Osteoclast-stimulating Activity Research Articles

IL-6, IL-1β, and TNF-α only in combination influence the osteoporotic phenotype in Crohn's patients via bone formation and bone resorption.

Crohn´s disease (CD) is associated with a higher prevalence of osteoporosis. The pathogenesis of bone affliction remains controversial, especially if inflammatory cytokines or glucocorticoid therapy are the main contributors. In postmenopausal osteoporosis, bone resorption is induced by IL-6, IL-1β and TNF-α. In contrast, in children with CD, IL-6 exclusively decreased bone formation without affecting bone resorption. The objective of this study was to further clarify the pathophysiology of bone affliction in adult patients with CD with the use of an osteoblast and osteoclast cell model. Inflammatory cytokines IL-6, IL-1β, and TNF-α were measured in adult CD patients' serum. Mean values of these cytokines were applied with or without dexamethasone to the human cell line SCP-1 (osteoblastic cell model). Also, the effect of cytokines on primary human osteoclast differentiation and activity was determined. The combined cytokine application increased the receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) ratio 2-fold after 2 and 14 days. Additional application of dexamethasone to SCP-1 cells further increased the RANKL/OPG ratio 3-fold, but decreased IL-6 and IL-1β expression to 10% and 50%, respectively. TNF-α expression was maximally suppressed to 16% by dexamethasone in the presence of cytokines. In osteoclasts, the combined cytokine treatment decreased expression of characteristic genes to approx. 30%, while increasing osteoclast resorption activity to 148%. In addition, a cytokine stimulated osteoblast cell culture-generated supernatant stimulated osteoclast resorption activity by 170%. Our results suggest that IL-6, IL-1β, and TNF-α only in combination induced osteoclaststimulating activity represented by the RANKL/OPG ratio in osteoblasts. Dexamethasone further increased this effect in osteoblasts, while decreasing cytokine expression. The results in osteoclasts support a direct and osteoblast-mediated effect on bone resorption. Our in vitro results differentiate for the first time the effect of cytokines on bone turnover as measured in adult CD patients from the additional dexamethasone effect on osteoblasts as part of the pathophysiology of osteoporosis.

Primary hyperoxaluria in an adult presenting with end‐stage renal failure together with hypercalcemia and hypothyroidism

Primary hyperoxaluria (PH) is a rare genetic disorder characterized by overproduction of oxalate due to specific enzyme deficiencies in glyoxylate metabolism. The primary clinical presentation is in the form of recurrent urolithiasis, progressive nephrocalcinosis, end-stage renal disease, and systemic oxalosis. Herein, we present a case of PH who was diagnosed at 47 years of age after 6 years on hemodialysis. He presented with fatigue, anorexia, weight loss, and was found to have cachexia, diffuse edema, hepatomegaly, ascites, hypercalcemia, hyperphosphatemia, hypoalbuminemia, low parathyroid hormone levels, lytic and resorptive areas in the vertebrae, diffusely increased echogenity of the liver, multiple renal stones, and bilateral nephrocalcinosis. Bone marrow biopsy showed calcium oxalate crystals and crystal granulomas. The liver biopsy could not be performed. The absence of an identifiable reason for secondary forms, the severity of the clinical presentation, and pathological findings led to the diagnosis of PH2. He died while waiting for a potential liver and kidney donor. The presented case is consistent with the literature as he had renal stone disease in the third decade and end-stage renal disease in the fifth decade. Hypercalcemia was thought to be due to osteoclast-stimulating activity of macrophages constituting the granuloma. Erythropoietin-resistant anemia and hypothyroidism were thought to be due to accumulation of oxalate in the bone marrow and thyroid gland, respectively. It is very important to keep in mind the possibility of PH when faced with a patient with nephrocalcinosis and oxalate stone disease.

Tissue inhibitor of metalloproteinases 1 and 2 directly stimulate the bone-resorbing activity of isolated mature osteoclasts.

