Determination of histamine and polyamines in calcified tissues of mice: Contribution of mast cells and histidine decarboxylase to the amount of histamine in the bone

Summary A simple method for determining histamine and polyamines in various tissues was devised. The method, however, could not be applied to calcified tissues, because the high concentration of Ca²⁺ in the extract interferes with the chromatographic separation of these amines. By treating the extracts from calcified tissues with K₂CO₃, we succeeded in removing the Ca²⁺, and the method could then be applied to determine the amines in bone tissues of mice. By using this method, we examined the contribution of mast cells and histidine decarboxylae (HDC) to the amount of histamine in the bone. The results indicate that (1) the HDC activity in the bone is the highest among the tissues of normal mice, and the histamine produced by the HDC in the bone is metabolized rapidly; (2) a major part of HDC in the bone is present in the bone marrow cells other than mast cells, and most of histamine in the bone is attributable to the histamine pooled in mast cells; (3) mast cells in the diaphysis are located largely along the endosteal lining; and (4) the method devised in this study may be useful for studying the roles of histamine (or mast cells) and polyamines in calcified tissues.     

Wortmannin,a potent inhibitor of phosphatidylinositol 3-kinase,inhibits osteoclastic bone resorption in vitro

Summary Phosphatidylinositol 3-kinase (Pl3-k) is involved in cellular signaling via the phosphoinositol pathway leading to mitogenesis in response to growth factors in proliferating cells, as well as cytoskeletal changes and secretory responses in terminally differentiated cells. The fungal metabolite, wortmannin, is a potent and selective inhibitor of Pl3-k at nanomolar concentrations. We show that wortmannin dose-dependently (0.001 – 1 M) inhibits bone resorption by isolated rat osteoclasts in the bone slice pit assay with an IC₅₀ of-5 nM. Wortmannin was not cytotoxic since osteoclast morphology and survival on bone slices was unaffected by concentrations up to 1 M. Since primary osteoclasts are terminally differentiated cells and osteoclast cytoplasmic spreading and morphology was unaffected by wortmannin, we suggest that Pl3-k signaling is involved in vesicle exocytosis and ruffled border membrane formation that are required for osteoclastic bone resorption to take place.     

Permeabilization of cells of hemopoietic origin by extracellular ATP4-: Elimination of osteoclasts,macrophages, and their precursors from isolated bone cell populations and fetal bone rudiments

Skeletal tissues contain, apart from cells of the osteogenic and chondrogenic lineage, cells of hemopoietic origin, e.g., macrophages, osteoclasts, and their precursors. In the present study we examined the sensitivity for extracellular ATP^4- of the above-mentioned cell types in freshly isolated, bone-derived cell populations and in explanted fetal metatarsal bones. Cells of hemopoietic origin reacted to the presence of ATP^4- with an increased permeability for impermeant cytotoxic molecules, e.g., ethidium bromide (EB), thiocyanate (KSCN), and an increased non-ion selective membrane conductance. As a consequence, these cells could be killed by a short treatment with adenosine-5triphosphate (ATP)+KSCN. On the other hand, cells of nonhemopoietic origin (e.g., osteoblasts, chondrocytes) were found to be insensitive to ATP^4- in this respect. These cells survived the treatment without apparent damage to their alkaline phosphatase activities, osteogenic potentials, and osteoclast induction capacities. The elimination of the endogenous cells of hemopoietic origin from bone tissue or cell populations derived therefrom offers the possibility to study the properties and functions of osteogenic or chondrogenic cells without interference by the presence of cells of hemopoietic origin. It also allows the study of interactions between osteogenic cells and selected cell populations of hemopoietic origin in coculture experiments.     

Effect of bisphosphonates on the increase in bone resorption induced by a low calcium diet

This study investigates whether bisphosphonate-treated rats are still able to adapt to low calcium supply through an increase in bone resorption assessed by measuring the urinary excretion of [³H]-tetracycline from chronically prelabeled rats. First it was shown that in this model, parathyroid hormone was responsible for the increase in bone resorption on the low calcium diet. In the second part, animals were treated with the three bisphosphonates—clodronate, alendronate, and ibandronate—given in two doses. Animals receiving a dose that already strongly inhibits bone resorption were still able to respond to a low calcium diet by increasing bone resorption, showing the potency of the latter as a stimulator of bone resorption. Higher doses were, however, able to blunt this response. As soon as the treatment was discontinued, this increase in bone resorption resumed with clodronate but not with alendronate or ibandronate.     

