Strain-Specific Variations in the Development of Dendritic Cells in Murine Bone-Marrow Cultures

AbstractHeme is an erythrocyte-derived toxin that drives disease progression in hemolytic anemias, such as sickle cell disease. During hemolysis, specialized bone marrow-derived macrophages with a high heme-metabolism capacity orchestrate disease adaptation by removing damaged erythrocytes and heme-protein complexes from the blood and supporting iron recycling for erythropoiesis. Since chronic heme-stress is noxious for macrophages, erythrophagocytes in the spleen are continuously replenished from bone marrow-derived progenitors. Here, we hypothesized that adaptation to heme stress progressively shifts differentiation trajectories of bone marrow progenitors to expand the capacity of heme-handling monocyte-derived macrophages at the expense of the homeostatic generation of dendritic cells, which emerge from shared myeloid precursors. This heme-induced redirection of differentiation trajectories may contribute to hemolysis-induced secondary immunodeficiency. We performed single-cell RNA-sequencing with directional RNA velocity analysis of GM-CSF-supplemented mouse bone marrow cultures to assess myeloid differentiation under heme stress. We found that heme-activated NRF2 signaling shifted the differentiation of bone marrow cells towards antioxidant, iron-recycling macrophages, suppressing the generation of dendritic cells in heme-exposed bone marrow cultures. Heme eliminated the capacity of GM-CSF-supplemented bone marrow cultures to activate antigen-specific CD4 T cells. The generation of functionally competent dendritic cells was restored by NRF2 loss. The heme-induced phenotype of macrophage expansion with concurrent dendritic cell depletion was reproduced in hemolytic mice with sickle cell disease and spherocytosis and associated with reduced dendritic cell functions in the spleen. Our data provide a novel mechanistic underpinning of hemolytic stress as a driver of hyposplenism-related secondary immunodeficiency.

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Differentiation of dendritic cells in cultures of rat bone marrow cells.

Journal of Experimental Medicine ◽

10.1084/jem.163.4.872 ◽

1986 ◽

Vol 163 (4) ◽

pp. 872-883 ◽

Cited By ~ 45

Author(s):

W E Bowers ◽

M R Berkowitz

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Bone Marrow Cells ◽

Sensitive Assay ◽

Ia Antigens ◽

Bone Marrow Cultures ◽

Nonadherent Cells ◽

Marrow Cultures ◽

Rat Bone ◽

Marrow Cells

Although dendritic cells (DC) originate from bone marrow, they were not observed in fresh preparations of bone marrow cells (BMC). Likewise, accessory activity was barely measurable in a sensitive assay for this potent function of DC. However, both DC and accessory activity developed when BMC were cultured for 5 d. Based on fractionation before culture, nearly all of the accessory activity could be attributed to only 5% of the total BMC recovered in a low-density (LD) fraction. The LD-DC precursors differed from mature DC in a number of important respects. Removal of Ia+ cells from the LD fraction by panning did not decrease the production of DC when the nonadherent cells were cultured. Thus, the cell from which the DC is derived does not express or minimally expresses Ia antigens, in contrast to the strongly Ia+ DC that is produced in bone marrow cultures. Irradiation of LD cells before culture prevented the development of DC. When irradiation was delayed by daily intervals, progressive increases in the number of DC resulted, up to the fifth day. These findings, together with preliminary autoradiographic data, indicate that cell division has occurred, in contrast to the DC, which does not divide. We conclude that bone marrow-derived DC arise in culture from the division of LD, Ia- precursors.

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Murine Plasmacytoid Pre-Dendritic Cells Generated from Flt3 Ligand-Supplemented Bone Marrow Cultures Are Immature APCs

The Journal of Immunology ◽

10.4049/jimmunol.169.12.6711 ◽

2002 ◽

Vol 169 (12) ◽

pp. 6711-6719 ◽

Cited By ~ 176

Author(s):

Pierre Brawand ◽

David R. Fitzpatrick ◽

Brad W. Greenfield ◽

Kenneth Brasel ◽

Charles R. Maliszewski ◽

...

