RESUMEN
The treatment of autoimmune disorders has been revolutionised by the introduction of biologics such as anti-tumour necrosis factor (anti-TNF). Although in rheumatoid arthritis patients a bone sparing effect of anti-TNF has been shown, the mechanism is not fully understood. Anti-TNF molecules block tumour necrosis factor (TNF) and prevent signalling via both TNF receptor 1 (TNFR1; p55) and TNF receptor 2 (TNFR2; p75). However, signalling via TNFR2 is reported to have protective effects in a number of cell and organ systems. Hence we set out to investigate if pharmacological inhibition of TNFR1 had differential effects compared to pan-TNF inhibition in both an in vitro cell-based model of human osteoclast activity and an in vivo mouse model of lipopolysaccharide (LPS)-induced osteolysis. For the in vitro experiments the anti-human TNFR1 domain antibody (dAb) DMS5541 was used, whereas for the in vivo mouse experiments the anti-mouse TNFR1 dAb DMS5540 was used. We show that selective blocking of TNFR1 signalling reduced osteoclast formation in the presence of TNF. Subcutaneous LPS injection over the calvaria leads to the development of osteolytic lesions within days due to inflammation driven osteoclast formation. In this model, murine TNFR2 genetically fused with mouse IgG1 Fc domain (mTNFR2.Fc), an anti-TNF, did not protect from bone loss in contrast to anti-TNFR1, which significantly reduced lesion development, inflammatory infiltrate, and osteoclast number and size. These results support further exploring the use of TNFR1-selective inhibition in inflammatory bone loss disorders such as osteomyelitis and peri-prosthetic aseptic loosening.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Osteoclastos/inmunología , Osteólisis/inmunología , Osteólisis/patología , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Animales , Recuento de Células , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Lipopolisacáridos , Ratones , Ratones Endogámicos C57BL , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Osteólisis/terapia , Resultado del TratamientoRESUMEN
IL-18 was identified due to its ability to induce interferon-gamma (IFNgamma) production by T cells. It is a pleiotropic factor that shares structural features with IL-1 and functional activities with IL-12. IL-18 has a role in T cell development, where it has been demonstrated to act cooperatively with IL-12 to regulate IFNgamma. In bone, IL-18 is mainly produced by macrophages, but is also expressed by osteoblasts and inhibits osteoclast formation through granulocyte-macrophage colony-stimulating factor (GM-CSF) and not IFNgamma production by T cells. We have investigated the effects of IL-18 on mature osteoclast activity and for potential actions on osteoblasts or chondrocytes. The effects of IL-18 on mature osteoclast activity were determined using two assays: isolated mature osteoclast cell culture and neonatal murine calvarial organ culture. IL-18 did not affect bone resorption in either assay system. The actions of IL-18 on osteogenic cells (primary cell cultures of fetal rat and neonatal mouse osteoblasts, as well as neonatal mouse calvarial organ culture) and primary chondrocytes (canine) were assessed by proliferation assays (quantification of cell numbers and thymidine incorporation). In each assay system, IL-18 acted as a mitogen to the osteogenic and chondrogenic cells. Since IL-18 signal transduction may involve IFNgamma or GM-CSF, we assessed their involvement in the IL-18 response. IL-18 did not induce IFNgamma production by primary osteoblasts, but, of greater significance, IFNgamma had the opposing action to IL-18 in that it inhibited the primary osteoblast cell proliferation. Although IL-18 rapidly induced GM-CSF production by primary osteoblasts, IL-18 was still mitogenic in osteoblast preparations established from GM-CSF-deficient mice. Combined, these studies indicate that IL-18 may have an autocrine/paracrine mitogen role for both osteogenic and chondrogenic cells, independent of the production of IFNgamma or GM-CSF.
Asunto(s)
Condrocitos/citología , Interleucina-18/farmacología , Mitógenos/farmacología , Osteoclastos/citología , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Interferón gamma/genética , Ratones , Técnicas de Cultivo de Órganos , Osteoclastos/efectos de los fármacos , Osteoclastos/fisiología , Ratas , Ratas WistarRESUMEN
There is increasing evidence that NF-kappaB is a major, if not the major transcription factor regulating inflammation and immunity. While this implies that blocking NF-kappaB might be therapeutically beneficial, it raises clear questions regarding the balance between efficacy and safety. In this brief review we discuss the effects of NF-kappaB blockade in rheumatoid arthritis, inflammation and immunity, and consider possible therapeutic targets within the NF-kappaB family.
