Osteoclast activity modulates B-cell development in the bone marrow

B-cell development is dependent on the interactions between B-cell precursors and bone marrow stromal cells, but the role of osteoclasts (OCLs) in this process remains unknown. B lymphocytopenia is a characteristic of osteopetrosis, suggesting a modulation of B lymphopoiesis by OCL activity. To address this question, we first rescued OCL function in osteopetrotic oc/oc mice by dendritic cell transfer, leading to a restoration of both bone phenotype and B-cell development. To further explore the link between OCL activity and B lymphopoiesis, we induced osteopetrosis in normal mice by injections of zoledronic acid (ZA), an inhibitor of bone resorption. B-cell number decreased specifically in the bone marrow of ZA-treated mice. ZA did not directly affect B-cell differentiation, proliferation and apoptosis, but induced a decrease in the expression of CXCL12 and IL-7 by stromal cells, associated with reduced osteoblastic engagement. Equivalent low osteoblastic engagement in oc/oc mice confirmed that it resulted from the reduced OCL activity rather than from a direct effect of ZA on osteoblasts. These dramatic alterations of the bone microenvironment were disadvantageous for B lymphopoiesis, leading to retention of B-cell progenitors outside of their bone marrow niches in the ZA-induced osteopetrotic model. Altogether, our data revealed that OCLs modulate B-cell development in the bone marrow by controlling the bone microenvironment and the fate of osteoblasts. They provide novel basis for the regulation of the retention of B cells in their niche by OCL activity.     

Migration of natural suppressor cells from bone marrow to peritoneal cavity by live BCG

Delayed-type hypersensitivity (DTH) responses were suppressed in mice inoculated with bone marrow cells from mice that had been injected with 10(8) colony-forming units (CFU) of live BCG. Upon analysis of this DTH-suppression by the use of a macrophage migration inhibition (MI) assay, the in vitro correlate of DTH, suppressor macrophages in the peritoneal cavity were found to play an important role in DTH suppression. However, neither suppression of DTH nor production of suppressor macrophages was observed in mice inoculated with bone marrow cells from mice that had been injected with methotrexate (MTX), a folic acid antagonist, and 10(8) CFU of live BCG. Moreover, suppressor cells against the MI activity of peritoneal exudate cells from BCG cell wall-immunized mice existed in bone marrow cells from normal mice, natural suppressor (NS) cells, and they were sensitive to MTX. In addition, these NS cells phagocytized carbonyl iron particles, were adherent to Sephadex G-10, and had Fc receptors, but they had no B or T cell markers, suggesting that these cells belonged to a macrophage compartment. From this evidence, we hypothesized that the origin of suppressor macrophages in the peritoneal cavity induced by live BCG injection was MTX-sensitive NS cells in bone marrow, and that these NS cells were stimulated by a small dose of live BCG trapped in bone marrow after i.v. injection of a high dose of live BCG and migrated from bone marrow to the peritoneal cavity.     

Osteoclast generation from human fetal bone marrow in cocultures with murine fetal long bones

Summary Osteoclast formation in vitro from human progenitor cells was studied in cocultures of periosteum-free murine long-bone rudiments and human fetal tissues. No osteoclasts were generated from chorionic villi or from fetal liver, but bone marrow and purified bone-marrow fractions gave rise to multinucleated cells that resorbed calcified cartilage matrix. These polykarya react very strongly for tartrate-resistant acid phosphatase (TrAP) and upon ultrastructural examination show large ruffled borders in areas of resorption. Resorption of murine calcified cartilage matrix by human osteoclasts was less than resorption by osteoclasts formed from murine fetal bone-marrow cells. Our results show that the murine long-bone rudiment can be used to generate osteoclasts from human sources of progenitor cells and to assess the biological activity of the formed osteoclasts. This coculture system thereby offers possibilities to study human osteoclast pathology in vitro. The use of TrAP as marker for osteoclasts in cell cultures is discussed.These investigations were supported in part by the Foundation for Medical Research (MEDIGON; grant no. 900-541-069), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO)     

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. Download video file.^{(65M, mp4)}     

The bone marrow origin of the cells encapsulating a foreign body in the peritoneal cavity of xenogeneic radiation chimeras]

The origin and ultrastructure of the cells, encapsulating foreign body in peritoneal cavity of xenogeneic (rat in mouse) radiation chimeras was studied. The donor nature of the cells was identified by their karyotype and by DNA hybridization with rat ID-element. Cells with ultrastructural characteristics of fibroblasts encapsulating foreign body in the peritoneal cavity of the xenogeneic radiation chimeras were shown to originate from the transplanted (donor) bone marrow.     

