Bone Marrow Culture System Research Articles

Prostaglandin E2-mediated anabolic effect of a novel inhibitor of phosphodiesterase 4, XT-611, in the in vitro bone marrow culture.

The mechanism of osteoblast formation by a novel PDE4 inhibitor, XT-611, was studied in the in vitro bone marrow culture system. The compound potentiated the osteoblast differentiation through accumulation of cyclic AMP after autocrine stimulation of EP4 receptor by PGE2 in pro-osteoblastic cells. We previously reported that inhibitors of phosphodiesterase (PDE)4 isoenzyme increase osteoblast formation in an in vitro bone marrow culture system and inhibit bone loss in animal osteoporosis models. Here we investigated the mechanism of the effect of a novel PDE4 inhibitor, 3,4-dipropyl-4,5,7,8-tetrahydro-3H-imidazo[1,2-i]-purin-5-one (XT-611), on osteoblast formation in the in vitro bone marrow culture system. Rodent bone marrow cells were cultured in the presence of 0.2 mM ascorbic acid phosphate ester, 1 mM beta-glycerophosphate, and 10 nM dexamethasone for 10 days. Drug treatments were done for 24 h on day 3 of culture. PDE4 inhibitors, including XT-611, but not PDE3 and PDE5 inhibitors, increased mineralized nodule formation in rat and mouse bone marrow cell cultures. During culture of the bone marrow cells, prostaglandin E2 (PGE2) production increased with a peak on day 4, but the increase was completely inhibited by indomethacin, an unselective cyclo-oxygenase (COX) inhibitor. Spontaneous and XT-611-induced mineralized-nodule formation was also inhibited by indomethacin and COX-2 inhibitors, in a similar potential. Alkaline phosphatase-positive nodule formation in the absence or presence of XT-611 was inhibited by an antagonist of EP4 receptor, AH23848B, and synergistically potentiated by 11-deoxy-PGE1, but it was not influenced by other EP antagonists and agonists examined. The expression of PDE4 and EP4 mRNAs was observed in bone marrow cells. The effect of XT-611 was also confirmed to involve an increase of cyclic AMP and the cyclic AMP-dependent protein kinase pathway. These results suggest that PGE2 stimulates differentiation of osteoblast progenitor cells through the EP4 receptor in an autocrine manner, and the PDE4 inhibitor potentiates the differentiation by inhibiting hydrolysis of cyclic AMP in the cells.

Inhibitory effect of titanium particles on osteoclast formation generated by treatment of mouse bone marrow cells with PGE2.

The present study was designed to evaluate the effect of titanium (Ti) particles with no endotoxin on osteoclast differentiation and osteoclast activity in in vitro experiments. Osteoclast formation as well as osteoclastic bone resorbing activity were examined using the mouse bone marrow culture system and purified rabbit osteoclasts treated with Ti particles (2.5-20 microgram cm-2). Ti particles, with no adherent endotoxin, inhibited osteoclastogenesis and receptor activator of NF-kappaB ligand (RANKL) expression in bone marrow cells treated with prostaglandin E2 (PGE2) (100 nM). The inhibitory effect of Ti particles was concentration-dependent (5-20 microgram cm-2), and was observed only on the generation of osteoclasts by PGE2, but not by 1,25-dihydroxyvitamin D3 or soluble RANKL. This suggests that Ti particles did not act uniformly on a common process in the generation of osteoclasts, but specifically on signal transduction for PGE2 in generating osteoclasts. In highly purified osteoclasts, Ti particles showed no effect on survival and bone resorbing activity. Ti particles inhibited osteoclast differentiation and RANKL expression in mouse bone marrow cells treated with PGE2, without affecting mature osteoclast survival or activity. Thus, Ti particles may alter the osteoclastogenetic action of PGE2, which is one of the regulatory factors of bone remodeling.

Receptor editing in CD45-deficient immature B cells.

