Detection of measles virus-induced apoptosis of human monocytic cell line (THP-1) by DNA fragmentation ELISA

Abstract Measles virus (wild strain, Toyoshima strain)-induced cell death is characterized by cell shrinkage, chromatin condensation, and nuclear fragmentation in a human monocytic cell line (THP-1). DNA fragmentation of measles virus-infected THP-1 cells was demonstrated by DNA agarose gel electrophoresis as well as by DNA fragmentation ELISA. When measles virus-infected THP-1 cells were cultured on monolayers of fibroblasts or human umbilical vein endothelial cells (HUVEC), the percentage of measles virus antigen-positive THP-1 cells and DNA fragmentation were significantly decreased. Addition of anti-intercellular adhesion molecule (ICAM)-1 (CD54) monoclonal antibody to culture of measles virus-infected THP-1 cells reduced significantly DNA fragmentation induced by measles virus. These findings suggest that inhibition of virus spread by fibroblasts and HUVEC reduces apoptosis, and ICAM-1 (CD54) may participate in the DNA fragmentation pathway.     

Signaling through toll-like receptor 7/8 induces the differentiation of human bone marrow CD34+ progenitor cells along the myeloid lineage

Toll-like receptors (TLRs) play a key role in pathogen recognition and regulation of the innate and adaptive immune responses. Although TLR expression and signaling have been investigated in blood cells, it is currently unknown whether their bone marrow ancestors express TLRs and respond to their ligands. Here we found that TLRs (e.g. TLR4, TLR7 and TLR8) were expressed by freshly isolated human bone marrow (BM) hematopoietic CD34+ progenitor cells. Incubation of these primitive cells with TLR ligands such as immunostimulatory small interfering RNAs and R848, a specific ligand for TLR7/8, induced cytokine production (e.g. IL1-beta, IL6, IL8, TNF-alpha, GM-CSF). Moreover, TLR7/8 signaling induced the differentiation of BM CD34+ progenitors into cells with the morphology of macrophages and monocytic dendritic precursors characterized by the expression of CD13, CD14 and/or CD11c markers. By contrast, R848 ligand did not induce the expression of glycophorin A, an early marker for erythropoiesis. Collectively, the data indicate for the first time that human BM CD34+ progenitor cells constitutively express functional TLR7/TLR8, whose ligation can induce leukopoiesis without the addition of any exogenous cytokines. Thus, TLR signaling may regulate BM cell development in humans.     

Low molecular weight polyethylenimine for efficient transfection of human hematopoietic and umbilical cord blood-derived CD34+ cells

With the emerging role of hematopoietic stem cells as potential gene and cell therapy vehicles, there is an increasing need for safe and effective nonviral gene delivery systems. Here, we report that gene transfer and transfection efficiency in human hematopoietic and cord blood CD34+ cells can be enhanced by the use of low molecular weight polyethylenimine (PEI). PEIs of various molecular weights (800-750,000) were tested, and our results showed that the uptake of plasmid DNA by hematopoietic TF-1 cells depended on the molecular weights and the N/P ratios. Treatment with PEI 2K (m.w. 2000) at an N/P ratio of 80/1 was most effective, increasing the uptake of plasmid DNA in TF-1 cells by 23-fold relative to Lipofectamine 2000. PEI 2K-enhanced transfection was similarly observed in hematopoietic K562, murine Sca-1+, and human cord blood CD34+ cells. Notably, in human CD34+ cells, a model gene transferred with PEI 2K showed 21,043- and 513-fold higher mRNA expression levels relative to the same construct transfected without PEI or with PEI 25 K, respectively. Moreover, PEI 2K-treated TF-1 and human CD34+ cells retained good viability. Collectively, these results indicate that PEI 2K at the optimal N/P ratio might be used to safely enhance gene delivery and transfection of hematopoietic and human CD34+ stem cells.     