Asunto(s)
Artritis Reumatoide/terapia , FN-kappa B/antagonistas & inhibidores , Adenoviridae/genética , Presentación de Antígeno , Artritis Reumatoide/inmunología , Técnicas de Cultivo de Célula , Células Dendríticas/inmunología , Vectores Genéticos , Humanos , Proteínas I-kappa B/genética , Inmunidad Celular , Prueba de Cultivo Mixto de Linfocitos , FN-kappa B/fisiología , Membrana Sinovial/inmunologíaRESUMEN
IL-12, like IL-18, was shown to potently inhibit osteoclast formation in cultures of cocultures of murine osteoblast and spleen cells, as well as in adult spleen cells treated with M-CSF and receptor activator of NF-kappaB ligand (RANKL). Neither IL-12 nor IL-18 was able to inhibit RANKL-induced osteoclast formation in cultured RAW264.7 cells, demonstrating that IL-12, like IL-18, was unable to act directly on osteoclastic precursors. IL-12, like IL-18, was found to act by T cells, since depletion of T cells from the adult spleen cell cultures ablated the inhibitory action of IL-12 and addition of either CD4 or CD8 T cells from C57BL/6 mice to RANKL-stimulated RAW264.7 cultures permitted IL-12 or IL-18 to be inhibitory. Additionally, IL-12 was still able to inhibit osteoclast formation in cocultures with osteoblasts and spleen cells from either GM-CSF R(-/-) mice or IFN-gamma R(-/-) mice, indicating that neither GM-CSF nor IFN-gamma was mediating osteoclast inhibition in these cultures. Combined, IL-18 and IL-12 synergistically inhibited osteoclast formation at concentrations 20- to 1000-fold less, respectively, than when added individually. A candidate inhibitor could not be demonstrated using neutralizing Abs to IL-4, IL-10, or IL-13 or from mRNA expression profiles among known cytokine inhibitors of osteoclastogenesis in response to IL-12 and IL-18 treatment, although the unknown inhibitory molecule was determined to be secreted from T cells.
Asunto(s)
Inhibidores de Crecimiento/fisiología , Interleucina-12/fisiología , Interleucina-18/fisiología , Osteoclastos/citología , Osteoclastos/inmunología , Animales , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/inmunología , Línea Celular , Células Cultivadas , Técnicas de Cocultivo , Regulación hacia Abajo/inmunología , Combinación de Medicamentos , Sinergismo Farmacológico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Inhibidores de Crecimiento/metabolismo , Interferón gamma/metabolismo , Interferón gamma/fisiología , Interleucina-12/metabolismo , Interleucina-18/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/deficiencia , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Receptor de Interferón gammaRESUMEN
Osteoclast formation in bone is supported by osteoblasts expressing receptor activator of NF-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) expression. Numerous osteotropic factors regulate expression levels of RANKL and the RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts, thereby affecting osteoclast differentiation. However, not only in RANKL widely expressed in soft tissues, but osteoclasts have been noted in extraskeletal lesions. We found that cultured skin fibroblastic cells express RANKL, M-CSF, and OPG messenger (mRNA). Stimulation by 1 alpha,25 dihydroxyvitamin D3 [1,25(OH)2D3] plus dexamethasone (Dex) augmented RANKL and diminished OPG mRNA expression in fibroblastic cells and caused the formation of numerous osteoclasts in cocultures of skin fibroblastic cells with hemopoietic cells or monocytes. The osteoclasts thus formed expressed tartrate-resistant acid phosphatase (TRAP) and calcitonin (CT) receptors and formed resorption pits in cortical bone. Osteoclast formation also was stimulated (in the presence of Dex) by prostaglandin E2 (PGE2), interleukin-11 (IL-11), IL-1, tumor necrosis factor-alpha (TNF-alpha), and parathyroid hormone-related protein (PTHrP), factors which also stimulate osteoclast formation supported by osteoblasts. In addition, granulocyte-macrophage-CSF (GM-CSF), transforming growth factor-beta (TGF-beta), and OPG inhibited osteoclast formation in skin fibroblastic cell-hemopoietic cell cocultures; CT reduced only osteoclast nuclearity. Fibroblastic stromal cells from other tissues (lung, respiratory diaphragm, spleen, and tumor) also supported osteoclast formation. Thus, RANKL-positive fibroblastic cells in extraskeletal tissues can support osteoclastogenesis if osteolytic factors and osteoclast precursors are present. Such mesenchymally derived cells may play a role in pathological osteolysis and may be involved in osteoclast formation in extraskeletal tissues.
