scholarly journals Neutrophils Promote Hematopoiesis Via a Novel Mechanism Involving Leucine Rich α-2 Glycoprotein (LRG)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2725-2725
Author(s):  
Lawrence J Druhan ◽  
Shimena Li ◽  
Sarah A Baxter ◽  
Amanda Lance ◽  
Andrea E Price ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Myeloid Cell ◽  
Hematopoietic Progenitor Cells ◽  
Human Neutrophils ◽  
Neutrophil Granulocyte ◽  
Granulocytic Differentiation ◽  
Hematopoietic Progenitor ◽  
Granule Proteins

Abstract Leucine-rich α2 glycoprotein (LRG), the founding member of the leucine-rich repeat superfamily of proteins, was initially identified in serum more than 30 years ago, but its biologic function has remained elusive. A role for LRG has been implicated in inflammation and angiogenesis. Our laboratory previously identified cDNA and genomic clones for human and murine LRG, and showed that ectopically expressed LRG localizes to the granule compartment in transfected myeloid cell lines and promotes their granulocytic differentiation. We also demonstrated that expression of LRG is transcriptionally regulated during neutrophil granulocyte differentiation in a manner similar to that reported for genes encoding the different subsets of neutrophil granule proteins. The presence of LRG in primary neutrophils and a role for LRG in hematopoiesis, however, have not been previously described. Based on our prior studies in transfected myeloid cell lines, we considered the tantalizing possibility that LRG is a novel neutrophil granule protein that is secreted extracellularly upon neutrophil activation to modulate hematopoiesis. To investigate this, we examined LRG in primary human neutrophils isolated from healthy volunteers. Immunoblot analysis of whole cell lysates from neutrophils (97% purity) identified a higher molecular weight LRG species in neutrophils (62 kDa) compared to serum (50 kDa); our data demonstrate the difference in apparent molecular weight is due to differential glycosylation. Immunofluorescence microscopy using antibodies to human LRG and antibodies to the neutrophil granule proteins myeloperoxidase (MPO), lactoferrin (LF), and matrix metalloproteinase 9 (MMP-9, also known as gelatinase), along with fluorescently-labeled secondary antibodies, demonstrated the presence of LRG in the cytoplasm of neutrophils in a compartment corresponding to LF. ELISA and immunoblot analyses of subcellular fractions from isolated neutrophils prepared by nitrogen cavitation demonstrated the presence of LRG in LF-containing fractions as well as some MMP-9-containing fractions, consistent with localization of LRG to the secondary/tertiary granule compartment. Neutrophil exocytosis assays using ionomycin, phorbol-12-myristate 13-acetate, and f-Met-Leu-Phe as stimulants also indicated that LRG is co-released with LF and MMP9, but not with MPO. Notably, LRG secreted from activated neutrophils could bind cytochrome c as reported for LRG purified from serum. Recent reports that LRG can also bind to the TGFβR1 receptor on endothelial cells prompted us to investigate the effects of LRG on TGFβ signaling in hematopoietic cells. LRG significantly antagonized the inhibitory effect of TGFβ on HL-60 cell proliferation (n=3; p<0.05) and also on colony growth of human hematopoietic progenitor cells. When LRG was added to hematopoietic progenitor cells cultured in TGFβ-containing Methocult (SF H4436, serum free), a 50% increase in CFU-GMs was observed. Collectively, these data suggest a novel mechanism whereby neutrophils modulate hematopoiesis in the microenvironment via extracellular release of LRG, and invoke an additional role for neutrophils in innate immunity that has not previously been reported Disclosures No relevant conflicts of interest to declare.

scholarly journals Human interleukin-6 supports granulocytic differentiation of hematopoietic progenitor cells and acts synergistically with GM-CSF

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 666-670
Author(s):  
D Caracciolo ◽  
SC Clark ◽  
G Rovera
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Interleukin 6 ◽  
Dose Range ◽  
Leukemic Cell ◽  
Hematopoietic Progenitor Cells ◽  
Target Cells ◽  
Granulocytic Differentiation ◽  
Hematopoietic Progenitor ◽  
Gm Csf

