RESUMO
OBJECTIVE: Hyperbilirubinemia in patients with biliary atresia causes deciduous tooth injuries such as green pigmentation and dentin hypoplasia. In patients with biliary atresia who received liver transplantation, tooth structure appears to be recovered radiographically. Nevertheless, little is known about cellular mechanisms underlying bilirubin-induced damage and suppression of deciduous tooth formation. In this study, we examined the effects of bilirubin in stem cells from human exfoliated deciduous teeth (SHED) in vitro. MATERIALS AND METHODS: SHED were cultured under exposure to excess of bilirubin and then interruption of bilirubin stimulation. RESULTS: Bilirubin induced cell death and inhibited the odontogenic capacity of SHED by suppressing AKT and extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathways and enhancing nuclear factor kappa B p65 (NF-κB p65) pathway. The interruption of bilirubin stimulation reduced cell death and recovered the inhibited odontogenic capacity of bilirubin-damaged SHED. The bilirubin interruption also normalized the impaired AKT, ERK1/2, and NF-κB p65 signaling pathways. CONCLUSION: These findings suggest that tooth hypodontia in patients with hyperbilirubinemia might be due to bilirubin-induced cell death and dentinogenic dysfunction of odontogenic stem cells via AKT, ERK1/2, and NF-κB pathways and also suggested that bilirubin-induced impairments in odontogenic stem cells were reversible when bilirubin stimulation is interrupted.
Assuntos
Bilirrubina/farmacologia , Morte Celular/efeitos dos fármacos , Odontogênese/efeitos dos fármacos , Células-Tronco , Dente Decíduo/citologia , Atresia Biliar/sangue , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Pré-Escolar , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfoliação de Dente , Fator de Transcrição RelA/metabolismoRESUMO
BACKGROUND AND OBJECTIVE: Platelet-rich plasma is characterized by containing fundamental protein growth factors. Although many in vitro studies have documented the capability of platelet-rich plasma to induce the growth of osteoblasts or osteoblast-like cells, the effect of platelet-rich plasma on osteoclastogenesis has not yet been studied. The aim of the present study was to evaluate the effects of platelet-rich plasma and platelet-poor plasma on osteoclastogenesis with rat bone marrow cell culture. MATERIAL AND METHODS: Platelet-rich plasma and platelet-poor plasma were produced from the whole blood of rat. For cell culture, rat bone marrow cells were isolated from rat tibiae and then treated with 1,25alpha dihydroxy vitamin D(3) and with different concentrations of platelet-rich plasma or platelet-poor plasma. After 4 d of culture, rat bone marrow cells were stained with tartrate-resistant acid phosphatase (TRAP), and TRAP-positive cells that had more than three nuclei (TRAP-positive multinucleated cells) were counted as osteoclast-like cells. Osteoprotegerin, known as an osteoclastogenesis-related factor, cells was quantified using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Although platelet-poor plasma had no effect on the formation of TRAP-positive multinucleated cells, platelet-rich plasma decreased the number of TRAP-positive multinucleated cells in a dose-dependent manner. The amount of osteoprotegerin produced from rat bone marrow cells and from MC3T3-E1 cells was enhanced in platelet-rich plasma-treated groups. CONCLUSION: Under our experimental conditions, platelet-rich plasma decreased the formation of TRAP-positive multinucleated cells and increased the secretion of osteoprotegerin. This study suggests that platelet-rich plasma suppresses osteoclastogenesis, therefore inhibiting bone resorption. In addition we also demonstrated that platelet-rich plasma increased the secretion of osteoprotegerin, an inhibitor for osteoclast formation, thus suggesting that the enhancement of osteoprotegerin secretion induces this inhibitory effect.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoprotegerina/biossíntese , Plasma Rico em Plaquetas , Células 3T3 , Fosfatase Ácida/biossíntese , Animais , Células da Medula Óssea/metabolismo , Proliferação de Células , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Isoenzimas/biossíntese , Masculino , Camundongos , Osteoblastos/metabolismo , Osteoprotegerina/agonistas , Plasma , Ratos , Ratos Sprague-Dawley , Fosfatase Ácida Resistente a Tartarato , Vitamina D/análogos & derivados , Vitamina D/farmacologiaRESUMO
A quantum secret sharing (QSS) protocol based on a differential-phase-shift scheme is proposed, which quantum mechanically provides a full secret key to one party and partial keys to two other parties. A weak coherent pulse train is utilized instead of individual photons as in conventional schemes. Compared with previous QSS protocols, the present one features a simple setup, is suitable for fiber transmission, and offers the possibility for a high key creation rate. An experiment is also carried out to demonstrate the operation.