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Flt3 Ligand ◽

Bone Marrow Cultures ◽

Marrow Cultures

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Inhibin Suppresses and Activin Stimulates Osteoblastogenesis and Osteoclastogenesis in Murine Bone Marrow Cultures

Endocrinology ◽

10.1210/endo.143.1.8580 ◽

2002 ◽

Vol 143 (1) ◽

pp. 74-83 ◽

Cited By ~ 67

Author(s):

D. Gaddy-Kurten ◽

J. K. Coker ◽

E. Abe ◽

R. L. Jilka ◽

S. C. Manolagas

Keyword(s):

Bone Marrow ◽

Alkaline Phosphatase ◽

Bone Morphogenetic Protein ◽

Colony Formation ◽

Tartrate Resistant Acid Phosphatase ◽

Murine Bone Marrow ◽

Morphogenetic Protein ◽

Bone Marrow Cultures ◽

Marrow Cultures

Abstract Using primary murine bone marrow cell cultures, we demonstrate that inhibin suppresses osteoblastogenesis and osteoclastogenesis. In contrast, activin supports osteoblast formation (by alkaline phosphatase-positive and mineralized colony formation); and activin also stimulates osteoclast formation (as measured by staining tartrate-resistant acid phosphatase-positive multinucleated cells). Inhibin, the activin antagonist follistatin, and the bone morphogenetic protein antagonist noggin can all suppress endogenous activin accumulation in bone marrow cultures. Associated with this decrease in activin is the loss of mineralized osteoblastic colony formation (colony forming unit-osteoblast; CFU-OB). However, exogenous activin administration, even in the presence of noggin, permits both alkaline phosphatase-positive and CFU-OB colony formation in vitro. In contrast, the stimulatory effects of locally produced activin on osteoblast and osteoclast development are not likely to be dominant over the suppressive effects of gonadally derived inhibin. The suppressive effect of inhibin is maintained in the presence of either activin or bone morphogenetic protein, suggesting the presence of a distinct inhibin-specific receptor. Taken together, the direct regulation of osteoblastogenesis and osteoclastogenesis by inhibin and activin in vitro suggest that changes in the inhibin/activin ratio detected by bone marrow cells, during the perimenopausal transition, contribute to altered cell differentiation and may be associated with the increased bone resorption observed at this time.

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Hematopoietic regulatory factors produced in long-term murine bone marrow cultures and the effect of in vitro irradiation

Blood ◽

10.1182/blood.v64.2.516.bloodjournal642516 ◽

1984 ◽

Vol 64 (2) ◽

pp. 516-525 ◽

Cited By ~ 1

Author(s):

RJ Gualtieri ◽

RK Shadduck ◽

DG Baker ◽

PJ Quesenberry

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Factor Vii ◽

Murine Bone Marrow ◽

L Cell ◽

Bone Marrow Cultures ◽

Macrophage Colony ◽

Marrow Cultures

The nature of hematopoietic regulatory factors elaborated by the adherent (stromal) cells of long-term murine bone marrow cultures and the effect of in vitro stromal irradiation (XRT) on the production of these factors was investigated. Using an in situ stromal assay employing a double layer of semisolid agar, it was possible to demonstrate stromal elaboration of at least two colony-stimulating activities, ie, granulocyte/macrophage colony-stimulating activity (G/M- CSA) and megakaryocyte colony-stimulating activity (Meg-CSA). Exposure of the stroma to XRT resulted in dose-dependent elevations of both activities that correlated inversely with total myeloid cell mass as determined by concurrent reductions in total supernatant cell recoveries from irradiated cultures. Mixture experiments that combined control and irradiated stroma revealed that the hematopoietically active control stroma could block detection of XRT-related G/M-CSA elevations. These data implicate a local negative feedback mechanism in the regulation of hematopoiesis. Antiserum directed against purified L cell colony-stimulating factor (CSF) reduced granulocyte/macrophage colony formation in the target layer but did not effect the increased Meg-CSA. While a radioimmunoassay for L-cell type CSF was unable to detect significant differences in concentrated media from control and irradiated cultures, bioassays of these media revealed XRT-related G/M- CSA elevations. These results indicate that the G/M-CSA elaborated in these cultures is immunologically distinct from the Meg-CSA produced, and although distinct from L cell CSF, the G/M-CSA is crossreactive with the L cell CSF antiserum. Morphologic, histochemical, and factor VII antigen immunofluorescent studies were performed on the stromal cell population responsible for production of these stimulatory activities. In addition to “fat” cells, the stromal cells remaining after XRT were composed of two predominant cell populations. These included a major population of acid phosphatase and nonspecific esterase-positive macrophage-like cells and a minor population of factor VII antigen negative epithelioid cells.

