Evaluation of the expression of NADPH oxidase components during maturation of HL-60 cells to neutrophil lineage

To understand the expression of NADPH oxidase components during neutrophil maturation, we examined the expression of mRNAs and proteins for NADPH oxidase components, and the superoxide-producing activity using HL-60 cells incubated with dimethyl sulfoxide (DMSO). Northern blot and Western blot analyses revealed that gp91(phox), p67(phox), and p47(phox) were expressed after myelocyte stages, whereas p22(phox), p40(phox), and rac-2 were expressed from the promyelocyte stage. Furthermore, immunocytochemical staining of DMSO-induced HL-60 cells indicated that gp91(phox), p67(phox), and p47(phox) were detected only after myelocyte stages (myelocytes, metamyelocytes, band cells, and segmented cells), whereas p22(phox), p40(phox), and rac-2 were detected from the promyelocyte stage. In addition, nitro blue tetrazolium (NBT) assay showed that superoxide could be produced after myelocyte stages but not produced before promyelocyte stages. Moreover, almost the same results as those with DMSO-induced HL-60 cells were obtained using human bone-marrow cells by immunocytochemical staining and NBT assay, except that p22(phox) was detected by immunocytochemical staining after myelocyte stages in bone-marrow cells. Together, these observations indicate that all the components for NADPH oxidase are expressed, and the superoxide-producing activity is obtained after myelocyte stages during neutrophil maturation.     

The effect of IFN-gamma and TNF-alpha on the eosinophilic differentiation and NADPH oxidase activation of human HL-60 clone 15 cells.

The aim of this study was to investigate the effect of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on NADPH oxidase activity and gp91-phox gene expression in HL-60 clone 15 cells as they differentiate along the eosinophilic lineage. The results were compared to the eosoniphilic inducers interleukin-5 (IL-5) and butyric acid. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) or IL-5 (200 pM) caused a significant increase in the expression of the eosinophil peroxidase (EPO) and the major basic protein (MBP) genes. Similar results were observed when the cells were cultured with 0.5 mM butyric acid for 5 days. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) also caused a significant increase in superoxide release by HL-60 clone 15 cells after 2 days compared with control or with butyric acid-induced cells. After 5 days, these cytokines and butyric acid induced an even stronger release of superoxide. HL-60 clone 15 cells cultured with IFN-gamma and TNF-alpha for 2 days showed a significant increase in gp91-phox gene expression. We conclude that IFN-gamma and TNF-alpha are sufficient to induce the differentiation of HL-60 clone 15 cells to the eosinophilic lineage and to upregulate gp91-phox gene expression and activity of the NADPH oxidase system.     

Mechanism of the eosinophilic differentiation of HL-60 clone 15 cells induced by n-butyrate

n-Butyrate is one of the most powerful chemical inducers of the differentiation of human eosinophilic leukemia HL-60 clone 15 cells into mature eosinophils. We have recently reported that the mechanism by which HL-60 clone 15 cells differentiate into eosinophils by n-butyrate is that n-butyrate continuously inhibits histone deacetylase activity as a histone deacetylase inhibitor, resulting in continuous acetylation of histones. In this review, we discuss roles of histone acetyltransferase, histone deacetylase and histone deacetylase inhibitors in the differentiation of HL-60 clone 15 cells into eosinophils.     

Effects of Anaplasma phagocytophila on NADPH oxidase components in human neutrophils and HL-60 cells

The human granulocytic ehrlichiosis agent, Anaplasma phagocytophila, resides and multiplies exclusively in cytoplasmic vacuoles of granulocytes. A. phagocytophila rapidly inhibits the superoxide anion (O(2)(-)) generation by human neutrophils in response to various stimuli. To determine the inhibitory mechanism, the influence of A. phagocytophila on protein levels and localization of components of the NADPH oxidase were examined. A. phagocytophila decreased levels of p22(phox), but not gp91(phox), p47(phox), p67(phox), or P40(phox) reactive with each component-specific antibody in human peripheral blood neutrophils and HL-60 cells. Double immunofluorescence labeling revealed that p47(phox), p67(phox), Rac2, and p22(phox) did not colocalize with A. phagocytophila inclusions in neutrophils or HL-60 cells, and p22(phox) levels were also reduced. A. phagocytophila did not prevent either membrane translocation of cytoplasmic p47(phox) and p67(phox) or phosphorylation of p47(phox) upon stimulation by phorbol myristate acetate. The inhibitory signals for O(2)(-) generation was independent of several signals required for A. phagocytophila internalization. These results suggest that rapid alteration in p22(phox) induced by binding of A. phagocytophila to neutrophils is involved in the inhibition of O(2)(-) generation. Absence of colocalization of NADPH oxidase components with the inclusion further protects A. phagocytophila from oxidative damage.     