RESUMO
Transplantation studies have suggested that peripheral blood mononuclear cells contain precursors for osteoclasts. Thus we tested the capacity of peripheral blood monocytes to form osteoclasts in long-term culture. We have reported previously that mononuclear cells from feline, baboon, and human marrow form osteoclast-like cells in long term cultures. Further, the formation of these cells is increased in response to bone resorption stimulatory agents such as PTH, interleukin 1, and transforming growth factor alpha. We now report that these cells show characteristic cytoplasmic contraction with calcitonin and form resorption lacunae when cultured on sperm whale dentine. Thus, these bone marrow-derived multinucleated cells fulfill the functional criteria for osteoclasts. Although cultured peripheral blood monocytes can be induced to form multinucleated cells with 1,25-dihydroxyvitamin D3, these cells did not show similar responses to the osteotropic factors as multinucleated cells formed in the bone marrow cultures multinucleated cells. These results indicate that osteoclasts or cells closely related to osteoclasts form in long-term human bone marrow cultures. In contrast, few mononuclear cells in the peripheral blood appear capable of forming osteoclasts under the culture conditions used in these experiments.
Assuntos
Células da Medula Óssea , Osteoclastos/citologia , Medula Óssea/efeitos dos fármacos , Calcitonina/farmacologia , Calcitriol/farmacologia , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Humanos , Interleucina-1/farmacologia , Hormônio Paratireóideo/farmacologia , Fatores de Crescimento Transformadores/farmacologiaRESUMO
The HTLV-I provirus-encoded Tax protein induces NF-kappaB in Tax-transfected Jurkat T cells or HTLVL-I- infected T cells in vitro. Tax induction of NF-kappaB is presumed to be involved in proliferation and activation of primary leukemia cells in vivo. Recent studies have demonstrated that NF-kappaB activities in human T cells are mediated by at least four c-Rel-related DNA binding proteins - p50, p55, p75 and p85. We examined the significance of NF-kappaB induction in primary adult T cell leukemia cells and the induction kinetics of each of the four NF-kappaB species. Marked NF-kappaB activity was detected using an electrophoretic mobility shift assay (EMSA) in the primary cells of patients with acute disease, but little activity was noted in the cells of chronic patients. NF-kappaB activity was enhanced in a time-dependent manner in acute type cells cultured with mitogen-free medium; there was no induction of activity in chronic type cells. UV crosslinking demonstrated all four species of NFkappaB complex - high levels of p50 and lower levels of p55 and p75, in acute type cells; chronic type cells showed only the p50. As a control, normal resting T cells similarly showed only p50; control cells showed little change in activity when cultured without mitogenic stimulation, analogous to chronic type ATL. Northern blotting revealed enhancement of c-rel (encoding p85) and KBFI (encoding p50 and p55) expression in acute type cells during culture, while there was no significant enhancement of mRNAs in chronic type ATL cells or unstimulated normal T cells. Northern blotting also revealed that Tax is upregulated at the mRNA level in acute- but not chronic-type cells during culture. Expression of c-rel and KBF1 mRNAs in acute type cells appeared to be related to Tax mRNA expression. These results suggest that Tax is capable of inducing nuclear expression of all four NF-kappaB species in primary ATL cells of acute type patients, with marked effects on p55, p75, and p85. Tax induction of NF-kappaB species is regulated, at least in part, at a pretranslational level involving increases in c-rel and KBF1 mRNA.
