Yoshitomi Aida

Extracellular matrix regulates induction of alkaline phosphatase expression by ascorbic acid in human fibroblasts.

During wound healing and inflammation, fibroblasts express elevated alkaline phosphatase (ALP), but are not in contact with collagen fibrils in the fibronectin (FN)‐rich granulation tissue. We hypothesized that the extracellular matrix (ECM) environment might influence the induction of ALP in fibroblasts. Here we tested this hypothesis by studying the ALP‐inductive response of normal human gingival fibroblasts to ascorbic acid (AsA). AsA induced ALP activity and protein in cells in conventional monolayer culture. This induction was inhibited by blocking‐antibodies to the FN receptor α5β1 integrin and by the proline analog 3,4‐dehydroproline (DHP). DHP prevented cells from arranging FN fibrils into a pericellular network and reduced the activity of cell spreading on FN. Plating of cells on FN facilitated the up‐regulation by AsA of ALP expression, but did not substitute for AsA. In contrast, AsA did not cause ALP induction in cells cultured on and in polymerized type I collagen gels. Collagen fibrils inhibited the up‐regulation by AsA of ALP expression in cells plated on FN. These results indicate that the ECM regulates the induction of ALP expression by AsA in fibroblasts: FN enables them to express ALP in response to AsA through interaction with integrin α5β1, whereas type I collagen fibrils cause the suppression of ALP expression and overcome FN.

An analogue of lipid A and LPS from Rhodobacter sphaeroides inhibits neutrophil responses to LPS by blocking receptor recognition of LPS and by depleting LPS-binding protein in plasma.

When incubated with lipopolysaccharide (LPS) in the presence of plasma, neutrophils become primed for enhanced release of superoxide in response to triggering by formyl‐Met‐Leu‐Phe (fMLP). The effect of LPS on phagocytes is inhibited by a synthetic lipid A precursor, LA‐14‐PP (lipid IVa) or by LPS from Rhodobacter sphaeroides (Rs). We studied the mechanisms by which LA‐14‐PP or Rs‐LPS inhibited LPS‐induced responses. When neutrophils were exposed to LA‐14‐PP or Rs‐LPS for 3 min and then to Escherichia coli‐LPS, the antagonists inhibited priming for superoxide release, and also blocked up‐regulation of CD11b and adherence. This inhibition was dependent on plasma, was not overcome by higher amounts of E. coli‐LPS or plasma, and was not observed at 0°C, suggesting that E. coli‐LPS was not able to interact with its receptor or other cellular recognition molecule in neutrophils that had been exposed to the antagonists. The alternative possibility that LA‐14‐PP or Rs‐LPS depleted a plasma cofactor, resulting in inhibition of priming, was investigated by using LPS from Porphyromonas gingivalis (Pg) and Bordetella pertussis (Bp). These LPS primed neutrophils in a plasma‐dependent and CD14‐dependent manner, but were not blocked by LA‐14‐PP or Rs‐LPS. When sub‐optimal concentrations of plasma were exposed to LA‐14‐PP or Rs‐LPS, and then mixed with Pg‐IPS or Bp‐LPS, followed by incubation with neutrophils, priming and up‐regulation of GD11b were inhibited, and this inhibition was overcome by increasing the concentration of plasma. Binding of LPS‐binding protein (LBP) in plasma to immobilized E. coli‐LPS was inhibited by pre‐incubation of plasma with LA‐14‐PP or Rs‐LPS. Together with the result that treatment of plasma with anti‐LBP antibody abolished the cofactor activity of plasma, these results indicated that LA‐14‐PP and Rs‐LPS depleted LBP from plasma, resulting in inability of LPS to act on neutrophils. Thus LA‐14‐PP and Rs‐LPS inhibited the action of LPS on neutrophils by at least two mechanisms, blocking of LPS receptor recognition and depletion of the cofactor LBP.

Follistatin restricts bone morphogenetic protein (BMP)-2 action on the differentiation of osteoblasts in fetal rat mandibular cells.

We tested whether FS secretion might modulate BMP‐2 actions by measuring FS levels and counting bone numbers of rat mandibular cells. In the presence of Dex, BMP‐2 stimulated FS secretion at the early phase and augmented bone nodule by neutralizing with FS antibody. We concluded that BMP‐2 facilitates FS secretion, and the FS restricts BMP‐2 action on osteoblastogenesis.

Serum or Growth Factor Deprivation Induces the Expression of Alkaline Phosphatase in Human Gingival Fibroblasts

We have previously reported that the increased expression of alkaline phosphatase (ALP) activity is a phenotypic characteristic of gingival fibroblasts present in chronic inflammatory periodontal lesions. We hypothesized that ALP might be induced in gingival fibroblasts by environmental factors. In the present study, we investigated the factors influencing the induction of ALP expression in fibroblasts derived from healthy human gingiva. The withdrawal of serum from confluent cultures of fibroblasts increased the number of cells positive for ALP activity and protein, without their proliferation. Suramin, a growth factor antagonist, induced ALP expression in cells cultured with serum. Serum re-addition or exposure to platelet-derived growth factor-AB and/or insulin-like growth factor I suppressed ALP induction and caused cell growth. ALP-positive cells could survive for up to 6 weeks after serum deprivation, a condition inducing cell death via apoptosis. These results demonstrate that serum or growth factor deprivation induces the expression of ALP in gingival fibroblasts. ALP expression is negatively correlated with cell growth and accompanied by a change into serum-growth-factor-independent survival.

