Murine Bone Marrow Mononuclear Cells Research Articles

Effect of phosphodiester composition in polyphosphoesters on the inhibition of osteoclastic differentiation of murine bone marrow mononuclear cells

Osteoporosis is a common bone disorder characterized by reduced bone density and increased risk of fractures. The modulation of bone cell functions, particularly the inhibition of osteoclastic differentiation, plays a crucial role in osteoporosis treatment. Polyphosphoesters (PPEs) have shown the potential in reducing the function of osteoclast cells, but the effect of their chemical structure on osteoclastic differentiation remains largely unexplored. In this study, we evaluated the effect of PPE’s chemical structure on the inhibition of osteoclastic differentiation of murine bone marrow mononuclear cells (BMNCs). PPEs containing phosphotriester and phosphodiester units at varying compositions were synthesized. Cytotoxicity testing confirmed the biocompatibility of the copolymers at concentrations below 0.5 mg/mL. Isolated from long bones, BMNCs were cultured in a differentiation medium supplemented with different PPE concentrations. Osteoclast formation was assessed through tartrate-resistant acid phosphatase and phalloidin staining. A significant decrease in the size of osteoclast cells formed upon BMNC contact with PPEs was observed, with a more pronounced effect observed at higher PPE concentrations. In addition, an increased composition of phosphodiester units in the PPEs yielded a decreased density of differentiated osteoclasts. Furthermore, real-time PCR analysis of major osteoclastic markers provided gene expression data that correlated with microscopic observations, confirming the effect of phosphodiester units in suppressing osteoclast differentiation of BMNCs from the early stages. These findings highlight the potential of PPEs as polymers are capable of modulating bone cell functions through their chemical structures.

Checkpoint Inhibitor PD-1H/VISTA Mediates MMP-13 Induced Osteoclast Activation and Multiple Myeloma Bone Disease

Introduction Multiple myeloma (MM) bone disease remains one of the most devastating complications of this incurable cancer, causing bone fractures, pain, mobility issues and neurological deficits. MM cells produce osteoclast-activating factors that induce osteoclast activation, thereby leading to excessive bone resorption and lytic bone lesions1. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor that is highly secreted by MM cells. MMP-13 induces osteoclast fusion and bone-resorption via a mechanism independent of its proteolytic activity2. We recently reported that MMP-13 binds to checkpoint inhibitor programmed death-1 homolog (PD-1H/VISTA), a surface receptor that is expressed in osteoclasts at high levels3. Binding of MMP-13 to PD-1H/VISTA induces osteoclast fusion and bone resorption activity whereas knockdown or knockout of PD-1H/VISTA largely block MMP-13 mediated effects on osteoclasts3. However, the function of PD-1H inMM bone disease in vitro or in vivo has not been previously defined. Methods and Results To confirm the role of PD-1H in MMP-13 induced bone disease in MM, we first conducted MM-osteoclast trans-well co-culture assay using murine MM cell line, 5TGM1 cells, and bone marrow mononuclear cells from Pd-1h-/- or wild type (WT) mice. 5TGM1 control cells or MMP-13 knockdown 5TGM1 cells were seeded in the upper wells of the transwell plates; while WT or Pd-1h-/- bone marrow mononuclear cells were seeded in the lower wells and cultured for osteoclast differentiation assessed by TRAP staining. Results show that 5TGM1 induced differentiation of WT osteoclasts with significantly increased osteoclast size and nuclei number/osteoclast. Consistent with our previous results2, MMP-13 knockdown blocked the 5TGM1 MM cells-induced activation of WT osteoclasts. In contrast, neither 5TGM1 MM cells nor MMP-13 knockdown cells had significant effects on Pd-1h-/- osteoclasts. Hence, knockout of Pd-1h abrogated MMP-13 mediated MM induction of osteoclasts, indicating that MMP-13/PD-1H interactions are critically involved in MM-induced osteoclast activation. The in vivo role of PD-1H in MM bone disease was investigated using the intratibial 5TGM1 Rag2-/- MM bone disease mice model2. For this purpose, Pd-1h-/-Rag2-/- mice were generated by crossbreeding C57BL/6 Pd-1h-/- with C57BL/6 Rag2-/- mice. 3x105 firefly luciferase expressing 5TGM1 cells (5TGM1-luc) were intratibially injected into age and sex-paired Rag2-/- or Pd-1h-/-Rag2-/- mice (N=5). Tumor progression was monitored by weekly bioluminescence imaging (BLI). 3 weeks after tumor inoculation, tibiae were harvested for quantitative micro-CT, followed by histological analysis. Histological staining showed that intratibial injection of 5TGM1-luc MM cells induced extensive lytic lesions and trabecular bone loss in Rag2-/- mice. In contrast, in Pd-1h-/-Rag2-/- mice,the bone structure was maintained with markedly less bone loss. Morphological analyses of trabecular bone across proximal tibiae further indicated that in Rag2-/- mice, 5TGM1 induced significant changes in bone microarchitecture, with decreased bone volume fraction (bone volume/tissue volume), connective density, trabecular bone numbers, and trabecular bone thickness, as well as increased trabecular bone spacing (Table 1). In contrast, in Pd-1h-/-Rag2-/- mice, 5TGM1 failed to induce significant loss of trabecular bone, confirming the critical role of PD-1H in MM induced bone disease in vivo. Conclusions Taken together, our study, for the first time, reveal that checkpoint inhibitor PD-1H/VISTA is the critical receptor for MMP-13 in osteoclasts, thereby mediating MMP-13-induced osteoclast fusion, activation and bone resorption. MM-induced trabecular bone loss was significantly lower in Pd-1h-/-mice, demonstrating that PD-1H/VISTA plays a critical role in MMP-13-induced MM bone disease. Given the checkpoint role of PD-1H/VISTA in cancer immunosuppression, we further posit that targeting the interaction of MMP-13 and PD-1H may represent a novel therapeutic strategy to treat MM bone disease and modulate the MM immune environment.

