Inhibition of GM-CSF/IL-3/IL-5 signaling by antisense oligodeoxynucleotides targeting the common beta chain of their receptors.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 play a key role in allergic inflammation. They mediate their effect via receptors that consist of two distinct subunits, a cytokine-specific alpha subunit and a common beta subunit (betac) that transduces cell signaling. We sought to down-regulate the biologic activities of GM-CSF, IL-3, and IL-5 simultaneously by inhibiting betac mRNA expression with antisense technology. Experiments were performed with TF-1 cells (a human erythroleukemia cell line expressing GM-CSF, IL-3, and IL-5 receptors, which proliferates in response to these cytokines), monocytic U937 cells, which require these cytokines for differentiation, and purified human eosinophils. Cells were treated with antisense phosphorothioate oligodeoxynucleotides (ODN) targeting betac mRNA. In contrast to nontreated cells and cells treated by sense or mismatched ODN, antisense ODN inhibited betac mRNA expression and significantly decreased the level of cell surface betac protein expression on TF-1 and U937 cells. Receptor function was also affected. Antisense ODN were able to inhibit TF-1 cell proliferation in vitro in the presence of GM-CSF, IL-3, or IL-5 in the culture medium and eosinophil survival. We suggest that antisense ODN against betac may provide a new therapeutic alternative for the treatment of neoplastic or allergic diseases associated with eosinophilic inflammation.     

Proteolysis of C3 on U937 cell plasma membranes. Purification of cathepsin G

Covalent binding of C3 fragments to U937 cell membranes involved a cell surface-associated proteolytic activity. Two proteases able to cleave C3 were purified from U937 plasma membranes. Purification involved solubilization of the membranes and ion exchange chromatography. One of the purified proteases was identified as elastase, based upon a substrate specificity for benzyloxycarbonylalanine-o-nitrophenyl ester and complete inhibition by elastatinal and methoxysuccinyl-alanyl-alanyl-prolyl-valyl-chloromethyl-ketone. The other protease (m.w. 28,000) is cathepsin G, as deduced from the amino acid composition, the amino-terminal sequence, and the substrate specificity for succinyl-alanyl-alanyl-phenylalanine-p-nitroanilide. These two lysosomal proteases are present on the U937 cell surface, as confirmed by immunofluorescence analysis. Plasma membrane elastase and cathepsin G from U937 cells cleave C3 into C3a- and C3b-like fragments; further incubation leads to C3c- and C3dg-like fragments, as judged from SDS-PAGE analysis of the digests. Sequencing of the C3b-like fragment purified by reverse phase chromatography indicates that initial cleavage of C3 by purified cathepsin G occurs at two positions in the amino-terminal part of the alpha-chain, at a Arg-Ser bond located between residues 748 and 749 and at a Leu-Asp bond between residues 751 and 752. These proteases are, thus, able to generate, on the U937 surface, active fragments of C3, which are likely to be involved in cell-protein and cell-cell interactions.     

Autogenous production of interferon-beta switches on HLA genes during differentiation of histiocytic lymphoma U937 cells

The expression of class I HLA genes was measured during the in vitro differentiation of human U937 lymphoma cells towards macrophages. Following the onset of differentiation by phorbol myristate acetate the levels of cytoplasmic mRNA that hybridized with a [32P]HLA-B cDNA probe increased by a factor of nine. Elevation in HLA mRNA accumulation was followed by an increase in the rate of synthesis of HLA proteins and also by a dramatic increase in class I HLA cell surface antigen expression, as shown by cytofluorimetric analysis. The elevation in HLA mRNA and surface antigens could be prevented by adding antibodies against human interferon-beta (IFN-beta) to the culture medium at the onset of differentiation. Interferon antiviral activity was detected in the medium of differentiated U937 cells. The same anti-IFN-beta antibodies prevented the increase in (2'-5')oligo(A) synthetase activity which also takes place in differentiating U937 cells. Accumulation of the IFN-induced (2'-5')oligo(A) synthetase in U937 cells is preceded by an increase in its specific 1.6-kb mRNA as shown by hybridization to cloned (2'-5')-oligo(A) synthetase cDNA. The enzyme was preferentially found in the nuclear fraction of differentiating U937 cells. We suggest that an autogenous production of interferon-beta by the differentiating cells, switches on expression of the class I HLA genes as well as that of the (2'-5')oligo(A) synthetase.     

