Murine Bone Marrow Mononuclear Cells Research Articles

Independent Regulation of ABCB1 and ABCC Activities in Thymocytes and Bone Marrow Mononuclear Cells during Aging

Aging modifies a number of functional and phenotypic parameters of cells from the immune system. In this study, the activities of two members of the superfamily of ATP-binding cassette (ABC) transport proteins, ABCB1 and ABCC (measured by rhodamine 123 efflux and Fluo-3 efflux respectively), were compared in murine bone marrow cells and thymocytes of young (3-4 weeks old), adult (2-3 months old) and old (18 months old) mice. ABCB1 activity was shown to be age regulated in murine bone marrow mononuclear cells and thymocytes. In the bone marrow, the increased amount of cells with ABCB1 activity observed in old mice was restricted to the c-kit(-)Sca-1(+) and c-kit(+)Sca-1(+) subpopulations. Only a small percentage of c-kit(+) cells in the thymus had ABCB1 activity, and this subpopulation increased with age. In the thymus, old age augmented this activity in the CD4(-) CD8(-) double-negative cells and in the CD4(+) and CD8(+) single-positive populations. The activity of another ABC transporter, the ABCC-related activity, was also modified by age in the bone marrow. However, the age-related increase was observed in the subpopulations were ABCB1 was not modified, namely the non-progenitor population (c-kit(-)Sca-1(-)cells) and c-kit(+)Sca-1(-) cells. Nearly, all thymocytes expressed the ABCC1 molecule in an active form and aging did not affect this pattern. This study demonstrates an independent upregulation of ABCB1 and ABCC activities during the aging process. The increases were observed in different subsets of cells but followed a developmentally regulated pattern. The functions played by these transporters and alterations in aging are discussed.

Anti-CD25 Targeted Killing of Bicistronically Transduced Cells: A Novel Safety Mechanism Against Retroviral Genotoxicity

Gene therapy for Fabry disease, a deficiency in alpha-galactosidase A (alpha-gal A) activity, has the potential to provide a cure for the disorder with a single treatment. Despite modifications to existing vectors, concerns have arisen regarding the risk of genotoxicity associated with the use of retroviruses. To address safety concerns, we propose that expression of a cell surface protein, human CD25 (huCD25) in a bicistronic format, with any therapeutic gene such as alpha-gal A can provide a target that can be used to kill transduced cells selectively should transformative events occur. We show that an anti-CD25 antibody and immunotoxin can specifically target and eliminate transduced leukemia cells expressing CD25. In a murine leukemia model, antibody treatment reduced tumor burden 32-fold and increased survival compared with untreated mice. Furthermore, after a bone marrow transplant of therapeutically transduced cells into Fabry mice, antibody treatment reduced the number of retrovirally transduced huCD25-expressing cells in the peripheral blood. A systemic loss of transduced cells with functional consequences was also evident in the liver and spleen. This proof-of-principle study demonstrates that a targeted antibody can reduce tumor burden and selectively clear bicistronically transduced hematopoietic cells that express a target antigen, thus acting as a built-in safety mechanism.

Purification and growth of endothelial progenitor cells from murine bone marrow mononuclear cells

This study reports the culture and purification of murine bone marrow endothelial progenitor cells (EPCs) using endothelial cell-conditioned medium (EC-CM). Endothelial-like cells appeared at day 5 in culture of bone marrow mononuclear cells in the presence of EC-CM in the culture system, and these cells incorporated acetylated low-density lipoproteins (Ac-LDL) and reacted with endothelial-specific Ulex Europaeus Lectin. Continued incubation of these cells at low density with EC-CM for longer than 10 days resulted in the formation of endothelial cell colonies which gave rise to colonies of endothelial progeny and can be passed for many generations in the EC-CM culture system. Cells derived from these colonies expressed endothelial cell markers such as vWF and CD31, incorporated Dil-Ac-LDL, stained positive for Ulex Europaeus Lectin, formed capillary-like structures on Matrigel, and demonstrated a high proliferative capacity in culture. These bone marrow-derived adherent cells were identified as EPCs. The purification and the formation of EPC colonies by using EC-CM were associated with the cytokines secreted in the EC-CM. VEGF, bFGF, and GM-CSF in the EC-CM stimulated the proliferation and growth of EPCs, whereas AcSDKP (tetrapeptide NAc-Ser-Asp-Lys-Pro) in EC-CM suppressed the growth of mesenchymal stem cells (MSC) and fibroblasts. This approach is efficient for isolation/purification and outgrowth of bone marrow EPCs in vitro, a very important cell source in angiogenic therapies and regenerative medicine.