Asunto(s)
Proteínas Portadoras/genética , Fibroblastos/metabolismo , Glicoproteínas de Membrana/genética , FN-kappa B/metabolismo , Osteoclastos/fisiología , Células del Estroma/metabolismo , Animales , Calcitriol/farmacología , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Dexametasona/farmacología , Expresión Génica , Glicoproteínas/genética , Ligandos , Factor Estimulante de Colonias de Macrófagos/genética , Ratones , Osteoblastos/citología , Osteoclastos/citología , Osteoclastos/metabolismo , Osteoprotegerina , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Receptores Citoplasmáticos y Nucleares/genética , Receptores del Factor de Necrosis Tumoral , Células del Estroma/efectos de los fármacosRESUMEN
Osteoblastic stromal cells are capable of supporting osteoclast formation from hematopoietic precursors in the presence of osteotropic factors such as 1alpha,25(OH)(2)D(3), PTH, and IL-11. Osteoblastic stromal cells produce receptor activator of NF-kappaB ligand (RANKL), a type II membrane protein of the TNF ligand family, in response to these agents. Activated T lymphocytes also produce RANKL; however, the ability of this cell type to support osteoclast formation in vitro is unknown. Human PBMC-derived T cells, extracted using alphaCD3-coated magnetic beads, were cocultured with adherent murine spleen cells in the presence of Con A and a panel of cytokines. In the presence of Con A, bona fide osteoclasts were formed in vitro with activated T cells: IL-1alpha and TGFbeta further enhanced osteoclast numbers. PBMC-derived lymphocytes showed an increase in the mRNA expression of RANKL within 24 h of treatment with the same agents that were used to induce osteoclast formation. In synovial tissue sections with lymphoid infiltrates from RA patients, the expression of RANKL was demonstrated in CD3(+) T cells. The ability of activated T lymphocytes to support osteoclast formation may provide a mechanism for the potentiation of osteoclast formation and bone resorption in disease states such as rheumatoid arthritis.
Asunto(s)
Proteínas Portadoras/genética , Células Madre Hematopoyéticas/citología , Activación de Linfocitos , Glicoproteínas de Membrana/genética , Osteoclastos/citología , Linfocitos T/fisiología , Anciano , Animales , Animales Recién Nacidos , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Proteínas Portadoras/biosíntesis , Diferenciación Celular , Técnicas de Cocultivo , Concanavalina A/farmacología , Femenino , Regulación de la Expresión Génica , Humanos , Interleucina-2/farmacología , Masculino , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Ligando RANK , ARN Mensajero/genética , Receptor Activador del Factor Nuclear kappa-B , Células del Estroma/citología , Membrana Sinovial/inmunología , Membrana Sinovial/patología , Linfocitos T/inmunología , Linfocitos T/patología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/inmunología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
RANKL (receptor activator of NFkappaB ligand) is a membrane-associated osteoblastic molecule, and along with macrophage-colony-stimulating factor, is crucial for osteoclast formation. RANKL is known to be strongly expressed in osteoblasts and lymphoid tissues. We have sought to determine the skeletal and extraskeletal sites of production of RANKL mRNA and protein using the techniques of in situ hybridization and immunohistochemistry. Expression of RANKL mRNA and protein were determined in the developmental progression of endochondral bone formation in mouse, intramembranous bone formation in a rabbit model (mRNA only), in human giant cell tumors of bone, and at extraskeletal sites in the mouse. RANKL mRNA was expressed in prehypertrophic and hypertrophic chondrocytes at day E15 embryonic mouse long bone, and its expression was maintained at these sites throughout development. In newborn and adult mice, high levels of RANKL mRNA were expressed in mesenchymal cells of the periosteum and in mature osteoblasts, while megakaryocytes within the marrow microenvironment expressed RANKL mRNA from 1 week of age. Immunohistochemical analysis revealed a similar localization pattern of RANKL protein at the sites described. In the intramembranous bone formation model, RANKL mRNA was expressed in mesenchymal cells and in actively synthesizing osteoblasts, but not in flattened lining osteoblasts or late osteocytes. Expression of RANKL mRNA and protein in osteoclasts was variable with those within resorption lacunae showing the strongest signal/staining. Likewise, expression varied in osteoclasts from giant cell tumor of bone with a minority of tartrate-resistant acid phosphatase-positive multinucleated cells having no detectable RANKL mRNA or protein. In extraskeletal tissues, RANKL mRNA and protein were detected in the brain, heart, kidney, skeletal muscle, and skin throughout mouse development, suggesting the possibility of several other functions of the molecule. RANKL was also developmentally regulated, as evidenced by its expression in the intestine, liver, and lung at E15 and newborn mouse but not in the adult.