Recombinant human (rh) interleukin-6 (IL-6), in a dose range of 1 to 10 U/mL, was able to induce a low number of neutrophilic-granulocytic colonies in a CFU-GM clonogenic assay, using T cells and adherent cells, depleted low density marrow cells. A synergistic increase in the number of granulocytic colonies was observed when rhGM-CSF at suboptimal doses and IL-6 at effective doses were both present in the assay; the increase was only additive when either rhIL-1 alpha or rhIL- 3 was used together with IL-6. To determine whether the increase in colony number reflects the interactions of these factors on the same hematopoietic progenitor target cells or, instead, represents activation of accessory cells, we analyzed the effect of IL-6 on the proliferation and differentiation of three growth factor-dependent leukemic cell lines that respond with continuous proliferation to the presence of GM-CSF and IL-3 in culture. One of the three cell lines (AML-193) showed limited proliferation in the presence of IL-6 followed by terminal differentiation after 14 days into basophilic-granulocytic- like cells. A synergistic proliferative response was observed on the same cells treated with both GM-CSF and IL-6. These data support the hypothesis that IL-6 may have a direct effect on myeloid hematopoietic progenitor cells, and that GM-CSF interacts synergistically with IL-6 by acting on the same target cells.

scholarly journals Human interleukin-6 supports granulocytic differentiation of hematopoietic progenitor cells and acts synergistically with GM-CSF

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 666-670 ◽  
Author(s):  
D Caracciolo ◽  
SC Clark ◽  
G Rovera
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Interleukin 6 ◽  
Dose Range ◽  
Leukemic Cell ◽  
Hematopoietic Progenitor Cells ◽  
Target Cells ◽  
Granulocytic Differentiation ◽  
Hematopoietic Progenitor ◽  
Gm Csf

Abstract Recombinant human (rh) interleukin-6 (IL-6), in a dose range of 1 to 10 U/mL, was able to induce a low number of neutrophilic-granulocytic colonies in a CFU-GM clonogenic assay, using T cells and adherent cells, depleted low density marrow cells. A synergistic increase in the number of granulocytic colonies was observed when rhGM-CSF at suboptimal doses and IL-6 at effective doses were both present in the assay; the increase was only additive when either rhIL-1 alpha or rhIL- 3 was used together with IL-6. To determine whether the increase in colony number reflects the interactions of these factors on the same hematopoietic progenitor target cells or, instead, represents activation of accessory cells, we analyzed the effect of IL-6 on the proliferation and differentiation of three growth factor-dependent leukemic cell lines that respond with continuous proliferation to the presence of GM-CSF and IL-3 in culture. One of the three cell lines (AML-193) showed limited proliferation in the presence of IL-6 followed by terminal differentiation after 14 days into basophilic-granulocytic- like cells. A synergistic proliferative response was observed on the same cells treated with both GM-CSF and IL-6. These data support the hypothesis that IL-6 may have a direct effect on myeloid hematopoietic progenitor cells, and that GM-CSF interacts synergistically with IL-6 by acting on the same target cells.

scholarly journals Murine yolk sac endoderm- and mesoderm-derived cell lines support in vitro growth and differentiation of hematopoietic cells

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2436-2443 ◽  
Author(s):  
MC Yoder ◽  
VE Papaioannou ◽  
PP Breitfeld ◽  
DA Williams
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Yolk Sac ◽  
Hematopoietic Progenitor Cells ◽  
Visceral Endoderm ◽  
Hematopoietic Progenitor

Abstract The mechanisms involved in the induction of yolk sac mesoderm into blood islands and the role of visceral endoderm and mesoderm cells in regulating the restricted differentiation and proliferation of hematopoietic cells in the yolk sac remain largely unexplored. To better define the role of murine yolk sac microenvironment cells in supporting hematopoiesis, we established cell lines from day-9.5 gestation murine yolk sac visceral endoderm and mesoderm layers using a recombinant retrovirus vector containing Simian virus 40 large T- antigen cDNA. Obtained immortalized cell lines expressed morphologic and biosynthetic features characteristic of endoderm and mesoderm cells from freshly isolated yolk sacs. Similar to the differentiation of blood island hematopoietic cells in situ, differentiation of hematopoietic progenitor cells in vitro into neutrophils was restricted and macrophage production increased when bone marrow (BM) progenitor cells were cultured in direct contact with immortalized yolk sac cell lines as compared with culture on adult BM stromal cell lines. Yolk sac- derived cell lines also significantly stimulated the proliferation of hematopoietic progenitor cells compared with the adult BM stromal cell lines. Thus, yolk sac endoderm- and mesoderm-derived cells, expressing many features of normal yolk sac cells, alter the growth and differentiation of hematopoietic progenitor cells. These cells will prove useful in examining the cellular interactions between yolk sac endoderm and mesoderm involved in early hematopoietic stem cell proliferation and differentiation.

