Treatment Of Bone Marrow Cells Research Articles

The Coumarin Derivative 5'-Hydroxy Auraptene Suppresses Osteoclast Differentiation via Inhibiting MAPK and c-Fos/NFATc1 Pathways.

The phytochemical substances, coumarin derivatives, have demonstrated antiresorptive bone effects by suppressing osteoclast differentiation in vitro and in vivo. Recently, we have identified 5′-hydroxy auraptene (5′-HA), a coumarin derivative isolated from Lotus lalambensis Schweinf, as a novel stimulator for osteoblast differentiation. In this study, we investigated the effect of 5′-HA on osteoclast differentiation of mouse bone marrow (BM) cells. The effect of 5′-HA on BM cell proliferation and osteoclast differentiation was determined by measuring cell viability and tartrate-resistant acid phosphatase (TRAP) enzyme activity, quantification of TRAP+ multinucleated cells (TRAP+MNCs), and quantitative real-time PCR (qPCR) of osteoclastic gene expression. Regulation of NF-κB, c-Fos/NFATc1, and MAPK signaling pathways by 5′-HA during osteoclastogenesis was measured by the NF-κB reporter assay and Western blot analysis. 5′-HA significantly suppresses the receptor activator of NF-κB ligand (RANKL) induced osteoclast differentiation of BM cells in a dose-dependent manner. Consistently, treatment of BM cells with 5′-HA significantly inhibited RANKL-induced activation of NF-κB and c-Fos/NFATc1 pathways in a dose-dependent manner. Furthermore, RANKL-induced phosphorylation of ERK1/2, p-38, and JNK was significantly inhibited by 5′-HA in BM cells. In conclusion, we identified 5′-HA as a novel coumarin derivative that suppresses RANKL-induced osteoclastogenesis via inhibiting c-Fos/NFATc1 and MAPK signaling pathways.

Clostridium perfringens \u03b1-toxin impairs erythropoiesis by inhibition of erythroid differentiation

Clostridium perfringens α-toxin induces hemolysis of erythrocytes from various species, but it has not been elucidated whether the toxin affects erythropoiesis. In this study, we treated bone marrow cells (BMCs) from mice with purified α-toxin and found that TER119+ erythroblasts were greatly decreased by the treatment. A variant α-toxin defective in enzymatic activities, phospholipase C and sphingomyelinase, had no effect on the population of erythroblasts, demonstrating that the decrease in erythroblasts was dependent of its enzymatic activities. α-Toxin reduced the CD71+TER119+ and CD71–TER119+ cell populations but not the CD71+TER119− cell population. In addition, α-toxin decreased the number of colony-forming unit erythroid colonies but not burst-forming unit erythroid colonies, indicating that α-toxin preferentially reduced mature erythroid cells compared with immature cells. α-Toxin slightly increased annexinV+ cells in TER119+ cells. Additionally, simultaneous treatment of BMCs with α-toxin and erythropoietin greatly attenuated the reduction of TER119+ erythroblasts by α-toxin. Furthermore, hemin-induced differentiation of human K562 erythroleukemia cells was impaired by α-toxin, whereas the treatment exhibited no apparent cytotoxicity. These results suggested that α-toxin mainly inhibited erythroid differentiation. Together, our results provide new insights into the biological activities of α-toxin, which might be important to understand the pathogenesis of C. perfringens infection.

Clostridium perfringens α-Toxin Impairs Innate Immunity via Inhibition of Neutrophil Differentiation.

