Vedran Katavić

Role of B Lymphocytes in New Bone Formation

Although there may be a close relationship between B lymphocytes and osteoclasts, or bone resorbing cells, little is known about the role of B lymphocytes in bone formation. We compared in vivo new bone induction in mice homozygous for the B-cell deficient (μMT) gene knockout, which lack functional B lymphocytes, with bone induction in control wild-type (C57BL/6) mice. Our comparison used two models of new bone induction in vivo: endochondral osteoinduction by subcutaneous implantation of recombinant human bone morphogenetic protein (rhBMP-2) and osteogenic regeneration after tibial bone marrow ablation. The expression of bone-specific proteins (bone sialoprotein, osteopontin, and osteocalcin) and inflammatory/immunomodulatory cytokines (interleukin-1α and -1β, interleukin-6, and tumor necrosis factor-α) was assessed by Northern blot analysis or reverse transcription-polymerase chain reaction, respectively. Ossicles induced by rhBMP-2 were larger in volume and mass in μMT knockout mice, but relative volumes of the newly induced bone, cartilage, and bone marrow were similar in the two groups. Six days after tibial bone marrow ablation, μMT knockout mice resorbed the initial blood clot faster and formed more trabecular bone, paralleled by greater levels of bone sialoprotein mRNA than in the wild-type mice. μMT knockout and wild-type mice also differed in the expression pattern of inflammatory/immunomodulatory cytokines during the development of the newly induced bone, suggesting that a genetic lack of B lymphocytes may create a change in the immunological milieu at the site of new bone induction, which stimulates the initial accumulation and proliferation of mesenchymal progenitor.

The Fas/Fas Ligand System Inhibits Differentiation of Murine Osteoblasts but Has a Limited Role in Osteoblast and Osteoclast Apoptosis

Apoptosis through Fas/Fas ligand (FasL) is an important regulator of immune system homeostasis but its role in bone homeostasis is elusive. We systematically analyzed: 1) the expression of Fas/FasL during osteoblastogenesis and osteoclastogenesis in vitro, 2) the effect of FasL on apoptosis and osteoblastic/osteoclastic differentiation, and 3) osteoblastogenesis and osteoclastogenesis in mice deficient in Fas or FasL. The expression of Fas increased with osteoblastic differentiation. Addition of FasL weakly increased the proportion of apoptotic cells in both osteoclastogenic and osteoblastogenic cultures. In a CFU assay, FasL decreased the proportion of osteoblast colonies but did not affect the total number of colonies, indicating specific inhibitory effect of Fas/FasL on osteoblastic differentiation. The effect depended on the activation of caspase 8 and was specific, as addition of FasL to osteoblastogenic cultures significantly decreased gene expression for runt-related transcription factor 2 (Runx2) required for osteoblastic differentiation. Bone marrow from mice without functional Fas or FasL had similar osteoclastogenic potential as bone marrow from wild-type mice, but generated more osteoblast colonies ex vivo. These colonies had increased expression of the osteoblast genes Runx2, osteopontin, alkaline phosphatase, bone sialoprotein, osteocalcin, and osteoprotegerin. Our results indicate that Fas/FasL system primarily controls osteoblastic differentiation by inhibiting progenitor differentiation and not by inducing apoptosis. During osteoclastogenesis, the Fas/FasL system may have a limited effect on osteoclast progenitor apoptosis. The study suggests that Fas/FasL system plays a key role in osteoblastic differentiation and provides novel insight into the interactions between the immune system and bone.