Comparison of the mechanisms of bone resorption induced by 1α,25-dihydroxyvitamin D3 and lipopolysaccharides

Summary The mechanisms of increase in bone resorption induced by 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] and bacterial lipopolysaccharides (LPS) were compared in anin vitro dead bone assay and a living bone assay. 1α,25(OH)₂D₃ at concentrations of 0.05–5 ng/ml dose-dependently enhanced the ability of alveolar macrophages to release⁴⁵Ca from prelabeled dead bone particles (dead bone assay). In addition, the vitamin promoted fusion of the macrophages to form multinucleated cells and also enhanced glucose consumption, a marker of activation of macrophages. LPS at 0.05–5 μg/ml similarly enhanced the release of⁴⁵Ca from the dead bone particles and glucose consumption by alveolar macrophages, but it did not induce fusion of the cells at any concentration. Both 1α,25(OH)₂D₃ and LPS dose-dependently stimulated the release of⁴⁵Ca from fetal mouse calvaria prelabeled with⁴⁵Ca (living bone assay). Compared to control bone, there were several times as many osteoclasts per given length of trabecular bone surface in calvaria treated for 5 days with either 5 ng/ml of 1α,25(OH)₂D₃ or 5 μg/ml of LPS. Indomethacin (10⁻⁵ M) completely inhibited the LPS-induced increase of osteoclasts, but not the 1α,25(OH)₂D₃-induced increase. These results suggest that 1α,25(OH)₂D₃ and LPS similarly stimulate bone resorption by activating macrophages as well as by promoting fusion of precursor cells to form multinucleated cells. 1α,25(OH)₂D₃ induced formation of multinucleated cells with bone-resorbing activity directly, whereas LPS appeared to induce multinucleated cells through prostaglandin synthesis by some other types of cells present in living bone tissues.     

Osteoclastic resorption of3H-proline labelled bone,dentine and cementum in the rat

Six litter mate Wistar rats were injected intraperitoneally with 2 Ci/g body weight³H-proline at the age of 15 and 20 days. Six to seven weeks later resorption of cementum, dentine and bone was caused experimentally by luxation of the first right mandibular molar. The animals were killed 10 days later in glutaraldehyde perfusion fixation and 5 serial sections of the mandibles were processed for radioautography. Cementum, dentine and bone showed bands of labelling corresponding to the stage of odontogenesis and osteogenesis at the time of injection. Osteoclasts were found in contact with labelled cementum, dentine and bone but the number of grains over osteoclasts was insignificant.     

Effects of parathyroid hormone on the osteoclastic pool,bone resorption and formation in rat alveolar bone

Summary The osteoclast number and its relation to parathyroid hormone have been studied in rat alveolar bone by quantitative histology and fluorescent labeling. The osteoclast number decreases 60 h after parathyroidectomy and remains constant for the next 132 h. Parathyroid hormone administration to parathyroidectomized animals 96 h after the operation induces an increase in osteoclast number within 12 h to some-what above those of control animals. The elevated osteoclast counts remain constant for 60 h then rapidly fall over the next 24 h to the level seen in untreated parathyroidectomized animals. As determined by fluorescent labeling, normal alveolar bone resorption and formation were disturbed by parathyroidectomy, such that significant bone formation occurred for only 6 days after surgery, after which a quiescent state followed.     