Although granulopoiesis is accelerated to suppress bacteria during infection, some bacteria can still cause life-threatening infections, but the mechanism behind this remains unclear. In this study, we found that mature neutrophils in bone marrow cells (BMCs) were decreased in C. perfringens-infected mice and also after injection of virulence factor α-toxin. C. perfringens infection interfered with the replenishment of mature neutrophils in the peripheral circulation and the accumulation of neutrophils at C. perfringens-infected sites in an α-toxin-dependent manner. Measurements of bacterial colony-forming units in C. perfringens-infected muscle revealed that α-toxin inhibited a reduction in the load of C. perfringens. In vitro treatment of isolated BMCs with α-toxin (phospholipase C) revealed that α-toxin directly decreased mature neutrophils. α-Toxin did not influence the viability of isolated mature neutrophils, while simultaneous treatment of BMCs with granulocyte colony-stimulating factor attenuated the reduction of mature neutrophils by α-toxin. Together, our results illustrate that impairment of the innate immune system by the inhibition of neutrophil differentiation is crucial for the pathogenesis of C. perfringens to promote disease to a life-threatening infection, which provides new insight to understand how pathogenic bacteria evade the host immune system.

Estrogen enhances the functions of CD4(+)CD25(+)Foxp3(+) regulatory T cells that suppress osteoclast differentiation and bone resorption in vitro.

Cross-talk has been shown to occur between the immune system and bone metabolism pathways. In the present study, we investigated the impact of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells on osteoclastogenesis and bone resorption. Treg cells that were isolated and purified from peripheral blood mononuclear cells (PBMCs) of healthy adults inhibited both the differentiation of osteoclasts (OCs) from human embryo bone marrow cells (BMCs) and the pit formation in a dose-dependent manner. In cell cocultures, the production levels of both interleukin-10 (IL-10) and transforming growth factor-beta 1 (TGF-β1) were proportionally upregulated as the ratio of Treg cells to BMCs was increased, and the inhibition of OC differentiation and bone resorption by Treg cells was completely reversed by anti-IL-10 and anti-TGF-β1 antibodies. Treatment of BMC and Treg cell cocultures with 17β-estradiol (E2) at concentrations between 10⁻⁷ and 10⁻⁹ mol/l suppressed OC differentiation and bone resorption more efficiently than it did in cultures of BMCs alone; this enhanced suppression occurred via the stimulation of Treg cell IL-10 and TGF-β1 expression. These data suggest that Treg cells suppress OC differentiation and bone resorption by secreting IL-10 and TGF-β1. E2 enhances the suppressive effects of Treg cells on OC differentiation and bone resorption by stimulating IL-10 and TGF-β1 secretion from these cells. Therefore, Treg cell-derived IL-10 and TGF-β1 are likely involved in the regulation of E2 on bone metabolism and represent potential therapeutic targets for the treatment of postmenopausal osteoporosis (PMO).

Extracellular calcium increases CXCR4 expression on bone marrow‐derived cells and enhances pro‐angiogenesis therapy

Cell surface receptors play major roles in the mobilization and homing of progenitor cells from the bone marrow to peripheral tissues. CXCR4 is an important receptor that regulates homing of leucocytes and endothelial progenitors in response to the chemokine stromal cell-derived factor-1 (SDF-1). Ionic calcium is also known to regulate chemotaxis of selective bone marrow cells (BMCs) through the calcium-sensing receptor, CaR. Here we show that calcium regulates CXCR4 expression and BMC responses to SDF-1. CaCl2 treatment of BMC induced a time- and dose-dependent increase in both the transcription and cell surface expression of CXCR4. BMC subpopulations expressing VEGFR2+, CD34+ and cKit+/Sca-1+ were especially sensitive to calcium. The effects were blocked by calcium influx inhibitors, anti-CaR antibody and the protein synthesis inhibitor cycloheximide, but not by the CXCR4 antagonist AMD3100. Calcium treatment also enhanced SDF-1-mediated CXCR4 internalization. These changes were reflected in significantly improved chemotaxis by SDF-1, which was abolished by AMD3100 and by antibody against CXCR4. Calcium pre-treatment improved homing of CD34+ BMCs to ischemic muscle in vivo, and enhanced revascularization in ischemic mouse hindlimbs. Our results identify calcium as a positive regulator of CXCR4 expression that promotes stem cell mobilization, homing and therapy.