Tissue inhibitor metalloproteinases 1 (TIMP-1) and 2 have been reported to inhibit bone resorption. However, here, we report the direct action of both TIMP-1 and TIMP-2 on isolated rabbit mature osteoclasts to stimulate their bone-resorbing activity at significantly lower concentrations (approximately ng/ml) than those (approximately microg/ml) required for the inhibition of bone resorption. The cell population used in this study consisted of a mature osteoclast population with >95% purity. TIMP-1 (approximately 50 ng/ml) and TIMP-2 (approximately 8-10 ng/ml) increased the pit area excavated by the isolated mature osteoclasts. The stimulatory effects of TIMPs were abolished by simultaneous addition of anti-TIMP antibodies. At higher concentrations, the stimulation of bone resorption decreased reversely to the control level. The magnitude of the stimulatory effect of TIMP-2 was more than that of TIMP-1. Metalloproteinase inhibitors such as BE16627B and R94138 could not replace TIMPs with respect to the bone-resorbing activity, suggesting that the osteoclast-stimulating activity of TIMPs was independent of the inhibitory activity on matrix metalloproteinases (MMPs). TIMPs stimulated tyrosine phosphorylation of cellular proteins in the isolated mature osteoclasts. Both herbimycin A, an inhibitor of tyrosine kinases, and PD98059 and U0126, inhibitors of mitogen-activated protein kinase (MAPK), completely blocked the TIMP-induced stimulation of osteoclastic bone-resorbing activity. On the plasma membrane of osteoclasts, some TIMP-2-binding proteins were detected by a cross-linking experiment. These findings show that TIMPs directly stimulate the bone-resorbing activity of isolated mature osteoclasts at their physiological concentrations and that the stimulatory action of TIMPs is likely to be independent of their activities as inhibitors of MMPs.

Cell–cell contact between marrow stromal cells and myeloma cells via VCAM-1 and α4β1-integrin enhances production of osteoclast-stimulating activity

AbstractMyeloma is a unique hematologic malignancy that exclusively homes in the bone marrow and induces massive osteoclastic bone destruction presumably by producing cytokines that promote the differentiation of the hematopoietic progenitors to osteoclasts (osteoclastogenesis). It is recognized that neighboring bone marrow stromal cells influence the expression of the malignant phenotype in myeloma cells. This study examined the role of the interactions between myeloma cells and neighboring stromal cells in the production of osteoclastogenic factors to elucidate the mechanism underlying extensive osteoclastic bone destruction. A murine myeloma cell line 5TGM1, which causes severe osteolysis, expresses α4β1-integrin and tightly adheres to the mouse marrow stromal cell line ST2, which expresses the vascular cell adhesion molecule-1 (VCAM-1), a ligand for α4β1-integrin. Co-cultures of 5TGM1 with primary bone marrow cells generated tartrate-resistant acid phosphatase-positive multinucleated bone-resorbing osteoclasts. Co-cultures of 5TGM1 with ST2 showed increased production of bone-resorbing activity and neutralizing antibodies against VCAM-1 or α4β1-integrin inhibited this. The 5TGM1 cells contacting recombinant VCAM-1 produced increased osteoclastogenic and bone-resorbing activity. The activity was not blocked by the neutralizing antibody to known osteoclastogenic cytokines including interleukin (IL)-1, IL-6, tumor necrosis factor, or parathyroid hormone-related peptide. These data suggest that myeloma cells are responsible for producing osteoclastogenic activity and that establishment of direct contact with marrow stromal cells via α4β1-integrin/VCAM-1 increases the production of this activity by myeloma cells. They also suggest that the presence of stromal cells may provide a microenvironment that allows exclusive colonization of myeloma cells in the bone marrow.

Cell–cell contact between marrow stromal cells and myeloma cells via VCAM-1 and α4β1-integrin enhances production of osteoclast-stimulating activity