Anti-CD25 Targeted Killing of Bicistronically Transduced Cells: A Novel Safety Mechanism Against Retroviral Genotoxicity

Anti-CD25 Targeted Killing of Bicistronically Transduced Cells: A Novel Safety Mechanism Against Retroviral Genotoxicity

Angelica stimulates proliferation of murine bone marrow mononuclear cells by the MAPK pathway

Murine bone marrow mononuclear cells (MNC) were isolated and co-incubated with Angelica to investigate its effects on bone marrow cells and the underlying mechanism of action. Angelica stimulates MNC proliferation as determined by the 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Our results also suggest that the mechanism of action involves the phosphorylation of ERK1/2 and P38, two key proteins in the MAPK pathway. MAPK inhibitors, PD 98059 and SB 203580, block MNC proliferation caused by Angelica. Taken together, our results show that Angelica induces the proliferation of murine MNC by activating ERK1/2 and P38 MAPK proteins.

Furosin, an ellagitannin, suppresses RANKL-induced osteoclast differentiation and function through inhibition of MAP kinase activation and actin ring formation

Phenolic compounds including tannins and flavonoids have been implicated in suppression of osteoclast differentiation/function and prevention of bone diseases. However, the effects of hydrolysable tannins on bone metabolism remain to be elucidated. In this study, we found that furosin, a hydrolysable tannin, markedly decreased the differentiation of both murine bone marrow mononuclear cells and Raw264.7 cells into osteoclasts, as revealed by the reduced number of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells and decreased TRAP activity. Furosin appears to target at the early stage of osteoclastic differentiation while having no cytotoxic effect on osteoclast precursors. Analysis of the inhibitory mechanisms of furosin revealed that it inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced activation of p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK)/activating protein-1 (AP-1). Furthermore, furosin reduced resorption pit formation in osteoclasts, which was accompanied by disruption of the actin rings. Taken together, these results demonstrate that naturally occurring furosin has an inhibitory activity on both osteoclast differentiation and function through mechanisms involving inhibition of the RANKL-induced p38MAPK and JNK/AP-1 activation as well as actin ring formation.

A Methylcellulose Microculture Assay for the In Vitro Assessment of Drug Toxicity on Granulocyte/macrophage Progenitors (CFU-GM)

In a recent prevalidation study, the use of a methylcellulose colony-forming unit-granulocyte/macrophage (CFU-GM) macroassay for two independent in vitro tests (human and murine cell based) was suggested for quantifying the potential haematotoxicity of xenobiotics. In this paper, we describe the transfer of the macroassay to a 96-well plate microassay, in which the linearity of the response was studied (both in terms of CFU-GM and optical density [OD] versus the number of cells cultured), and the inhibitory concentration (IC) values for doxorubicin, 5-fluorouracil and taxol were determined and compared with those obtained by using the original macroassay. Fresh murine bone marrow and human umbilical cord blood mononuclear cells were used as a source of myeloid progenitors. The cells were cultured in methylcellulose containing granulocyte/macrophage-colony-stimulating factor, and in the presence of increasing drug concentrations. The cloning capacity of the progenitors was measured both as the number of colonies counted manually (CFU-GM), and as OD evaluated with an automated plate reader in an MTT test. Our results show that, in the microassay, up to 20 colonies/well could be easily counted, and that this range (20 to zero) gave a regression line from which IC values were calculated, which were very close to those obtained by using the macroassay (where the range of colony numbers was from 100 to zero). The test did not give good results when the OD (instead of the colony count) was used as the endpoint, because, although a high coefficient of determination was obtained, the OD values ranged from 0.6 to zero and the IC values determined were not comparable to those obtained by manual counts. The use of the microassay dramatically reduces the quantity of methylcellulose needed, and permits hundreds of cultures to be processed in the same experiment, contributing to significant reductions in both the work involved and the cost. A further important benefit is a reduction of the amount of drug needed for testing, which is crucial for screening new molecules, when many different toxicological tests have to be carried out. The microassay is therefore a useful and reproducible tool for screening compounds (chemicals, drugs and xenobiotics) for potential haematotoxicity directly on human myeloid progenitors, and could contribute significantly to reducing the use of animals in toxicity testing.