Asunto(s)
Huesos/metabolismo , Proteínas Portadoras/biosíntesis , Glicoproteínas de Membrana/biosíntesis , ARN Mensajero/metabolismo , Secuencia de Aminoácidos , Animales , Desarrollo Óseo/fisiología , Médula Ósea/metabolismo , Huesos/embriología , Proteínas Portadoras/genética , Femenino , Tumor Óseo de Células Gigantes/patología , Células Gigantes/metabolismo , Células Gigantes/patología , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Osteoblastos/metabolismo , Osteoclastos/citología , Osteoclastos/metabolismo , Embarazo , Ligando RANK , ARN Mensajero/biosíntesis , Conejos , Receptor Activador del Factor Nuclear kappa-B , Especificidad de la EspecieRESUMEN
Breast cancers commonly cause osteolytic metastases in bone, a process that is dependent upon osteoclast-mediated bone resorption. Recently the osteoclast differentiation factor (ODF), better termed RANKL (receptor activator of NF-kappaB ligand), expressed by osteoblasts has been cloned as well as its cognate signaling receptor, receptor activator of NFkappaB (RANK), and a secreted decoy receptor osteoprotegerin (OPG) that limits RANKL's biological action. We determined that the breast cancer cell lines MDA-MB-231, MCF-7, and T47D as well as primary breast cancers do not express RANKL but express OPG and RANK. MCF-7, MDA-MB-231, and T47D cells did not act as surrogate osteoblasts to support osteoclast formation in coculture experiments, a result consistent with the fact that they do not express RANKL. When MCF-7 cells overexpressing PTH-related protein (PTHrP) were added to cocultures of murine osteoblasts and hematopoietic cells, osteoclast formation resulted without the addition of any osteotropic agents; cocultures with MCF-7 or MCF-7 cells transfected with pcDNAIneo required exogenous agents for osteoclast formation. When MCF-7 cells overexpressing PTHrP were cultured with murine osteoblasts, osteoblastic RANKL messenger RNA (mRNA) levels were enhanced and osteoblastic OPG mRNA levels diminished; MCF-7 parental cells had no effect on RANKL or OPG mRNA levels when cultured with osteoblastic cells. Using a murine model of breast cancer metastasis to bone, we established that MCF-7 cells that overexpress PTHrP caused significantly more bone metastases, which were associated with increased osteoclast formation, elevated plasma PTHrP concentrations and hypercalcaemia compared with parental or empty vector controls.
Asunto(s)
Neoplasias Mamarias Animales/fisiopatología , Osteoblastos/fisiología , Osteoclastos/citología , Receptores Citoplasmáticos y Nucleares , Animales , Proteínas Portadoras/genética , División Celular/fisiología , Técnicas de Cocultivo , Femenino , Glicoproteínas/genética , Masculino , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Osteoblastos/metabolismo , Osteólisis/fisiopatología , Osteoprotegerina , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/metabolismo , Proteínas/fisiología , Ligando RANK , ARN Mensajero/metabolismo , Receptor Activador del Factor Nuclear kappa-B , Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores del Factor de Necrosis Tumoral/genética , Células Tumorales CultivadasRESUMEN
Just over two years ago the newest member of the interleukin family of cytokines, IL-18, was molecularly cloned. IL-18 was originally identified as a result of its ability to induce interferon gamma production, however with the advent of its cloning and the production of recombinant protein a number of other biological actions have since been identified. Recently the receptor for IL-18 was also characterised. Due to the structural and biological properties shared between IL-18 and IL-1 and their respective receptors, questions relating to IL-18 activities are being answered at a rapid pace. This article addresses the biology of IL-18 in both disease and non-disease states.
Asunto(s)
Interleucina-18 , Secuencia de Aminoácidos , Animales , Autoinmunidad , Diabetes Mellitus Tipo 1/inmunología , Expresión Génica , Humanos , Interleucina-1/genética , Interleucina-1/fisiología , Interleucina-18/genética , Interleucina-18/fisiología , Subunidad alfa del Receptor de Interleucina-18 , Datos de Secuencia Molecular , Neoplasias Experimentales/inmunología , Osteoclastos/citología , Receptores de Interleucina/fisiología , Receptores de Interleucina-18 , Linfocitos T/inmunologíaRESUMEN
The osteoclastogenic factor of osteoblastic origin has recently been elucidated as a novel Tumor Necrosis Factor (TNF)-ligand family member, termed osteoclast differentiation factor (ODF). Using a semiquantitative RT-PCR approach, we sought to determine the mRNA expression of ODF and its decoy receptor, osteoprotegerin (OPG), in a selection of osteoblastic cell lines and in response to three factors representative of different signal transduction pathways, vitamin D receptor, protein kinase A or gp130. Each osteotropic agent, either 1,25-(OH)2D3, PTH or IL-11, promoted an increase in the ratio of ODF:OPG, with maximal stimulation occurring at 24 h, 4 h, and 8 h, respectively, and furthermore each was shown to act in a dose-dependent manner. This report establishes that osteoblastic cell lines incapable of supporting osteoclast formation have markedly reduced ODF expression and also illustrates the importance of the relative abundance of ODF compared with the levels of OPG for the induction of osteoclastogenesis.