Effect of hypoxic treatment on bone marrow cells that are able to migrate to the injured liver

Restricted numbers and poor regenerative properties limit the use of adult stem cells. We tested the effect of hypoxic treatment as a method by which to increase cell migration. Bone marrow cells (BMCs) were cultured under oxygen saturations of 0.1, 3, and 20% for 24h. After hypoxic treatment, BMCs of apoptotic fraction were decreased. The expression of CXCR4 was noticeably increased in the hypoxia-treated BMCs and their migration in response to SDF-1alpha was enhanced compared with cells cultured under normoxic condition. Hypoxic BMCs had a higher degree of engraftment to the CCl(4)-injured liver than the normoxic cells. Hypoxic treatment of BMCs may have merits in decreasing apoptosis of those cells as well as in enhancing cellular migration to SDF-1alpha, the chemokine which binds to BMCs expressed CXCR4 and to the injured tissue, such as CCl(4) damaged liver.

Activation of protein kinase A (PKA) by 8-Cl-cAMP as a novel approach for antileukaemic therapy.

Activation of PKA by cAMP agonists, such as 8-Cl-cAMP activation, selectively causes rapid apoptosis in v-abl transformed fibroblasts by inhibiting the Raf-1 kinase. Here we investigated whether 8-Cl-cAMP is useful for the treatment of chronic myelogenous leukaemia (CML), which is hallmarked by the expression of the p210bcr/abl oncogene. Autologous bone marrow transplantation is a feasible alternative for patients with no suitable donor, but hampered by the risk of relapse due to the persistence of leukaemia cells in the transplant. To study the effects of 8-Cl-cAMP on primary leukaemic cells, bone marrow cells (BMCs) from eight CML patients (one at diagnosis, three in chronic and four in accelerated phase) were treated. Ex vivo treatment of BMCs obtained in chronic phase of CML with 100 μM 8-Cl-cAMP for 24–48 h led to the selective purging of Philadelphia Chromosome (Ph1 chromosome) without toxic side effects on BMCs from healthy donors as measured by colony-forming unit (CFU) assays. BMCs from patients in accelerated phase showed selective, but incomplete elimination of Ph1 chromosome positive colony forming cells. The mechanism of 8-Cl-cAMP was investigated in FDCP-mix cells transformed by p210bcr/abl, a cell culture model for CML. The results showed that 8-Cl-cAMP reduced DNA synthesis and viability independent of Raf inhibition as Raf inhibitors had no effect. MEK inhibitors interfered with DNA synthesis, but not with viability. In summary, our results indicate that 8-Cl-cAMP could be useful to purge malignant cells from the bone marrow of patients with CML and certain other forms of leukaemias.

Interleukin-4 Inhibits Growth of Multiple Myelomas by Suppressing Interleukin-6 Expression

Unfractionated bone marrow (BM) cells obtained from patients with multiple myeloma (MM) exhibit high levels of interleukin (IL)-6. Secretion of IL-6 by these cells as well as spontaneous plasma cell proliferation can be abrogated by neutralizing anti-IL-6 monoclonal antibody (MoAb). Treatment of BM cells with recombinant human (rh)IL-4 at doses of 50 to 250 U/mL blocked endogenous IL-6 synthesis in a dose-dependent fashion and was associated with significant reduction of plasma cell growth that could be reversed by exogenous rhIL-6. Enrichment of BM cells from MM patients for plasma cells and adherent cells and analysis of IL-6 mRNA in these subpopulations by means of quantitative polymerase chain reaction (PCR) showed that adherent BM cells accounted for most of the synthesis of IL-6 transcripts, whereas plasma cells displayed negligible levels of IL-6 mRNA only. These results suggest therapeutic evaluation of rhIL-4 in patients with plasma cell neoplasms.