Differentiation of human stromal bone marrow cell precursors in monolayer culture

The colonies of human bone marrow fibroblasts in monolayer culture have been studied. It has been shown that there are two types of colonies in the cultures: monolayer and multilayer ones, both having alkaline phosphatase-positive cells. In monolayer colonies one can observe calcium deposition indicative of osteogenic differentiation of human bone marrow stromal cells.     

Skeletal muscle precursors do not arise from bone marrow cells

Cell and Tissue Research - This paper tests the hypothesis that bone marrow stem cells can give rise to circulating muscle precursor cells. Irradiated host mice were reconstituted with bone marrow...     

Osteoclast development in marrow cultured in calvaria-conditioned media

The precise signals responsible for recruitment and differentiation of osteoclasts (OCs) from their mononuclear precursors are poorly understood. Marrow mononuclear cells, a reputed source of OC precursors, fuse in culture, forming multinucleated cells. These cells, although similar to OCs, differ from osteoclasts in cell-surface morphology and are not recognized by an OC-specific monoclonal antibody. We have used the expression of an osteoclast-specific membrane epitope designated by monoclonal antibody 121F to delineate OCs from marrow-derived giant cells (MAGC). In this report we describe a series of experiments designed to better define the role of the bone environment in the osteoclast differentiation process. Periosteum-free calvariae from hatchling chicks or their conditioned media were combined with adherent Day 1 cultured marrow cells. The time course of OC marker expression was monitored by ELISA and the requirement for live bone and PTH was investigated. Freshly isolated marrow, MAGC, and calvariae were devoid of OC expression. Antigen expression developed in cultured MAGC after 4 days of coplating with either live bone or live bone-conditioned media. The presence of PTH in the cocultures or conditioned media from PTH-treated calvariae did not significantly alter the level of expression. These data indicate that live bone is, in part, responsible for the production of osteoclasts from mononuclear precursors.     

Development of T cells in vitro from precursors in mouse bone marrow

Bone marrow cells from 6- to 8-week-old athymic nude mice were depleted of nylon-wool adherent cells and cultured in vitro at low cell numbers (300 cells/well) in medium supplemented with a supernatant from a thymoma cell line. About 1% of cultured cells grew. Pooled cultures contained cells expressing CD3 (52%), CD4 (37%), CD8 (11%), Thy 1.2 (72%), MAC-1 (43%) and J11d (86%) but no cells expressing sIg. They also contained cells expressing mRNA for the alpha, beta, gamma, and delta chains of the T cell receptor as assessed with C region probes using a sensitive dot blot assay. These cells appear to develop from progenitors which are CD3-. When pooled Day 10 cultures were depleted of nylon-wool adherent cells, the remaining cells were nearly all J11d+, Thy 1.2+, MAC-1-, CD3+, and either CD4+CD8+; CD4+CD8-; CD4-CD8+, or CD4-CD8-; i.e., their surface marker patterns were reminiscent of those of thymocytes. We conclude that our culture system is enabling bone marrow precursors to commence differentiation down the T cell lineage in the absence of a thymic environment.     

Different radiosensitivities of mast-cell precursors in the bone marrow and skin of mice

Although tissue mast cells are derived from the bone marrow, some descendants of bone marrow-derived precursors retain the ability to proliferate and differentiate into mast cells even after localization in the skin. The purpose of the present study was to determine the D0 values for mast-cell precursors in the bone marrow and those localized in the skin. Bone marrow cells were removed from (WB X C57BL/6)F1-+/+ mice after various doses of irradiation and injected into the skin of the congenic W/Wv mice which were genetically without mast cells. Radiosensitivity of mast-cell precursors in the bone marrow was evaluated by determining the proportion of the injection sites at which mast cells did not appear. For the assay of the radiosensitivity of mast-cell precursors localized in the skin, pieces of skin were removed from beige C57BL/6 (bgJ/bgJ. Chediak-Higashi syndrome) mice after various doses of irradiation and grafted onto the back of the normal C57BL/6 mice. Radiosensitivity of mast-cell precursors in the skin was evaluated by determining the decrease of beige-type mast cells which possessed giant granules. Mast-cell precursors in the bone marrow were much more radiosensitive than those localized in the skin. D0 value was about 100 rad for the former and about 800 rad for the latter.     

