Glucocorticoids inhibit IgE receptor expression on the human monocyte cell line U937.

Cells of a human monocyte-like cell line (U937) were analysed for IgE Fc receptors (Fc epsilon R) before and after glucocorticoid treatment. Specific binding of human myeloma IgE (Sha) was measured by 125I-labelled IgE, and by fluorescein-labelled IgE monitored by flow cytometry. Treatment of cells with dexamethasone or other steroids with glucocorticoid activity caused a significant decrease in Fc epsilon R expression. The inhibition was dose dependent, with a half-maximal effect at 20 nM dexamethasone, a concentration which is near to the dissociation constant of glucocorticoid receptors for dexamethasone. Inhibition of Fc epsilon R was significant beginning 8 h following glucocorticoid treatment and reached a plateau at 24 hr. The Ka for IgE binding was similar for control and dexamethasone-treated cells, while the number of IgE binding sites was decreased by 50-60%. Culture supernatants from dexamethasone-treated U937 cells which were concentrated 10-fold and depleted of free steroid did not affect Fc epsilon R expression. These results demonstrate that glucocorticoids can directly decrease the number of Fc epsilon R. This effect could participate in the glucocorticoid-induced suppression of IgE-mediated allergic reactions.     

Regulation of Fc epsilon receptor expression on a human monoblast cell line U937.

Fc epsilon receptor (Fc epsilon R) expression on several human cell lines (U937, RPMI 8866, HL 60, THP-1, and Molt 4) and its regulation were examined by immunofluorescent analysis using a monoclonal anti-human Fc epsilon R antibody, H107. Phorbol ester (PMA), recombinant gamma interferon (IFN-gamma) and H107 itself enhanced Fc epsilon R expression on a FC epsilon R positive cell line U937, whereas these reagents did not induce FC epsilon R expression on the Fc epsilon R negative cell lines, Molt 4, HL 60 and THP-1. Dexamethasone not only suppressed by 50% the spontaneous Fc epsilon R expression on U937 cells but also completely inhibited the enhancement of their Fc epsilon R expression on U937 cells induced by PMA, IFN-gamma or H107. Dexamethasone caused a little suppression of Fc epsilon R expression by RPMI 8866 cells. The results showed that Fc epsilon R expression on a human monoblast cell line U937 was up- or down-regulated by a variety of physiological or pharmacological agents. These experimental systems provide a good model for the investigation of the regulatory mechanisms of Fc epsilon R expression.     

Poliovirus induces apoptosis in the human U937 promonocytic cell line

The human promonocytic U937 cell line, which is moderately susceptible to poliovirus infection, has been used to investigate the induction of apoptosis by this virus. Infection of U937 cells with poliovirus induces morphological changes typical of apoptosis. Poliovirus-resistant U937 cells (PRU) have been isolated that are resistant to apoptosis induced by poliovirus, but that undergo apoptosis after treatment with TNF plus cycloheximide. Despite the fact that poliovirus triggers nitric oxide production in U937 cells, the inhibitor of inducible nitric oxide (NO) synthase, N(omega)-monomethyl-l-arginine, did not hinder apoptosis after infection, suggesting that NO does not play a direct role in this process. Finally, poliovirus infection of U937 cells led to the cleavage of pro-caspase-3 and poly(ADP-ribose)polymerase, indicating the activation of the CPP32 ICE-like cysteine protease in the induction of apoptosis. Our findings suggest that cellular death takes place in U937 cells productively infected by poliovirus as a result of apoptosis and involves caspase activation.     

Hydrogen sulfide induces the synthesis of proinflammatory cytokines in human monocyte cell line U937 via the ERK-NF-kappaB pathway