A 3D construct based on mesenchymal stromal cells, collagen microspheres and plasma clot supports the survival, proliferation and differentiation of hematopoietic cells in vivo.

The hematopoietic niche is a specialized microenvironment that supports the survival, proliferation and differentiation of hematopoietic stem progenitor cells (HSPCs). Three-dimensional (3D) models mimicking hematopoiesis might allow in vitro and in vivo studies of the hematopoietic (HP) process. Here, we investigate the capacity of a 3D construct based on non-adherent murine bone marrow mononuclear cells (NA-BMMNCs), mesenchymal stromal cells (MSCs) and collagen microspheres (CMs), all embedded into plasma clot (PC) to support in vitro and in vivo hematopoiesis. Confocal analysis of the 3D hematopoietic construct (3D-HPC), cultured for 24h, showed MSC lining the CM and the NA-BMMNCs closely associated with MSC. In vivo hematopoiesis was examined in 3D-HPC subcutaneously implanted in mice and harvested at different intervals. Hematopoiesis in the 3D-HPC was evaluated by histology, cell morphology, flow cytometry, confocal microscopy and hematopoietic colony formation assay. 3D-HPC implants were integrated and vascularized in the host tissue, after 3months of implantation. Histological studies showed the presence of hematopoietic tissue with the presence of mature blood cells. Cells from 3D-HPC showed viability greater than 90%, expressed HSPCs markers, and formed hematopoietic colonies, in vitro. Confocal microscopy studies showed that MSCs adhered to the CM and NA-BMMNCs were scattered across the 3D-HPC area and in close association with MSC. In conclusion, the 3D-HPC mimics a hematopoietic niche supporting the survival, proliferation and differentiation of HSPCs, in vivo. 3D-HPC may allow evaluation of regulatory mechanisms involved in hematopoiesis.

Murine osteoclastogenesis suppression using conditioned media produced by melanoma or activated and non-activated Jurkat-E6 cells.

Conditioned medium (CM) (cell secretome) is a cocktail of growth factors, cytokines, and other soluble mediators secreted by cells into a culture medium. These growth factors are fundamental in many cellular processes such as cell growth, differentiation, and others and the composition of these factors is individual for each cell type. Osteoclasts are large multinucleated cells that are responsible for bone resorption. Immune and cancer cells are known to produce different growth factors, which are able to induce or inhibit osteoclast differentiation. Herein, we evaluated the effect of CM obtained from the supernatant of activated and non-activated Jukart-E6 cells, as well as from one murine (B16-F10) and one human melanoma cell line (SK-MEL-28). To induce osteoclast differentiation, murine bone marrow mononuclear cells were cultured in the presence and absence of differentiation factors (DF), such as macrophage colony-stimulating factor, prostaglandin E2, receptor activator of nuclear factor-κB ligand, and CM. We measured the concentration of interleukin 6, tumor necrosis factor-α and interferon γ (IFN-γ) in CM that can inhibit or induce osteoclastogenesis. Our study demonstrated that CM obtained from each cell line suppresses or inhibits osteoclasts formation at early and intermediate stages of differentiation in the absence or presence of DF. CM obtained from activated Jurkat-E6 cells demonstrates a stronger effect when compared with CM from naïve Jurkat-E6 cells or human and murine melanoma cells. Moreover, CM obtained from activated Jurkat-E6 cells shows higher secretion of IFN-γ, which is an inhibitor of osteoclastogenesis, in comparison with CM obtained from the three other cell lines. On the other hand, CM derived from B16-F10 cells showed a smaller inhibitory effect when compared with CM derived from the other cells.

Novel Evidence That Extracellular Adenosine Triphosphate (ATP), As a Purinergic Signaling Mediator, Activates Mobilization By Engaging a P2X4 Ligand-Gated Cation Channel Receptor Expressed on the Surface of Hematopoietic and Innate Immunity Cells