Characterization of distinct conventional and plasmacytoid dendritic cell-committed precursors in murine bone marrow

The developmental pathways and differentiation relationship of dendritic cell (DC) subsets remain unclear. We report that murine CD11c(+)MHC II(-) bone marrow cells, which are immediate DC precursors of CD8 alpha(+), CD8 alpha(-), and B220(+) DC in vivo, can be separated into B220(+) and B220(-) DC precursor subpopulations. Purified B220(-) DC precursors expand, and generate exclusively mature CD11c(+)CD11b(+)B220(-) DC in vitro and after adoptive transfer. B220(+) DC precursors, which resemble plasmacytoid pre-DC, have a lower proliferative potential than B220(-) DC precursors and generate both CD11b(-) B220(+) and CD11b(+)B220(-) DC populations. Both DC precursor populations can give rise to CD8 alpha(+) and CD8 alpha(-) DC subtypes. Our findings indicate that CD11c(+)MHC II(-)B220(+) and CD11c(+)MHC II(-)B220(-) bone marrow cells are distinct DC lineage-restricted precursors.     

Characterization of natural killer cells and their precursors in the murine bone marrow

We have fractionated murine bone marrow cells according to their density on bovine serum albumin (BSA) gradient and studied (a) the NK activity against YAC-1 targets, (b) the proportion of asialo GM1+ lymphocytes, (c) and the presence of large granular lymphocytes (LGL) in the different fractions (A, B, C, D). The NK activity was found mainly in the C fraction, but the proportion of asialo GM1+ cells was the same in every fraction. No LGLs were found in the bone marrow. Cells from the various fractions were also transplanted into irradiated recipients. Seven days later the highest NK activity was found in the spleens of mice injected with cells from the A + B fractions indicating that the immediate precursors for NK cells reside in the low density fractions of the BSA gradient. Mice transplanted with C or D fractions needed longer time to develop normal NK levels. The treatment of bone marrow cells before transplantation with anti-asialo GM1+ complement did not inhibit the development of NK activity, so it can be concluded that the precursor for NK is asialo GM1-.     

Characterization of early B lymphocyte precursors present in long-term bone marrow cultures

Lymphoid precursor cells are present in long-term bone marrow cultures (LTBMC), but their differentiation into mature lymphocytes is blocked. A quantitative assay for B cell precursors in LTBMC, which gives a linear relationship between the number of grafted LTBMC cells and the frequency of B cell colony forming units (CFU-B) in the spleen and bone marrow of immunodeficient CBA/N mice 19 days after reconstitution, is described. Characterization of the B cell precursor indicates that this assay is detecting a very early precursor and not a B lymphocyte or a late pre-B cell. This conclusion is based on the observations that a) pre-B cells transformable by Abelson murine leukemia virus are not present in LTBMC by 3 days postrecharge and CFU-B are absent by 6 days postrecharge; b) late B cell progenitors capable of rapid repopulation of irradiated CBA/N mice are not present in LTBMC, since a lag in the kinetics of B cell reconstitution in animals grafted with LTBMC cells is observed compared with fresh bone marrow cells; c) the B cell precursors in LTBMC have high proliferative potential, since they can stably repopulate recipient mice for at least 8 wk postreconstitution and through two serial passages in irradiated CBA/N recipients; and d) the B cell precursors are large, rapidly sedimenting cells as determined by velocity sedimentation. The serial transplantation experiment further shows that a split is often observed between lymphoid and myeloid reconstituting ability of LTBMC cells. The LTBMC B cell precursor may be a pluripotent stem cell or a lymphoid stem cell, although its differentiative potential remains to be determined.     

The analysis of immature lymphoid precursors stored in longterm bone marrow culture

The long-term bone marrow culture system developed by Dexter (MBMC) is known to store immature lymphoid precursors capable of differentiating into mature B cells in irradiated or immunodeficient mice. It has been suggested that pre-B cells are not generated under such culture conditions, but that opinion was not based on any systematic analyses. In the present study under carefully controlled conditions, we observed that pre-B and pro-B cells were eliminated from the late stage of primary MBMC, and the former were not generated in recharged MBMC. Under appropriate conditions, these immature precursors in recharged MBMC generated in vitro immunoglobulin-positive (Ig+) cells to differentiate into antibody-forming cells upon stimulation with lipopolysaccharide (LPS). LPS-reactive B cells were observed in every 10th of the Ig+ cells, the frequency being essentially the same as that observed in normal B cells in different tissues. The immature B cell precursors generating LPS reactive cells were expressed in recharged MBMC at the frequency of 4.2 x 10(-6). A staining experiment showed that cells bearing AA4.1 were stored at the frequency of 10(-4)-10(-5). This frequency is thought to be similar to that of lymphoid precursors in recharged MBMC committed to differentiate along B lineage cells. Based on these results, we discussed the stage, nature, and mode of differentiation of immature lymphoid precursors stored in MBMC.     