Hydrogen sulfide (H2S) is now considered an endogenous, gaseous mediator, which has been demonstrated to be involved in many inflammatory states. However, the mechanism of its proinflammatory function remains unknown. In the present study, we used IFN-gamma-primed human monocytic cell line U937 to investigate the effects of H2S in vitro on monocytes. We found that treatment with the H2S donor, sodium hydrosulfide, led to significant increases in the mRNA expression and protein production of TNF-alpha, IL-1beta, and IL-6 in U937 cells. H2S-triggered monocyte activation was confirmed further by the up-regulation of CD11b expression on the cell surface. We also observed that H2S could induce a rapid degradation of IkappaBalpha and subsequent activation of NF-kappaB p65, and this effect was attenuated by Bay 11-7082, a specific inhibitor of NF-kappaB. Furthermore, pretreatment of cells with Bay 11-7082 substantially inhibited the secretion of TNF-alpha, IL-1beta, and IL-6 induced by H2S. We also found that H2S stimulated the phosphorylation and activation of ERK1/2, but not of p38 MAPK and JNK, and pretreatment with PD98059, a selective MEK1 antagonist, could inhibit H2S-induced NF-kappaB activation markedly. Together, our findings suggest for the first time that H2S stimulates the activation of human monocytes with the generation of proinflammatory cytokines, and this response is, at least partially, through the ERK-NF-kappaB signaling pathway.     

Poliovirus modulates Bcl-xl expression in the human U937 promonocytic cell line

Poliovirus induces bcl-2-independent apoptosis in the human U937 promonocytic cell line [28]. Here we describe that this cell death, induced after viral infection, correlates with the modulation of the protooncoprotein Bcl-xl. Furthermore, poliovirus infection decreases the detected Bcl-xl in a U937 clone that overexpresses this protein (U937bcl-xl). Although in U937bcl-xl cells, Bcl-xl was not as highly regulated as in parental U937 cells, correlation between Bcl-xl modulation and the cleavage of poly(ADP-ribose)polymerase could be observed. Nevertheless, the induction of shutoff after infection of transfected control U937neo or U937bcl-xl clones was not significantly altered. Finally, production of new viral particles was slightly restricted in Bcl-xl-overexpressing U937 cells. Taken together, these results suggest that Bcl-xl is modulated during the induction of apoptosis in the promonocytic cell line U937 after poliovirus infection, although modulation of this protooncogene was not sufficient to modify the course of infection.     

Quantitative measurement of Fc receptor activity on human peripheral blood monocytes and the monocyte-like cell line, U937, by laser flow cytometry

The expression of high affinity Fc receptors for IgG (FcRI) on the cell line U937 has been measured by flow cytometry, using fluorescein isothiocyanate-conjugated human IgG (FITC-IgG) calibrated spectrofluorimetrically against lasergrade fluorescein (Fl). A standard curve is presented, relating the effective fluorescence in terms of fluorescein equivalents per IgG molecule, to the degree of conjugation of FITC-IgG. The flow cytometer was calibrated with commercially available fluorescein-coupled latex beads. The quantitation of FcRI, in terms of sites per cell and affinity constants, was compared with a radioligand assay performed concurrently on the same cell population. Good agreement between the two assays was observed. The Fc receptors on peripheral blood monocytes were measured in unpurified lysed blood by gating on forward/side scatter. Monomer IgG binding to monocyte FcRII or FcRIII cannot be measured in direct IgG radioligand analyses because of the low affinity of these receptors and their low numbers per cell. However, flow cytometry may be employed for measuring both high and low affinity ligand-FcR interactions, using monomer FITC-IgG.     

Internalization and degradation of human recombinant interferon-gamma in the human histiocytic lymphoma cell line, U937: relationship to Fc receptor enhancement and antiproliferation

This report describes the association between the intracellular fate of human recombinant interferon-gamma (rIFN-gamma) and the induction of enhanced numbers of Fc receptors and an antiproliferative effect in the human monocyte-like cell line, U937. Full receptor occupancy of Bolton-Hunter labeled 125I-rIFN-gamma occurred within 10 min at 37 degrees C. However, only 50% of those molecules bound were internalized within 30 min. Residency of rIFN-gamma within the cell was 60-120 min. Eventually, 60-70% of those molecules initially bound to the cell were completely degraded within monensin-sensitive compartments of the cell as measured by the presence of trichloroacetic acid-soluble radioactivity in the medium. Exposure of rIFN-gamma to cell extracts resulted in a shift in its pI from 8 to 6, presumably due to proteolytic cleavage of carboxy-terminal basic amino acids. A single brief exposure (5-15 min) of U937 cells to rIFN-gamma resulted in enhanced numbers of receptors for the Fc portion of 125I-IgG1 as measured 24 hr later. Eighty percent of a maximal Fc receptor response occurred at only 30% receptor occupancy (50 U/ml). In contrast, repeated daily exposure of U937 cells to moderate concentrations of rIFN-gamma (125-250 U/ml) was necessary to induce an antiproliferative effect. These data suggest that a given response of the cell to rIFN-gamma may require different intensities of the signal. This may reflect the overall complexity of the response generated.     