Short- and long-term effects of a single dose of bisphosphonates on retinoid-induced bone resorption in thyroparathyroidectomized rats

Summary The inhibitory effect of a single subcutaneous (s.c.) dose of three different bisphosphonates (Bps)—4-amino-1-hydroxybutylidene-1,1-bisphosphonate (AHBuBP), 2-(2-pyridinyl)-ethylidene-1,1-bisphosphonate (2-PEBP), and dichloromethylene-bisphosphonate (Cl₂MBP)—was studied in a model of retinoid-induced bone resorption, which consists of assessing the hypercalcemic effect of the arotinoid Ro 13-6298 given s.c. for three days in thyroparathyroidectomized (TPTX) rats. The retinoid was given on day 0, 1, and 2. Bps were administered together with or at different times prior to the first dose of retinoid. A dose-dependent inhibition was obtained with all three compounds. AHBuBP produced complete inhibition which remained for 3 weeks at 0.1 mg P/kg. The dose-response curves were identical when the compound was given on the first day of retinoid administration (day 0) or 6 days earlier. With 2-PEBP, the dose-response curve was the same as that for AHBuBP when given on day 0. When given 6 days earlier, the curve was shifted to 30 times less potency. Cl₂MBP was about 100 times less potent than AHBuBP when given on day 0, with 3 mg P/kg producing complete inhibition. When given 6 days earlier, the curve was also shifted to 10 times less potency, and even 30 mg P/kg failed to produce complete inhibition. With 10 mg P/kg, the inhibitory effect was maintained partially for up to 3 weeks. This study shows that in this model of bone resorption the inhibitory effect of a single dose of certain Bps is effective for at least 3 weeks and that the compounds vary in their activity over time.     

Biological and clinical assessment of a new bisphosphonate, (chloro-4 phenyl)thiomethylene bisphosphonate, in the treatment of Paget's disease of bone

Several Biophosphonates have been used as therapeutic agents for Paget's bone disease. (Chloro-4 phenyl)thiomethylene-bisphosphonate (CIPsMBP) has recently been shown to have significant antiosteoclastic activity while an affect of CIPsMBP on mineralization was only observed at high doses. We tested this drug for 6 months in 23 pagetic patients distributed in three groups. Gr 1 (n = 5) receiving 200 mg/day showed a decrease of serum alkaline phosphatase (SAP) to 42 ± 4% (p < 0.01) of initial value (100%) while hydroxyprolinuria/creatinuria ratio (OH/Cr) dropped to 69 ± 8% of baseline. In 4 patients receiving 400 mg/day, SAP improved to 48 ± 9% of initial value (p < 0.01) and OH/Cr to 40 ± 3% (p < 0.01). In the last group (n = 14) receiving 200 mg/day for 3 months, and 400 mg/day thereafter up to the 6th month SAP decreased to 53 ± 4% and OH/Cr to 62 ± 6% of initial value (p < 0.01).

Clinical improvement was significant from the first month of treatment. No resistance (mean decrease of SAP lower than 30%) was recorded and no radiological or clinical evidence of mineralization defect appeared. The clinical and biological tolerance was excellent throughout the study.     

Identification,characterization, and isolation of a common progenitor for osteoclasts,macrophages, and dendritic cells from murine bone marrow and periphery

Osteoclasts are specialized bone‐resorbing cells that derive from monocyte precursors. We have identified three populations of cells with high osteoclastogenic potential in murine bone marrow, which expressed the phenotype B220^‐CD3^‐CD11b^?/low CD115⁺ and either CD117^hi, CD117^intermediate, or CD117^low. We have evaluated these populations for their ability to also generate macrophages and dendritic cells. At a single‐cell level, the population expressing higher CD117 levels was able to generate bone‐resorbing osteoclasts, phagocytic macrophages, and antigen‐presenting dendritic cells in vitro with efficiencies of more than 90%, indicating that there exists a common developmental pathway for these cell types. Cells with osteoclastogenic potential also exist in blood and peripheral hematopoietic organs. Their functional meaning and/or their relationship with bone marrow progenitors is not well established. Hence, we characterized murine peripheral cell populations for their ability to form osteoclasts, macrophages, and dendritic cells in vitro. The spleen and peripheral blood monocyte progenitors share phenotypic markers with bone marrow progenitors but differ in their expression of CD11b, which was low in bone marrow but high in periphery. We propose that circulating monocyte progenitors are derived from a common bone marrow osteoclasts/macrophage/dendritic cell progenitor (OcMDC), which we have now characterized at a clonal level. However, the lineage relationship between the bone marrow and peripheral monocyte progenitors has yet to be defined. © 2013 American Society for Bone and Mineral Research.     