FMLP刺激HL—60细胞NADPH氧化酶激活的信号转导途径

目的 澄清中性粒细胞中[Ca^2 ]i及某些激酶在NADPH氧化酶激活中的作用和NADPH氧化酶激活的信号转导途径。方法 利用中性粒细胞样HL-60细胞,研究[Ca^2 ]i和某些激酶对fMLP刺激NADPH氧化酶激活的影响。结果 用10umol/L BAPTA-AM去除[Ca^2 ]i后使O2^-生成明显减少;8umol/L的PKC抑制物GF109203x显著地抑制了O2^-产生;50umol/L的p38抑制物SB203580、50umol/L的ERK抑制物PD098059和0.1umol/L的PI3K抑制物Wortmannin使O2^-产生受到不同程度抑制;PKC、PI3K、p38和ERK激活对[Ca^2 ]i升高没有影响;[Ca^2 ]i、PKC和PI3K对p38激活有一定作用,而ERK和Akt激活主要受PI3-K的调控。结论 试验说明[Ca^2 ]i依赖途径（PKC）和[Ca^2 ]i非依赖途径（PI3K、p38和ERK）对NADPH氧化酶激活都起着重要作用。     

Histamine-induced differentiation of eosinophilic subclone of HL-60 cells

Inflammation Research -     

Characterization of cytohesin-1 monoclonal antibodies: expression in neutrophils and during granulocytic maturation of HL-60 cells

ADP-ribosylation factors (Arf) are small GTP-binding proteins involved in vesicular transport and the activation of phospholipase D (PLD). The conversion of Arf-GDP to Arf-GTP is promoted in vivo by guanine nucleotide exchange factors such as ARNO or cytohesin-1. In order to examine the expression of ARNO and cytohesin-1 in human granulocytes, we generated specific polyclonal and monoclonal antibodies (mAbs). We also overexpressed GFP-ARNO and GFP-cytohesin-1 in RBL-2H3 cells to characterize the specificity and the ability of cytohesin-1 mAbs to immunoprecipitate cytohesin-1. Among the hybridomas secreting cytohesin-1 mAbs, only the clones 2E11, 1E4, 3C8, 6F5, 4C7, 7A3 and 8F7 were found to be specific for cytohesin-1. Furthermore, mAb 2E11 immunoprecipitated GFP-cytohesin-1 but not GFP-ARNO under native conditions. In contrast, mAbs 5D8, 4C3, 2G8, 6G11, 4C3, 6D4, 7B4 and 6F8 detected both cytohesin-1 and ARNO as monitored by immunoblotting. Although mAb 6G11 detected both proteins, this antibody immunoprecipitated GFP-ARNO but not GFP-cytohesin-1 under native conditions. Another antibody, mAb 10A12, also selectively immunoprecipitated GFP-ARNO under native conditions, but the epitope recognized by this mAb is unlikely to be linear as no signal was obtained by immunoblotting. Immunoprecipitation with a cytohesin-1 polyclonal antibody and blotting with cytohesin-1 specific mAbs revealed that cytohesin-1 is highly expressed in neutrophils. Cytohesin-1 can be detected in HL-60 cells but the endogenous protein levels were low in undifferentiated cells. Using the specific cytohesin-1 mAb 2E11 we observed a marked increase in levels of cytohesin-1 expression during dibutyryl-cyclic AMP-induced granulocytic differentiation of HL-60 cells. These data suggest that cytohesin-1, which may have important functions in neutrophil physiology, can be useful as a potential marker for granulocytic differentiation.     