Assuntos
Vírus Linfotrópico T Tipo 1 Humano/genética , Leucemia-Linfoma de Células T do Adulto/genética , NF-kappa B/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas/biossíntese , Ativação Transcricional , Doença Aguda , Idoso , Idoso de 80 Anos ou mais , Doença Crônica , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Progressão da Doença , Feminino , Regulação Leucêmica da Expressão Gênica , Regulação Viral da Expressão Gênica , Produtos do Gene tax/fisiologia , Genes pX , Humanos , Células Jurkat , Cinética , Leucemia-Linfoma de Células T do Adulto/patologia , Masculino , Pessoa de Meia-Idade , NF-kappa B/biossíntese , NF-kappa B/genética , Subunidade p50 de NF-kappa B , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-rel , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transfecção , Células Tumorais CultivadasRESUMO
CD21, which is expressed on B cells, is also expressed on human T lymphotropic virus-type I (HTLV-I)-infected T cell lines. CD21 also serves as a receptor of Epstein-Barr virus (EBV). We evaluated the mechanism of CD21 induction on HTLV-I-infected T cells and its clinical significance in the leukemogenesis of adult T cell leukemia (ATL). CD21 induction was detected at very low levels in T cell lines (Jurkat and CEM cells), and in non- or low-Tax-producing HTLV-I-infected T cell lines (Oh13T, S1T, and Su9T01 cells). In contrast, marked induction of CD21 was detected in high-Tax-producing HTLV-I-infected T cell lines (K3T, F6T, and MT-2). A Jurkat T cell clone stably transfected with tax-expressing cDNA expressed a significant amount of CD21 on the cell surface. These results strongly suggest that HTLV-I Tax induces CD21 on T cells. On two-color analysis, CD21 expression was detected in CD4+ T cells of the primary ATL cells from a subset of patients, suggesting that EBV infection may be associated with the leukemogenesis of ATL, at least in part. However, no genome of EBV was detected in the genomic DNA of six HTLV-I-infected T cell lines or the primary ATL cells separated from all patients, indicating the irrelevance of EBV infection to ATL leukemogenesis.
Assuntos
Produtos do Gene tax/metabolismo , Infecções por HTLV-I/virologia , Infecções por Herpesviridae/virologia , Herpesvirus Humano 4 , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Leucemia de Células T/virologia , Receptores de Complemento 3d/metabolismo , Linfócitos T/virologia , Infecções Tumorais por Vírus/virologia , DNA Viral/metabolismo , Humanos , Imunofenotipagem , Leucemia de Células T/etiologia , Linfonodos/imunologia , Linfonodos/virologia , Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
Vesnarinone is a positive inotropic agent used for treating congestive heart failure. We evaluated its ex vivo effects on myeloid leukemia cell lines and primary acute myelogenous leukemia cells. Vesnarinone inhibited the incorporation of radiolabeled thymidine by a myeloid cell line, HL60, in a dose-dependent manner at concentrations ranging from 0.1 to 30 microg/mL. A maximum 40% suppression was seen at a concentration of 10 microg/mL. Determination of viable cell counts by trypan blue dye exclusion method demonstrated vesnarinone to be cytocidal for HL60 cells. Vesnarinone induced DNA fragmentation as detected by electrophresis in HL60 cells after 72-hour culture; this effect was not inhibited by G-CSF. The apoptosis induced by vesnarinone was also detected by the in situ end-labeling method. Northern blot analysis showed a reduction of c-myc mRNA expression in HL60 cells by vesnarinone. However, immunostaining assay showed no change in the expression of Fas and Bcl-2 proteins. We next examined the effect of vesnarinone on primary myeloid leukemia cells derived from 10 patients: 3 cases of M1, 2 of M2, 3 of M3, 1 of M4, and 1 of M6, by the French-American-British classification. Vesnarinone inhibited the incorporation of thymidine in all cells, with a mean suppression of 58.1%. DNA electrophoresis showed induction of DNA fragmentation in cultured cells with vesnarinone for 72 hours in 8 of the 10 patients with primary leukemia. However, bone marrow mononuclear cells from healthy controls showed no growth suppression or DNA fragmentation in response to vesnarinone. These results suggest that vesnarinone may be useful in treating myeloid leukemia.