B cell receptor signaling threshold regulates negative selection of autoreactive B cells and determines the mechanism of B cell tolerance. Using mice carrying immunoglobulin transgene specific for MHC class I antigen K(k) (3-83 Tg mice), and IL-7-driven bone marrow (BM) culture system, we have previously shown that receptor editing is a major mechanism in B cell tolerance. To test the role of BCR signaling competence on the induction of tolerance-mediated receptor editing, we crossed the 3-83 Tg mice with mice deficient in CD45, a protein tyrosine phosphatase that functions asa positive regulator of the BCR signaling. We found that in the absence of self-antigen allelic exclusion is efficiently imposed in 3-83 Tg CD45(-/-) mice, although numbers of peripheral B cells are reduced. Using our BM culture system, we show here that immature 3-83 Tg CD45(-/-) B cells encountering self-antigen are developmentally arrested and undergo secondary light chain recombination and receptor editing, not different than CD45-sufficient cells. Thus, lack of CD45 does not abolish the receptor editing competence in immature B cells encountering high avidity membrane-bound antigen.

Osteoclastogenic potential of bone marrow cells increases with age in elderly women with fracture

The most reliable explanation for decreasing bone mass in elderly women is an imbalance of osteoclastic resorption and osteoblastic formation resulting from a relative increase in osteoclastic resorption. However, it is not clear whether an increase in osteoclastic bone resorption with age is due to increased osteoclast formation or to osteoclastic bone resorption activity. In this study, using a human bone marrow culture system, we attempt to clarify the increase in osteoclast formation with age. The mononuclear cell-rich fraction from bone marrow, obtained from the proximal region of the femur from female elderly patients with fracture, were cultured for 14 days in the presence of 1,25 dihydroxyvitamin D 3. Tartrate-resistant acid phosphatase-positive multinucleated cells were counted as osteoclasts. In our investigation, human osteoclast formation in the bone marrow culture increased with age in elderly women (age 64–96 years). The osteoclast formation was positively correlated with macrophage-colony stimulation factor and prostaglandin E 2 production in bone marrow culture. Also, osteoclast formation ex vivo was negatively correlated with bone mineral density of the lumbar spine (L2–L4). The above results indicate that the osteoclastogenic potential of bone marrow cells increases with aging in elderly women with fracture, and suggest that a decrease in bone mass of elderly women may be due to an increase in osteoclast population associated with aging.

Cell-to-Cell adhesion via intercellular adhesion molecule-1 and leukocyte function-associated antigen-1 pathway is involved in 1alpha,25(OH)2D3, PTH and IL-1alpha-induced osteoclast differentiation and bone resorption.

Cell-to-cell interaction is required for the differentiation of osteoclast precursors as well as for osteoclast function. The present study was undertaken to determine whether 1,25 dihydroxyvitamin D3 (1,25D), PTH, IL-1alpha and PGE2 depend on cell-to-cell interactions through the intercellular adhesion molecule (ICAM)-1/leukocyte function-associated antigen (LFA)-1 pathway in osteoclast formation and bone resorption. We found that mouse osteoblasts expressed ICAM-1 and that the expression was increased by treatment with PTH, IL-1alpha or 1,25D, but not by PGE2. In resorption assays measuring either 45Ca release from bone organ cultures or pit formation in bone cell cultures, 1,25D-, PTH- and IL-1alpha-stimulated resorption was inhibited by anti-ICAM-1 monoclonal antibody (mAb) and/or anti-LFA-1 mAb, while basal and PGE2-stimulated bone resorbing activities were not affected by these mAbs. Furthermore, in a mouse bone marrow culture system, stimulation of osteoclast-like (OCL) cell formation by 1,25D (10 nM), PTH (10 ng/ml) or IL-1alpha (10 ng/ml) was inhibited by the addition of anti-ICAM-1 mAb and/or anti-LFA-1 mAb. In a coculture system of murine spleen cells and osteoblasts, the ICAM-1/LFA-1 interaction was also involved in 1,25D-, PTH- and IL-1alpha-stimulated TRAP-positive MNC formation. However, anti-ICAM-1 mAb and anti-LFA-1 mAb did not alter either 1,25D- or PTH-stimulated receptor activator of NF-kappaB ligand (RANKL) mRNA transcription in bone marrow cultures. Taken together, we here propose that ICAM-1-mediated cell-to-cell adhesion of osteoblasts and osteoclast precursors is involved in RANKL-dependent osteoclast maturation stimulated by 1,25D, PTH, and IL-1alpha.