Myeloma is a unique hematologic malignancy that exclusively homes in the bone marrow and induces massive osteoclastic bone destruction presumably by producing cytokines that promote the differentiation of the hematopoietic progenitors to osteoclasts (osteoclastogenesis). It is recognized that neighboring bone marrow stromal cells influence the expression of the malignant phenotype in myeloma cells. This study examined the role of the interactions between myeloma cells and neighboring stromal cells in the production of osteoclastogenic factors to elucidate the mechanism underlying extensive osteoclastic bone destruction. A murine myeloma cell line 5TGM1, which causes severe osteolysis, expresses alpha(4)beta(1)-integrin and tightly adheres to the mouse marrow stromal cell line ST2, which expresses the vascular cell adhesion molecule-1 (VCAM-1), a ligand for alpha(4)beta(1)-integrin. Co-cultures of 5TGM1 with primary bone marrow cells generated tartrate-resistant acid phosphatase-positive multinucleated bone-resorbing osteoclasts. Co-cultures of 5TGM1 with ST2 showed increased production of bone-resorbing activity and neutralizing antibodies against VCAM-1 or alpha(4)beta(1)-integrin inhibited this. The 5TGM1 cells contacting recombinant VCAM-1 produced increased osteoclastogenic and bone-resorbing activity. The activity was not blocked by the neutralizing antibody to known osteoclastogenic cytokines including interleukin (IL)-1, IL-6, tumor necrosis factor, or parathyroid hormone-related peptide. These data suggest that myeloma cells are responsible for producing osteoclastogenic activity and that establishment of direct contact with marrow stromal cells via alpha(4)beta(1)-integrin/VCAM-1 increases the production of this activity by myeloma cells. They also suggest that the presence of stromal cells may provide a microenvironment that allows exclusive colonization of myeloma cells in the bone marrow. (Blood. 2000;96:1953-1960)

Tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) stimulate osteoclastic bone resorption.

As both tissue inhibitor of metalloproteinases-1 (TIMP-1) and TIMP-2 have been reported to inhibit bone resorption, we examined whether TIMP-1 or TIMP-2 in fetal calf serum (FCS), with which culture media were supplemented, affected osteoclastic bone resorption in vitro. Contrary to our expectation, almost complete suppression of osteoclastic bone resorption was observed when both TIMP-1 and TIMP-2 were removed from the FCS. Bone resorption was, however, almost fully restored by the addition of recombinant TIMPs. TIMPs stimulate bone resorption at significantly lower concentrations (approximately ng/ml) than those (approximately micrograms/ml) required to inhibit bone resorption. To understand the mechanism of TIMP-dependent bone resorption, we counted and compared the number of tartrate-resistant acid phosphatase-(TRAP-) positive and multinuclear cells in cultures containing either 10% FCS or TIMP-1-free and/or TIMP-2-free FCS. There was essentially no difference in number among these, suggesting that the TIMP role seems to be related to the functional expression of osteoclasts. Metalloproteinase inhibitors, either BE16627B[L-N-(N-hydroxy-2-isobutylsuccinynamoyl)-seryl-L-val ine] or R94138 ¿N-methyl-(3S)-2-[(2R)-2-hydroxycarbamoylmethylundecanoyl] hexahydropyridazine-3-carboxamide¿, could not replace TIMPs, suggesting that the osteoclast-stimulating activity of TIMPs cannot be ascribed to merely their inhibitory effect on matrix metalloproteinases.

Bone resorbing activity released from zymosan-activated mouse peritoneal macrophages - the role of prostanoids and interleukin-1

To study the effect of zymosan on the release of osteoclast stimulating activity from macrophages. Calvarial bones from neonatal mice and peritoneal macrophages were incubated in the absence and presence of zymosan for 72 h and supernatants harvested for subsequent analysis of bone resorbing activities and prostaglandin concentration. Bone resorption was assessed in vitro by analysing the release of 45Ca and 3H from neonatal mouse calvarial bones prelabelled in vivo by injections of [45Ca]CaCl2 or [3H]-proline. Prostaglandin E2 (PGE2) and I2 (PGI2) were analyzed using radioimmunoassays. Supernatants from macrophages treated with zymosan stimulated the release of 45Ca and 3H. The amount of bone resorbing activity present in the macrophage supernatants was dependent on the concentration of zymosan (0.1-100 microg/ml), as well as the number of macrophages present. The 45Ca release induced by zymosan treated macrophages was inhibited by three different inhibitors of osteoclastic bone resorption (calcitonin, acetazolamide, amino bisphosphonate). The bone resorbing activity released by the zymosan-activated macrophages was lost after ultrafiltration using a filter with a molecular weight cut off of 30,000 Daltons, but retained when using a filter with a cut off of 3000 Daltons. Time-course studies of the production of bone resorbing activity in macrophages showed that activity increased during the first hour of exposure to zymosan and then reached a plateau for 96 h. PGE2 and PGI2 release from macrophages was increased during the first three hours of exposure to zymosan. This prostanoid production, together with bone resorbing activity, was abolished by indomethacin. The bone resorbing activity present 3-72 h after zymosan exposure, however, was not inhibited by indomethacin. Bone resorption stimulated by conditioned media from zymosan treated macrophages after 3 h was inhibited by 60-75% in the presence of anti IL-1alpha, 0-20% by anti IL-1beta, and completely by antisera neutralizing both IL-1alpha and IL-1beta. In addition, an IL-1 receptor antagonist abolished the stimulatory effect of conditioned media from zymosan treated macrophages. These data indicate that treatment of mouse peritoneal macrophages with zymosan results in production of activities capable of stimulating bone resorption in vitro. The activity released initially appears to be due to a zymosan induced burst of prostanoid production, while the activity released during prolonged exposure to zymosan is due primarily to IL-1alpha.