A human cell model for dynamic testing of MR contrast agents

To determine the initial feasibility of using magnetic resonance (MR) imaging to detect early atherosclerosis, we investigated inflammatory cells labeled with a positive contrast agent in an endothelial cell-based testing system. The human monocytic cell line THP-1 was labeled by overnight incubation with a gadolinium colloid (Gado CELLTrack) prior to determination of the in vitro release profile from T1-weighted MR images. Next, MR signals arising from both a synthetic model of THP-1/human umbilical vein endothelial cell (HUVEC) accumulation and the dynamic adhesion of THP-1 cells to activated HUVECs under flow were obtained. THP-1 cells were found to be successfully--but not optimally--labeled with gadolinium colloid, and MR images demonstrated increased signal from labeled cells in both the synthetic and dynamic THP-1/HUVEC models. The observed THP-1 contrast release profile was rapid, suggesting the need for an agent that is optimized for retention in the target cells for use in further studies. Detection of labeled THP-1 cells was accomplished with no signal enhancement from unlabeled cells. These achievements demonstrate the feasibility of targeting early atherosclerosis with MR imaging, and suggest that using an in vitro system like the one described provides a rapid, efficient, and cost-effective way to support the development and evaluation of novel MR contrast agents.     

Antibody-mediated platelet phagocytosis by human macrophages is inhibited by siRNA specific for sequences in the SH2 tyrosine kinase, Syk

Immune thrombocytopenia depends upon Fc receptor-mediated phagocytosis that involves signaling through the SH2 tyrosine kinase, Syk. We designed small interfering (siRNA) sequences complementary to Syk coding regions to decrease the expression of Syk in the human macrophage cell line, THP-1. To evaluate the functional effect of siRNA on phagocytosis, we developed a new in vitro assay for antibody-mediated platelet ingestion by THP-1 cells. Incubation of THP-1 cells at 37 °C with fluorescence-labeled platelets and anti-platelet antibody promoted ingestion of platelets that could be quantitated by flow cytometry. Transfection of THP-1 cells with Syk-specific siRNA resulted in a reduction in the amount of FcγRII-associated Syk protein. Coincident with decreased Syk expression, we observed inhibition of antibody-mediated platelet ingestion. These results confirm a key role for Syk in antibody-mediated phagocytosis and suggest Syk-specific siRNA as a possible therapeutic candidate for immune thrombocytopenia.     

Comparison of the size of membrane microparticles of different cellular origin by dynamic light scattering

Size of membrane microparticles (MPs) from blood plasma and MPs produced in vitro by activated endothelial cells (ECs), monocytes, THP-1 monocytic cells, granulocytes, and platelets was evaluated by dynamic light scattering. MPs were sedimented from the culture media, cell supernatants, and plasma at 20 000 g for 30 min. Average diameters of all types of MPs ranged from 300 to 600 nm. Plasma MPs had the smallest size. Close sizes were registered for MPs from platelets and THP-1 cells. MPs from monocytes were larger, and MPs from granulocytes and ECs were the largest ones. The data obtained indicate that the size of membrane MPs depends on the type of their cell-producers.     

Mechanism of cigarette smoke condensate induced adhesion of human monocytes to cultured endothelial cells