Effects of a variety of cytokines and inducing agents on vascular permeability factor mRNA levels in U937 cells.

Vascular permeability factor (VPF) is an approximately 40-kDa disulfide-linked dimeric glycoprotein that is active in increasing blood vessel permeability, endothelial cell growth and angiogenesis. Little is known about VPF gene regulation. In this study, we investigated the effects of a variety of cytokines and inducing agents on VPF mRNA levels in the monocyte-like U937 cell line. Transforming growth factor-beta 1 caused a 1.8-fold increase in VPF mRNA levels after 4 hours, followed by a decline to basal levels by 18 hours. Phorbol 12-myristate 13-acetate, a potent inducer of the differentiation of U937 cells, caused a 12.5-fold increase in VPF mRNA levels at 24 hours, coinciding with the differentiation of these monocyte-like cells into macrophage-like cells.     

Spontaneous production of a suppressor factor by a human macrophage-like cell line U937. II. Suppression of antigen- and mitogen-induced blastogenesis, IL 2 production and IL 2 receptor expression in T lymphocytes

U937, a human macrophage-like cell line, spontaneously produces a factor which inhibited blastogenic responses of human blood T lymphocytes stimulated with tuberculin-purified protein derivative (PPD) or phytohemagglutinin (PHA). We investigated the mechanism of suppressor action of the U937 factor. The U937 suppressor factor inhibited interleukin 2 (IL 2) production by human blood T lymphocytes stimulated with PPD or PHA. IL 1 did not overcome the inhibitory action of the U937 factor on PPD-induced IL 2 production by human blood T lymphocytes. The U937 factor also inhibited the production of IL 2 by a human leukemic cell line, JURKAT, stimulated with PHA. The U937 suppressor factor interfered with the expression of Tac antigen (IL 2 receptor) on PPD- or PHA-stimulated blood T lymphocytes. The inhibitory activity of the U937 factor on Tac expression was not affected by the addition of IL 2 or a crude lymphokine-containing T cell supernatant. Tac expression was more sensitive than IL 2 production to inhibition by U937-conditioned medium. The U937 suppressor factor was precipitable by 33 to 67% saturated ammonium sulfate and was inactivated at pH 2 or pH 11. Sephacryl S-200 Gel filtration analysis of U937 culture supernatants revealed that the inhibitory activities for blastogenesis, IL 2 production, and Tac expression co-purified in fractions with an apparent m.w. between 67,000 and 130,000. These data indicate that U937 spontaneously produces a macromolecular suppressive factor with major locus of action on the production of IL 2 and the expression of the IL 2 receptor.     

Glycated human serum albumin enhances macrophage inflammatory protein-1beta mRNA expression through protein kinase C-delta and NADPH oxidase in macrophage-like differentiated U937 cells

BACKGROUND: In a previous report (Higai K et al., Biol Pharm Bull, 2007), glycated human serum albumin (Glc-HSA) was found to induce interleukin-8 (IL-8) mRNA expression in human monocyte-derived U937 cells through a reactive oxygen species (ROS)-dependent pathway; however, Glc-HSA signaling has not been elucidated in macrophages. METHODS: U937 cells were differentiated by treatment with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) for 2 days and the macrophage-like differentiated U937 (differentiated U937) cells were stimulated with Glc-HSA and glycolaldehyde dimer-modified HSA (GA-HSA) in the presence of various signaling inhibitors. Macrophage inflammatory protein-1beta (MIP-1beta) mRNA expression was determined by real-time PCR. Intracellular ROS generation was estimated by confocal laser microscopy. RESULTS: Glc-HSA and GA-HSA markedly enhanced MIP-1beta mRNA expression in differentiated U937 cells. Enhanced MIP-1beta mRNA expression was completely suppressed by the ROS scavenger N-acetyl-l-cysteine, the NADPH oxidase inhibitors diphenylene iodonium and apocynin, and the protein kinase C (PKC)-delta inhibitor rottlerin. Furthermore, ROS generation was suppressed completely by rottlerin but not by the PKC-gamma inhibitor Ro318425 or the PKC-alpha, -beta1 and -micro inhibitor Go6976. CONCLUSION: Glc-HSA and GA-HSA enhance MIP-1beta mRNA expression in differentiated U937 cells through PKC-delta-dependent activation of NADPH oxidase.     