Bisphosphonates induce osteoblasts to secrete an inhibitor of osteoclast-mediated resorption.

Current knowledge indicates that osteoblasts play an integral role in osteoclastic bone resorption through an osteoclast-stimulating activity produced by osteoblasts in response to resorption-promoting osteotropic factors. Previously, we have shown that the inhibitory action of the bisphosphonates on bone resorption in part is mediated by osteoblasts. The aim of the present study was to investigate whether the bisphosphonate-generated inhibition is due to these compounds decreasing the synthesis of the osteoclast-stimulating activity or is the result of osteoblasts synthesizing an osteoclast resorption inhibitor. Using the osteoblastic cell line CRP 10/30, which produces osteoclast- stimulating activity, constitutively and employing isolated rat osteoclasts cultured on ivory, evidence was obtained indicating that the bisphosphonates ibandronate and alendronate at a concentration of 10(-7) M induce osteoblasts to synthesize an osteoclast inhibitor that reduces pit formation by more than 50%. The inhibitor is heat and proteinase labile and has a molecular mass between 1-10 kDa. The reduction of resorption pits is paralleled by a decrease in tartrate-resistant acid phosphatase-positive mono- and multinucleated cells, whereas the mean area resorbed per pit was not changed, suggesting that the inhibitor affects osteoclast formation and/or survival and probably not the osteoclast resorption activity. Rat preosteoblastic cells and rat dermal fibroblasts were found not to produce the inhibitor. In conclusion, osteoblasts aside from their role of mediating osteoclastic resorption promoters are also involved in inhibiting bone resorption through the synthesis of an osteoclast resorption inhibitor.

Constitutive expression of osteoclast-stimulating activity by normal clonal osteoblast-like cells: effects of parathyroid hormone and 1,25-dihydroxyvitamin D3.

Osteoblasts are known to produce osteoclast-stimulating activity (OSA). The aim of the current study was to relate the expression of OSA to the osteoblastic phenotype and examine its regulation by calciotropic hormones. The study was performed with the normal osteoblastic cell clone CRP 10/30 and the preosteoblastic clone CRP 4/7. OSA was determined with the well described isolated osteoclast pit assay, using sperm whale dentine as substrate. In contrast to previous studies, the assay was carried out at pH 7.36, rather than at pH 6.4 or 6.9. The results indicate that over 24 h, CRP 10/30 cells produce constitutively OSA, which compared to controls corresponds to an about 7-fold increase in resorption pits. There was considerably less activity expressed by either CRP 4/7 cells or fibroblasts. OSA proved to be heat labile, and its mol wt was estimated to be over 10 kilodaltons. While PTH-(1-34) did not influence the synthesis of OSA, the number of pits formed by osteoclasts incubated with medium conditioned by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-treated CRP 10/30 cells was increased 3-fold above baseline values. A similar increase was obtained with 1.25-(OH)2D3 added directly to CRP 10/30-conditioned medium. These results could not be duplicated with 1,25-(OH)2D3 added to either control medium or medium conditioned by CRP 4/7 cells or fibroblasts. The present study shows that normal clonal bone cells synthesize constitutively OSA, which is not regulated by PTH or 1,25-(OH)2D3. Furthermore, the results suggest that the synthesis of bone cell-derived OSA is limited to cells expressing the mature osteoblastic phenotype. Finally, CRP 10/30-conditioned medium appears to permit 1,25-(OH)2D3 to function on osteoclasts.