Specific Signals Generated by the Cytoplasmic Domain of the Granulocyte Colony-Stimulating Factor (G-CSF) Receptor Are Not Required for G-CSF–Dependent Granulocytic Differentiation

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 353-361 ◽  
Author(s):  
Jason Jacob ◽  
Jeffery S. Haug ◽  
Sofia Raptis ◽  
Daniel C. Link
Keyword(s):  
Progenitor Cells ◽  
Ectopic Expression ◽  
Cytoplasmic Domain ◽  
Hematopoietic Progenitor Cells ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Granulocytic Differentiation ◽  
Hematopoietic Progenitor ◽  
Stimulating Factor ◽  
Factor G

Abstract Granulocyte colony-stimulating factor (G-CSF) is the principal growth factor regulating the production of neutrophils, yet its role in lineage commitment and terminal differentiation of hematopoietic progenitor cells is controversial. In this study, we describe a system to study the role of G-CSF receptor (G-CSFR) signals in granulocytic differentiation using retroviral transduction of G-CSFR–deficient, primary hematopoietic progenitor cells. We show that ectopic expression of wild-type G-CSFR in hematopoietic progenitor cells supports G-CSF–dependent differentiation of these cells into mature granulocytes, macrophages, megakaryocytes, and erythroid cells. Furthermore, we show that two mutant G-CSFR proteins, a truncation mutant that deletes the carboxy-terminal 96 amino acids and a chimeric receptor containing the extracellular and transmembrane domains of the G-CSFR fused to the cytoplasmic domain of the erythropoietin receptor, are able to support the production of morphologically mature, chloroacetate esterase-positive, Gr-1/Mac-1–positive neutrophils in response to G-CSF. These results demonstrate that ectopic expression of the G-CSFR in hematopoietic progenitor cells allows for multilineage differentiation and suggest that unique signals generated by the cytoplasmic domain of the G-CSFR are not required for G-CSF–dependent granulocytic differentiation.

Knockdown of HPRT Enables Selection of Genetically Modified Human Hematopoietic Progenitor Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3772-3772
Author(s):  
Rashmi Choudhary ◽  
Brian Freed ◽  
James DeGregori ◽  
Christopher C. Porter
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Hematopoietic Progenitor Cells ◽  
Off Label Use ◽  
Hematopoietic Progenitor ◽  
Off Label ◽  
Human Cd34 ◽  
Dose Dependent ◽  
Hematopoietic Cell Lines