Ex Vivo Hematopoiesis and Lymphopoiesis

Bone marrow is the hematopoietic tissue and a primary lymphoid organ. Its intricate, three-dimensional architecture facilitates cell-cell and cell-matrix interactions and provides a microenvironment that supports self-renewal and multilineal differentiation. Traditional in vitro culture systems (flask cultures) are limited by the absence of normal marrow spatial organization and cellular interactions with extracellular matrices. They deviate from the three-dimensional marrow structure, induce artificial lipogenesis and cell flattening, and produce only limited cell lineages. Our hypotheses are: 1) the marrow’s function in promoting multilineal hemopoiesis depends on its three-dimensional structure and 2) this function can be (substantially) reproduced in vitro only if its three-dimensional structure is preserved. We have developed a novel human ex vivo bone marrow model that mimics bone marrow both structurally and functionally. By providing an artificial scaffolding, we were able to obtain a three- dimensional growth configuration with high cell density and intimate physical contact between hematopoietic and stromal cells. Characterization of the stroma developed demonstrated that an anatomical tissue-like structure was obtained. More importantly, the three-dimensional culture system supported multilineal hematopoiesis, including the development of myeloid, erythroid, and lymphoid lineages. The ability to engineer a structural and functional human bone marrow mimicry ex vivo provides the possibilities of generating lineage-specific cells from the hematopoietic stem cells for adoptive cellular therapies. In this presentation, the B-lymphocyte development in our three-dimensional bone marrow culture system will be compared with B-lymphopoiesis in the traditional two-dimensional culture system.

Evaluation Of Myelotoxicity In Cotton Rats (Sigmodon hispidus) Exposed To Environmental Contaminants. I. In Vitro Bone-Marrow Progenitor Culture

Bone marrow is extremely sensitive to toxicants, and in vitro culture of bone-marrow progenitor cells has been shown to be a sensitive indicator of bone-marrow injury in laboratory rodents. The ability of a bone-marrow progenitor cell assay to detect myelotoxicity in a wild rodent model (cotton rat, Sigmodon hispidus) that inhabits many contaminated ecosystems in the southern United States was examined. Responsiveness of progenitor cells to recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and cotton rat lung-conditioned medium (LCM) was determined to optimize culture conditions for cotton rats. Myelotoxicity was induced in cotton rats by treating animals with either cyclophosphamide (8 or 80 mg/kg) or dexamethasone (500 µg/kg) over a 5-d period. Administration of a high dose of cyclophosphamide caused nearly total suppression of colony formation of granulocyte-macrophage progenitor cells (CFU-GM). Marked histological changes in both the bone marrow and spleen were also observed in cotton rats treated with a high dose of cyclophosphamide. Although histological lesions were not apparent, the number of CFU-GM in the bone marrow of low-dose cyclophosphamide- and dexamethasone-treated cotton rats was significantly suppressed compared to controls. The number of CFU-GM was consistently higher using LCM than recombinant murine GM-CSF. This reproducible, quantitative, in vitro bone-marrow progenitor cell culture system was a sensitive indicator of myelotoxicity in wild cotton rats and should be useful for monitoring chronic exposures to low levels of environmental toxicants in wild rodent populations.

Ig Light Chain Receptor Editing in Anergic B Cells

Receptor editing in the bone marrow (BM) serves to modify the Ag receptor specificity of immature self-reactive B cells, while anergy functionally silences self-reactive clones. Here, we demonstrate that anergic B cells in hen egg lysozyme Ig (HEL-Ig)/soluble HEL double transgenic mice show evidence of having undergone receptor editing in vivo, as demonstrated by the presence of elevated levels of endogenous kappa light chain rearrangements in the BM and spleen. In an in vitro IL-7-driven BM culture system, HEL-Ig BM B cells grown in the presence of soluble HEL down-regulated surface IgM expression and also showed induction of new endogenous kappa light chain rearrangements. Using a panel of soluble protein ligands with reduced affinity for the HEL-Ig receptor, the editing response was shown to correlate in a dose-dependent fashion with the strength of signaling through the B cell receptor. The finding that the level of B cell receptor cross-linking sufficient to induce anergy in B cells is also capable of engaging the machinery required for receptor editing suggests an intimate relationship between these two mechanisms in maintaining B cell tolerance.