The surface antigen CD45R identifies a population of estrogen- regulated murine marrow cells that contain osteoclast precursors

We examined the osteoclastogenic potential of murine bone marrow cells that were fractionated according to their expression of the surface antigen CD45R. Osteoclast-like cells (OCL) with many authentic osteoclast characteristics readily formed in purified CD45R(+) murine bone marrow cell cultures after treatment with receptor activator of nuclear factor kappaB ligand (RANKL) and M-CSF. Ovariectomy (Ovx) caused a 1.5- to 2-fold increase in OCL number in unfractionated and CD45R(+) murine bone marrow cell cultures without affecting OCL formation in CD45R(-) marrow cells. Limiting dilution assays confirmed that Ovx caused an increase in osteoclast precursor cell number in CD45R(+) but not CD45R(-) cells. Mice deficient in the type 1 IL-1 receptor (IL-1R1 KO) do not lose bone mass after Ovx. We found that unfractionated, CD45R(+), and CD45R(-) bone marrow cells from IL-1R1 KO mice showed no increase in OCL formation in vitro after Ovx. In both the wild-type (WT) and the IL-1R1 KO mice Ovx was associated with a 2-fold increase in pre-B-lymphocytes. About 1.3-3.5% of murine marrow cells expressed surface RANK (the receptor for RANKL) while about 11.9-15% of murine bone marrow cells expressed c-Fms (the receptor for M-CSF). There was little effect of Ovx on cells expressing either RANK or c-Fms. These results demonstrate that CD45R expression identifies a subset of murine bone marrow cells whose ability to form OCL in vivo is regulated by estrogen in WT but not IL-1R1 KO cells. The effects of estrogen on bone mass may be related to these responses.

Activated T lymphocytes suppress osteoclastogenesis by diverting early monocyte/macrophage progenitor lineage commitment towards dendritic cell differentiation through down-regulation of receptor activator of nuclear factor-kappaB and c-Fos

Activated T lymphocytes either stimulate or inhibit osteoclastogenesis from haematopoietic progenitors in different experimental models. To address this controversy, we used several modes of T lymphocyte activation in osteoclast differentiation − mitogen‐pulse, anti‐CD3/CD28 stimulation and in vivo and in vitro alloactivation. Osteoclast‐like cells were generated from non‐adherent immature haematopoietic monocyte/macrophage progenitors in murine bone‐marrow in the presence of receptor activator of nuclear factor (NF)‐κB ligand (RANKL) and monocyte–macrophage colony‐stimulating factor (M‐CSF). All modes of in vivo and in vitro T lymphocyte activation and both CD4+ and CD8+ subpopulations produced similar inhibitory effects on osteoclastogenesis paralleled by enhanced dendritic cell (DC) differentiation. Osteoclast‐inhibitory effect was associated with T lymphocyte activation and not proliferation, and could be replaced by their culture supernatants. The stage of osteoclast differentiation was crucial for the inhibitory action of activated T lymphocytes on osteoclastogenesis, because the suppressive effect was visible only on early osteoclast progenitors but not on committed osteoclasts. Inhibition was associated specifically with increased granulocyte–macrophage colony‐stimulating factor (GM‐CSF) expression by the mechanism of progenitor commitment toward lineages other than osteoclast because activated T lymphocytes down‐regulated RANK, CD115, c‐Fos and calcitonin receptor expression, and increased differentiation towards CD11c‐positive DC. An activated T lymphocyte inhibitory role in osteoclastogenesis, confirmed in vitro and in vivo, mediated through GM‐CSF release, may be used to counteract activated bone resorption mediated by T lymphocyte‐derived cytokines in inflammatory and immune disorders. We also demonstrated the importance of alloactivation in osteoclast differentiation and the ability of cyclosporin A to abrogate T lymphocyte inhibition of osteoclastogenesis, thereby confirming the functional link between alloreaction and bone metabolism.