Interleukin-4 inhibits growth of multiple myelomas by suppressing interleukin-6 expression

Unfractionated bone marrow (BM) cells obtained from patients with multiple myeloma (MM) exhibit high levels of interleukin (IL)-6. Secretion of IL-6 by these cells as well as spontaneous plasma cell proliferation can be abrogated by neutralizing anti-IL-6 monoclonal antibody (MoAb). Treatment of BM cells with recombinant human (rh)IL-4 at doses of 50 to 250 U/mL blocked endogenous IL-6 synthesis in a dose- dependent fashion and was associated with significant reduction of plasma cell growth that could be reversed by exogenous rhIL-6. Enrichment of BM cells from MM patients for plasma cells and adherent cells and analysis of IL-6 mRNA in these subpopulations by means of quantitative polymerase chain reaction (PCR) showed that adherent BM cells accounted for most of the synthesis of IL-6 transcripts, whereas plasma cells displayed negligible levels of IL-6 mRNA only. These results suggest therapeutic evaluation of rhIL-4 in patients with plasma cell neoplasms.

Depletion of human lymphocytes from peripheral blood and bone marrow by affinity ligands conjugated to agarose-polyacrolein microsphere beads.

Protein-A or goat anti-mouse-Ig (GAMIg) covalently bound to agarose-polyacrolein microsphere beads (APAMB) were employed for the removal of T cells from human peripheral blood leukocytes (PBL) and bone marrow (BM). The cell suspensions were treated with a monoclonal anti-T cell antibody (Leu-1) or monoclonal antilymphocyte antibody (CAMPATH-1) and passed through the conjugated APAMB columns. Cell separation efficacy was determined by assaying the number and function of T cells in the final cell preparation in comparison with a sample of unseparated cells. The number of cells that form rosettes (E-RFC) with sheep red blood cells (SRBC) in a sample of PBL treated with anti-Leu-1 antibodies and subsequently passed once through GAMIg-conjugated APAMB dropped from a range of 41.5-86.0% to a range of 1.6-13.3%. The in vitro response to concanavalin-A (Con-A) dropped to a range of 0.7-27.2% (GAMIg) and a range of 1.2-21.8% (protein-A column) of the response of untreated PBL. Treatment with CAMPATH-1 antibody and passage through a protein-A-conjugated APAMB reduced E-RFC from a range of 55.6-57.4% to a range of 3.2-3.9% and abolished the Con-A induced proliferative responsiveness to background levels. Treatment of BM cells with CAMPATH-1 and passage of the cells through either GAMIg or protein-A conjugated APAMB columns resulted in reduction of E-RFC from a range of 12.4-17.7% to a range of 0-1% and from a range of 17.7-19% to a range of 1.6-3.2%, respectively. Viability of BM precursors, determined by the CFU-GM assay in semisolid medium, was not affected by these cell separation procedures. The data suggest that protein-A or GAMIg-conjugated APAMB columns may be a useful tool for separation of BM cell suspensions into specific cell subsets that can be defined by monoclonal antibodies.

The immunoregulatory role of bone marrow: II. Characterization of a suppressor cell inhibiting the in vitro antibody response

The addition of bone marrow cells (BMC) to spleen cell cultures suppressed the antibody response in a dose-dependent manner. This suppression required viable cells. Treatment of BMC with anti-thymocyte serum did not affect the suppressive activity and BMC, but not spleen cells, from nude mice inhibited the antibody response to the same degree as marrow from normal littermates. BMC which had been depleted of macrophages with antimacrophage serum or carbonyl iron showed increased suppressor activity. Furthermore, fractionation of BMC by velocity sedimentation and resetting revealed the suppressor cell to be a medium-to-large Fc receptor-positive lymphocyte. Absence of detectable B or T cell markers on the suppressor cell indicates this cell to be an Fc-positive null lymphocyte, possibly a precursor cell, which inhibits the response of mature lymphocytes