Assuntos
Antineoplásicos/toxicidade , Apoptose/fisiologia , Células HL-60/efeitos dos fármacos , Leucemia Mieloide Aguda/patologia , Quinolinas/toxicidade , Adulto , Idoso , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Fragmentação do DNA , Relação Dose-Resposta a Droga , Feminino , Humanos , Leucemia Mieloide Aguda/classificação , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-bcl-2/análise , Pirazinas , Células Tumorais Cultivadas , Receptor fas/análiseRESUMO
It is unclear how mechanical stress influences bone cells. Mechanical stress causes fluid shear stress (FSS) in the bone. Osteoblast lineage cells are thought to sense FSS and regulate bone remodeling. We therefore investigated the effects of FSS on human osteoblast-like osteosarcoma cells: SaOS-2 cells in vitro. The conditioned medium of the SaOS-2 cells after 24 h of FSS (24 h-FSS CM) showed such osteoclastic phenotype inductions as significantly increasing the number of tartrate-resistant acid phosphatase (TRAP) positive multinuclear cells in rat bone marrow cells and TRAP-positive cells in human preosteoclastic cells: FLG 29.1 cells. An enzyme-linked immunosorbent assay showed interleukin-11 (IL-11) protein to increase 7-fold in the 24 h-FSS CM. A Northern analysis showed that IL-11 mRNA increased 4-fold in the SaOS-2 cells after 6 h-FSS; however, no IL-6 mRNA expression was detected. Furthermore, the anti-human IL-11 antibody significantly neutralized the osteoclastic phenotype induction of the 24 h-FSS CM. The IL-11 mRNA up-regulation in SaOS-2 cells by the 6 h-FSS was not inhibited by the anti-human transforming growth factor-beta1 antibody, but it was significantly inhibited by indomethacin. An enzymeimmunoassay showed prostaglandin E2 to increase 7-fold in the 1 h-FSS CM. These findings thus suggest that FSS induces osteoblasts to produce IL-11 (mediated by prostaglandins) and thus stimulates bone remodeling.
Assuntos
Remodelação Óssea/efeitos dos fármacos , Interleucina-11/biossíntese , Osteoblastos/metabolismo , Fosfatase Ácida , Animais , Anticorpos/farmacologia , Meios de Cultivo Condicionados , Dinoprostona/metabolismo , Proteínas Filagrinas , Regulação da Expressão Gênica , Histocitoquímica , Humanos , Indometacina/farmacologia , Interleucina-11/genética , Interleucina-11/imunologia , Isoenzimas , Osteoblastos/efeitos dos fármacos , RNA Mensageiro/biossíntese , Ratos , Estresse Mecânico , Fosfatase Ácida Resistente a Tartarato , Fator de Crescimento Transformador beta/farmacologia , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacosRESUMO
Osteoclast precursors have not been well characterized because there are no known markers that can detect them. We have used osteoclast-like cells formed in vitro to develop a panel of specific antibodies that react with mature osteoclasts, osteoclast precursors, and other cells in the osteoclast lineage. Monoclonal antibody Kn22 reacted strongly with osteoclast-like multinucleated cells formed in long term marrow cultures and reacted very strongly with freshly isolated bone-derived baboon osteoclasts. Using immune cell panning, Kn22 enriched precursors for osteoclasts. The majority of multinucleated cells (71%) formed from fresh marrow mononuclear cells adherent to Kn22 strongly reacted with a monoclonal antibody that recognizes mature osteoclasts (23c6) and responded appropriately to calcitonin. In contrast, only 23% of multinucleated cells formed from marrow mononuclear cells that were not bound by Kn22 formed osteoclast-like cells. The majority (77%) of these multinucleated cells did not strongly react with the osteoclast-specific monoclonal antibody 23c6 or respond to calcitonin. Thus, we have developed a panel of monoclonal antibodies that recognize cells in the osteoclast lineage. One of these antibodies, Kn22, is unique in that it identifies an osteoclast precursor. The 50K antigen detected by Kn22 appears to be a membrane protein present on osteoclast precursors and osteoclasts that has not been previously identified.