Cigarette smoking is ranked among the leading risk factors in the etiology of atherosclerotic vascular disease. The mechanisms, however, that link cigarette smoking to increased incidence of atherosclerosis are not understood. The adherence of circulating monocytes to the endothelium, migration into the subendothelium, and subsequent formation of foam cells are principal initial events in the development of atherosclerosis. We therefore determined whether cigarette smoke caused increased adherence of monocytes to endothelial cells and the cellular mechanism of this increased adherence. Cigrette smoke condensate (CSC), the particulate fraction of cigarette smoke derived from 2R1 standard research cigarettes, at a concentration of 25–30 μg/ml (average yield of CSC is 26.1 mg/cigarette), augmented (70–90%) basal adherence of human peripheral blood monocytes to a cultured monolayer of endothelial cells derived from bovine aorta (BAEC) and human umbilical vein (HUVEC). There was a concomitant increase in the expression of CD11b ligand on the surface of monocytes as determined by flow cytometry, utilizing FITC conjugated Mab MO-1 (CD11b). However, nicotine (1–15 μg/ml) and cadmium sulfate (10 μg/ml), constituents of CSC, individually or in combination had no effect either on CD11b expression or adherence of monocytes to endothelial cells. Treatment of HUVEC with CSC for 60 min also resulted in an increased expression of ICAM-1 and ELAM-1 as determined by mean fluorescence intensity of ICAM-1 and ELAM-1 labeled cells in flow cytometric analysis. The CSC induced expression of CD11b in monocytes was optimal at 25–30 min and was inhibited by protein kinase C inhibitors, staurosporine and H-7, and also by baicalein, a lipoxygenase inhibitor. Similarly, CSC induced ICAM-1 and ELAM-1 expression in HUVEC was inhibited by protein kinase C inhibitors. CSC stimulated the adherence of human monocytes but not the monocytic cell lines HL-60, U937, and THP-1 to endothelial cells. The CSC stimulated adherence of human monocytes was inhibited (80%) by MAb to CD11b and 50% by Mab to ICAM-1 and ELAM-1. These results suggest that cigarettee smoke particulate constituents activate protein kinase C, leading to increased surface expression of adhesive ligand CD11b on peripheral blood monocytes and counter receptor(s) ICAM-1 and ELAM-1 in endothelial cells. The expression of ligand and counter receptor leads to potentiated adherence of monocytes to endothelial cells, an initial event in the pathogenesis of cigarette smoke induced inflammatory response in the vessel wall. © 1994 Wiley-Liss, Inc.     

Participation of beta2-integrins CD11b/CD18 and CD11c/CD18 in adhesion and migration of cells on fibrinogen

The role of beta2-integrins CD11b/CD18 and CD 11c/CD 18 in adhesion and migration of leukocytes on fibrinogen was studied. The monoclonal antibodies against CD11b inhibited the spontaneous adhesion of monocytic THP-1 cells on fibrinogen, whereas antibodies to CD11c more effectively inhibited the adhesion stimulated by chemokine MCP-1. By the RNA-interference method the clones of THP-1 with reduced expression of CD11b and general beta2-subunit CD18 were obtained. MCP-I stimulated the adhesion to fibrinogen of THP-1 cells of wild-type and mutant cells with reduced expression of CD11b (THP-1-CD11b-low), but not of cells with low expression of CD18 (THP-1-CD18-low). THP-1-CD18-low cells were also characterized by the impaired chemotaxis in presence of MCP-1. The data obtained suggest that spontaneous cell adhesion to fibrinogen is mediated to a greater extent by CD11b/CD18 integrins, while chemokine-stimulated adhesion and migration is mostly dependent on CD11c/CD18 molecules.     

THP-1 cells form foam cells in response to coculture with lipoproteins but not platelets

The human monocytic leukemia cell line, THP-1, shares many properties with human monocyte-derived macrophages and might be a useful model for studying foam cell formation in vitro. Therefore, we examined the ability of THP-1 cells to accumulate cholesteryl esters, the hallmark feature of foam cells, in response to culture with native low density lipoprotein (LDL), modified LDL, and platelets. THP-1 cells stored more cholesteryl esters than macrophages in response to 200 micrograms/ml of LDL. Down-regulation of LDL receptors occurred in macrophages at lower LDL concentrations than in THP-1 cells. Phorbol ester-treated THP-1 cells stored more cholesteryl esters than human macrophages in response to 25-200 micrograms/ml of acetylated LDL. Because we have previously demonstrated that activated platelets enhanced macrophage cholesteryl ester storage, we examined the ability of THP-1 cells to store cholesteryl esters in response to coculture with platelets. Compared with macrophages, dividing THP-1 cells and phorbol ester-treated THP-1 cells accumulated only 50% and 33% as much cholesteryl esters, respectively. Furthermore, although platelets induced a 90% reduction in cholesterol synthesis in macrophages by day 5, cholesterol synthesis in THP-1 cells and phorbol ester-treated THP-1 cells was inhibited less than 50% by platelets. Nevertheless, both THP-1 cells and macrophages responded to platelets by increasing their secretion of apolipoprotein E. Therefore, we conclude that dividing THP-1 cells and phorbol ester-treated THP-1 cells are capable of forming foam cells in response to physiologic doses of both LDL and acetylated LDL, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lentivirus vector-mediated hematopoietic stem cell gene transfer of common gamma-chain cytokine receptor in rhesus macaques