Vitamin D regulates transferrin receptor expression by bone marrow macrophage precursors

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is known to prompt monocytic differentiation of a variety of leukemic lines. We previously extended these observations to non-transformed bone marrow macrophage precursors by demonstrating that the steroid enhances plasma membrane expression of the macrophage-specific mannose-fucose receptor (Clohisy et al., J Biol Chem 262:15922-15929, 1987). Because this membrane protein is involved in non-opsonin mediated endocytosis, these observations raised the possibility that 1,25(OH)2D3 globally upregulates endocytic receptors. The present study, aimed at addressing this issue, turns to the transferrin receptor as a paradigm for endocytic receptors and explores the impact of 1,25(OH)2D3 on its expression. We found that in contrast to the mannose-fucose receptor, plasma membrane transferrin receptor expression by bone marrow-derived macrophage precursors declines by at least 30% in a dose-dependent fashion with exposure to 1,25(OH)2D3. The effect reflects diminished receptor capacity with no change in Kd, and is independent of cell cycle. Moreover, while Vmax of receptor-ligand internalization mirrors plasma membrane occupancy, Kuptake remains unaltered in the presence of vitamin D3, indicating that the down-regulating event does not reflect on enhanced rate of endocytosis. Further, pulse chase experiments show parallel cell surface, intra-cellular, and medium redistribution of radioligand with time steroid-treated and control cells. In a similar vein, while total cell-associated radioligand falls in the presence of vitamin D3, the percentage of intracellular and surface bound counts at equilibrium are constant in both groups. Finally, immunoprecipitation studies reveal that the down-regulating effects of 1,25(OH)2D3 cannot be explained by inhibition of transferrin receptor synthesis. Thus, the decrease in total cellular transferrin binding sites is likely to represent either enhanced degradation or synthesis of "cryptic" receptors which fail to recognize 125I-transferrin.     

HSP10 selective preference for myeloid and megakaryocytic precursors in normal human bone marrow

Heat shock proteins (HSPs) constitute a heterogeneous family of proteins involved in cell homeostasis. During cell life they are involved in harmful insults, as well as in immune and inflammatory reactions. It is known that they regulate gene expression, and cell proliferation, differentiation and death. HSP60 is a mitochondrial chaperonin, highly preserved during evolution, responsible of protein folding. Its function is strictly dependent on HSP10 in both prokaryotic and eukaryotic elements. We investigated the presence and the expression of HSP60 and HSP10 in a series of 20 normal human bone marrow specimens (NHBM) by the means of immunohistochemistry. NHBM showed no expression of HSP60, probably due to its being below the detectable threshold, as already demonstrated in other normal human tissues. By contrast, HSP10 showed a selective positivity for myeloid and megakaryocytic lineages. The positivity was restricted to precursor cells, while mature elements were constantly negative. We postulate that HSP10 plays a role in bone marrow cell differentiation other than being a mitochondrial co-chaperonin. The present data emphasize the role of HSP10 during cellular homeostasis and encourage further investigations in this field.     

The influence of histamine on precursors of granulocytic leukocytes in murine bone marrow

The effect of histamine on granulocytic progenitor cells in murine bone marrow was studied in vitro. When bone marrow cells were cultured for three days with the drug, 10(-8) M to 10(-5) M of histamine stimulated differentiation and proliferation of myeloid precursor cells. Subsequently, the number of descendant cells, such as metamyelocytes and neutrophils, increased dose-dependently. Co-existence of equimolar H2 blockers such as cimetidine and ranitidine completely suppressed this effect of histamine, though this was not the case with an H1 blocker/histamine combination. Significant increase in 3H-thymidine incorporation was observed almost exclusively in myeloblasts, promyelocytes and myelocytes after exposure to histamine at concentrations higher than 10(-8) M. Also, selective incorporation of 3H-histamine into bone marrow cells was observed in myeloblasts and promyelocytes, but histamine incorporation was not influenced by the presence of either of histamine agonists or antagonists. While histamine, via H2 receptors, selectively increased the number of granulocytic colony forming units in culture (CFU-C), it had no such effect on macrophage colonies. Considering these findings, it was concluded that histamine promotes proliferation and differentiation of granulocytic myeloid cells via 1) H2 receptors in the CFU-C stage and 2) histamine receptors which are neither H1 nor H2 in the stages of myeloblast and promyelocyte differentiation.     