Regulated expression of the Mac-1, LFA-1, p150,95 glycoprotein family during leukocyte differentiation

The regulation of Mac-1, LFA-1, and p150,95 expression during leukocyte differentiation was examined. LFA-1 was present on almost all cell types studied. Both Mac-1 and p150,95 were present on the more mature cells of the myelomonocytic series, but only p150,95 was detected on some B cell lines and cloned cytotoxic T lymphocytes. Phorbol myristate acetate (PMA) stimulation of B chronic lymphocytic leukemia cells dramatically increased p150,95 expression. The resultant Mac-1, LFA-1, p150,95 phenotype resembled hairy cell leukemia, a B cell plasmacytoid leukemia. The promonocytic cell line U937 and the promyeloblastic cell line HL-60 expressed only LFA-1. Monocytic differentiation of U937 cells was stimulated by PMA, and induced the concomitant expression of Mac-1 and p150,95, with more p150,95 induced than Mac-1. Granulocyte/macrophage colony-stimulating factor (GM-CSF) stimulation of U937 cells gave similar results. PMA-stimulated monocytic differentiation of the HL-60 cell line also induced expression of both Mac-1 and p150,95. The number of p150,95 molecules on PMA-stimulated U937 and HL-60 cells were 5 X 10(5) and 3 X 10(5), respectively. Retinoic acid stimulated myeloid differentiation of HL-60 cells and induced expression of both Mac-1 and p150,95. These cells acquired a Mac-1, LFA-1, p150,95 profile that resembled that of granulocytes, with more Mac-1 than p150,95 induced. GM-CSF stimulation of HL-60 cells induced a similar Mac-1 and p150,95 phenotype. The contributions of Mac-1, LFA-1, and p150,95 to aggregation of PMA-differentiated U937 cells were assessed. Monoclonal antibodies to the beta subunit and the LFA-1 alpha subunit, but not those to p150,95 or Mac-1 alpha subunit, inhibited this homotypic adherence.     

Decrease in CuZn-superoxide dismutase mRNA level during differentiation of human monocytic and promyelotic leukemia cells

Change in the level of CuZn-superoxide dismutase (SOD) mRNA was examined using a molecular probe during differentiation of human monocytic leukemia U937 cells or promyelotic leukemia HL-60 cells induced by either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or dimethylsulfoxide (DMSO). CuZn-SOD mRNA levels were found to decrease during the course of differentiation, and this response is specific for differentiation, since the treatment of human B cell leukemia cells or normal diploid fibroblasts with TPA failed to have any effect on the level of CuZn-SOD mRNA. The activity of CuZn-SOD in U937 cells also decreased during differentiation, but following that of the CuZn-SOD mRNA level. The expression of the CuZn-SOD gene is thus concluded to diminish during the differentiation of HL-60 and U937 cells.     

白血病耐药细胞系U937/ADR的建立及其生物学性状

目的：建立白血病耐药细胞系U937/ADR模型,并检测其多药耐药相关基因及其生物学性状的改变。方法：以大剂量阿霉素(IC50浓度),短时间(2h)暴露法诱导人白血病细胞系U937细胞的阿霉素耐药性。检测细胞的生长曲线,计算阿霉素耐药倍数,流式细胞术分析细胞周期分布;罗丹明123检测药物外排功能;荧光定量PCR（FQ-PCR）检测MDR1、MRP1、NF-Κb、Bcl-2、Bax mRNA水平变化;Western blot 检测Akt、p-Akt、P65、P-gp、MRP1和Bcl-2蛋白水平变化。结果：成功构建耐阿霉素U937/ADR细胞系,对阿霉素耐药指数为亲代U937细胞的11倍,U937/ADR群体倍增时间为43.6h,高于亲代细胞8.9h;流式细胞分析显示与U937细胞相比,U937/ADR的G0/G1期细胞增多,而G2/M期细胞减少。并对多种化疗药物产生交叉耐药性。罗丹明123外排试验显示,U937/ADR细胞外排明显增加。U937/ADR细胞MDR1、NF-Κb、Bcl-2 mRNA表达水平明显增加,P-gp及p-Akt、P65表达水平增加。结论：成功构建的U937/ADR细胞系其生物学特性明显不同与亲代U937细胞,对多种化疗药物产生多药耐药,高表达多药耐药蛋白P-gp,同时激活p-Akt及NF-Kb。     

Cell-specific differences in activation of NF-kappa B regulatory elements of human immunodeficiency virus and beta interferon promoters by tumor necrosis factor.