Nonhuman primate model systems of autologous CD34+ cell transplant are the most effective means to assess the safety and capabilities of lentivirus vectors. Toward this end, we tested the efficiency of marking, gene expression, and transplant of bone marrow and peripheral blood CD34+ cells using a self-inactivating lentivirus vector (CS-Rh-MLV-E) bearing an internal murine leukemia virus long terminal repeat derived from a murine retrovirus adapted to replicate in rhesus macaques. In vitro cytokine stimulation was not required to achieve efficient transduction of CD34+ cells resulting in marking and gene expression of the reporter gene encoding enhanced green fluorescent protein (EGFP) following transplant of the CD34+ cells. Monkeys transplanted with mobilized peripheral blood CD34+ cells resulted in EGFP expression in 1 to 10% of multilineage peripheral blood cells, including red blood cells and platelets, stable for 15 months to date. The relative level of gene expression utilizing this vector is 2- to 10-fold greater than that utilizing a non-self-inactivating lentivirus vector bearing the cytomegalovirus immediate-early promoter. In contrast, in animals transplanted with autologous bone marrow CD34+ cells, multilineage EGFP expression was evident initially but diminished over time. We further tested our lentivirus vector system by demonstrating gene transfer of the human common gamma-chain cytokine receptor gene (gamma(c)), deficient in X-linked SCID patients and recently successfully used to treat disease. Marking was 0.42 and.001 HIV-1 vector DNA copy per 100 cells in two animals. To date, all EGFP- and gamma(c)-transplanted animals are healthy. This system may prove useful for expression of therapeutic genes in human hematopoietic cells.     

Insulin-like growth factor binding protein-2 and neurotrophin 3 synergize together to promote the expansion of hematopoietic cells ex vivo

Co-culture of Umbilical Cord Blood (UCB) CD34+ cells with irradiated Mesenchymal Stem Cells (MSCs) without contact increase the expansion of Hematopoietic Progenitor Cells (HPC). Neurotrophin-3 (NT-3) and insulin-like growth factor binding protein-2 (IGFBP-2) are two factors whose expressions were significantly elevated in conditioned media derived from irradiated MSCs. To determine whether these factors are partly responsible for the growth promoting potential of MSCs, we investigated their impact on the growth and differentiation of UCB-CD34+ cells. Addition of either factor alone had little impact on cell growth, however both factors synergized together to increase the expansion of total nucleated cells, erythroids, megakaryocytes (Mk) and CD34+ cells. However, in contrast to MSCs they failed to significantly improve the expansion of hematopoietic progenitors. Consistent with the impact of these factors on hematopoietic cells, both synergized to activate ERK1/2 and AKT in primary human UCB cells. In conclusion, the study demonstrates for the first time that a neurotrophin factor can synergize with IGFBP-2 to promote hematopoietic cell expansion.     