Karyotypic analysis of the human monoblastic cell line U937

Karyotypes of three sublines of the human cell line U937 showed considerable variation, but all contained four consistent marker chromosomes (i.e., 3q−, 11q−, 16p+, and 17p− chromosomes). The 11q− chromosome appeared to be derived from either an interstitial deletion in bands 11q21–23 or from a translocation with an unidentified chromosome. The presence of this chromosome was of particular interest because rearrangements of chromosome #11 at band 11q23 are often associated with malignancies of the monocytic lineage. The possible significance of these findings is discussed.     

Dibutyryl cyclic AMP stimulation of a monocyte-like cell line, U937: a model for monocyte chemotaxis and Fc receptor-related functions.

Treatment of the U937 cell line with 1 mM dibutyryl cyclic AMP (Bt2cAMP) resulted in a reduction in cell size and inhibition of DNA synthesis, and morphologically the cells appeared similar to macrophages. Electron micrographs indicated an increase in intracellular apparatus, whilst histochemical studies revealed smaller, denser nuclei and a greater intensity of non-specific esterase staining. Ia-like antigens (HLA-DR and HLA-DC) and complement receptor CR1 were not detected on U937 cells by monoclonal antibodies, nor were they induced by Bt2cAMP. CR3 was present in small amounts on U937 cells, and stimulation with Bt2cAMP increased the expression of this molecule in the cytoplasm and on the cell surface. Leu M3, a monocyte-specific antibody, was weakly reactive on both unstimulated and stimulated cells, whereas transferrin receptors, present on 90% of U937 cells, were lost after 48-hr stimulation with Bt2cAMP. JW6 and NH6, two monoclonal antibodies raised in our laboratory and found to be against immature monocytic antigens, showed decreased expression on stimulation. Monomer IgG binding via Fc receptors decreased on stimulated cells, and a monoclonal antibody (32.2) specific for FcRI confirmed this to be due to a decrease in the number of high-affinity receptors, rather than a decrease in IgG-binding affinity. In contrast, expression of the low-affinity FcRII, monitored by monoclonal antibody IV3, increased dramatically after stimulation. Other functional changes included the production of superoxide anions and the induction of non-specific phagocytosis. Two dimensional gel analysis, of detergent soluble proteins from unstimulated and 48-hr stimulated U937 cells, showed many differences in protein expression. A detailed investigation of these changes will facilitate a better understanding of the molecular mechanisms involved in the differentiation of U937 cells.     

Prostaglandin biosynthesis by a human macrophage-like cell line, U937

The human macrophage-like cell line, U937, produced significant amounts of prostaglandin (PG) E2 when incubated with exogenous arachidonic acid (AA). The synthesis of PGE2 was completely inhibited by pretreatment with indomethacin (20 micrograms/ml). Another major metabolite, unidentified, which was released during incubation with AA, was not inhibited by indomethacin, but was decreased by nordihydroguaiaretic acid (NDGA) (10(-5)M) or BW755C (10(-4)M). These results confirm the presence of cyclooxygenase and perhaps lipoxygenase activities in this macrophage-like cell line. Challenge of U937 cells with zymosan, opsonized zymosan, phorbolmyristate acetate (PMA), heat-aggregated human IgG (AHG), or calcium ionophore A23187 failed to stimulate synthesis and release of either PGE2 or the above mentioned metabolite. The inability of U937 cells to release endogenous AA from cell lipid for PG synthesis constitutes an important functional difference between these cells and normal macrophages.     