Increased Bone Mass Is a Part of the Generalized Lymphoproliferative Disorder Phenotype in the Mouse

We investigated the bone phenotype of mice with generalized lymphoproliferative disorder (gld) due to a defect in the Fas ligand-mediated apoptotic pathway. C57BL/6-gld mice had greater whole body bone mineral density and greater trabecular bone volume than their wild-type controls. gld mice lost 5-fold less trabecular bone and had less osteoclasts on bone surfaces after ovariectomy-induced bone resorption. They also formed more bone in a model of osteogenic regeneration after bone marrow ablation, had less osteoclasts on bone surfaces and less apoptotic osteoblasts. gld and wild-type mice had similar numbers of osteoclasts in bone marrow cultures, but marrow stromal fibroblasts from gld mice formed more alkaline phosphatase-positive colonies. Bone diaphyseal shafts and bone marrow stromal fibroblasts produced more osteoprotegerin mRNA and protein than wild-type mice. These findings provide evidence that the disturbance of the bone system is a part of generalized lymphoproliferative syndrome and indicates the possible role of osteoprotegerin as a regulatory link between the bone and immune system.

Induction of osteoclast progenitors in inflammatory conditions: key to bone destruction in arthritis

The inflammatory milieu favors recruitment and activation of osteoclasts, and leads to bone destruction as a serious complication associated with arthritis and with other inflammatory processes. The frequency and activity of osteoclast progenitors (OCPs) correspond to arthritis severity, and may be used to monitor disease progression and bone resorption, indicating the need for detailed characterization of the discrete OCP subpopulations. Collectively, current studies suggest that the most potent murine bone marrow OCP population can be identified among lymphoid negative population within the immature myeloid lineage cells, as B220−CD3−CD11b–/loCD115+CD117+CX3CR1+ and possibly also Ter119−CD11c−CD135loLy6C+RANK−. In peripheral blood the OCP population bears the monocytoid phenotype B220−CD3−NK1.1−CD11b+Ly6ChiCD115+CX3CR1+, presumably expressing RANK in committed OCPs. Much less is known about human OCPs and their regulation in arthritis, but the circulating OCP subset is, most probably, comprised among the lymphoid negative population (CD3−CD19−CD56−), within immature monocyte subset (CD11b+CD14+CD16−), expressing receptors for M-CSF and RANKL (CD115+RANK+). Our preliminary data confirmed positive association between the proportion of peripheral blood OCPs, defined as CD3−CD19−CD56−CD11b+CD14+, and the disease activity score (DAS28) in the follow-up samples from patients with psoriatic arthritis receiving anti-TNF therapy. In addition, we reviewed cytokines and chemokines which, directly or indirectly, activate OCPs and enhance their differentiation potential, thus mediating osteoresorption. Control of the activity and migratory behaviour of OCPs as well as the identification of crucial bone/joint chemotactic mediators represent promising therapeutic targets in arthritis.

Fas receptor is required for estrogen deficiency-induced bone loss in mice

Bone mass is determined by bone cell differentiation, activity, and death, which mainly occur through apoptosis. Apoptosis can be triggered by death receptor Fas (CD95), expressed on osteoblasts and osteoclasts and may be regulated by estrogen. We have previously shown that signaling through Fas inhibits osteoblast differentiation. In this study we analyzed Fas as a possible mediator of bone loss induced by estrogen withdrawal. At 4 weeks after ovariectomy (OVX), Fas gene expression was greater in osteoblasts and lower in osteoclasts in ovariectomized C57BL/6J (wild type (wt)) mice compared with sham-operated animals. OVX was unable to induce bone loss in mice with a gene knockout for Fas (Fas –/– mice). The number of osteoclasts increased in wt mice after OVX, whereas it remained unchanged in Fas –/– mice. OVX induced greater stimulation of osteoblastogenesis in Fas –/– than in wt mice, with higher expression of osteoblast-specific genes. Direct effects on bone cell differentiation and apoptosis in vivo were confirmed in vitro, in which addition of estradiol decreased Fas expression and partially abrogated the apoptotic and differentiation-inhibitory effect of Fas in osteoblast lineage cells, while having no effect on Fas-induced apoptosis in osteoclast lineage cells. In conclusion, the Fas receptor has an important role in the pathogenesis of postmenopausal osteoporosis by mediating apoptosis and inhibiting differentiation of osteoblast lineage cells. Modulation of Fas effects on bone cells may be used as a therapeutic target in the treatment of osteoresorptive disorders.