Protein tyrosine kinase inhibitors block the stimulatory actions of phosphotyrosine phosphatase inhibitors to increase cell proliferation, alkaline phosphatase activity, and collagen synthesis in normal human bone cells

The present study sought to test whether inhibition of phosphotyrosine phosphatases (PTPs) would stimulate proliferation and differentiation of normal bone cells, and whether the PTP inhibitor-mediated effects would be blocked by protein tyrosine kinase (PTK) inhibitors. Three inhibitors [phenylarsine oxide (PAO), orthovanadate (VO(4)), and molybdate (MoO(4))] and two normal human bone cells with different basal differentiation status (i.e., mandible- and vertebra-derived bone cells) were used. Cell proliferation was determined with [(3)H]thymidine incorporation, and confirmed by cell counting. Bone cell differentiation was assessed by increases in alkaline phosphatase (ALP) specific activity and collagen synthesis. The three test PTP inhibitors each stimulated [(3)H]thymidine incorporation in both human bone cell types in a biphasic, dose-dependent manner with optimal doses of 20 nM PAO, 1 microM VO(4) and 2 microM MoO(4), respectively. These PTP inhibitors at mitogenic doses each significantly and reproducibly increased ALP specific activity and collagen synthesis. To determine whether the stimulatory effects of PTP inhibitors could be blocked by PTK inhibitors, the effects of tyrphostin A51 and erbstatin, two potent PTK inhibitors, on the actions of PTK inhibitors on [(3)H]thymidine incorporation and ALP specific activity were evaluated. Both tyrphostin A51 and erbstatin, which by themselves alone significantly inhibited human bone cell proliferation and increased ALP specific activity, completely abolished the stimulatory effects of each of the three test PTP inhibitors on bone cell proliferation and ALP specific activity. In conclusion, these findings confirm the premise that inhibition of PTP activities in normal human bone cells could lead to increases in cell proliferation and differentiation, effects that are independent of basal differentiation status of the cells. More importantly, this study demonstrates for the first time that the stimulatory actions of the PTP inhibitor on bone cell proliferation and ALP could be blocked by a PTK inhibitor, suggesting that the osteogenic effects of PTP inhibitors may depend on PTK activities, presumably to increase basal tyrosyl phosphorylation level. Accordingly, one should interpret results of studies using PTK inhibitors with caution in that an inhibition by a PTK inhibitor does not necessarily indicate the requirement of PTK activities, as it could also suggest involvement of an inhibition of PTPs.     

Disodium 1-hydroxy-3-(1-pyrrolidinyl)-propylidene-1,1-bisphosphonate (EB-1053) is a potent inhibitor of bone resorption in vitro and in vivo.

The ability of the new nitrogen-containing bisphosphonate disodium-1-hydroxy-3-(1-pyrrolidinyl)-propylidene-1,1-bisphosphona te (EB-1053) to inhibit osteoclastic resorption was examined in vitro and in vivo. Results were compared to those obtained with 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (pamidronate or APD). In vitro, when tested in osteoclast precursor-dependent systems (fetal mouse metacarpals and a coculture system), EB-1053 suppressed 45Ca release effectively and was found to be about 10 times more potent than pamidronate (ED50 = 2.5 x 10(-7) versus 2.5 x 10(-6) M, respectively). The EB-1053-inhibited osteoclastic resorption could be reversed by treatment with parathyroid hormone (PTH). In vivo, daily subcutaneous injections of EB-1053 to young growing rats for 7 days increased metaphyseal bone mass in tibiae dose dependently. In these experiments EB-1053 was about 50 times more potent than pamidronate. These studies show that EB-1053 is a very potent bisphosphonate that has potential use in the treatment of skeletal disorders.     