Cellular uptake and reactive oxygen species modulation of cerium oxide nanoparticles in human monocyte cell line U937

Cerium oxide nanoparticles (nanoceria) are promising materials for intracellular oxygen free radical scavenging providing a potential therapy for reactive oxygen species (ROS)-mediated inflammatory processes. In this study rhombohedral-shaped nanoceria were synthesized by flame spray pyrolysis with tuneable particle diameters between 3 and 94?nm by changing the liquid precursor flow rate. Monocytes and macrophages are major players in inflammatory processes as their production of ROS species has important downstream effects on cell signalling. Therefore, this study examined the ability of the nanoceria to be internalised by the human monocytic cell line, U937, and scavenge intracellular ROS. U937 cells activated in the presence of phorbol 12-myristate 13-acetate (PMA) were found to be more responsive to the nanoceria than U937 cells, which may not be surprising given the role of monocyte/macrophages in phagocytosing foreign material. The smaller particles were found to contain more crystal lattice defects with which to scavenge ROS, however a greater proportion of both the U937 and activated U937 cell populations responded to the larger particles. Hence all nanoceria particle sizes examined in this study were equally effective in scavenging intracellular ROS.     

上调c-myc基因的表达对U937白血病细胞的影响

 目的：构建稳定表达外源c-myc基因的人单核细胞白血病细胞株U937,并初步分析其特性。方法：首先构建重组质粒MSCV-c-myc-IRES-GFP （MMIG）载体,分别用MMIG及空载体MSCV-IRES-GFP（MIG）包装病毒,并感染U937细胞,用流式细胞术分选绿色荧光细胞,获得 U937/GFP和U937/MYC细胞,用荧光显微镜及流式细胞术检测GFP阳性率,用Western blotting测定细胞中c-Myc、survivin、X连锁凋亡抑制蛋白（XIAP）和Bcl-2的蛋白表达水平,流式细胞术检测U937/GFP和U937/MYC细胞周期,并用MTT法测定U937/GFP和U937/MYC细胞的生长情况,克隆形成实验检测克隆形成能力。结果：荧光显微镜观察,MIG和MMIG病毒感染后,2种细胞均表达绿色荧光蛋白;流式细胞术结果显示,MIG病毒感染的细胞荧光率为26.0%,MMIG病毒感染的细胞荧光率为277%;Western blotting的结果显示,c-Myc蛋白在MMIG病毒感染的细胞中表达水平升高。流式细胞术分选后,荧光显微镜观察可见绿色荧光蛋白表达明显增多,U937/GFP和U937/MYC细胞绿色荧光蛋白表达率分别达98.7%和93.7%。 U937/MYC细胞中c-Myc蛋白表达较U937/GFP细胞显著升高,c-Myc蛋白下游的survivin表达增多,而凋亡相关蛋白XIAP及Bcl-2的表达则没有明显变化。细胞周期检测显示,U937/MYC细胞处于S期的细胞数增多。MTT实验结果显示,U937/MYC细胞的生长速率较U937/GFP细胞增快。U937/MYC细胞的克隆形成能力较U937/GFP细胞强。结论：成功构建了c-myc基因高表达的U937稳定细胞株U937/MYC。在U937/MYC细胞中,c-Myc及其下游的survivin表达明显上调,处于细胞周期S期的细胞数增多、细胞生长加快、克隆形成能力增强,提示c-Myc可能通过增强自我更新能力、加快细胞周期、促进相关抗凋亡蛋白表达从而提高细胞的存活率。     

Beta-2-adrenoceptor agonists up-regulate the in vitro Fc epsilon receptor II/CD23 expression on, and release from, the promonocytic cell line U937 and human blood monocytes.

The possible regulatory role of beta 2-adrenoceptor agonists in the modulation of CD23 on the human promonocytic cell line U937 and on human monocytes was investigated. Incubation for 48 h in the presence of interleukin-4 (IL-4; 30 U/ml) induced optimal expression and release of CD23 on both U937 cells and human monocytes. When a beta 2-adrenoceptor agonist, either salbutamol or fenoterol, was added to U937 cells or monocytes both the IL-4-induced CD23 expression and release were markedly up-regulated. This effect was dose-dependent, starting at 10 nM and reaching a maximum at 1 microM final concentration of either salbutamol or fenoterol. The potentiating effect of salbutamol and fenoterol on CD23 expression and release was not observed when a beta-adrenoceptor antagonist, either D,L-propranolol (1 microM) or butoxamine (1 microM), was added to the incubation medium. The alpha-adrenoceptor agonist norepinephrine (1 microM) was ineffective in enhancing the IL-4-induced expression and release of CD23 from U937 cells or human monocytes, demonstrating the specificity of the beta 2-adrenoceptor-mediated effect. In the absence of IL-4, a moderate but significant increase in the CD23 expression on U937 cells and human monocytes by these drugs was observed, as compared to the basal values. These results indicate that, besides their bronchodilator effect, beta 2-adrenoceptor agonists may regulate IgE-dependent processes.     