Erythroid precursors studied by mouse bone marrow cultivation in a plasma clot]

Two types of erythroid precursors were found by cultivation of the mouse bone marrow in the plasma clot with mouse serum and without adding exogenic erythropoietin to the culture medium. The first precursor had properties similar to the erythroid colony-forming unit (CFUe) previously described while the second resembles in its properties the erythroid burst-forming unit (BFUe). Optimal concentration of mouse serum in the culture medium was 10-15%. Clone nature of the colonies and bursts described is confirmed by linear dependence of their number on the cell concentration in the culture.     

Maturation-dependent adhesion of human B cell precursors to the bone marrow microenvironment

Murine B cell precursors can be induced to proliferate in culture if allowed to bind to bone marrow derived adherent cells prepared under specific conditions. We studied the binding of human B cell precursor subpopulations to various in vitro microenvironments to determine which conditions may potentially be suitable models for human B precursor differentiation. Using the markers CD10, CD34, and CD20, B lineage populations of increasing maturation were quantitated: CD10+/CD34+, CD10+/CD20-, CD10+/CD20+, and CD10-/CD20+ cells in marrow, and CD10-/CD20+ mature B cells in peripheral blood. The adhesion of subpopulations of blood and marrow-derived light density cells to adherent cell layers or matrix was studied following a 2-h incubation in 24-well plates. The absolute number of bound B lineage cells was determined by cell counts and flow cytometry analysis. The adherence of B lineage cells to passaged human marrow fibroblasts (BM-FB) was highest in the most immature CD10+/CD34+ cells (34.3 +/- 4.2%), decreasing steadily with each stage of maturation to the peripheral blood B cells (11.2 +/- 2.4%). Increased adhesion of CD10+ B cell precursors relative to CD10-/CD20+ marrow B cells was confirmed by adhesion studies using sorted cells. The two most immature B lineage cells (CD10+CD34+ and CD10+/CD20-) showed more adherence to BM-FB than any other cell type tested, except for monocytes. Only B lineage precursor cells, erythroid precursors and CD10-/CD34+ cells showed significantly greater binding to BM-FB than to plastic. B lineage precursors bound equally well to primary and passaged human marrow fibroblasts, but bound significantly less well to passaged human foreskin fibroblasts, primary human marrow stroma, extracellular matrix of marrow fibroblasts, or fibronectin. These results suggest that specific binding to marrow fibroblasts is part of the differentiation program of early B lineage precursors. This binding activity gradually and predictably decreases during B lineage differentiation, in contrast to expression of other binding receptors, such as LFA-1 and CD44, which increase during B lineage maturation.     

Studies on bone marrow histogenesis: Morphometric and autoradiographic studies of regenerating marrow stroma in extramedullary autoimplants and after evacuation of marrow cavity

Summary The marrow cavity of the rat tibia was mechanically evacuated and autoimplanted to the subcutaneous tissue. The regenerative process which restored the integrity of marrow stroma and hemopoiesis, was morphometrically evaluated in whole mount of tibia. Following evacuation, the clot filled the cavity. The granulation tissue then appeared and expanded, penetrating and replacing the clot. The fibroblasts of the granulation tissue differentiated into osteoblasts forming osteoid bone. Within its interstices, the primordial marrow consisting of loose connective tissue and vascular sinuses appeared and hemopoiesis resumed. Expansion of hemopoiesis resulted in the resorption of bone and within three weeks the tibial cavity was restored to the pre-evacuation state.Autoradiography indicated that the labeling index was initially high in fibroblasts and osteoblasts but was subsequently reduced while it increased in osteocytes, cells of Haversian canals, stromal and hemopoietic cells of marrow. The finding is in disagreement with the view that the regenerative process originates from the Haversian canal. When the label was introduced on day 4 post-operatively, it subsequently appeared in osteocytes, cells of Haversian canal, stromal elements of the marrow, but not in the hemopoietic cells. This indicates complete dissociation of marrow stroma and hemopoietic stem cell.Supported by NASA Contract NSG 9061. Mehdi Tavassoli is the recipient of a CRD Award AM-70551