Exercise training raises expression of the cytosolic components of NADPH oxidase in rat neutrophils

Exercise activates neutrophil burst and this effect is dependent on training status and exercise intensity. In this study, the chronic effect of treadmill exercise on phagocytosis, production of reactive oxygen metabolites and expression of NADPH oxidase components in rat neutrophils was investigated. Neutrophils were obtained by intraperitoneal lavage with PBS. After 11 weeks of training the exercised group showed increased phagocytosis capacity (49%) and production of reactive oxygen metabolites (6.6-fold) when compared with neutrophils from the sedentary group. Exercised had no effect on expression of the membrane components of NADPH oxidase (p22 ^phox , gp91 ^phox ). In contrast, there was an increase of the p47 ^phox mRNA levels (by 126%), the cytosolic component of the enzyme. In addition, exercise increased the protein content of p47 ^phox (by 22%) and of p67 ^phox (by 2.8-fold) in neutrophils. Evidence is then presented that training to moderate exercise increases phagocytosis and production of reactive oxygen metabolites and the expression of p47 ^phox and p67 ^phox in neutrophils. Therefore, moderate exercise might enable neutrophils to respond more efficiently when exposed to pathogens.     

Changes in susceptibility to Fas-mediated apoptosis during differentiation of HL-60 cells

The Fas-mediated pathway has been implicated as an important cellular pathway mediating apoptosis in diverse cell types. We conducted studies to examine the susceptibility to Fas-mediated apoptosis of HL-60 cells treated with differentiation-inducing factors such as dimethyl sulfoxide (DMSO), retinoic acid (RA), and 1alpha, 25 dihydroxyvitamin D3 (VD3). Although the expression of Fas antigen (Ag) and its mRNA showed a marked increase in HL-60 cells with cell differentiation, that of Bcl-2 protein and its mRNA revealed the reverse. The expression of caspase proteins such as caspases-3 and -8 was also enhanced during cell differentiation. DNA fragmentation, annexin V binding, and caspase activities increased in differentiated HL-60 cells with the addition of anti-Fas Ag antibody. These findings were more clearly demonstrated in DMSO- or RA-induced neutrophil-like cells than in VD3-induced monocyte-like cells. Therefore, susceptibility to Fas-mediated apoptosis showed an increase with differentiation of HL-60 cells, especially in the neutrophil lineage. These results suggest that the difference of susceptibility to Fas-mediated apoptosis among cell populations depends on the expression of Fas Ag, Bcl-2, and caspases. Cell maturation and susceptibility to Fas-mediated apoptosis may be linked in hematopoietic cells.     

The loss of IAP expression during HL-60 cell differentiation is caspase-independent

Human promyelocytic leukaemia cells (HL-60) differentiate into neutrophil-like cells that die spontaneously by apoptosis when treated with retinoic acid (RA). Inhibitors of apoptosis proteins (IAP) bind to and inhibit caspases 3, 7, and 9 activity and the induction of apoptosis. In this study, we demonstrate that undifferentiated HL-60 cells express IAP. During their differentiation, IAP expression is decreased at the mRNA and protein levels. In addition, we show that there is a corresponding increase in the expression and functional activity of active caspases 3 and 9. This activity was associated with the cleavage of XIAP, NAIP, and cIAP-2. Most importantly, we demonstrate that blocking caspase activity does not alter the decrease in IAP protein expression during differentiation but prevents caspase activation, IAP cleavage, and the induction of apoptosis. This result shows that the loss of IAP expression is independent of the induction of apoptosis and is solely related to the differentiation process. However, IAP cleavage is caspase-dependent. Terminal differentiation results in an altered apoptotic phenotype that is associated with the induction of HL-60 cell apoptosis.     