Generation of murine dendritic cells from flt3-ligand–supplemented bone marrow cultures

AbstractMurine dendritic cells (DCs) can be classified into at least 2 subsets, “myeloid-related” (CD11bbright, CD8α−) and “lymphoid-related” (CD11bdull, CD8α+), but the absolute relationship between the 2 remains unclear. Methods of generating DCs from bone marrow (BM) precursors in vitro typically employ granulocyte-macrophage colony-stimulating factor (GM-CSF) as the principal growth factor, and the resultant DCs exhibit a myeloidlike phenotype. Here we describe a flt3-ligand (FL)–dependent BM culture system that generated DCs with more diverse phenotypic characteristics. Murine BM cells cultured at high density in recombinant human FL for 9 days developed into small lymphoid-sized cells, most of which expressed CD11c, CD86, and major histocompatibility complex (MHC) class II. The CD11c+ population could be divided into 2 populations on the basis of the level of expression of CD11b, which may represent the putative myeloid- and lymphoid-related subsets. The FL in vitro–derived DCs, when treated with interferon-α or lipopolysaccharide during the final 24 hours of culture, expressed an activated phenotype that included up-regulation of MHC class II, CD1d, CD8α, CD80, CD86, and CD40. The FL-derived DCs also exhibited potent antigen-processing and antigen-presenting capacity. Neutralizing anti–interleukin-6 (IL-6) antibody, but not anti–GM-CSF, significantly reduced the number of DCs generated in vitro with FL, suggesting that IL-6 has a role in the development of DCs from BM precursors. Stem cell factor, which exhibits some of the same bioactivities as FL, was unable to replace FL to promote DC development in vitro. This culture system will facilitate detailed analysis of murine DC development.

Generation of murine dendritic cells from flt3-ligand–supplemented bone marrow cultures

Murine dendritic cells (DCs) can be classified into at least 2 subsets, “myeloid-related” (CD11bbright, CD8α−) and “lymphoid-related” (CD11bdull, CD8α+), but the absolute relationship between the 2 remains unclear. Methods of generating DCs from bone marrow (BM) precursors in vitro typically employ granulocyte-macrophage colony-stimulating factor (GM-CSF) as the principal growth factor, and the resultant DCs exhibit a myeloidlike phenotype. Here we describe a flt3-ligand (FL)–dependent BM culture system that generated DCs with more diverse phenotypic characteristics. Murine BM cells cultured at high density in recombinant human FL for 9 days developed into small lymphoid-sized cells, most of which expressed CD11c, CD86, and major histocompatibility complex (MHC) class II. The CD11c+ population could be divided into 2 populations on the basis of the level of expression of CD11b, which may represent the putative myeloid- and lymphoid-related subsets. The FL in vitro–derived DCs, when treated with interferon-α or lipopolysaccharide during the final 24 hours of culture, expressed an activated phenotype that included up-regulation of MHC class II, CD1d, CD8α, CD80, CD86, and CD40. The FL-derived DCs also exhibited potent antigen-processing and antigen-presenting capacity. Neutralizing anti–interleukin-6 (IL-6) antibody, but not anti–GM-CSF, significantly reduced the number of DCs generated in vitro with FL, suggesting that IL-6 has a role in the development of DCs from BM precursors. Stem cell factor, which exhibits some of the same bioactivities as FL, was unable to replace FL to promote DC development in vitro. This culture system will facilitate detailed analysis of murine DC development.