Development of Mineralized Nodules in Fetal Rat Mandibular Osteogenic Precursor Cells: Requirement for Dexamethasone but Not for β-Glycerophosphate

Abstract: We have reported that a cell population obtained from fetal rat mandible with neutral protease (Pro I) has a unique differentiation sequence in which the elevation of alkaline phosphatase (ALPase), calcium accumulation, and collagen synthesis occurs simultaneously. In this report, we further characterized Pro I-released population of cells by studying the effect of dexamethasone (Dex) or β-glycerophosphate (β-GP) on the formation of bone nodules. The formation of bone nodules in Pro I-released population of cells (ProIRPC) was augmented by the addition of Dex (10−7 M) from days 3 to 14, suggesting that Pro IRPC contained osteoprogenitor (OP) cells. A 24-hour pulse treatment of ProIRPC released population of cells with Dex on days 9 and 12 resulted in an increase in the number of nodules but treatment on days 3, 6, or 15 did not. The number of bone nodules formed in Pro IRPC pulse treated with Dex on day 9 was comparable with that in Pro IRPC treated with Dex from days 3 to 14. Dex caused an earlier elevation of ALPase, in which maximal expression was observed on day 10. β-GP caused a prolonged elevation of ALPase, but did not affect the formation of bone nodules. Unlike Pro I-released population of cells, rat calvarial cells did not form mineralized nodules without β-GP, and showed that a Dex-responsive period on bone nodule formation in rat calvarial cells was at preconfluency (days 0 and 1). Thus, it appeared that the Dex-induced differentiation of early OP cells in Pro IRPCs occurred during the limited period from day 9 to day 12. Pro IRPC was found to have an unique characteristic that bone nodule formation was not affected by β-GP.

Local anesthetics inhibit priming of neutrophils by lipopolysaccharide for enhanced release of superoxide: suppression of cytochrome b558 expression by disparate mechanisms

Local anesthetics have anti‐inflammatory effects in vivo and inhibit neutrophil functions in vitro, but how these agents act on neutrophils remains unclear. Phagocytosis and bactericidal activity of neutrophils are enhanced by exposure to bacterial components such as lipopolysaccharide (LPS); this process is termed priming, which for enhanced release of superoxide (O2−) causes mobilization of intracellular granules that contain cytochrome b558, a component of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We studied whether local anesthetics affected LPS priming for enhanced release of O2− in response to triggering by the chemotactic peptide N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP), and we investigated which element in the LPS signaling pathway might be the target of local anesthetics. Neutrophils were incubated with 10 ng/ml LPS and 1% plasma ± local anesthetics, washed, and triggered with fMLP. Local anesthetics all inhibited LPS priming, and 50% inhibition was at 0.1 mM tetracaine, 0.5 mM bupivacaine, 3.0 mM lidocaine, or 4.0 mM procaine. Local anesthetics inhibited LPS‐induced mobilization of specific granules and secretory vesicles. Local anesthetics inhibited LPS‐induced up‐regulation of cytochrome b558 but not LPS‐induced translocation of p47phox. Inhibition of priming by local anesthetics was reversed by washing and incubating for 5 min. Tetracaine alone, but not the other local anesthetics, inhibited LPS activation of p38 mitogen‐activated protein kinase (MAPK) and MAPK kinase 3 (kinases in the LPS signaling pathway). The p38 MAPK inhibitors SB203580 and PD169316 also blocked LPS priming. Thus, tetracaine and the other local anesthetics inhibit by disparate mechanisms, but all the local anesthetics impaired up‐regulation of cytochrome b558 and all impaired priming of NADPH oxidase by LPS.

Neutrophils responded to immobilized lipopolysaccharide in the absence of lipopolysaccharide‐binding protein

Lipopolysaccharide (LPS) in solution primes neutrophils for enhanced release of superoxide in response to N‐formyl‐methionyl‐leucyl‐phenylalanine. We show that LPS immobilized on polystyrene or polypropylene acted on neutrophils by a mechanism different from that of LPS in solution. Coating the surface with 1% plasma, either before coating with LPS (plasma/LPS) or after coating with LPS (LPS/plasma), was essential to induce the LPS response in neutrophils. However, plasma could be replaced by fibrinogen, type I collagen or type IV collagen, or, to a lesser extent, by fibronectin or vitronectin, which was not true for LPS in solution. About 20% of the LPS added was immobilized on the plastic surfaces, based on its ability to adsorb anti‐LPS antibody after extensive washing. The amount of soluble LPS that might have been released from surfaces during the incubation with neutrophils was too low to account for the priming by immobilized LPS. About 13–20 min was needed for neutrophils to become primed after incubation with immobilized LPS. Immobilized LPS induced up‐regulation of CD11b/CD18 and latent alkaline phosphatase and also enhanced the adhesive response of neutrophils. Priming by immobilized LPS was inhibited by anti‐CD14 antibody or by treatment of neutrophils with the LPS antagonist LA‐14‐PP. When immobilized LPS was treated with anti‐LPS‐binding protein (LBP) antibody, the response of neutrophils to LPS/plasma was inhibited but the response to plasma/LPS or fibrinogen/LPS was not. Thus, the LPS in plasma/LPS or fibrinogen/LPS acted on neutrophils in an LBP‐independent manner. We conclude that the CD14‐dependent LPS receptor system of neutrophils was capable of working in the absence of LBP, but only when LPS was immobilized on a surface coated with protein. J. Leukoc. Biol. 64: 177–184; 1998.