Background . Adenosine triphosphate (ATP) is an important nucleotide involved in intracellular energy transfer, but it is also released from activated cells into the extracellular space as a crucial component of the purinergic signaling network. Purinergic signaling is an ancient form of extracellular signaling that is mediated by ATP and other extracellular nucleotides (EXNs). Purinergic receptors for EXNs are expressed on the surface of all cells in the body; are represented by several families of P1, P2X, and P2Y receptors; and are among the most abundant receptors in living organisms. Of all these receptors, the P2X receptor family is most highly specific for ATP signaling and consists of seven members (P2X1-7). We recently reported that by activating the P2X7 receptor ATP activates the Nlrp3 inflammasome in hematopoietic cells and significantly enhances G-CSF-induced mobilization of hematopoietic stem progenitor cells (HSPCs, Leukemia 2018, 32:1920-1931). Hypothesis. Since P2X7-KO mice still mobilize some HSPCs, we hypothesized that, in addition to the P2X7 receptor, other receptors from this family also play a role in the mobilization process.Materials and Methods. RT-PCR and FACS were employed to phenotype human and murine bone marrow (BM) mononuclear cells (MNCs) as well as sorted HSCs for expression of P2X receptors. Mobilization studies were performed in wild type mice in which the P2X4 receptor was blocked by the specific small-molecule inhibitor PSB12054 and in P2X7-KO animals. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. We also evaluated activation of the Nlrp3 inflammasome as well as other inflammasomes expressed in hematopoietic cells, including Aim2, Nlrp1a, Nlrp1b, and Nlrc4. Results. We found that, of all the receptors of the P2X family, two members, P2X7 and P2X4, are highly expressed on the surface of murine and human MNCs and HSPCs (Figure 1a). Activation of both receptors on the surface of innate immunity CD11b+Gr-1+ cells resulted in activation of the Nlrp3, Nlrp1a, Nlrp1b, and Aim2 inflammasomes. Inhibition of the P2X4 receptor by a specific small-molecule inhibitor decreased G-CSF- and AMD3100-induced mobilization in wild type mice (Figure 1b). Interestingly, we observed differences during AMD3100 mobilization, namely that while P2X7-KO deficiency resulted in decreased G-CSF-induced mobilization and did not affect AMD3100 mobilization efficiency, P2X4 inhibition, by contrast, led to a profound decrease in both G-CSF- and AMD3100-induced mobilization efficiency. Conclusions. Our results further support an important role for purinergic signaling in the mobilization of HSPCs. We also demonstrated that P2X7 and P2X4 receptors, which are highly expressed on human and murine hematopoietic cells, are involved in G-CSF-induced mobilization by activating the Nlrp3, Nlrp1a, Nlrp1b and Aim2 inflammasomes. By contrast, AMD3100-induced mobilization requires expression of P2X4 but not P2X7 receptors, which implies the involvement of different mechanisms and indicates that P2X7 deficiency can be compensated by the P2X4 receptor. Currently, we are performing RNASeq studies to identify differences in the G-CSF- and AMD3100-induced pathways in the mobilization process. These investigations will shed more light on the molecular pathways involved in the egress of HSPCs from BM into PB and will help to design better mobilization protocols in cases of poor mobilizers. Figure 1 Disclosures No relevant conflicts of interest to declare.

Knockdown of Bone Morphogenetic Proteins Type 1a Receptor (BMPR1a) in Breast Cancer Cells Protects Bone from Breast Cancer-Induced Osteolysis by Suppressing RANKL Expression

Background/Aims: Bone morphogenetic proteins (BMPs) and BMP receptors widely participate in osteolytic metastasis of breast cancer, while their role in tumor-stromal interaction is largely unknown. In this study, we investigated whether BMP receptor type 1a (BMPR1a) can alter the interaction between metastatic cancer cells and osteoclast precursors. Methods: Adenovirus-mediated RNA interference was used to interrupt target genes of human breast cancer cell lines and nude mice were injected intratibially with the cancer cells. Tumor-bearing mice were examined by bioluminescence imaging and microCT. Sections of metastatic legs were measured by a series of staining methods. Murine bone marrow mononuclear cells or RAW264.7 cells were cultured with conditioned media of breast cancer cells. RT-PCR, Western blotting and ELISA were used to test mRNA and protein expressions of target molecules. Results: Expression of BMPR1a of MDA-MB-231-luc cells at tumor-bone interface was apparently stronger than that of cancer cells distant from the interface. Mice injected with BMPR1a-knockdown MDA-MB-231-luc cells showed reduced tumor growth and bone destruction compared with control groups. Knockdown (KD) of BMPR1a of MDA-MB-231-luc cells or MCF-7 cells decreased the level of receptor activator for NF-κB ligand (RANKL). Level of RANKL in MDA-MB-231-luc cells or MCF-7 cells was reduced by p38 inhibitor. Compared with control group, knockdown of p38 of breast cancer cells decreased cancer-induced osteoclastogenesis. Conclusion: Knockdown of BMPR1a of breast cancer cells suppresses their production of RANKL via p38 pathway and inhibits cancer-induced osteoclastogenesis, which indicates that BMPR1a might be a possible target in breast cancer-induced osteolytic metastasis.

EphA receptors and ephrin-A ligands are upregulated by monocytic differentiation/maturation and promote cell adhesion and protrusion formation in HL60 monocytes

BackgroundEph signaling is known to induce contrasting cell behaviors such as promoting and inhibiting cell adhesion/spreading by altering F-actin organization and influencing integrin activities. We have previously demonstrated that EphA2 stimulation by ephrin-A1 promotes cell adhesion through interaction with integrins and integrin ligands in two monocyte/macrophage cell lines. Although mature mononuclear leukocytes express several members of the EphA/ephrin-A subclass, their expression has not been examined in monocytes undergoing during differentiation and maturation.ResultsUsing RT-PCR, we have shown that EphA2, ephrin-A1, and ephrin-A2 expression was upregulated in murine bone marrow mononuclear cells during monocyte maturation. Moreover, EphA2 and EphA4 expression was induced, and ephrin-A4 expression was upregulated, in a human promyelocytic leukemia cell line, HL60, along with monocyte differentiation toward the classical CD14++CD16− monocyte subset. Using RT-PCR and flow cytometry, we have also shown that expression levels of αL, αM, αX, and β2 integrin subunits were upregulated in HL60 cells along with monocyte differentiation while those of α4, α5, α6, and β1 subunits were unchanged. Using a cell attachment stripe assay, we have shown that stimulation by EphA as well as ephrin-A, likely promoted adhesion to an integrin ligand-coated surface in HL60 monocytes. Moreover, EphA and ephrin-A stimulation likely promoted the formation of protrusions in HL60 monocytes.ConclusionsNotably, this study is the first analysis of EphA/ephrin-A expression during monocytic differentiation/maturation and of ephrin-A stimulation affecting monocyte adhesion to an integrin ligand-coated surface. Thus, we propose that monocyte adhesion via integrin activation and the formation of protrusions is likely promoted by stimulation of EphA as well as of ephrin-A.