单核细胞、NK细胞和T细胞在猪-猕猴延迟性异种移植排斥反应中的作用

目的观察单核细胞、NK细胞和T细胞在猪-猕猴延迟性异种移植排斥反应(DXR)中的作用。方法建立湖北白猪-云南猕猴的腹腔异位心脏移植模型，实验分为2组：对照组(n=5)，不使用中华眼睛蛇毒因(Y-CVF)；实验组(n=4)应用Y-CVF完全清除受者体内补体。2组受体猴均采用环孢素A(CsA)，环磷酰胺(CTX)和甲基强的松龙(MP)三联免疫抑制治疗。免疫组织化学方法检测移植心组织中细胞间黏附分子(ICAM)-1、肿瘤坏死因子(TNF)-α、单核细胞、NK细胞和T细胞的表达。结果对照组3个移植心在15～60min内发生超急性排斥反应(HAR)，另2个分别存活22h及6d，移植心均未见明显的炎性细胞浸润及ICAM-1和TNF-α的表达。实验组移植心存活时间分别为8、10、13和13d，移植物浸润细胞中可见大量的单核细胞(50％)，少量的NK细胞(8％～10％)，CD4^ T细胞(15％)和C08^ T细胞(25％)。移植物血管内皮细胞表面出现ICAM-1的表达上调，移植物间质中出现TNF-α的表达增加。结论单核细胞、NK细胞和T细胞介导的移植物损伤，在应用Y-CVF处理的猪-猕猴DXR发生中发挥重要作用。     

Comparison of the effects of a potent synthetic analog of bovine parathyroid hormone with native bPTH-(1-84) and synthetic bPTH-(1-34) on bone resorption and collagen synthesis

Summary An analog of bovine PTH [nle-8, nle-18, tyr-34 bPTH-(1-34) amide, (PTH-Ana)] which is a potent stimulator of renal adenylate cyclase has been compared with the native hormone bPTH-(1-84) and the biologically active amino terminal portion, bPTH-(1-34), for its effects on bone resorption and bone collagen synthesis in organ culture. All three compounds stimulated the release of previously incorporated⁴⁵Ca from cultured fetal rat long bone shafts with similar dose-response curves at 10⁻⁹ to 3 × 10⁻⁸ M. All three compounds inhibited bone collagen synthesis as measured by incorporation of proline into collagenase digestible protein, whereas incorporation into noncollagen protein was not inhibited. The effects were dose related and decreases in percent collagen synthesis were significant at 10⁻⁹ M. Thus PTH-Ana appears to have the same effects on bone resorption and collagen synthesis as bPTH-(1-84) and (1-34) and is likely to be a valid probe for investigating PTH receptors in bone as well as in kidney.     

The effects of COX-1 and COX-2 inhibitors on prostaglandin synthesis and the formation of heterotopic bone in a rat model

Introduction Traumatic heterotopic ossification (HO) is a common clinical condition associated with various orthopedic procedures that involve injury to soft tissues near bone. In this study, we tested the hypothesis that the prophylactic effects of NSAID’s in the treatment of HO are mediated via inhibition of the COX-2 enzyme. Here we describe a rat model that simulates HO in the human that was used to test the above hypothesis. Materials and methods Heterotopic ossification was surgically induced in the quadriceps by injury to the muscle and femoral periosteum and transplantation of donor bone marrow cells containing osteoprogenitors into the site of injury. HO was imaged and quantified by micro-CT scanning of femurs removed from sacrificed animals at 6 weeks post-injury, three-dimensional computer reconstructions of the scanned bones and computer-assisted morphometric analysis. Prostaglandin E₂ (PGE₂) synthesis was quantified using an enzyme immunoassay system. The effects of a nonselective COX inhibitor or specific inhibitors of COX-1 or COX-2 following oral administration on the content of ectopic bone and PGE₂ were also measured. Results Micro-CT and histological analyses demonstrated that all of the femurs in operated limbs developed HO in the vastus lateralis muscle belly of the quadriceps close to the anterior femur. Only the COX-1,2 nonselective and COX-2 inhibitors significantly decreased HO formation (by about one-third in each case; P < 0.05). PGE₂ synthesis at the site of injury was increased 50- and 100-fold (to 25 ng/g tissue) within 1 and 7 days, respectively, post-injury with the levels declining to near baseline within 2 weeks of surgery. Both the COX-1,2 nonselective and COX-2 inhibitors significantly decreased PGE₂ levels to 25% of control HO levels within 24 h of the first administration, even at low dosages. The COX-1 inhibitor only produced the same effect after 1 week of administration. Conclusion These findings suggest that although inhibitors of COX-2 or COX-1 reduced PGE₂ synthesis, only the COX-2 enzyme plays a role in the mechanism of traumatic HO.     