Differentiation and mineralization in osteogenic precursor cells derived from fetal rat mandibular bone

SummaryThe process of mineralization in cells prepared either by neutral protease digestion (Pro I) or by collagenase digestion (fifth cycle, Col V) from fetal rat mandible was studied in vitro. Alkaline phosphatase (ALPase) activity of cells in Pro I was low on day 3, increased rapidly from day 8, and reached a maximum on day 16, whereas that in Col V was high on day 2, then declined and thereafter elevated to reach a maximum on day 13. Both cell populations synthesized type I collagen in cell matrix and medium. Type III collagen was observed in cell matrix of Pro I on day 14 and 21. There was α2 band of type V collagen in cell matrix of Pro I on day 21. Calcium deposition could be detected from day 14 in Pro I and from day 19 in Col V. The von Kossapositive nodules were found on day 17 in Pro I and day 21 in Col V, respectively. The extracellular matrix in Pro I electron-microscopically consisted of well-banded collagen fibrils with a large number of calcified spherules. An elevation of ALPase activity, collagen synthesis, and mineral deposition occurred sequentially with a time lapse in Col V, and almost simultaneously in Pro I. The number of mineralized nodules was correlated with the density of plated cells in Pro I, but not in Col V. Dexamethasone caused an increase in the number of mineralized nodules in Pro I, but not in Col V, suggesting that Pro I contained osteoprogenitor cells. Thus, the mode of mineralization in cells derived from the mandible may differ depending on the presence of osteoprogenitor cells.

1-Hydroxyethylidene-1,1-bisphosphonate decreases the postovariectomy enhanced interleukin 1 secretion from peritoneal macrophages in adult rats

SummaryBisphosphonates are potent inhibitors of bone resorption. In previous studies, we have shown that ovariectomy accelerated bone resorption and 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) inhibits ovariectomy-accelerated bone resorption in female Wistar adult rats. As interleukin 1 (IL-1) stimulates bone resorptionin vitro andin vivo, we have investigated the effects of ovariectomy and HEBP administeredin vivo on IL-1 secretion from peritoneal macrophages in adult rats. Ovariectomy or sham surgery were performed in female Wistar rats at 40 weeks of age. Overiectomized and sham-operated rats were administered with HEBP (10 mg) or saline, 10 times in total, from 43 to 46 weeks of age. Paraffin oil-induced peritoneal macrophages at 46 weeks of age were cultured for 24 hours. Lipopolysaccharide (LPS)-stimulated peritoneal macrophages from ovariectomized rats secreted more IL-1 than sham-operated rats. HEBP decreased LPS-stimulated IL-1 secretion from peritoneal macrophages in ovariectomized rats, but not in sham-operated rats.In vivo administration of HEBP decreased IL-1 secretion only in postovariectomy hyperresorptive states. These results suggest that alterations in LPS-stimulated IL-1 secretion from oil-induced peritoneal macrophages may be responsible, at least in part, for the postovariectomy acceleration in bone resorption and its inhibition by HEBP.

Effects of disodium ethane-1-hydroxy-1, 1-diphosphonate (EHDP) on interleukin 1 production by macrophages

The effects of disodium ethane‐1‐hydroxy‐1,1‐diphosphonate (EHDP) on the in vitro functions of guinea pig macrophages were studied. A high dose (1 mg/ml) of EHDP inhibited interleukin 1 (IL 1) production by oil‐induced peritoneal macrophages stimulated with muramyl dipeptide (MDP), lipopolysaccharide (LPS), phorbol myristic acetate (PMA), heat‐aggregated IgG2 or calcium ionophore A23187. On the other hand, low doses (<0.125 mg/ml) of EHDP augmented the MDP induced IL 1 production by macrophages. This biphasic effect was also observed when macrophages were exposed to EHDP at 37 C for 24 hr and then stimulated with IL 1 inducers. Superoxide anion generation induced by formyl peptide or PMA was not affected by preincubation of the macrophages with doses of EHDP up to 1 mg/ml. Adherence and spreading of macrophages was inhibited by EHDP in a dose dependent manner without affecting cell viability. These results demonstrated that EHDP acted on macrophages directly and modulated IL 1 production in vitro.