Activity of tissue factor in microparticles produced <Emphasis Type="Italic">in vitro</Emphasis> by endothelial cells,monocytes, granulocytes,and platelets

Activity of tissue factor (TF) in membrane microparticles (MPs) produced in vitro by endothelial cells (ECs), monocytes, THP-1 monocytic cells, granulocytes, and platelets was investigated. ECs were isolated from human umbilical vein, and monocytes, granulocytes, and platelets–from the blood of healthy donors. ECs, monocytes, and THP-1 cells were activated by bacterial lipopolysaccharide, granulocytes–by lipopolysaccharide or phorbol myristate acetate, and platelets - by SFLLRN, thrombin receptor-activating peptide. MPs were sedimented from the culture medium or supernatant of activated cells at 20,000g for 30 min. Coagulation activity of MPs was analyzed in a modified recalcification assay by assessing their effects on coagulation of donor plasma depleted of endogenous MPs (by centrifuging at 20,000g for 90 min). MPs from all cell types accelerated plasma coagulation. Antibodies blocking TF activity prolonged coagulation lagphase in the presence of MPs from ECs, monocytes, and THP-1 cells (by 2.7-, 2.0-, and 1.8-fold, respectively), but did not influence coagulation in the presence of MPs from granulocytes and platelets. In accordance with these data, TF activity measured by its ability to activate factor X was found in MPs from ECs, monocytes, and THP-1 cells, but not in MPs from granulocytes and platelets. The data obtained indicate that active TF is present in MPs produced in vitro by ECs, monocytes, and THP-1 cells, but not in MPs derived from granulocytes and platelets.     

Indoxyl sulfate induces leukocyte-endothelial interactions through up-regulation of E-selectin

Despite a positive correlation between chronic kidney disease and atherosclerosis, the causative role of uremic toxins in leukocyte-endothelial interactions has not been reported. We thus examined the effects of indoxyl sulfate, a uremic toxin, on leukocyte adhesion to activated endothelial cells and the underlying mechanisms. Pretreatment of human umbilical vein endothelial cells (HUVEC) with indoxyl sulfate significantly enhanced the adhesion of human monocytic cells (THP-1 cell line) to TNF-α-activated HUVEC under physiological flow conditions. Treatment with indoxyl sulfate enhanced the expression level of E-selectin, but not that of ICAM-1 or VCAM-1, in HUVEC. Indoxyl sulfate treatment enhanced the activation of JNK, p38 MAPK, and NF-κB in TNF-α-activated HUVEC. Inhibitors of JNK and NF-κB attenuated indoxyl sulfate-induced E-selectin expression in HUVEC and subsequent THP-1 adhesion. Furthermore, treatment with the NAD(P)H oxidase inhibitor apocynin and the glutathione donor N-acetylcysteine inhibited indoxyl sulfate-induced enhancement of THP-1 adhesion to HUVEC. Next, we examined the in vivo effect of indoxyl sulfate in nephrectomized chronic kidney disease model mice. Indoxyl sulfate-induced leukocyte adhesion to the femoral artery was significantly reduced by anti-E-selectin antibody treatment. These findings suggest that indoxyl sulfate enhances leukocyte-endothelial interactions through up-regulation of E-selectin, presumably via the JNK- and NF-κB-dependent pathway.     

Eltrombopag, a thrombopoietin receptor agonist, enhances human umbilical cord blood hematopoietic stem/primitive progenitor cell expansion and promotes multi-lineage hematopoiesis

Umbilical cord blood (UCB) transplantation has emerged as a promising therapy, but it is challenged by scarcity of stem cells. Eltrombopag is a non-peptide, thrombopoietin (TPO) receptor agonist, which selectively activates c-Mpl in humans and chimpanzees. We investigated eltrombopag's effects on human UCB hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) expansion, and its effects on hematopoiesis in vivo. Eltrombopag selectively augmented the expansion of human CD45+, CD34+, and CD41+ cells in bone marrow compartment without effects on mouse bone marrow cells in the NOD/SCID mice xenotransplant model. Consequently, eltrombopag increased peripheral human platelets and white blood cells. We further examined effects in the STAT and AKT signaling pathways in serum-free cultures. Eltrombopag expanded human CD34+ CD38-, CD34+, and CD41+ cells. Both eltrombopag and recombinant human TPO (rhTPO) induced phosphorylation of STAT5 of CD34+ CD41-, CD34- CD41+, and CD34- CD41- cells. rhTPO preferentially induced pSTAT3, pAKT, and more pSTAT5 in CD34- C41+ cells, while eltrombopag had no effects on pSTAT3. In conclusion, eltrombopag enhanced expansion of HSCs/HPCs of human UCB in vivo and in vitro, and promoted multi-lineage hematopoiesis through the expansion of bone marrow HSCs/HPCs of human UCB in vivo. Eltrombopag differed somewhat from rhTPO in the signal transduction pathways by favoring earlier HSC/HPC populations.     