Abstract Abstract 3772 Genetic modification of autologous hematopoietic stem cells (HSC) has the potential for effective treatment of a wide variety of inherited blood disorders. However, HSC gene therapy has shown limited clinical efficacy (with notable exceptions), in part because of the small proportion of engrafted genetically corrected HSCs. The use of drug-resistance genes to enable selection for transduced HSCs has been explored, but with limited success. Previous studies from our laboratory have indicated that murine HSC can be selected with 6-Thioguanine (6TG), a relatively non-toxic drug used in the treatment of leukemias, after knocking down the expression of hypoxanthine-guanine phosphoribosyltransferase (HPRT), an enzyme that metabolizes 6TG to its active state. We sought to determine if these findings can be translated to human hematopoietic cells. In the present study, we transduced human myeloid (Molm13, MV4-11) and lymphoid cell lines (Reh) with lentiviral vectors expressing shRNA constructs targeting HPRT or a non-targeted control sequence (Ctrl). Two of the most promising constructs directed against HPRT (491 and 50) were studied in more detail to determine which is most effective. Cells were selected in puromycin and cell lysates analyzed for HPRT gene expression. Reverse-transcription, real-time PCR (RT qPCR) and western blotting demonstrated that construct 491 was most efficient in knocking down HPRT in human hematopoietic cell lines compared to construct 50 (and Ctrl). To determine whether knockdown of HPRT provided resistance to 6TG, cells were cultured in the absence or presence of different doses of 6TG and live cell concentrations were determined. While Ctrl transduced cells decreased in a dose dependent manner after 72h of 6TG treatment, cells transduced with constructs 491 and 50 were relatively resistant to 6TG. IC50 values for construct 491 were significantly higher (114μM for Molm13 and 46μM for Reh cell lines) than construct 50 (1μM for Molm13 and 10μM for Reh) in comparison to control transduced cells (0.4μM for Molm13 and 3.5μM for Reh). We assessed cell death in human hematopoietic cell lines by annexin V staining after exposure to 6TG at 48 and 72h. As expected, control transduced cells died of apoptosis upon 6TG treatment, while 491 and 50 transduced cells were resistant. Furthermore, 491 transduced cells were more resistant to apoptosis than 50 transduced cells. Based on these results, construct 491 was used to transduce human CD34+ progenitor cells isolated from umbilical cord blood along with control shRNA. Transduction efficiency varied from 25–35% as determined by %GFP expression by flow cytometry. Sorted GFP+ cells showed reduced expression of HPRT in 491 transduced cells compared to controls, as measured by RT qPCR. Similar to the effects in cell lines, in vitro proliferation of control transduced CD34+ cells diminished in response to increasing 6TG concentrations. There was an increase in the percentage of GFP+ cells in 6TG treated 491 transduced cells compared to untreated controls in a dose dependent fashion, indicating a selective advantage conferred to 491 transduced cells in the presence of 6TG. Importantly, 491 transduced cells continued to proliferate despite treatment with 6TG. Like 6TG, cisplatin requires mismatch repair (MMR) for cytotoxicity. To determine if HPRT knockdown had off-target effects impairing MMR, transduced cells were also treated with cisplatin. Both control and 491 transduced cells stopped proliferating in the presence of cisplatin indicating that MMR remained intact. These data indicate that human hematopoietic progenitor cells can be selected in vitro by knock-down of HPRT and treatment with 6TG. Xenografts of Ctrl and 491 transduced human CD34+ cord blood cells have been generated and are being treated with 6TG to determine if human cells can be selected with 6TG in vivo. Disclosures: Off Label Use: Off label use of 6-thioguanine will be suggested.

Cannabinoids Derivatives Exert a Potent Antileukemic Effect By Modifying the Pattern of Membrane Sphingolipids

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4718-4718
Author(s):  
Mayte Medrano ◽  
María Victoria Barbado-Gonzalez ◽  
Nuria Campillo ◽  
Francisco Hidalgo ◽  
Teresa Caballero-Velazquez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Leukemic Cells ◽  
Hematopoietic Progenitor Cells ◽  
Cb2 Receptor ◽  
Hematopoietic Progenitor ◽  
Healthy Donors ◽  
Win 55

Abstract Endocannabinoid system is a set of ligands, receptors and endogenous enzymes which modulate a variety of physiological effects. There are two well-characterized cannabinoid receptors, CB1 (mainly expressed in Central Nervous System) and CB2 (mainly in hematopoietic cells). Here, we tested the effect of the cannabinoid WIN-55 212-2 in acute myeloid leukemia (AML) in vitro, ex vivo and in vivo and studied the molecular signaling pathways involved in this effect. Moreover, we synthesized a new family of twelve cannabinoids that are specific to CB2 receptor. For their design and synthesis, computational techniques of docking, analytical and spectroscopic techniques such as mass spectrometry (MS) were used. To assess the anti-leukemia effect of the different cannabinoids, we analyzed cell viability by MTT and flow cytometry using six human AML cell lines, primary cells from healthy donors (hematopoietic progenitor cells (HPC) and lymphocytes) and blasts from AML patients. Cannabinoids induced a potent proapoptotic effect on AML cell lines and on primary leukemic cells, which was not observed in normal HPC and lymphocytes from healthy donors. Fragmentation of PARP and activation of caspases 2, 3, 8 and 9 were confirmed by western-blot. Other proteins involved in the effect of cannabinoids were p-AKT, p-ERK 1/2, p-38 and p- JNK. Also studies on p-PERK, p-IRE1 and CHOP confirmed an increased endoplasmic reticulum stress upon exposure to cannabinoids. Mitochondrial damage was analyzed by flow cytometry using TMRE and by MitoSOXTMRed. These assays confirmed a very early mitochondrial damage in leukemic cells which was not observed in normal hematopoietic progenitor cells. Moreover, we analyzed the ceramide levels, a membrane lipid associated with death/survival cell processes by HPLC and immunohistochemistry. Remarkably, we observed significant differences in the amounts of certain subtypes of ceramides in untreated versus treated leukemic cells. The proapoptotic effect of cannabinoids on AML cells was abolished upon co-culture with either CB2 receptor antagonists or with pancaspase inhibitors. Finally, NOD/scid/IL-2R gammae null (NSG) mice were xenotransplanted with HL60 cell line. We confirmed disease infiltration in bone marrow (BM) by BM aspirates and flow cytometry assays. Once the presence of leukemic cells was confirmed, treatment with vehicle, WIN-55 cannabinoid at a dose of 5 mg/kg/day or citarabine (ARA-C) at 50 mg/kg during 5 days was administered. We observed a significantly increased survival among mice treated with WIN-55 cannabinoid as compared to both the control group and the group treated with ARA-C. In addition, we tested in vivo the effect of these compounds on normal hematopoiesis by treating healthy BALB-C mice. We confirmed that cannabinoids did not affect the viability of the different populations of hematopoietic progenitors (LK, GMP, CMP) and, moreover, an increased platelet count was observed in treated mice. Our findings indicate that cannabinoids display a highly selective proapoptotic effect against leukemic cells. Several pathways are involved in this effect, the modification in the ceramide pattern playing a main role. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Toxicity of antimony trioxide nanoparticles on human hematopoietic progenitor cells and comparison to cell lines