Regulation of the Interleukin-6 gene expression during monocytic differentiation of HL-60 cells by chromatin remodeling and methylation

The pro-inflammatory cytokine Interleukin (IL)-6 is involved in the proliferation and differentiation of leukocytes and non-immune cells, but its overproduction is associated with inflammatory and autoimmune disorders. The main producers of IL-6 are mature monocytes, whereas progenitor cells and the promyeloid cell line HL-60 do not synthesize IL-6. In contrast, HL-60 cells differentiated into monocytic cells were able to express IL-6 after lipopolysaccharide (LPS) stimulation. This study investigated the chromatin structure of the IL-6 promoter and the effect of methylation on IL-6 gene regulation during monopoiesis. The results show that the proximal IL-6 promoter regions I to III (+13/−329) were inaccessible in undifferentiated HL-60 cells but became significantly accessible in differentiated HL-60 cells stimulated with LPS. Region IL-6 VI (−1099/−1142) remained closed, but the upstream region IL-6 VII (−2564/−2877) relaxed after differentiation and LPS treatment. The opening of IL-6 IV (−309/−521) and IL-6 V (−500/−722), containing DNA and histone methylation sites, was differentiation-dependent only. Demethylation experiments using 5-aza-2′-deoxycytidine (AZA) followed by LPS stimulation revealed a significant enhanced IL-6 mRNA expression and protein release by HL-60 cells. AZA treatment resulted in significant increased IL-6 promoter accessibilities, identifying methylation as an important repressor of IL-6 gene regulation in promyeloid cells. The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) had no effect on IL-6 promoter accessibility.     

干扰HMGA2基因表达抑制HL60细胞的增殖及浸润

目的:探索干扰高迁移率族蛋白A2(high mobility group protein A2,HMGA2)基因的表达对HL-60白血病细胞体外增殖、浸润的影响.方法:实验分为3组,实验组为稳定转染慢病毒干扰HMGA2基因表达载体的HL-60细胞;阴性对照组为稳定转染慢病毒空载体HL-60细胞;空白对照组为未转染的HL60细胞.3组分别接种BALB/c裸鼠建立种植瘤模型,检测接种后各个时间点裸鼠外周血和骨髓中白血病细胞的比例(肿瘤负荷)、生活质量,计算接种40 d后各组小鼠的肝脾指数.结果:RT-PCR及Western印迹证明干扰RNA慢病毒表达载体可明显降低HL-60细胞靶基因的表达.接种后骨髓涂片结果显示裸鼠白血病模型成功建立.接种后21,28,40 d,实验组HL-60细胞在小鼠外周血和骨髓中的比例均明显低于阴性对照组及空白对照组,差异有统计学意义(P<0.05),其白血病细胞比例随接种时间延长而增加的速度亦明显变缓;且实验组小鼠萎糜少动、腹部膨隆、皮下结节等并发症出现时间明显较空白对照组及阴性对照组延迟;接种40 d后实验组小鼠肝脾指数分别为66.76±5.56和20.57±0.75,均明显低于其余两组,差异有统计学意义(P<0.05).结论:干扰HMGA2基因表达,可明显抑制HL60白血病细胞在小鼠体内增殖、浸润.     

喜树碱诱导的HL-60细胞凋亡过程中线粒体的变化

目的:研究喜树碱(CPT)诱导的人早幼粒细胞性白血病细胞HL-60凋亡过程中线粒体的质量和膜电势的变化。方法:以CPT诱导HL-60细胞凋亡为模型,利用膜联蛋白V(annexinV)-FITC/PI双染流式细胞术,研究HL-60细胞的凋亡和坏死。用DiOC6(3)染色流式细胞术,检测线粒体的膜电势(△ψm)。用NAO染色流式细胞术,检测线粒体的质量。结果:在4×10-6mol/LCPT的诱导下,HL-60细胞(12h)早期的凋亡率为(12.75±4.61)%,对照组为(2.88±2.49)%,二者相比较差异显著(P<0.01);CPT组坏死比率为(3.48±1.67)%,对照组为(0.71±1.10)%(P<0.01)。PI染色的结果显示,HL-60细胞(12h)晚期凋亡细胞的百分率,CPT组为(3.52±1.07)%,对照组为(0.46±1.06)%(P<0.01)。同时观察到,G2/M期细胞出现阻滞,G2/M期细胞的百分率,对照组为(22.46±2.19)%,CPT组为(13.45±1.91)%(P<0.01)。在12h时间点,CPT组线粒体的质量显著低于对照组(P<0.01),低线粒体质量的细胞所占百分率,对照组为(4.53±1.26)%,CPT组为(25.74±2.09)%。同时,CPT组线粒体的膜电势显著下降(P<0.01),CPT组线粒体膜电势降低的比率为(17.71±5.23)%,对照组为(1.64±2.00)%。结论:CPT诱导HL-60细胞凋亡过程中,线粒体的质量和膜电势均有所下降,但其去极化作用增强。     