Assuntos
Anticorpos Monoclonais/imunologia , Células da Medula Óssea , Osteoclastos/imunologia , Animais , Especificidade de Anticorpos , Medula Óssea/imunologia , Linhagem Celular , Células Cultivadas , Reações Cruzadas , Feminino , Humanos , Hibridomas/citologia , Immunoblotting , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Leucócitos Mononucleares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Osteoclastos/citologia , Células Tumorais CultivadasRESUMO
The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) at 10(-9) M stimulated the formation of osteoclast-like multinucleated cells (MNCs) in the presence of 1 alpha,25-dihydroxyvitamin D3 in rat bone marrow cultures. However, at 10(-7) M, it clearly inhibited 1 alpha,25-dihydroxyvitamin D3-dependent osteoclast-like MNC formation at 6 days of culture. In cultures treated with 10(-7) M TPA, numerous MNCs that lack the marker enzyme tartrate-resistant acid phosphatase (TRAP) were formed. These TRAP-negative MNCs had neither receptors for calcitonin nor dentine-resorbing activity. The reactivity of the cells against antirat macrophage antibodies was completely different from that of authentic osteoclasts. These data suggest that TRAP-negative MNCs formed in the presence of 10(-7) M TPA are macrophage polykaryons. Time-course studies showed that 10(-7) M TPA stimulated osteoclast-like MNC formation at 4 days of culture, but these osteoclast-like MNCs were converted to TRAP-negative MNCs. Furthermore, 1-(5-isoquinolinyl-sulfonyl)2-methylpiperazine (H-7), a protein kinase-C inhibitor, inhibited osteoclast-like MNC formation in a dose-dependent fashion. These results suggest that activation of protein kinase-C may play a role in osteoclast differentiation.
Assuntos
Células da Medula Óssea , Diferenciação Celular/efeitos dos fármacos , Macrófagos/citologia , Osteoclastos/citologia , Acetato de Tetradecanoilforbol/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Fosfatase Ácida/metabolismo , Animais , Biomarcadores , Medula Óssea/efeitos dos fármacos , Reabsorção Óssea , Calcitonina/metabolismo , Calcitriol/farmacologia , Carcinógenos/farmacologia , Células Cultivadas , Isoquinolinas/farmacologia , Cinética , Macrófagos/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Modelos Biológicos , Osteoclastos/efeitos dos fármacos , Osteoclastos/ultraestrutura , Ésteres de Forbol/farmacologia , Piperazinas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Endogâmicos , Receptores da Calcitonina , Receptores de Superfície Celular/metabolismoRESUMO
Differentiation of osteoclasts is defined by the transcription factors expressed in response to bone microenvironments. In this work, we examined the effects of an expressional blockage of Egr-1 and/or WT1 on the differentiation of osteoclasts using specific antisense oligodeoxynucleotides (ODN). In a culture system forming preosteoclast-like cells (POC) from rat bone marrow cells depleted of marrow stromal cells, POC formation was markedly stimulated by the addition of Egr-1 antisense ODN compared to that in cultures in which sense ODN was added, whereas Egr-1 antisense ODN inhibited the formation of macrophage-like cells. The formation of multinucleated osteoclast-like cells was also stimulated by the addition of Egr-1 antisense ODN in whole bone marrow cultures. In contrast, WT1 antisense ODN did not affect POC formation induced by the treatment with Egr-1 antisense ODN; however, WT1 antisense ODN dramatically suppressed the formation of osteoclast-like multinucleated cells induced by the blockage of Egr-1 expression using Egr-1 antisense ODN. These data suggest that Egr-1 acts as the suppressor, not as the inducer, in osteoclastogenesis. The findings also suggested that WT1 could be involved in the multinucleation step of osteoclastogenesis, at least when Egr-1 expression was blocked.
Assuntos
Células da Medula Óssea , Proteínas de Ligação a DNA/química , Proteínas Imediatamente Precoces , Oligonucleotídeos Antissenso/farmacologia , Osteoclastos/citologia , Proteínas Oncogênicas de Retroviridae/química , Fatores de Transcrição/química , Dedos de Zinco/genética , Animais , Sequência de Bases , Medula Óssea/química , Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , DNA/análise , DNA/química , DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Proteína 1 de Resposta de Crescimento Precoce , Masculino , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/genética , Osteoclastos/química , Osteoclastos/fisiologia , Ratos , Ratos Sprague-Dawley , Proteínas Oncogênicas de Retroviridae/genética , Proteínas Oncogênicas de Retroviridae/fisiologia , Células-Tronco/química , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Proteínas WT1RESUMO
We investigated the effect of glucocorticoid on the generation of osteoclasts. In mouse bone marrow culture systems, dexamethasone, a synthetic glucocorticoid analog, enhanced osteoclast-like cell formation induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in a dose-dependent manner. Conversely, dexamethasone inhibited the endogenous production of granulocyte-macrophage colony-stimulating factor (GM-CSF) in bone marrow cultures. GM-CSF, when added exogenously, suppressed not only the osteoclast-like cell formation induced by 1,25-(OH)2D3, but also the stimulatory effect of dexamethasone, and addition of anti-GM-CSF neutralizing antibody to the cultures significantly increased the osteoclast-like cell formation induced by 1,25-(OH)2D3. These observations suggest that dexamethasone directly affects bone marrow cells and enhances osteoclast generation by inhibiting the endogenous production of GM-CSF, which may function as a negative regulator of osteoclast formation.