Effects of OPG and rankl in murine bone marrow and organ culture systems

Effects of OPG and rankl in murine bone marrow and organ culture systems

CROSS-SPECIES COMPATIBILITY OF INTERCELLULAR ADHESION MOLECULE-1 (CD54) WITH ITS LIGANDS1

The molecular interactions of intercellular adhesion molecule-1 (ICAM-1; CD54) are potentially important in several situations in the context of pig-to-human xenotransplantation. If porcine bone marrow is to be used for the induction of xenograft tolerance in humans, the role that has been suggested for ICAM-1 in the interactions of haematopoietic stem cells makes its cross-species compatibility important. Similarly, the potential role of ICAM-1 interactions in graft rejection makes it an important molecule to study. An in vitro static cell-to-cell adhesion study was used to look at the successful interaction of ICAM-1 with its ligands across the pig-human species barrier in both directions. A second in vitro system, the standard long-term bone marrow culture (LT-BMC), was used to study the functional role of ICAM-1 in haematopoiesis. Human ICAM-1 was able to adhere to ligands on porcine cells, including one or more ligand that contains CD18. Conversely, human CD18-containing ligands mediated adherence to porcine cells. Using the long-term bone marrow culture system, there was no evidence that blocking the interactions of ICAM-1 inhibited hematopoiesis, either in the human-human or pig-human combinations of precursor cells and marrow stroma. ICAM-1 is able to interact with at least some of its ligands across the species barrier, in both pig-human and human-pig combinations. However, the interactions of ICAM-1 do not appear to be central to hematopoiesis, at least in the model system used.

PHYSIOLOGY AND THE EFFECT OF SOME DRUGS ON OSTEOCLAST

The osteoclast cellular process is essential for bone growth, remodeling, and repair, and is, under normal conditions, tightly coupled to the process of bone formation by the osteoblast. It is the balance that determines skeletal mass and shape at any point in time. Some drugs effect the osteoclast formation and differentiation. Biphosphonate and non-steroid anti-inflammatory drugs (NSAIDs_ are known to reduce the resorption process of the bone in vitro. We worked on those two different drugs to see their effects and influences on bone remodeling, especially bone resoprtion using a bone marrow culture system. This culture system was found to be suitable for the recruitment and differentiation process of osteoclast-like cells from their progenitors. Biphosphonate and two NSAIDs that were used in this study using 1,25(OH) 2 D 3 as an osteoclast stimulani, proved to be very powerful inhibitor of bone resorption. Their mechanisms of action of bone resorption inhibition using bone marrow culture system are almost similar, which is during the later stage of differentiation. The improvement of our means of therapeutic intervention with antiresorptive agents will depend on our better understanding of the molecular basis of bone resorption.

Inhibition of experimental bone resorption and osteoclast formation and survival by 2-aminoethanesulphonic acid

It is known that bone resorption is mediated by osteoclasts, and lipopolysaccharide (LPS) and inflammatory mediators such as interleukin-1 (IL-1) and prostaglandin E 2 (PGE 2) induce osteoclast differentiation from haemopoietic cells, 2-aminoethanesulphonic acid, which is known as taurine, is an important nutrient and is added to most synthetic human infant milk formulas. In this study, it was found that 2-aminoethanesulphonic acid inhibits the stimulation of bone resorption mediated by LPS of the periodontopathic microorganism Actinobacillus actinomycetemcomitans Y4 in organ cultures of newborn mouse calvaria. The effect of 2-aminoethanesulphonic acid on the development and survival of osteoclast-like multinucleated cells produced in a mouse bone-marrow culture system was also examined. 2-aminoethanesulphonic acid (100 μg/ml) suppressed the formation of these osteoclast-like cells in the presence of LPS of A. actinomycetemcomitans Y4, IL-1α or PGE 2 in mouse marrow cultures. On the other hand, 2-aminoethanesulphonic acid did not inhibit 1α, 25-dihydroxyvitamin D 3-mediated osteoclast differentiation. Although IL-1α elongated the survival of the osteoclast-like cells, 2-aminoethanesulphonic acid blocked the supportive effect of IL-1α on osteoclast survival. 2-aminoethanesulphonic acid showed no effect on the growth of mouse osteoblasts. Finally, it was found that 2-aminoethanesulphonic acid inhibited alveolar bone resorption in experimental periodontitis in hamsters. These results suggest that 2-aminoethanesulphonic acid is an effective agent in preventing inflammatory bone resorption in periodontal diseases.

Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD34-positive cell proliferation and differentiation in vitro

We examined the supportive function of stromal cells from patients with refractory anemia (RA) of myelodysplastic syndrome (MDS) on CD34-positive hematopoietic cell proliferation and differentiation using a long-term bone marrow culture (LTMC) system. Primary marrow stromal cells were obtained from 11 MDS RA patients and 12 healthy volunteers, and freshly prepared CD34-positive bone marrow cells from a normal subject were inoculated onto the stroma. There seems to be three broad patterns of hematopoietic cell growth in the LTMCs. In one group, hematopoietic cells were maintained at near normal levels (type A). In the second group, the number of hematopoietic cells increased within the first 5–10 days of culture, but declined to low levels at 15–20 days of culture as compared with normal control (type B). In the third group, the incidence of hematopoietic cells steadily declined from the beginning of the culture (type C). Furthermore, apoptotic change of hematopoietic cells was very frequently observed in cultures with the type C stroma, which were especially defective for supporting CD34+cell proliferation and differentiation. The expression of CD95 on hematopoietic cells was induced by the type C stroma, however, production of fas ligand by the stromal cells was not observed. These findings suggest a lack of hematopoietic supportive function in some cases of MDS RA and also indicate that there is heterogeneity of stromal function among MDS RA patients.

In VivoDisturbance of Hematopoiesis in Mice Persistently Infected with Murine Cytomegalovirus: Impairment of Stromal Cell Function

Although the pathogenic effects of a primary cytomegalovirus (CMV) infection on hematopoiesis has been largely investigated so far, the effects of a persistent or latent infection have yet to be elucidated. The effects of persistent CMV infection on hematopoiesis thus were examined using BALB/c mice at 4 weeks postinfection with 0.2 LD50of murine CMV (MCMV) infection as a persistent infection model. The parameters of constitutive hematopoiesis of MCMV persistently infected mice were completely identical to those of the control. However, the inductive hematopoiesis, examined by the autologous marrow reconstitution after 5-fluorouracil administration, was significantly impaired in the MCMV persistently infected mice (P< 0.05). In a colony-forming unit-spleen assay and a long-term bone marrow culture system, a decreased capacity of bone marrow stromal cells to support hematopoiesis was observed in the MCMV-infected mice in comparison with the controls. The existence of MCMV DNA in the adherent cells of long-term bone marrow culture from the MCMV-infected mice were confirmed by a polymerase chain reaction but not in the nonadherent cells. Furthermore, the increased expression level of tumor necrosis factor-α by stromal cells was also observed by semiquantitative reverse transcriptase-polymerase chain reaction. These results therefore strongly suggest that MCMV remains to infect the stromal cells while also inhibiting inductive hematopoiesis through the impairment of the stromal cell functions in the MCMV persistently infected mice.

Involvement of prostaglandin E2 and interleukin-1 alpha in the differentiation and survival of osteoclasts induced by lipopolysaccharide from Actinobacillus actinomycetemcomitans Y4.

Lipopolysaccharide (LPS) is a bacterial cell component that plays multifunctional roles in inflammatory reactions. LPS from various periodontal pathogens is supposed to be a major virulence factor of periodontal diseases. In the present study, we demonstrated that LPS from periodontopathic bacterium Actinobacillus actinomycetemcomitans Y4 (Y4 LPS) stimulated osteoclast formation in mouse bone marrow culture systems. Addition of anti-interleukin-1 alpha (IL-1 alpha) antibody or indomethacin in the marrow cultures resulted in the suppression of osteoclast differentiation. Quantitative analyses revealed that Y4 LPS stimulated the production of IL-1 alpha and prostaglandin E2 (PGE2) by bone marrow cells. Furthermore, an immunoblot analysis showed that Y4 LPS stimulated bone marrow cells to upregulate the expression of cyclooxygenase-2, a rate-limiting enzyme for the conversion of arachidonic acid to prostanoids. These findings suggest that both IL-1 alpha and PGE2 are involved in the LPS-mediated osteoclast differentiation. In addition, we found that Y4 LPS supported the survival of osteoclasts. Addition of anti-IL-1 alpha antibody in the osteoclast culture resulted in a reduction of osteoclast survival. Indomethacin, however, showed no effect on osteoclast survival. These findings suggest that the increased PGE2 and IL-1 alpha synthesis by bone marrow cells may play an important role in the differentiation and survival of osteoclasts induced by A. actinomycetemcomitans LPS.