Human monocyte cell line (U937) releases suppressive IgG-binding factor(s)

Fc gamma receptor-bearing U937 cells, when incubated in serum-free buffer, were found to release spontaneously a suppressor material for pokeweed mitogen-drigen IgG synthesis which could be retained on Sepharose 4B-IgG immunosorbents. Immunosorbents coupled with IgM or F (ab')2 fragments of IgG were unable to retain the inhibitory activity of U937-derived material, suggesting a binding specificity for the Fc gamma fragment of IgG. This suppressor material corresponds therefore by definition to an IgG-binding factor (IgG-BF). The mechanism for in vitro suppression of the antibody response by U937-derived IgG-BF was investigated. It did not interfere with cell proliferation and displayed maximum effect when added at day 3 of the culture period. Tested for its effect on IgG, IgM and IgA synthesis, IgG-BF suppressed antibody production following a pattern specific for IgG. Finally gel filtration of the suppressor material gave rise to two peaks of inhibitory activity with an apparent molecular mass comprised between 30 to 40 kDa and 60 to 73 kDa, respectively.     

Regulation of the expression of the low-affinity IgE receptor (Fc epsilon RII) in the human monocyte-like cell line U-937 by phorbol esters and IgE

The expression of the low-affinity receptor for IgE Fc epsilon RII) in the human monocyte-like U-937 cell line can be upregulated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and by IgE. TPA induces terminal differentiation of U-937 cells and causes a four- to fivefold increase in the number of Fc epsilon RII. TPA also modulates the expression of several other membrane markers of U-937 cells. IgE alone has a modest effect on the expression of Fc epsilon RII (about a 10% increase), while simultaneous treatment of U-937 cells with TPA and IgE has a cooperative effect, causing an eightfold increase in the number of Fc epsilon RII. Cycloheximide strongly suppresses the expression of Fc epsilon RII, both in TPA-stimulated and unstimulated cells; this effect can be partly reversed by culturing the cells in the presence of IgE. These results suggest that TPA induces the expression of newly synthesized receptors, while IgE causes an accumulation of preformed receptors.     

Replication of human cytomegalovirus in the cells of the U937 monocytoid cell line

Human cytomegalovirus (HCMV) infection in immunocopromized hosts sometimes occurs as a result of reactivation. Cells of the monocytemacrophage linkage are suggested to be a site of latency and persistence for HCMV. The human monocytic cell line U937 was infected with the AD169 strain and a clinical isolate of HCMV. The expression of surface antigens on the cells was assessed by flow cytometry. The polymerase chain reaction (PCR) was used to detect viral DNA from infected cells. CMV immediate early antigen, early antigen, and late antigen (LA) were detected from both clinical isolate- and AD169-inoculated U937 cells by flow cytometry. CMV DNA which code major immediate early gene (US3) and LA gene (US14) were detected from the clinical isolateinoculated U937 over a period of 31 days as tested by PCR. These U937 cells proliferated as well as uninfected U937 cell, but only a small number of AD169-inoculated U937 cells survived after 14 days of inoculation. Interleukin-2 activities were detected in the media on days 24–40 after inoculation with AD169. This chronic CMV infection model of U937 might be utilized to study the mechanisms of persistence and reactivation.     

Differentiation of the U937 macrophage cell line removes an early block of HSV-1 infection.