Autophagy Promotes the Repair of Radiation-Induced DNA Damage in Bone Marrow Hematopoietic Cells via Enhanced STAT3 Signaling.

Autophagy protects hematopoietic cells from radiation damage in part by promoting DNA damage repair. However, the molecular mechanisms by which autophagy regulates DNA damage repair remain largely elusive. Here, we report that this radioprotective effect of autophagy depends on STAT3 signaling in murine bone marrow mononuclear cells (BM-MNCs). Specifically, we found that STAT3 activation and nuclear translocation in BM-MNCs were increased by activation of autophagy with an mTOR inhibitor and decreased by knockout of the autophagy gene Atg7. The autophagic regulation of STAT3 activation is likely mediated by induction of KAP1 degradation, because we showed that KAP1 directly interacted with STAT3 in the cytoplasm and knockdown of KAP1 increased the phosphorylation and nuclear translocation of STAT3. Subsequently, activated STAT3 transcriptionally upregulated the expression of BRCA1, which increased the ability of BM-MNCs to repair radiation-induced DNA damage. This novel finding that activation of autophagy can promote DNA damage repair in BM-MNCs via the ATG-KAP1-STAT3-BRCA1 pathway suggests that autophagy plays an important role in maintaining genomic integrity of BM-MNCs and its activation may confer protection of BM-MNCs against radiation-induced genotoxic stress.

Transient Ex Vivo Inhibition of Heme Oxygenase 1 (HO-1) in Hematopoietic Stem/Progenitor Cells (HSPCs) By Small-Molecule Inhibitors Enhances Their Migratory Responsiveness to Bone Marrow (BM)-Secreted Chemoattractants a Novel and Simple Strategy to Improve Homing of HSPCs

Background. Heme oxygenase 1 (HO-1) is an inducible stress-response enzyme that not only catalyzes the degradation of heme (e.g., released from erythrocytes) but also has an important function in various physiological and pathophysiological states associated with cellular stress, such as ischemic/reperfusion injury. HO-1 has a well-documented anti-inflammatory potential and inhibits complement cascade (ComC)-mediated inflammatory responses. Moreover, HO-1 has been reported to have a negative effect on adhesion and migration of neutrophils in acute inflammation in a model of peritonitis. Radiation chimeras created after transplantation with HSPCs having a mutation in one of the alleles of HO-1 engrafted much faster; however, a persistent decrease in HO-1 activity in these animals resulted in their enhanced sensitivity to stress and susceptibility to irradiation (Blood 2008, 112, 4494-4502). Moreover, we recently demonstrated that HO-1-deficient HSPCs show enhanced in vitro migration up an SDF-1 gradient (Stem Cell Rev & Rep. 2015, 11, 110-118).Hypothesis. Based on these findings, we hypothesized that transient inhibition of HO-1 by non-toxic, small-molecule inhibitors would enhance in vivo migration of HSPCs to bone marrow (BM)-derived chemoattractants and thus would facilitate their homing and accelerate hematopoietic recoveryMaterials and Methods. To address this issue, we first generated several human hematopoietic cell lines in which HO-1 was upregulated or downregulated. We also exposed murine and human BM-derived cells to small-molecule inhibitors or activators of HO-1 and performed dose and timing toxicity studies. Next, murine BM mononuclear cells (MNCs) and human umbilical cord blood (UCB) MNCs were exposed to the small-molecule HO-1 inhibitor Sn(IV) protoporphyrin IX dichloride (SnPP) and tested for their chemotactic response in Transwell migration assays to all currently known HSPC chemoattractants, including stromal-derived factor 1 (SDF-1), sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), and the extracellular nucleotides ATP and UTP. For in vivo assays, lethally irradiated mice were transplanted with BM MNCs exposed or not exposed to SnPP, and in recipient animals we evaluated i) the number of day-12 colony-forming units in spleen (CFU-S) and colony-forming units for granulocyte/macrophage (CFU-GM) progenitors in BM and ii) the kinetics of peripheral blood (PB) count recovery by measuring the number of leucocytes, lymphocytes, and platelets. We also performed competitive repopulation studies with a limited number of transplanted BM MNCs using the CD45.1 and CD45.2 congenic mouse models.Results and Conclusions. We demonstrate here that HO-1 is a negative regulator of HSPC migration, and thus, by transiently inhibiting its activity in HSPCs with the non-toxic small-molecule inhibitor (SnPP), it is possible to accelerate homing and subsequent engraftment of HSPCs. We propose that this simple and inexpensive strategy could be employed in the clinical setting to improve seeding efficiency of transplanted HSPCs and their engraftment, particularly in those situations in which the number of HSPCs available for transplant is limited (e.g., from UCB or grafts harvested from poor mobilizers). DisclosuresNo relevant conflicts of interest to declare.