Asunto(s)
Proteínas Portadoras , Citocinas/biosíntesis , Glicoproteínas/farmacología , Glicoproteínas de Membrana/biosíntesis , Osteoblastos/efectos de los fármacos , Osteoclastos/metabolismo , Receptores Citoplasmáticos y Nucleares , Células del Estroma/efectos de los fármacos , Animales , Animales Recién Nacidos , Calcitriol/farmacología , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Interleucina-11/farmacología , Ratones , Ratones Endogámicos C57BL , Osteoclastos/efectos de los fármacos , Osteoprotegerina , Hormona Paratiroidea/farmacología , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Receptores de Calcitriol/fisiología , Receptores del Factor de Necrosis Tumoral , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
IL-18 inhibits osteoclast (OCL) formation in vitro independent of IFN-gamma production, and this was abolished by the addition of neutralizing antibodies to GM-CSF. We now establish that IL-18 was unable to inhibit OCL formation in cocultures using GM-CSF-deficient mice (GM-CSF -/-). Reciprocal cocultures using either wild-type osteoblasts with GM-CSF -/- spleen cells or GM-CSF -/- osteoblasts with wild-type spleen cells were examined. Wild-type spleen cells were required to elicit a response to IL-18 indicating that cells of splenic origin were the IL-18 target. As T cells comprise a large proportion of the spleen cell population, the role of T cells in osteoclastogenesis was examined. Total T cells were removed and repleted in various combinations. Addition of wild-type T cells to a GM-CSF -/- coculture restored IL-18 inhibition of osteoclastogenesis. Major subsets of T cells, CD4+ and CD8+, were also individually depleted. Addition of either CD4+ or CD8+ wild-type T cells restored IL-18 action in a GM-CSF -/- background, while IL-18 was ineffective when either CD4+ or CD8+ GM-CSF -/- T cells were added to a wild-type coculture. These results highlight the involvement of T cells in IL-18-induced OCL inhibition and provide evidence for a new OCL inhibitory pathway whereby IL-18 inhibits OCL formation due to action upon T cells promoting the release of GM-CSF, which in turn acts upon OCL precursors.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Citocinas/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Inductores de Interferón/farmacología , Osteoclastos/efectos de los fármacos , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Células Cultivadas , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Interleucina-18 , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoclastos/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
We have established by differential display polymerase chain reaction of mRNA that interleukin (IL)-18 is expressed by osteoblastic stromal cells. The stromal cell populations used for comparison differed in their ability to promote osteoclast-like multinucleated cell (OCL) formation. mRNA for IL-18 was found to be expressed in greater abundance in lines that were unable to support OCL formation than in supportive cells. Recombinant IL-18 was found to inhibit OCL formation in cocultures of osteoblasts and hemopoietic cells of spleen or bone marrow origin. IL-18 inhibited OCL formation in the presence of osteoclastogenic agents including 1alpha,25-dihydroxyvitamin D3, prostaglandin E2, parathyroid hormone, IL-1, and IL-11. The inhibitory effect of IL-18 was limited to the early phase of the cocultures, which coincides with proliferation of hemopoietic precursors. IL-18 has been reported to induce interferon-gamma (IFN-gamma) and granulocyte/macrophage colony-stimulating factor (GM-CSF) production in T cells, and both agents also inhibit OCL formation in vitro. Neutralizing antibodies to GM-CSF were able to rescue IL-18 inhibition of OCL formation, whereas neutralizing antibodies to IFN-gamma did not. In cocultures with osteoblasts and spleen cells from IFN-gamma receptor type II-deficient mice, IL-18 was found to inhibit OCL formation, indicating that IL-18 acted independently of IFN-gamma production: IFN-gamma had no effect in these cocultures. Additionally, in cocultures in which spleen cells were derived from receptor-deficient mice and osteoblasts were from wild-type mice and vice versa, we identified that the target cells for IFN-gamma inhibition of OCL formation were the hemopoietic cells. The work provides evidence that IL-18 is expressed by osteoblasts and inhibits OCL formation via GM-CSF production and not via IFN-gamma production.