IL-24基因修饰增强CIK细胞对HL-60细胞的细胞毒作用

目的:研究IL-24基因修饰的CIK细胞与同源树突状细胞共培养后对白血病细胞的杀伤作用及其机制.方法:从健康人外周血单个核细胞中常规诱导DC和CIK 细胞,电穿孔法将IL-24基因导入CIK细胞中(获得细胞为CIK-IL24),RT-PCR 和ELISA法检测CIK细胞中IL-24基因的表达,FCM和ELISA法检测转基因前后CIK表型及分泌细胞因子能力的变化,将CIK 细胞和同源DC共培养,FCM法检测共培养的DC-CIK细胞对HL-60细胞细胞毒活性的变化.结果:通过电穿孔法成功将IL-24基因导入CIK细胞,与对照组相比,转IL-24基因后CIK细胞中CD3~+、CD3~+CD56~+细胞的比例无明显改变,CD4~+CD25~+细胞比例显著下降.IL-24可上调CD3+CD56+细胞表面粘附分子CD54、CXCR4的表达,转染IL-24基因后CIK分泌TNF-α和IFN-γ的能力显著增强,与DC共同作用HL-60细胞时转染IL-24基因后的CIK细胞细胞毒活性明显增强.结论:通过IL-24基因修饰,明显增强了CIK细胞对HL-60细胞的杀伤能力,其机制与IL-24促进CIK分泌TNF-α、IFN-γ,上调CIK细胞表面粘附分子的表达,减少CD4~+CD25~+调节性T细胞比例等密切相关.     

Modulation of the expression of CD4 on HL-60 cells by exposure to 1,25-dihydroxyvitamin D3

The CD4 molecule functions as a receptor for the binding and infectivity of the human immunodeficiency virus (HIV). It is of interest, therefore, to develop procedures for its down-regulation. In the present study, the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the expression of cell surface antigens of the HL-60 promyelocytic leukemia cell line was analyzed. Exposure of HL-60 cells to 1,25(OH)2D3 resulted in down-regulation of CD4 as assessed by their staining with the Leu-3a monoclonal antibody (MoAb). This treatment increased the staining of HL-60 cells with the monocyte-specific 63D3 MoAb. In contrast to the rapid elimination of cell surface CD4 by exposure of HL-60 to phorbol myristate acetate (PMA), the maximal reduction of CD4 by 1,25(OH)2D3 was attained within 48 h after the beginning of the exposure.     

KLF2和KLF15基因在hBMSCs定向分化中的动态表达

目的研究KLF2和KLF15在人骨髓间充质干细胞(hBMSCs)成脂、成骨和成肌分化过程中的表达水平及变化趋势,探讨KLF2和KLF15在hBMSCs定向分化过程中可能的作用方式。方法密度梯度离心法分离hBMSCs,流式细胞仪检测细胞表面标志物,取第4代细胞分别进行成脂、成骨和成肌诱导并以油红O、茜素红及免疫荧光染色进行鉴定。通过实时定量PCR和免疫荧光分别从mRNA水平和蛋白水平检测相关标志物、KLF2、KLF15和GLUT4的表达。结果体外培养的hBMSCs表达间充质干细胞表面标志CD29、CD90,并在特定诱导剂作用下能够定向分化为脂肪细胞、成骨细胞和成肌细胞,染色鉴定结果为阳性,并分别检测到相关标志基因的表达;hBMSCs成脂、成骨和成肌过程中KLF2、KLF15和GLUT4的表达均呈动态变化。结论 KLF2和KLF15与hBMSCs成脂、成骨和成肌分化的启动和维持有关;KLF2和KLF15可能通过GLUT4调节能量代谢从而影响hBMSCs定向分化。