Interleukin-6 does not mediate the stimulation by prostaglandin E2, parathyroid hormone,or 1,25 dihydroxyvitamin D3 of osteoclast differentiation and bone resorption in neonatal mouse parietal bones

The cytokine interleukin-6 (IL-6) was produced by neonatal mouse parietal bones during a 6- or 48-hour culture period in response to prostaglandin E₂ (PGE₂) and bovine parathyroid hormone (PTH) 1-34 fragment but not 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. At the same time there was an increase in tartrate-resistant, acid phosphatasepositive osteoclasts (TRAP+OC) with all three osteotropic effectors over 6 hours, and an increase in ⁴⁵Ca release over 48 hours. TRAP+OC numbers on PGE₂-stimulated bones were positively correlated with IL-6 concentration. Our aim was to determine if IL-6 mediated this response. Recombinant human IL-6 (rhIL-6) was added to parietal bones in culture at concentrations within the range that PGE₂ or PTH would produce during incubation. However, over 6 or 48 hours, rhIL-6 did not stimulate TRAP+OC to increase in number nor did it cause an increase in calcium release over 48 hours. Adding an antibody against mouse IL-6 to bone cultures stimulated with PTH or PGE₂ neutralized the resulting IL-6 bioactivity by up to 92% but did not inhibit TRAP+OC formation. We conclude that although IL-6 is produced in response to two important stimulators of bone resorption, it does not mediate osteoclast differentiation or bone resorption in this model.Part of this work has been presented as an abstract to the Bone and Tooth Society Winter Meeting on 6/12/93 at The Royal College of Obstetricians and Gynaecologists, London.     

Comparative study of deflazacort,a new synthetic corticosteroid,and dexamethasone on the synthesis of collagen in different rat bone cell populations and rabbit articular chondrocytes

Summary Deflazacort is a new synthetic glucocorticoid which is an oxazoline derivative of prednisolone. In previous studies, it was shown that deflazacort, depending on the test model used, not only showed considerably more antiinflammatory potency than prednisolone in animals but also caused less deleterious effects on bone mineral metabolism than equivalent amounts of other glucocorticoids in man. In this study, we have compared the effects of deflazacort with those of dexamethasone on the synthesis of collagen in various rat bone cell populations and chondrocytes. Three bone cell populations were prepared by sequential time-dependent collagenase treatment of 1-day-old rat calvaria. Each cell population was further purified on a Percoll gradient (10–90%) yielding three populations of which two are different in alkaline and acid phosphatase and response to parathyroid hormone. A 3-day treatment of bone cell populations with deflazacort and dexamethasone (10⁻¹¹-10⁻⁵ M) revealed that both glucocorticoids, although at different concentrations, inhibited collagen synthesis. 21-desacetyl-deflazacort (5β, 11β, 16β)- 11,21-dihydroxy-2′-methyl-5-H-pregna-1-enol[17,16-d]oxazole-3,20-dione), the presumably active form of the steroid, which is formedin vivo after administration, produced nearly identical results as its precursor. Glucocorticoid concentrations at which inhibition was initially observed were 10⁻⁹ M and 10⁻⁷ M for dexamethasone and deflazacort respectively. Inhibition of collagen synthesis was significantly impaired only in cells isolated from bone during early tissue digestion, and not in those obtained during extensive collagenase treatment. Chondrocytes isolated from articular cartilage of 3-month-old rabbits and grown in primary cultures did not respond to either steroid. However, when passed and grown in secondary culture, collagen synthesis was inhibited considerably more by dexamethasone than by deflazacort. It is apparent that both deflazacort and dexamethasone, although at different concentrations, inhibit collagen synthesis in a differential manner. The lesser deterrent effect of deflazacort may be of clinical importance.