Constitutive expression of osteoclast-stimulating activity by normal clonal osteoblast-like cells: effects of parathyroid hormone and 1,25- dihydroxyvitamin D3

Osteoblasts are known to produce osteoclast-stimulating activity (OSA). The aim of the current study was to relate the expression of OSA to the osteoblastic phenotype and examine its regulation by calciotropic hormones. The study was performed with the normal osteoblastic cell clone CRP 10/30 and the preosteoblastic clone CRP 4/7. OSA was determined with the well described isolated osteoclast pit assay, using sperm whale dentine as substrate. In contrast to previous studies, the assay was carried out at pH 7.36, rather than at pH 6.4 or 6.9. The results indicate that over 24 h, CRP 10/30 cells produce constitutively OSA, which compared to controls corresponds to an about 7-fold increase in resorption pits. There was considerably less activity expressed by either CRP 4/7 cells or fibroblasts. OSA proved to be heat labile, and its mol wt was estimated to be over 10 kilodaltons. While PTH-(1-34) did not influence the synthesis of OSA, the number of pits formed by osteoclasts incubated with medium conditioned by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-treated CRP 10/30 cells was increased 3-fold above baseline values. A similar increase was obtained with 1.25-(OH)2D3 added directly to CRP 10/30-conditioned medium. These results could not be duplicated with 1,25-(OH)2D3 added to either control medium or medium conditioned by CRP 4/7 cells or fibroblasts. The present study shows that normal clonal bone cells synthesize constitutively OSA, which is not regulated by PTH or 1,25-(OH)2D3. Furthermore, the results suggest that the synthesis of bone cell-derived OSA is limited to cells expressing the mature osteoblastic phenotype. Finally, CRP 10/30-conditioned medium appears to permit 1,25-(OH)2D3 to function on osteoclasts.

Bacteroides gingivalis stimulates bone resorption via interleukin-1 production by mononuclear cells The relative role for B. gingivalis endotoxin

Abstract. Supernatants of human peripheral blood mononuclear cells cultured in the presence of B. gingivalis, showed a strong osteoclast stimulating activity as measured by 45Ca release from fetal mouse long bones in vitro. These supernatants also contained a high concentration of bioactive and immunoreactive interleukin-l (IL-1), but tumor necrosis factor (TNFa), another osteoclast-activating cytokine, was not detected. Osteoclast activation by the supernatants was inhibited by an antibody against IL-1. whereas ultrapure human IL-1 mimicked the effect of the supernatant. The ability of B. gingivalis to induce IL-1 and OAF production was heat sensitive, as 20 min heating of the bacteria at 120°C caused a 50% loss of activity. In addition, purified B. gingivalis lipopolysaccharide (LPS) had little IL-1 inducing capacity, compared with LPS of Escherichia coli. These data suggest that human peripheral blood cells confronted with B. gingivalis produce large amounts of IL-1 which has strong osteoclast stimulating activity. However, in contrast with E. coli LPS, B. gingivalis LPS does not seem to be the major inducing agent. Thus other bacterial components must be responsible for the observed IL-1 and OAF induction.

Bacteroides gingivalisstimulates bone resorption via interleukin‐1 production by mononuclear cells The relative role forB. gingivalisendotoxin

Supernatants of human peripheral blood mononuclear cells cultured in the presence of B. gingivalis, showed a strong osteoclast stimulating activity as measured by 45Ca release from fetal mouse long bones in vitro. These supernatants also contained a high concentration of bioactive and immunoreactive interleukin-1 (IL-1), but tumor necrosis factor (TNFa), another osteoclast-activating cytokine, was not detected. Osteoclast activation by the supernatants was inhibited by an antibody against IL-1, whereas ultrapure human IL-1 mimicked the effect of the supernatant. The ability of B. gingivalis to induce IL-1 and OAF production was heat sensitive, as 20 min heating of the bacteria at 120 degrees C caused a 50% loss of activity. In addition, purified B. gingivalis lipopolysaccharide (LPS) had little IL-1 inducing capacity, compared with LPS of Escherichia coli. These data suggest that human peripheral blood cells confronted with B. gingivalis produce large amounts of IL-1 which has strong osteoclast stimulating activity. However, in contrast with E. coli LPS, B. gingivalis LPS does not seem to be the major inducing agent. Thus other bacterial components must be responsible for the observed IL-1 and OAF induction.