Three aspects of the involvement of tumor necrosis factor in human immunodeficiency virus (HIV) pathogenesis were examined. Tumor necrosis factor alpha (TNF-alpha) mRNA production was analyzed by polymerase chain reaction amplification in monocytic U937 cells and in a chronically HIV infected U937 cell line (U9-IIIB). TNF-alpha RNA was undetectable in U937 cells, whereas a low constitutive level was detected in U9-IIIB cells. Paramyxovirus infection induced a 5- to 10-fold increase in the steady-state level of TNF-alpha RNA in U9-IIIB cells compared with U937 cells, suggesting that HIV-infected monocytic cells produced higher levels of TNF-alpha than did normal cells after a secondary virus infection. The effects of TNF-alpha on gene expression were examined by transient expression assays using reporter chloramphenicol acetyltransferase plasmids linked to regulatory elements from the HIV long terminal repeat (LTR) and the beta interferon promoter. In U937 and Jurkat T lymphoid cells, the inducibility of the different hybrid promoters by TNF-alpha or phorbol ester varied in a cell type- and promoter context-specific manner; the levels of gene activity of NF-kappa B-containing plasmids correlated directly with induction of NF-kappa B DNA-binding activity. Although the intact beta interferon promoter was only weakly stimulated by phorbol ester or TNF-alpha, multimers of the PRDII NF-kappa B-binding domain were inducible by both agents. TNF-alpha was able to increase expression of the HIV LTR in T cells, but in monocytic cells, TNF-alpha did not induce the HIV LTR above a constitutive level of activity. This level of NF-kappa B-independent activity appears to be sufficient for virus multiplication, since TNF-alpha treatment had no effect on the kinetics of de novo HIV type 1 (HIV-1) infection and viral RNA production in U937 cells. However, in Jurkat cells, TNF-alpha dramatically enhanced the spread of HIV-1 through the cell population and increased viral RNA synthesis, indicating that in T cells HIV-1 multiplication was stimulated by TNF-alpha treatment.     

Spontaneous production of a suppressor factor by the human macrophage-like cell line U937. I. Suppression of interleukin 1, interleukin 2, and mitogen-induced blastogenesis in mouse thymocytes

The human macrophage-like cell line U937 spontaneously produced a nondialyzable factor that inhibited interleukin 1 (IL 1), interleukin 2 (IL 2), and phytohemagglutinin (PHA)-induced blastogenesis in mouse thymocytes. The suppression by U937 supernatant factor occurred independently of the concentration of IL 1 or PHA, indicating that it was noncompetitive. The U937 suppressor factor was not cytotoxic for thymocytes, nor did it affect the spontaneous proliferation of T lymphoblastoid cell lines and U937. Physicochemical characterization showed that the U937 suppressor factor was nondialyzable, partially inactivated by heat treatment (56 degrees C), ammonium sulfate (67% saturation) precipitable, sensitive to pH 2.5, and resistant to freeze-thawing. Molecular weight of the factor inhibiting co-mitogenic IL 1 activity was approximately 85,000, as estimated by gel filtration. The U937 cell line may provide a model for the study of mechanisms and mediators of immunosuppression by mononuclear phagocytes.     

Augmentation of Interferon Production after Cell-Differentiation of U937 Cells by TPA

Persistent infections with mumps virus were established in several human lymphoid cells of T-cell origin (Molt-4, TALL-1, and CCRF-CEM) and human monocyte cells (U937 and THP-1). 2′,5′-Oligoadenylate synthetase (2–5AS) activity was demonstrated to be only slightly induced by interferon (IFN) or TPA (12-O-tetradecanoyl-phorbol-13-acetate) treatment in these cells. Treatment of the persistently infected cells with IFN or TPA did not stimulate an increase in the amount of synthetase mRNA. Induction of cell differentiation and augmentation of IFN production by TPA were demonstrated in U937 cells persistently infected with mumps virus (U937-MP). Similar results for IFN production were obtained from differentiated U937 cells. It is suggested that cell differentiation of U937 cells might be associated with the development of IFN inducibility.