Ionizing radiation and busulfan inhibit murine bone marrow cell hematopoietic function via apoptosis-dependent and -independent mechanisms

Objective Ionizing radiation (IR) and busulfan (BU) are commonly used as preconditioning regimens for bone marrow transplantation (BMT). We examined whether induction of apoptosis in murine bone marrow (BM) hematopoietic cells contributes to IR- and BU-induced suppression of their hematopoietic function. Methods The hematopoietic functions of hematopoietic stem cells (HSCs) and progenitors were analyzed by the cobblestone area-forming cell (CAFC) assay. Apoptosis was determined by measuring 3,3′-dihexyloxacarbocyanine iodide (DiCO 6) uptake, annexin V staining, and/or sub-G 0/1 cells. Four cell types were studied: murine BM mononuclear cells (BM-MNCs), linage-negative hematopoietic cells (Lin − cells), Lin − Scal + c-kit + cells, and Lin − Scal − c-kit + cells by flow cytometry. Results Exposure of BM-MNCs to IR (4 Gy) or incubation of the cells with BU (30 μM) resulted in a significant reduction in CAFC frequency ( p<0.001). The survival fractions of various day-types of CAFC for the irradiated cells were less than 10%, while that for BU-treated cells was 71.3% on day 7 and progressively declined to 5.3% on day 35. Interestingly, IR significantly induced apoptosis in BM-MNCs, Lin − cells, HSCs, and progenitors, whereas BU failed to increase apoptosis in these cells. In addition, preincubation of BM-MNCs with z-Val-Ala-Asp (OCH3)-fluoromethylketone, methyl ester (z-VAD) attenuated IR-induced reduction in CAFC but not that induced by BU. Conclusion IR and BU differentially suppress the hematopoietic function of HSCs and progenitors by fundamentally different mechanisms. IR inhibits the function primarily by the induction of HSC and progenitor apoptosis. In contrast, BU suppresses HSC and progenitor function via an apoptosis-independent mechanism.

Macrophage-Stimulating Protein Cooperates with Erythropoietin to Induce Colony Formation and MAP Kinase Activation in Primary Erythroid Progenitor Cells

We have shown that Fv2, the Friend virus susceptibility 2 locus, encodes a naturally occurring amino-terminally truncated form of the STK receptor tyrosine kinase (Sf-Stk). Sf-Stk appears to interact with the viral glycoprotein gp55 and drive erythropoietin (Epo)-independent expansion of Friend virus-infected erythroblasts. Presumably, Sf-Stk provides signals that cooperate with EpoR signaling to induce the polyclonal expansion of infected cells. In this report, we show that macrophage-stimulating protein (MSP), the ligand for full-length STK, can also cooperate with Epo to enhance burst-forming units-erythroid (BFU-E) formation. To evaluate the signals induced by MSP/STK in primary erythroid progenitor cells, we adapted a method for the expansion of murine bone marrow mononuclear cells. The expanded progenitor cells express STK and respond to MSP in a colony assay. Furthermore, we demonstrate that low doses of MSP and Epo stimulation of the expanded cells cooperate to induce the phosphorylation of MAP kinase. Using the MEK inhibitor PD98059, we show that the activation of ERK is required for the enhanced BFU-E formation in response to MSP. These findings suggest that MSP has the ability to enhance erythroid colony formation in response to Epo, and that this response is dependent on the ability of MSP to induce the MAP kinase pathway.