IL-16 signaling specifically induces STAT6 activation through CD4

Biologic activities of IL-16 have been well described (e.g., chemotaxis of CD4+ cells, CD25 upregulation, secretion of IL-1b, IL-4 and TNF-a secretion) but very few signaling events have been described. To gain a better understanding of how the biologic activities of IL-16 are regulated following receptor engagement (CD4) we have analyzed the activation state of numerous STAT proteins in primary human peripheral blood mononuclear cells (PBMCs) and the human monocytic cell line THP-1 following IL-16 stimulation. Of the four STAT proteins tested, only STAT6 was activated (phosphorylated) in a dose-dependant manner by IL-16. The activation of STAT6 was completely abolished when IL-16 was pre-incubated with soluble CD4 (the IL-16 cell surface receptor), demonstrating the need for CD4 engagement in STAT6 activation. These results are the first to demonstrate a link between IL-16 and STAT6 activation.     

Forced expression of HoxB4 enhances hematopoietic differentiation by human embryonic stem cells

HoxB4 has been shown to enhance hematopoietic engraftment by hematopoietic stem cells (HSC) from differentiating mouse embryonic stem cell (mESC) cultures. Here we examined the effect of ectopic expression of HoxB4 in differentiated human embryonic stem cells (hESCs). Stable HoxB4-expressing hESCs were established by lentiviral transduction, and the forced expression of HoxB4 did not affect stem cell features. HoxB4-expressing hESC-derived CD34+ cells generated higher numbers of erythroid and blast-like colonies than controls. The number of CD34+ cells increased but CD45+ and KDR+ cell numbers were not significantly affected. When the hESC derived CD34+ cells were transplanted into NOD/SCID beta 2m-/- mice, the ectopic expression of HoxB4 did not alter their repopulating capacity. Our findings show that overexpression of HoxB4 in differentiating hESCs increases hematopoietic colony formation and hematopoietic cell formation in vitro, but does not affect in vivo repopulation in adult mice hosts.     

IFN-gamma and 1,25(OH)2D3 induce on THP-1 cells distinct patterns of cell surface antigen expression, cytokine production, and responsiveness to contact with activated T cells.

Differentiation and maturation of monocytes are accompanied by the expression of specific surface glycoproteins, the secretion of cytokines, and the capacity to respond to ligands. These changes may be influenced by interactions with hormones, soluble lymphocytic products, or direct contact with lymphocytes. We have studied two distinct pathways in the differentiation of a human monocytic cell line, THP-1: one being induced by IFN-gamma and the other by 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). In THP-1 cells, IFN-gamma induces cell surface expression of HLA-DR and CD54 and production of IL-1 beta, TNF-alpha, and IL-6. In contrast, 1,25(OH)2D3 increases cell surface expression of CD11b and CD14, but fails to stimulate cytokine production. Direct contact of THP-1 with stimulated fixed T cells markedly induces IL-1 beta, TNF-alpha, and IL-6 production by THP-1. Production is higher when THP-1 have been previously exposed to 1,25(OH)2D3 as compared to prior exposure to IFN-gamma. mAb raised against certain relevant cell surface glycoproteins on THP-1 were tested for their ability to block the response of THP-1 to T cells. Antibodies to CD11a, CD11b, and CD11c, alone or in combination, only partially blocked IL-1 beta production by THP-1, whereas antibodies to CD54 and CD14 did not. Thus other unknown structures on the THP-1 cells may be involved in the induction of THP-1 cytokine production by T cell contact.