A simple in vitro method for evaluating dendritic cell-based vaccinations.

BackgroundDendritic cell (DC) therapy is a promising therapy for cancer-targeting treatments. Recently, DCs have been used for treatment of some cancers. We aimed to develop an in vitro assay to evaluate DC therapy in cancer treatment using a breast cancer model.MethodsDCs were induced from murine bone marrow mononuclear cells in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with GM-CSF (20 ng/mL) and IL-4 (20 ng/mL). Immature DCs were primed with breast cancer stem cell (BCSC)-derived antigens. BCSCs were sorted from 4T1 cell lines based on aldehyde dehydrogenase expression. A mixture of DCs and cytotoxic T lymphocytes (CTLs) were used to evaluate the inhibitory effect of antigen-primed DCs on BCSCs. BCSC proliferation and doubling time were recorded based on impedance-based cell analysis using the xCELLigence system. The specification of inhibitory effects of DCs and CTLs was also evaluated using the same system.ResultsThe results showed that impedance-based analysis of BCSCs reflected cytotoxicity and inhibitory effects of DCs and CTLs at 72 hours. Differences in ratios of DC:CTL changed the cytotoxicity of DCs and CTLs.ConclusionThis study successfully used impedance-based cell analysis as a new in vitro assay to evaluate DC efficacy in cancer immunotherapy. We hope this technique will contribute to the development and improvement of immunotherapies in the near future.

The tissue inhibitor of metalloproteinases-1 improves migration and adhesion of hematopoietic stem and progenitor cells

Homing and engraftment of hematopoietic stem and progenitor cells (HSPCs) during bone marrow transplantation are critically dependent on integrins such as β1-integrin. In the present study, we show that β1-integrin and the tetraspanin CD63 form a cell surface receptor complex for the soluble serum protein tissue inhibitor of metalloproteinases-1 (TIMP-1) on human CD34⁺ HSPCs. Through binding to this receptor complex, TIMP-1 activates β1-integrin, increases adhesion and migration of human CD34⁺ cells, and protects these cells from induced apoptosis. TIMP-1 stimulation in murine bone marrow mononuclear cells also promotes migration and adhesion; this is associated with augmented homing of murine mononuclear cells and of murine LSK⁺ cells during bone marrow transplantation. These results not only indicate that TIMP-1 is conducive to HSPC homing; they also identify CD63 and β1-integrin as a TIMP-1 receptor complex on HSPCs.

Regulation of Flvcr by Macrophages in Response to Lipopolysaccharide

Abstract 2111Pathogenic bacteria must acquire iron from their hosts to survive and have evolved multiple mechanisms to capture iron or iron-containing heme from the bloodstream or tissues. In response, mammals have developed defense mechanisms to keep iron from pathogens. For example, in response to inflammatory cytokines, hepcidin secreted by the liver binds to the iron exporter ferroportin (FPN1), leading to FPN1 internalization and degradation, decreasing gastrointestinal iron absorption and increasing macrophage iron storage. Much of the body's iron stores are complexed in heme. The Feline leukemia virus, subgroup C (FeLV-C) receptor, FLVCR, is a heme export protein. We showed previously that FLVCR is required for the normal development of the erythroid [Science (2008)319:825] and T cell lineages [Blood (ASH Annual Meeting Abstracts)114:913,2009].Although macrophages express high levels of FLVCR, the role of FLVCR in regulating heme-iron after infection remains unexplored. Other heme regulatory proteins, such as heme oxygenase-1 (HMOX1), a heme-degrading enzyme, are known to be transcriptionally regulated in macrophages in response to infection. We hypothesized that macrophages dynamically regulate Flvcr in response to bacterial infection. To test this hypothesis, we stimulated J774, a murine macrophage cell line, with lipopolysaccharide (LPS from E. coli O111:B4) at varying concentrations and durations. LPS, an outer membrane component from gram-negative bacteria, binds to Toll-like receptor 4 (TLR4) on macrophages and activates downstream signaling pathways. Using multiplex quantitative reverse transcription polymerase chain reaction (qRT-PCR), we measured mRNA levels of Flvcr, Hmox1, and Fpn1. We found that J774 cells down-regulated Flvcr transcript levels in response to LPS with a maximal decrease (69%) seen at 6–8 hours of stimulation. While the extent of Flvcr down-regulation was dose-responsive, a significant decrease (57%) occurred even with the lowest LPS dose (10 ng/ml). Macrophages decreased Fpn1 expression (71%) and increased Hmox1 expression (55%) in response to LPS stimulation as previously reported. Similar results were obtained with LPS from a different bacterial source (Salmonella minnesota Re595). We also performed these studies using primary macrophages cultured from murine bone marrow mononuclear cells and observed a similar decrease in Flvcr and Fpn1 (64 and 72%) and an increase in Hmox1 (40%) transcripts after stimulation with both O111:B4 and Re595 LPS.While Fpn1 transcriptional regulation by heme and oxidative stress has been studied, the mechanism by which LPS regulates Fpn1 transcription is less clear. The similar pattern and kinetics of LPS-induced Flvcr and Fpn1 expression changes raise the possibility that the same regulatory mechanism is responsible. Analysis of the human and mouse Flvcr promoter regions revealed several putative LPS downstream transcription factor binding sites including NF-κB, AP1, and C/EBPβ. In addition to transcriptional regulation, LPS downstream signaling could alter Flvcr and Fpn1 mRNA stability and translation, so we compared the 5’ untranslated regions (UTR) and 3’UTR of murine Flvcr and Fpn1. We found little similarity between the 5’UTR of Flvcr and the 5’UTR of Fpn1, known to contain an iron-responsive element (IRE) and be regulated by iron via iron regulatory proteins (IRP). However, alignment of the 3’UTR from Flvcr and Fpn1 showed similarity (pair wise score 65). Both the Flvcr and Fpn1 3’UTR are predicted to have a high degree of secondary structure based on their large negative fold energies (−421.25 and −300.74 kcal/mol), further suggesting that these 3’UTR may have a regulatory function. Studies are underway to determine the roles of the Flvcr promoter, 5’UTR, and 3’UTR in LPS-induced down-regulation.This work suggests that LPS-induced down-regulation of Flvcr and Fpn1 might act in concert to decrease heme and iron export from macrophages and sequester iron from bacterial pathogens. Heme export control through FLVCR could serve as a novel mechanism of iron regulation in response to infection. Disclosures:No relevant conflicts of interest to declare.