GATA-2/estrogen receptor chimera regulates cytokine-dependent growth of hematopoietic cells through accumulation of p21WAF1 and p27Kip1 proteins

GATA-2 is considered to be essential for the development, maintenance, and function of hematopoietic stem cells (HSCs). However, it was also reported that GATA-2 inhibits the growth of HSCs. To examine the role of GATA-2 in the growth of hematopoietic cells, we introduced an estradiol-inducible form of GATA-2 (GATA-2/estrogen receptor [ER]) into interleukin 3 (IL-3)-dependent cell lines, Ba/F3, 32D, and FDC-P1. Estradiol-induced GATA-2 suppressed c-myc mRNA expression and inhibited IL-3-dependent growth in these clones. As for this mechanism, GATA-2 was found to inhibit ubiquitin/proteasome-dependent degradation of p21(WAF1) and p27(Kip1) and to induce their accumulation by repressing the expression of Skp2 and Cul1, both of which are components of the ubiquitin ligase for p21(WAF1) and p27(Kip1). Overexpression of c-myc restored the expression of Skp2 and Cul1 mRNA, reduced the amounts of p21(WAF1) and p27(Kip1) proteins, and canceled GATA-2-induced growth suppression, suggesting that down-regulation of c-myc expression may be primarily responsible for GATA-2-induced growth suppression. Next, we transduced retrovirus containing GATA-2/ER into murine bone marrow mononuclear cells (MNCs) and stem/progenitor (Sca-1(+)Lin(-)) cells. GATA-2/ER suppressed cytokine-dependent growth of MNCs and Sca-1(+)Lin(-) cells by about 70%, which was also accompanied by the reduced expression of c-myc, Skp2, and Cul1 mRNA and the accumulation of p21(WAF1) and p27(Kip1) proteins. In addition, the amount of GATA-2 protein was found to decline in hematopoietic stem/progenitor cells that were promoted to enter cell cycle by the stimulation with cytokines. These results suggest that GATA-2 may regulate expression levels of p21(WAF1) and p27(Kip1), thereby contributing to the quiescence of hematopoietic stem/progenitor cells.

Induction of Multinucleated Giant Cell Formation from In Vitro Culture of Human Monocytes with Interleukin-3 and Interferon-γ: Comparison with Other Stimulating Factors

One of the hallmarks of the granulomatous response to infection is the formation of multinucleated giant cells (MGC.) In an effort to study MGC, we examined the fusion-promoting effects of a variety of stimulating factors on human peripheral blood monocytes cultured on plastic surfaces in serum-supplemented media. MGC formation was minimally to moderately enhanced by interferon-gamma (IFN-gamma), interleukin (IL)-3, granulocyte/macrophage colony-stimulating factor (GM-CSF), 1,25-dihydroxycholecalciferol (1,25-(OH)2D3), retinoic acid (RA), and IL-6. IL-4 (which has been reported to promote MGC formation from murine macrophages) had an inhibitory effect. IFN-gamma was not required for MGC formation but it significantly increased the fusion-promoting activity of GM-CSF, 1,25-(OH)2D3, RA, and IL-6, IL-3, a hematopoietic growth factor, has been recently shown to induce osteoclast formation from murine bone marrow mononuclear cells. The most striking effect was seen with the combination of IL-3 and IFN-gamma. Fusion index is defined as a percentage of nuclei found within MGC, and an index of 67% at 1 wk was found. The formation of some very large cells with 50 to 100 nuclei was noted. Both Langhans' and foreign-body type cells were seen. Transmission electron micrographs clearly demonstrate the absence of plasma membrane between nuclei. Induction of MGC from peripheral human blood monocytes by IL-3 and IFN-gamma provides an in vitro system for the study of the formation and function of these cells.