Involvement of lymphocyte function-associated antigen-1 and intercellular adhesion molecule-1 in osteoclastogenesis: a possible role in direct interaction between osteoclast precursors.

In our search for molecules involved in the process of osteoclast differentiation, we examined the surface phenotypes of the preosteoclast-like cells and osteoclast-like multinucleated cells (MNCs) formed in bone marrow cultures, using monoclonal antibodies recognizing different antigen molecules expressed on hematopoietic cells. Among these cell surface antigens, lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) were highly expressed on mononuclear cells in the cultures for forming preosteoclast-like mononuclear cells. The double detection of these two antigen molecules with osteoclast-specific antigen and with calcitonin receptor, using a fluorescence-activated cell sorter or autoradiography technique, revealed that LFA-1 and ICAM-1 were expressed on the preosteoclasts. The expression of ICAM-1 was detected on both preosteoclasts and osteoclast-like MNCs, whereas the expression of LFA-1 was restricted to preosteoclasts. We designed a peptide with the sequence of the binding site of ICAM-1 against the ligand LFA-1. In the whole bone marrow culture system for forming osteoclast-like MNCs, a significant inhibition of MNC formation was observed by the addition of this peptide. These results strongly suggest the involvement of an LFA-1/ICAM-1-interaction in osteoclastogenesis.

Laminin, a major basement membrane component of the blood vessel, as a negative regulator of osteoclastogenesis.

Laminin, the major basement membrane glycoprotein of the blood vessel, inducing many cellular responses, inhibited the differentiation of osteoclasts in a rat bone marrow culture system when immobilized on the surface of the culture wells, showing that laminin acts as a negative regulator of osteoclast differentiation in a nonsolubilized form. Laminin inhibited the process of preosteoclast formation from early progenitor cells in bone marrow. This laminin-mediated inhibition of osteoclastogenesis was blocked by the addition of laminin fragment YIGSR, indicating that the inhibitory effect of laminin was mediated via laminin receptors. This finding suggests a significant role of basement membrane laminin of the blood vessels as a negative regulator of osteoclastogenesis.

Prostaglandin E2, interleukin 1alpha, and tumor necrosis factor-alpha increase human osteoclast formation and bone resorption in vitro.

Prostaglandin E2 (PGE2) and the cytokines interleukin (IL) 1alpha and tumor necrosis factor (TNF)alpha increase bone resorption in vivo, but the effect of these agents on osteoclastic bone resorption has never been studied in an in vitro human system. Our recently described human bone marrow culture system, in which osteoclasts are generated (vitronectin and calcitonin receptor-positive cells which resorb bone), was used to study the effects of these agents. Addition of indomethacin to macrophage colony-stimulating factor (M-CSF)-treated cultures nearly abolished osteoclast parameters, indicating that prostaglandins are virtually essential for human osteoclast formation. Additionally, PGE2 dose responsively increased osteoclast numbers and bone resorption. The effects of M-CSF and PGE2 are independent, as demonstrated by unaltered PGE2 concentrations in culture supernatants in spite of the dose-responsive increase in osteoclast parameters in response to M-CSF. The generation of osteoclasts in the presence of PGE2 occurred in favor of CD 14-positive macrophage formation. IL 1alpha and TNFalpha increased osteoclast parameters in a dose-responsive manner. Maximum stimulation yielded culture supernatant levels of PGE2 approximately the same as those concentrations of exogenous PGE2 that dramatically induced osteoclast formation. This osteoclast-inducing effect was inhibited both by indomethacin and by the specific inhibitor of inducible prostaglandin G/H synthase, NS398, and this was reversed by addition of exogenous PGE2. These results demonstrate unequivocally that IL 1alpha and TNFalpha enhance human osteoclast formation and suggest that they mediate their effects through PGE2.