Increased bone resorption and osteopenia are a part of the lymphoproliferative phenotype of mice with systemic over-expression of interleukin-7 gene driven by MHC class II promoter

Mice with interleukin (IL)-7 transgene under the control of E(alpha) promoter over-express IL-7 in MHC class II-positive cells and develop specific immune phenotype, marked by an increase in CD45R(+) cells in both the bone marrow and peripheral blood. We show that IL-7 transgenic mice have a bone phenotype characterized by an age-related loss of trabecular bone in both axial and long bones. Osteopenia was the result of increased number of active osteoclasts on the surface of trabecular bone. Furthermore, IL-7 transgenic mice showed increased osteoclastic but unchanged osteoblastic potential of the bone marrow in vitro. IL-7 over-expression also created osteoclastogenic microenvironment within the bone marrow which promoted the commitment of precursors towards the osteoclast lineage. These findings are important for immunological disturbances where IL-7 is involved and where alterations in the immune system are accompanied by changes in bone metabolism, such as multiple myeloma, rheumatoid arthritis and postmenopausal osteoporosis.

Alteration of newly induced endochondral bone formation in adult mice without tumour necrosis factor receptor 1.

Tumour necrosis factor (TNF)‐α, a major proinflammatory cytokine, exerts its role on bone cells through two receptors (TNFR1 and TNFR2). TNFR1, but not TNFR2, is expressed by osteoblasts and its function in bone formation in vivo is not fully understood. We compared in vivo new bone formation in TNFR1‐deficient (TNFR1–/–) mice and wild‐type mice, using two models of bone formation: intramembranous ossification following tibial marrow ablation and endochondral ossification induced by bone morphogenetic protein (BMP)‐2. Intramembranous osteogenesis in TNFR1–/– mice did not differ from the wild‐type mice either in histomorphometric parameters or mRNA expression of bone‐related markers and inflammatory cytokines. During endochondral osteogenesis, TNFR1–/– mice formed more cartilage (at post‐implantation day 9), followed by more bone and bone marrow (at day 12). mRNAs for BMP‐2, ‐4 and ‐7 were increased during the endochondral differentiation sequence in TNFR1–/– mice. The expression of receptor activator of NF‐κ B ligand (RANKL) and receptor activator of NF‐κ B (RANK), as assessed by quantitative reverse transcription polymerase chain reaction (RT‐PCR), was also increased significantly during endochondral ossification in TNFR1–/– mice. In conclusion, signalling through the TNFR1 seems to be a negative regulator of new tissue formation during endochondral but not intramembranous osteogenesis in an adult organism. BMPs and RANKL and its receptor RANK may be involved in the change of local environment in the absence of TNFR1 signalling.

Targeting Fas in osteoresorptive disorders.

Importance of the field: Fas receptor is a mediator of the external apoptotic pathway in many cells and tissues. It is proposed that Fas receptor mediates osteoresorptive effects of estrogen deficiency and local/systemic inflammation. Areas covered in this review: This review covers the past two decades of research on the expression and function of the Fas–Fas ligand system on bone cells, involvement in the pathogenesis of osteoresorption and potential therapeutic modulation. What the reader will gain: We review the structure, biological function and intracellular signaling pathways of the Fas–Fas ligand system emphasizing the role of the non-apoptotic signaling pathways in bone cells, particularly osteoblast differentiation. We also present data on the in vitro expression and function of the Fas–Fas ligand system on osteoblast/osteoclast lineage cells, animal and human studies confirming its involvement in osteoresorptive disorders and potential therapeutic approaches to modulate its function. Take home message: Tissue specific therapeutic approaches need to be established to modify the Fas–Fas ligand system in osteoresorptive disorders as systemic targeting has many side effects. The most promising approach would be to target Fas signaling molecules coupled with osteoblast/osteoclast differentiation pathways, but a precise definition of these targets is still needed.