In vitro reconstruction of a three-dimensional mouse hematopoietic microenvironment in the pore of polyurethane foam

Hematopoietic stem cells exist in specific niches in the bone marrow, and generate either more stem cells or differentiated hematopoietic progeny. In such microenvironments, cell-cell and cell-matrix interactions are as important as soluble factors such as cytokines. To provide a similar environment for in vitro studies, a three-dimensional culture technique is necessary. In this manuscript, we report the development of a three-dimensional culture system for murine bone marrow mononuclear cells (mBMMNCs) using polyurethane foam (PUF) as a scaffold. The mBMMNCs were inoculated into two kinds of PUF disks with different surface properties, and cultured without exogenous growth factors. After seeding the inside of the PUF pores with mBMMNCs, PUF disks were capable of supporting adherent cell growth and continuous cell production for up to 90days. On days 21-24, most nonadherent cells were CD45 positive, and some of the cells were of the erythroid type. From comparisons of the cell growth in each PUF material, the mBMMNC culture in PUF-W1 produced more cells than the PUF-R4 culture. However, the mBMMNC culture in PUF-W1 had no advantages over PUF-R4 with regard to the maintenance of immature hematopoietic cells. The results of scanning electron microscopy and colony-forming assays confirmed the value of the different three dimensional cultures.

Abstract 4953: Del-1, a New Beta2-integrin Ligand, Which Inhibits Mobilization, Adhesion and Homing of Progensitor Cells

Progenitor cells (PC) are recruited to ischemic tissues and improve neovascularization. Beta2-integrins are essential for adhesion and homing of PC to ischemic tissues. Developmental Endothelial Locus-1 (Del-1) is an extracellular matrix protein that binds alphaVbeta3-and beta2-integrins and is up-regulated during ischemia. Therefore, we investigated the role of Del-1 for angiogenesis and homing functions of PC. Del-1-deficient mice (Del-1−/−) displayed a significantly increased angiogenic response in ischemic muscles in comparison to the wild type (WT) mice in the model of hind limb ischemia. However, when we assessed the role of Del-1 in HUVEC in vitro, silencing of Del-1 by siRNA did not affect angiogenic sprouting. Moreover, the ischemic muscles of Del-1−/− mice displayed a higher infiltration with CD45+ cells than WT mice, suggesting that Del-1-deficiency may induce activation of homing of PC and inflammatory cells to ischemic tissues. Interestingly, in adhesion assays human endothelial progenitor cells (EPC) and murine Lin- PC bound to Del-1 via beta2-integrins, but not via the alphaVbeta3-integrins. Furthermore, soluble Del-1 significantly inhibited the adhesion of EPC to HUVEC monolayers and to the major beta2-integrin-ligand, ICAM-1. Moreover, WT murine bone marrow (BM) mononuclear cells displayed higher adhesion rates on Del-1-deficient murine lung endothelial cells (LEC) than on WT LEC. In order to investigate the role of Del-1 for in vivo homing of PC, we intravenously injected murine fluorescence-labeled WT Lin- BM PC in WT and Del-1−/− mice 2 days after the induction of hind limb ischemia. Interestingly, the homing of injected Lin- cells to ischemic muscles was significantly increased in Del-1−/− in comparison to WT mice (200±30 % increase). Moreover, Del-1−/− mice displayed a significantly increased GCSF-induced mobilization of Sca1+ cKit+ PC in the peripheral blood in comparison to WT mice. Taken together, Del-1 is a new beta2-integrin ligand, which blocks PC mobilization and beta2-integrin-dependent adhesion and homing of PC to ischemic tissues. It is conceivable, that endogenous Del-1 may reduce ischemia-induced neovascularization through an inhibitory effect on the beta2-integrins of PC and inflammatory cells.

