of PMN. The inhibitory effect of piroxicam on FMLP-induced O2- generation was dependent on the concentration of stimuli and was reversed by increasing the extracellular calcium concentration. In addition, piroxicam had no effect on the activity of a chymotrypsin-like esterase, N-acetyl-phenylalanine-beta-naphthyl esterase, isolated from rabbit PMN. These results suggest that at least some of the anti-inflammatory effects of piroxicam may be mediated by affecting PMN functions, and the inhibition of O2- generation of PMN by piroxicam may be related to its capacity to modulate the association of calcium with these cells.

[Action of piroxicam on allergic inflammation].

Effects of piroxicam on allergic inflammation were investigated with allergic air pouch inflammation and antigen-induced arthritis in rats. In allergic air pouch inflammation, piroxicam exerted a dose-dependent inhibition (1-10 mg/kg, p.o.) of the exudate production, the migration of leukocytes and the release of lysosomal enzyme into the exudate; and its potency was superior to that of indomethacin and equivalent to that observed with prednisolone. In contrast with this, the suppressive effect of piroxicam on non-allergic air pouch inflammation was as weak as indomethacin. Prednisolone showed a similar effect on both types of air pouch inflammation. In antigen-induced arthritis, piroxicam showed a dose-dependent (0.3-3 mg/kg, p.o.) inhibitory effect on knee joint swelling and an improving action on the functional disorder of the inflamed joint. On this model, piroxicam was 3 to 4 times more active than both indomethacin and prednisolone. In non-allergic joint inflammation induced with croton oil in rats, however, the anti-inflammatory potency of piroxicam was almost equal to those of indomethacin and prednisolone. Piroxicam showed more potent inhibition than indomethacin on heterologous passive cutaneous anaphylaxis in rats, but showed only a slight inhibition on the increased vascular permeability caused by histamine and bradykinin. Piroxicam had no influence upon the plaque-forming cell response and the delayed hypersensitivity reaction in mice; furthermore, the hemolytic activity of complement in guinea-pig serum was scarcely affected by piroxicam in vitro. These results indicate that piroxicam possesses prominent efficiency on allergic inflammation and may function on several activities of inflammatory cells.

A possible role of arachidonate metabolism in allergic air pouch inflammation in rats. Anti-inflammatory effect of indomethacin and dexamethasone and the level of prostaglandin E2 in the exudate.

The effect of indomethacin and dexamethasone on an allergic inflammation in rats, a novel model of allergic inflammation of an air pouch type, was examined. Indomethacin and dexamethasone exerted a dose-dependent inhibition of both the accumulation of inflammatory exudate and the migration of leukocytes into the exudate. And although prostaglandin E2 levels in the exudate were lowered to the same extent by treatment with indomethacin and dexamethasone, inhibition of both exudate accumulation and and leukocyte migration was more pronounced after treatment with dexamethasone. The difference in the effectiveness of indomethacin and dexamethasone in terms of inhibition of arachidonate metabolism in the allergic air pouch inflammation are discussed.