Assuntos
Medula Óssea/metabolismo , Dexametasona/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Osteoclastos/efeitos dos fármacos , Fosfatase Ácida/metabolismo , Animais , Calcitriol/farmacologia , Técnicas de Cultura , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Osteoclastos/metabolismoRESUMO
Lipopolysaccharide (LPS) is a potent bone resorbing factor. We investigated the effect of LPS on osteoclast formation in three types of cultures. LPS inhibited osteoclast formation induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], in a dose-dependent manner, in cultures of whole bone marrow cells without dexamethasone. LPS increased the amount of granulocyte-macrophage colony stimulating factor (GM-CSF) in the culture supernatant, and anti-GM-CSF antiserum almost abolished the inhibition of osteoclast formation by LPS, thereby indicating that GM-CSF generated by treatment with LPS may be responsible for the inhibition of osteoclast formation. In cultures with dexamethasone, the amount of GM-CSF was decreased to one-third of that with 1,25(OH)2D3 alone and was not changed by treatment with LPS. In this culture system, LPS enhanced osteoclast formation. In the coculture system of nonadherent bone marrow cells and a stromal cell line in the presence of 1,25(OH)2D3 and dexamethasone, where no detectable GM-CSF was present in the supernatant, LPS markedly enhanced osteoclast formation, whereas exogenously added GM-CSF (100 pg/ml) almost completely inhibited osteoclast formation. LPS stimulated pit formation on dentin slices by the osteoclast-like cells formed by in vitro culture system.
Assuntos
Células da Medula Óssea , Diferenciação Celular/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Lipopolissacarídeos/farmacologia , Osteoclastos/citologia , Animais , Medula Óssea/efeitos dos fármacos , Reabsorção Óssea , Calcitriol/farmacologia , Adesão Celular , Separação Celular , Células Cultivadas , Dexametasona/farmacologia , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C3H , Osteoclastos/efeitos dos fármacos , Proteínas Recombinantes/farmacologiaRESUMO
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.
Assuntos
Molécula 1 de Adesão Intercelular/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Osteoclastos/citologia , Animais , Anticorpos Monoclonais , Células da Medula Óssea/citologia , Comunicação Celular/fisiologia , Divisão Celular/fisiologia , Imuno-Histoquímica/métodos , Masculino , Monócitos/citologia , Osteoclastos/fisiologia , Fenótipo , Ratos , Ratos Sprague-Dawley , Células-Tronco/fisiologiaRESUMO
Rat bone marrow cultures containing 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] formed multinucleated cells (MNCs) that had many characteristics of osteoclasts. These MNCs, which have a tartrate-resistant acid phosphatase (TRAP) activity, could be classified into two morphological types: one type had smooth cellular margins (smooth-margined MNCs) and the other type had irregular spike-like margins (stellate MNCs). When bone marrow cells depleted of authentic osteoclasts were seeded and cultured on dentine slices, only low numbers of resorption lacunae could be detected. However, when preformed MNCs were detached by trypsinization and replated on dentine slices, numerous resorption lacunae were observed by scanning electron microscopy on these slices. Formation of lacunae occurred reproducibly during the five to ten days of culture. We also examined the effect of retinoic acid on TRAP-positive MNC formation in this bone marrow culture system. Although RA inhibited total TRAP-positive MNC formation, it increased the ratio of stellate MNCs to smooth-margined MNC, suggesting that RA may have the ability to regulate the formation of active osteoclasts.