Application of magnetic poly(styrene–glycidyl methacrylate) microspheres for immunomagnetic separation of bone marrow cells

Surface-functionalized magnetic poly(styrene–glycidyl methacrylate) (PS–GMA) microspheres were prepared and coupled with Sca-1 antibody for cell selection from murine bone marrow mononuclear cells (MNCs). Biotinylated Sca-1 antibody could be directly coupled to avidin-bound magnetic microspheres. Alternatively, oxidized goat anti-mouse antibody was covalently bound onto the amino group-containing magnetic microspheres in a site-directed manner, and the resultant conjugate was coupled with non-modified Sca-1 antibody. Using the indirect antibody-bound magnetic microspheres, the purity of isolated Sca-1 + cells increased with bead-to-cell ratio. Using a bead-to-cell ratio of 10 beads/cell, a purity of 85% Sca-1 + cells corresponding to a 17-fold enrichment was achieved.

GM-CSF accelerates proliferation of endothelial progenitor cells from murine bone marrow mononuclear cells in vitro

Objective: To test whether the GM-CSF accelerates the proliferation of bone marrow endothelial progenitor cells (BM EPCs). Methods: BM EPCs were induced by endothelial cell conditioned medium (EC-CM). The effect of different concentrations of GM-CSF on the proliferation of BM EPCs was evaluated by the formation of EC-cols, MTT assay, and cell cycle assay. The single progenitor cell growth curves were quantified. Results: The data indicated that GM-CSF accelerated the proliferation of BM EPCs both in colony numbers and colony size. MTT confirmed the effect of GM-CSF on accelerating the proliferation of BM EPCs. The single colony experiments showed that EC-cols expressed different proliferation capacity, suggesting that the EC-cols with different proliferation potentials might have been derived from different levels of immature progenitors. The cell cycle assay showed that the rate of cells entering into S phase was 9.3% in the group treated with GM-CSF and 2.1% in the controls. Furthermore, these cells displayed the specific endothelial cell markers and formed capillary-like structures. Conclusions: GM-CSF accelerates proliferation of BM EPCs. The potential beneficial of GM-CSF in the application of treating vascular ischemic patients is promising.

Overexpression of an isoform of AML1 in acute leukemia and its potential role in leukemogenesis

AML1/RUNX1 is a critical transcription factor in hematopoietic cell differentiation and proliferation. From the AML1 gene, at least three isoforms, AML1a, AML1b and AML1c, are produced through alternative splicing. AML1a interferes with the function of AML1b/1c, which are often called AML1. In this study, we found a higher expression level of AML1a in acute lymphoblastic leukemia and acute myeloid leukemia (AML)-M2 patients in comparison to the controls. Additionally, AML1a represses transcription of promoter of macrophage colony-stimulating factor receptor mediated by AML1b, indicating that AML1a antagonized the effect of AML1b. To investigate the role of AML1a in hematopoiesis and leukemogenesis in vivo, murine bone marrow mononuclear cells were transduced with AML1a and then transplanted into lethally irradiated mice, which developed lymphoblastic leukemia after transplantation. Taken together, these results indicate that overexpression of AML1a may be an important contributing factor to leukemogenesis.

Identification and Characterization of Sur3γ and Sur3γV, Two Novel Splice Variants of Survivin in HSPC

Survivin is a member of the inhibitor-of-apoptosis (IAP) family of endogenous caspase inhibitors. We have previously shown that Survivin is expressed in primitive hematopoietic stem and progenitor (HSPC) cells and required for HSC maintenance and for entry into cell cycle. Several alternative Survivin splice variants have been described that are associated with differential prognosis in patients with cancer and leukemia, however, the function of alternative splice variants in normal and cancer cells is not fully understood. Using rapid amplification of cDNA ends (RACE) with mRNA from HL-60 leukemia cells we have identified two novel alternative splice variants, Survivin-3γ (Sur3γ) and Survivin-3γ-Variant (Sur3γV) that differ by only 4 bases, however due to changes in the reading frame, they result in proteins with very different C-termini, with Sur3γV being the predominant variant. We have characterized the expression of Sur3γV and Sur3γ in normal HSPC, leukemia and solid cancer cells and evaluated their anti-apoptotic function in cancer cell lines and primary hematopoietic cells. In contrast to cell lines and most cancers, where SurWT is the predominant transcript, Sur3γV is expressed at higher levels than SurWT in a number of normal human tissues including bone marrow, pericardium, placenta, seminal vesicles, testis and uterus. In placenta and bone marrow, Sur3γV mRNA is expressed at 25-fold and 65-fold higher levels respectively, than wild-type Survivin (SurWT). Sur3γV is expressed at ~16-fold higher levels in CD34+ HSPC and at ~80-fold higher level in primary CD34+ CD38− HSPC. In CD34+ cells, stimulation with SCF, TPO, FLT3L was associated with 30 – 55 fold increase in expression of SurWT, however expression of Sur3γ and Sur3γV were not altered, suggesting that these variants may be associated with HSPC maintenance/quiescence. Sur3γV and SurWT are expressed at equivalent levels in AML however, in blast crisis CML, Sur3γV is expressed at 5-fold higher level than SurWT. Over expression of Sur3γ and Sur3γV in Yac1, a mouse lymphoma cell line, confers ~ 2-fold increased resistance to Taxol similar to that observed using Survivin-WT. Similarly, Yac-1 cells over expressing Sur3γV or Sur3γ were equally resistant to Etoposide as cells over expressing SurWT. Over-expression of Sur3γ and Sur3γV using the bi-cistronic MIEG vector in primary murine bone marrow mononuclear cells results in 1.7-fold increase in total CFU-GM, similar to SurWT. In addition, over-expression of Sur3γV and Sur3γ protected CFU-GM from apoptosis induced by delayed growth factor addition resulting in a 1.6 fold increase in the number of CFU-GM colonies detected. These studies clearly indicate that both Sur3γ and Sur3γV possess anti-apoptotic function at least equivalent to SurWT. In summary, we have identified 2 novel splice variants of Survivin that differ from one another by 4 bases. Unlike other Survivin splice variants that are expressed at low levels, Sur3γV is expressed at a higher level than SurWT in primitive HSPC and in some primary leukemia samples. Expression of Sur3γ and Sur3γV is not altered by hematopoietic growth factors that stimulate HSC proliferation. Over-expression of Sur3γ and Sur3γV protects cells from chemotherapeutic drugs and ectopic expression of Sur3γ and 3γV stimulates HPC proliferation and protects primary mouse HSPC from apoptosis. These studies also suggest that differential expression of Survivin splice variants may play a critical role in HSC function and differentiation.