In the human macrophage-like cell line U937, which is resistant to infection with herpes simplex virus type 1 (HSV-1), it was previously shown that resistance can be overcome by inducing differentiation of the cells by treatment with phorbol 12-myristate 13-acetate (PMA). The present data show that differentiation, and not PMA treatment alone, enabled HSV-1 replication, because vitamin D3 and mezerein were also able to cause U937 cells to differentiate to a state permissive for HSV-1 infection. Additionally, a portion of the undifferentiated cells underwent a productive infection when treated with PMA 2 days after infection, suggesting persistence of HSV-1 in these cells. The nonpermissiveness of the undifferentiated cells was further defined. Resistance did not involve differences in virus uptake, because the amounts of viral DNA in the infected cells and nuclei of differentiated and undifferentiated U937 cells were not significantly different early after infection. However, only very low levels of RNA for HSV-1 immediate-early, early, and late genes could be detected in the undifferentiated U937 cells by Northern blot analysis compared with the differentiated U937 cells. These data suggest that the primary block in HSV-1 replication in undifferentiated U937 cells occurred after transport of the viral DNA to the cell nucleus but prior to steady-state accumulation of viral RNA for immediate-early genes.     

Growth of measles virus in a human macrophagelike cell line: U937

Measles virus infection was established in U937, a continuous human macrophagelike cell line. Unlike cultured human peripheral macrophages, infection resulted in prominent giant cell formation, indicating that these cells are susceptible to viral-induced fusion. Although a high proportion of cells in culture contained measles viral antigen by immunofluorescent assay a relatively small amount of infectious virus was produced. In contrast to continuously cultured human lymphoblastoid cell lines, infection of U937 was lytic, and persistent infection could not be established. The U937 cell line may be useful for further studies of viral interaction with macrophages, including those related to the induction of cell fusion by measles or other syncytium-forming viruses.     

Expression of complement factor H on the cell surface of the human monocytic cell line U937

Binding assays and immunocytochemical staining with monoclonal antibodies against the human serum complement protein factor H indicate that factor H antigen is present on the surface of more than 95% of the cells of the human monocytic cell line U937. The antigen is uniformly distributed and there are 10 000-15 000 copies/cell. Factor H antigen is strongly associated with the cell surface and is not removed by hypotonic or hypertonic washes. Factor H antigen has been isolated from surface radioiodinated and 35S biosynthetically labeled cells using polyclonal anti-factor H-Sepharose columns. The antigen is indistinguishable from serum factor H in molecular weight. Secretion of factor H by U937 cells was not detected using sensitive tests in which factor H secretion by monocytes was apparent. Phorbol myristate acetate stimulation of the cells had no effect on the average number of factor H molecules expressed. We conclude that factor H is synthesized by U937 cells, but is not secreted, and remains strongly associated with the cell surface. The surface-bound factor H may function as a C3b receptor.     

Modulation of complement receptors of a human monocyte cell line, U-937, during incubation with phorbol myristate acetate: expression of an iC3b-specific receptor (CR3)

The human monocyte line, U-937, derived from an individual with histiocytic lymphoma was studied for the expression of surface C3 receptors, after cultivation in the presence of phorbol myristate acetate (PMA) or T lymphocyte-conditioned medium. Receptors were detected by using EAC4b, EAC3b, EC3b, EAC3bi and EAC3d intermediates. U-937 cells, in exponential growth phase, poorly bound the intermediates; after exposure to PMA or T lymphocyte-conditioned medium, U-937 cells strongly bound both EAC3b and EAC3bi since about 50% of cells rosetted with these intermediates. This binding was totally inhibited by EDTA and by Mac-1 monoclonal antibody, suggesting the presence of only CR3 receptor types on these cells. Although U-937 cells formed rosettes with EAC3b, there was no evidence for the presence of CR1 receptors since no rosette was observed either with EAC4b or with EC3b intermediates (EC3b were prepared by coupling purified C3b to erythrocytes with N-succinimidyl 3-(2-pyridyldithio)propionate. As small amounts of factor H were present on EAC3b intermediates, incubation of EAC3b with U-937 cells induced their transformation into EAC3bi and their binding to CR3. Moreover, U-937 cells did not promote the cleavage of C3b in the presence of factor I alone, suggesting that these cells did not bear a sufficient amount of functionally active CR1. These results demonstrated that U-937 cells predominantly expressed CR3. The study of the kinetics of EAC3bi rosette formation demonstrated that CR3 expression is closely related to PMA activation. We suggest that CR3 activity could result from a phosphorylation of existing receptors.