Assuntos
Dentina/metabolismo , Células Gigantes/metabolismo , Osteoclastos/metabolismo , Animais , Medula Óssea , Reabsorção Óssea/metabolismo , Calcitriol/farmacologia , Células Cultivadas , Células Gigantes/efeitos dos fármacos , Células Gigantes/ultraestrutura , Masculino , Microscopia Eletrônica de Varredura , Osteoclastos/efeitos dos fármacos , Osteoclastos/ultraestrutura , Ratos , Ratos Endogâmicos , Tretinoína/farmacologia , Tripsina/metabolismoRESUMO
A member of the tumor necrosis factor (TNF) family, receptor activator of nuclear factor kappaB ligand (RANKL; also known as ODF, OPGL, and TRANCE), plays critical roles in osteoclast differentiation and activation in the presence of macrophage colony-stimulating factor (M-CSF). Recently, TNF-alpha has also been shown to induce the formation of multinucleated osteoclast-like cells (MNCs) in the presence of M-CSF from mouse macrophages. We demonstrated that mononuclear preosteoclast-like cells (POCs) were formed in the presence of conditioned medium of osteoblastic cells in a rat bone marrow culture depleted of stromal cells. Using this culture system, in this study we examined whether TNF-alpha affects differentiation into POCs from hematopoietic progenitor cells. Human TNF-alpha (hTNF-alpha) markedly stimulated the formation of POCs. Moreover, a concentration as low as 0.005 ng/mL of hTNF-alpha increased the level of mRNA for calcitonin receptor (CTR) and cathepsin-K of POCs. The POCs induced by hTNF-alpha formed MNCs, which showed dentine-resorbing activity after coculture with primary osteoblasts. Stimulation was observed after 24 h of treatment with hTNF-alpha only on day 1 or day 2 of the culture. After 24 h of hTNF-alpha treatment, expression of the receptor activator of nuclear factor kappaB (RANK) mRNA was markedly increased. The addition of soluble RANKL (sRANKL) to the preformed POCs efficiently induced MNCs. Interestingly, treatment of bone marrow cells with hTNF-alpha and sRANKL synergistically augmented the formation of MNCs. This formation was abolished by the addition of human osteoprotegerin (hOPG). These results suggest that cooperation of TNF-alpha and RANKL is important for osteoclastogenesis.
Assuntos
Remodelação Óssea/fisiologia , Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Interações Medicamentosas/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Glicoproteínas de Membrana/metabolismo , Osteoclastos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Remodelação Óssea/efeitos dos fármacos , Osso e Ossos/citologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/farmacologia , Catepsinas/genética , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Glicoproteínas/metabolismo , Glicoproteínas/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/farmacologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoprotegerina , Ligante RANK , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Ativador de Fator Nuclear kappa-B , Receptores da Calcitonina/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Fator de Necrose Tumoral , Células Estromais/citologia , Células Estromais/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
We studied the ultrastructural distribution of fibronectin in the smooth muscle layer of mouse small intestine with affinity-purified antibodies using the immunogold technique. Fibronectin was present over the pericellular area extending from the cell membrane to the extracellular matrix beyond the basal lamina. Distribution of the glycoprotein over the pericellular area was heterogeneous, i.e., it was localized more abundantly in the narrow space between smooth muscle cells, the gaps having a width of 60-80 nm where the two dense bands in adjacent cells matched each other. Such localization suggests that fibronectin contributes to cell adhesion. Within the basement membrane, gold label was localized both in lamina lucida and lamina densa, more densely in the latter than in the former. Fibronectin was also co-distributed with collagen fibers in the extracellular matrix. Within smooth muscle cells, gold particles were observed on rough endoplasmic reticulum and secretory vesicle-like structures. These results suggest that smooth muscle cells synthesize fibronectin and secrete it as a component of the basal lamina and extracellular matrix.