Toxicology ◽  
2009 ◽  
Vol 262 (2) ◽  
pp. 121-129 ◽  
Author(s):  
Lisa Bregoli ◽  
Francesca Chiarini ◽  
Andrea Gambarelli ◽  
Gianluca Sighinolfi ◽  
Antonietta M. Gatti ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Hematopoietic Progenitor Cells ◽  
Antimony Trioxide ◽  
Hematopoietic Progenitor

scholarly journals Lineage-Specific Regulation of Hematopoiesis by HOX-B8 (HOX-2.4): Inhibition of Granulocytic Differentiation and Potentiation of Monocytic Differentiation

Blood ◽  
1997 ◽  
Vol 90 (5) ◽  
pp. 1840-1849 ◽  
Author(s):  
Kandasamy Krishnaraju ◽  
Barbara Hoffman ◽  
Dan A. Liebermann
Keyword(s):  
Progenitor Cells ◽  
Ectopic Expression ◽  
Homeobox Gene ◽  
Hematopoietic Progenitor Cells ◽  
Colony Stimulating Factor ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation ◽  
Hematopoietic Progenitor ◽  
Specific Regulation ◽  
Stimulating Factor

Abstract Homeobox proteins comprise a major class of transcription factors, which have been implicated in normal hematopoiesis and leukemogenesis. Notable in this context is the homeobox gene HOX-B8 (formerly known as HOX-2.4), which was shown to cooperate with hematokines to induce leukemia, and to enhance self-renewal of immature myeloid progenitors when expressed alone. How HOX-B8 may affect lineage specific development of hematopoietic progenitor cells is unknown. Here it is shown that ectopic expression of HOX-B8 specifically inhibited dimethyl sulfoxide (DMSO)-induced granulocytic differentiation of autonomously proliferating HL-60 myeloid progenitor cells. HOX-B8 also inhibited the granulocyte colony-stimulating factor (G-CSF )–induced granulocytic developmental program of factor dependent 32Dcl3 hematopoietic progenitors, including survival, proliferation, and differentiation, as evident by rapid apoptosis of the cells following removal of interleukin-3 (IL-3) and addition of G-CSF. In sharp contrast, HOX-B8 had no effect on macrophage differentiation of M1 and HL-60 cells induced by IL-6 and phorbol-12-myristate-13-acetate, respectively. Moreover, HOX-B8 expression endowed the 32Dcl3 cells with the ability to be induced by granulocyte-macrophage colony-stimulating factor (GM-CSF ) for terminal differentiation exclusively along the macrophage lineage; this effect was at least partially mediated via expression of the zinc finger transcription factor Egr-1. Thus, ectopic expression of HOX-B8 in hematopoietic progenitor cells appears to differentially affect lineage specific development, negatively regulating granulocyte development and positively regulating macrophage development.

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