Abstract 3915: Del-1, A New β2-Integrin Ligand, Which Inhibits Adhesion and Homing of Progenitor Cells

Progenitor cells (PC) are recruited to ischemic tissues and improve neovascularization. Beta2-integrins are essential for adhesion, transmigration and homing of PC to ischemic tissues. Developmental Endothelial Locus-1 (Del-1) is an extracellular matrix protein, that binds alphaVbeta3- and alphaVbeta5-integrins and is up-regulated during ischemia. Therefore, we investigated the role of endogenous Del-1 for angiogenesis and homing functions of PC. The Del-1-deficient mice (Del-1 −/− ) displayed a significantly increased angiogenic response in ischemic muscles in comparison to the wild type (WT) mice in the model of hind limb ischemia. However, when we assessed the role of Del-1 in HUVEC in vitro , silencing of Del-1 by siRNA did not affect angiogenic sprouting. Moreover, the ischemic muscles of Del-1 −/− mice displayed a higher infiltration with CD45 + hematopoietic cells than WT mice, suggesting that Del-1 may have an inhibitory effect on homing of PC and inflammatory cells to ischemic tissues. Interestingly, in adhesion assays human endothelial progenitor cells (EPC) and murine Lin − progenitor cells bound to Del-1 via beta2-integrins, but not via the alphaVbeta3- and alphaVbeta5- integrins. Furthermore, soluble Del-1 significantly inhibited the adhesion of EPC to HUVEC monolayers and to the major beta2-integrin-ligand, ICAM-1, raising the possibility that Del-1 is a beta2-integrin-inhibitor. Indeed, WT murine bone marrow mononuclear cells displayed higher adhesion rates on Del-1-deficient murine lung endothelial cells (LEC) than on WT LEC. In order to investigate the role of Del-1 for in vivo homing of PC, we intravenously injected murine fluorescence-labeled WT Lin − bone marrow PC in WT and Del-1 −/− mice 2 days after the induction of hind limb ischemia. Interestingly, the homing of injected Lin − cells to ischemic muscles was significantly increased in Del-1 −/− in comparison to WT mice (200± 30 % increase). Taken together, endogenous Del-1 is a new beta2-integrin ligand, which blocks beta2-integrin-dependent adhesion and homing of PC to ischemic tissues. It is conceivable, that endogenous Del-1 may reduce ischemia-induced neovascularization through an inhibitory effect on the beta2-integrins of progenitor and inflammatory cells.

Macrophage Inflammatory Protein–1δ: A Novel Osteoclast Stimulating Factor Secreted by Renal Cell Carcinoma Bone Metastasis

Approximately 30% of patients with renal cell carcinoma (RCC) develop bone metastasis, which is characterized by extensive osteolysis leading to severe bone pain and pathologic fracture. Although the mechanism of RCC-induced osteolysis is unknown, studies of bone metastasis have shown that tumor-induced changes in bone remodeling are likely mediated by alterations in the bone microenvironment. Here, we report the discovery of a novel osteoclast stimulatory factor secreted by RCC bone metastasis (RBM). Through microarray analysis, we found expression of the chemokine, macrophage inflammatory protein-1 delta (MIP-1 delta), to be increased in RBM versus patient-matched primary RCC tissues and confirmed this finding by quantitative reverse transcription-PCR (qRT-PCR) and ELISA (P < 0.05). Furthermore, MIP-1 delta expression in RBM tissues was significantly (P < 0.001) higher than in human bone marrow, suggesting a potential alteration of the bone microenvironment. The receptors for MIP-1 delta, CCR1 and CCR3, were expressed in both osteoclast precursors and mature, bone-resorbing osteoclasts as shown by qRT-PCR and Western analysis. In functional studies, MIP-1 delta stimulated chemotaxis of two osteoclast precursor cell types: murine bone marrow mononuclear cells (BM-MNC) and RAW 264.7 cells. Furthermore, MIP-1 delta treatment of murine calvaria caused increased bone resorption as determined by measurement of released calcium. Correspondingly, MIP-1 delta significantly enhanced osteoclast formation and activity in response to RANKL in both BM-MNC and RAW 264.7 cells. Taken together, these data suggest that MIP-1 delta expression is increased in RBM relative to RCC and bone marrow, and may promote RBM-induced osteolysis by stimulating the recruitment and differentiation of osteoclast precursors into mature osteoclasts.