Assuntos
Fibronectinas/análise , Músculo Liso/análise , Animais , Ouro , Histocitoquímica , Técnicas Imunológicas , Intestino Delgado/análise , Intestino Delgado/ultraestrutura , Camundongos , Microscopia Eletrônica , Músculo Liso/ultraestrutura , Proteína Estafilocócica ARESUMO
Although calcitonin has been clinically utilized as a primary treatment for several metabolic bone diseases, its inhibitory effects against osteoclastic function diminish after several days owing to the calcitonin 'escape phenomenon'. We have previously found a unique cell-surface antigen (Kat1-antigen) expressed on rat osteoclasts. Here we show evidence that, in the presence of calcitonin, the Kat1-antigen is involved in osteoclastogenesis. Treatment of bone marrow cultures for forming osteoclast-like cells with anti-Kat1-antigen monoclonal antibody (mAb Kat1) provoked a marked stimulation of osteoclast-like cell formation only in the presence of calcitonin but not in its absence. Osteoclastogenesis stimulated by the receptor activator of nuclear factor kappa B (NF-kappaB) ligand/osteoclast differentiation factor was further augmented by mAb Kat1 in the presence of calcitonin. Furthermore, even in the presence of the osteoprotegerin/osteoclast inhibitory factor, mAb Kat1 induced osteoclast-like cell formation. Our current data suggest that the Kat1-antigen is a molecule that is distinct from receptor activator of NF-kappaB. The presence of the unique Kat1-antigen on cells in the osteoclast lineage appears to contribute to the fine regulation of osteoclastogenesis in vivo. Expression of this cell-surface molecule in cells in the osteoclast lineage may partly explain the mechanism responsible for the escape phenomenon.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Superfície/metabolismo , Calcitonina/metabolismo , Osteoclastos/citologia , Análise de Variância , Animais , Antígenos de Superfície/imunologia , Biomarcadores/análise , Western Blotting , Células da Medula Óssea , Proteínas de Transporte/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Glicoproteínas de Membrana/farmacologia , Osteoclastos/efeitos dos fármacos , Ligante RANK , Ratos , Ratos Sprague-DawleyRESUMO
The binding of interleukin-2 (IL-2) to its receptor on normal T cells induces nuclear expression of nuclear factor kappaB (NF-kappaB), activation of the IL-2 receptor (IL-2R) alpha chain gene, and cell proliferation. In the present study, the role of IL-2R signaling in the growth of CD8+ T cell prolymphocytic leukemia (T-PLL) cells has been investigated. Flow cytometry revealed that primary leukemia cells from a patient with CD8+ T-PLL expressed IL-2Ralpha and beta chains, and the cells showed a proliferative response and an increase in IL-2Ralpha expression on culture with exogeneous IL-2. Northern blot analysis failed to detect IL-2 mRNA, suggesting that IL-2 may act in a paracrine manner in vivo. Electrophoretic mobility-shift assays revealed that recombinant IL-2 increased NF-kappaB binding activity in nuclear extracts of the leukemia cells, and Northern blot analysis showed that IL-2 increased the abundance of mRNAs encoding the NF-kappaB components c-Rel and KBF1 in these cells. IL-2 binding analysis demonstrated that IL-2 markedly increased the number of low affinity IL-2Rs on the leukemia cells, without an effect on the number of high-affinity IL-2Rs. These results show that IL-2 is capable of inducing the nuclear expression of NF-kappaB in primary CD8+ T-PLL cells, and that this effect is mediated, at least in part, at a pretranslational level.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Interleucina-2/farmacologia , Leucemia Prolinfocítica/metabolismo , Leucemia Prolinfocítica/patologia , Leucemia de Células T/metabolismo , Leucemia de Células T/patologia , NF-kappa B/biossíntese , Receptores de Interleucina-2/biossíntese , Northern Blotting , Linfócitos T CD8-Positivos/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Citometria de Fluxo , Humanos , Interleucina-2/biossíntese , Interleucina-2/metabolismo , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-rel , RNA Mensageiro/metabolismo , Receptores de Interleucina-2/metabolismo , Células Tumorais CultivadasRESUMO
We report a case of a 70-year-old man with a hybrid leukemia treated successfully with granulocyte-colony stimulating factor (G-CSF) combined with a cytocine arabinoside regimen through the induction of differentiation of leukemic cells into monocytoid cells resulting in apoptosis. The leukemic cells demonstrated a TCR-gamma rearrangement, and expressed CD2, CD7, CD33 and G-CSF receptors but not CD11b on the cell surface nor non-specific esterase in cytoplasm. Several days following the administration of G-CSF, the cells with monocytoid characteristics such as CD11b and cytoplasmic non-specific esterase appeared in the peripheral blood replacing the blastic cells. The cells were shown to be derived from the same clone of the leukemic cell because of the identical TCR-gamma gene rearrangement. The short-term culture of leukemic cells with G-CSF induced the differentiation into a monocyte lineage, resulting in apoptosis. Although there is no denying the possibility that cytosine arabinoside is partly responsible, our results strongly suggest that G-CSF plays the main role in differentiation of leukemic cells into a monocyte lineage inducing apoptosis in vivo in this patient.