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No stimulative effect of adipocytes on hematopoiesis in long-term human bone marrow cultures

Blood ◽

10.1182/blood.v61.4.770.bloodjournal614770 ◽

1983 ◽

Vol 61 (4) ◽

pp. 770-774

Author(s):

I Touw ◽

B Lowenberg

Keyword(s):

Bone Marrow ◽

Human Bone ◽

Human Bone Marrow ◽

Adherent Cell ◽

Murine Bone Marrow ◽

Bone Marrow Cultures ◽

Human Bone Marrow Cultures ◽

Marrow Cultures

Long-term cultures of human bone marrow were established for 5–13 wk to study the role of adipocytes in sustaining hematopoiesis. At weekly intervals, the numbers of nucleated cells and granulocyte-macrophage progenitor cells (GM-CFU) in culture were estimated in relation to the numbers of fat-containing cells present in the adherent stroma layer. In these quantifications, the numbers of GM-CFU trapped in the adherent cell layer were considered separately. It was found that the presence of adipocytes did not correlate with more active hematopoiesis. Fat cells appeared at late stages when successful cultures were being exhausted or early in cultures with poor activity. These observations suggest that human marrow continuous hematopoiesis in vitro, unlike hematopoiesis in the analogous murine bone marrow cultures, does not depend on the presence of adipocytes.

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Extracellular matrix production by the adherent cells of long-term murine bone marrow cultures

Blood ◽

10.1182/blood.v61.3.540.540 ◽

1983 ◽

Vol 61 (3) ◽

pp. 540-547 ◽

Cited By ~ 110

Author(s):

KS Zuckerman ◽

MS Wicha

Keyword(s):

Bone Marrow ◽

Extracellular Matrix ◽

Stromal Cells ◽

Extracellular Matrix Proteins ◽

Matrix Proteins ◽

Immunofluorescent Staining ◽

Murine Bone Marrow ◽

Bone Marrow Cultures ◽

Marrow Cultures

Abstract We have studied the deposition of extracellular matrix proteins in the adherent stroma of long-term murine bone marrow cultures. Stable hematopoiesis was maintained for greater than 12 wk. At selected intervals, culture dishes were sacrificed by removing all nonadherent cells and air drying the dishes. The adherent stromal layer was analyzed for the presence of intracellular and extracellular collagen, fibronectin, and laminin using double immunofluorescent staining with specific antisera against these matrix components. In cultures examined during the first 2 wk, large numbers of stromal cells contained collagen, fibronectin, and laminin. Over the next 2 wk, an extensive extracellular network of fibronectin, laminin, and collagen was deposited on the dishes, which persisted throughout the life of the cultures. In contrast to a previous report, we detected substantial numbers of endothelial cells by means of immunofluorescent staining of stromal cells with antisera to type IV collagen, laminin, and factor VIII antigen. Although deposition of these extracellular matrix proteins coincides with onset of active hematopoietic cell production, the relative roles of the stromal cells and the extracellular matrix in supporting hematopoiesis in murine bone marrow cell cultures remain to be determined.

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