OBJECTIVETo investigate the cytotoxicity and its mechanism of ZnO nanoparticles on human leukemic monocyte lymphoma cell line U937.

METHODSFour different size ZnO (10, 30, 60, 500 nm) were carefully characterized. The survival rate and viability were measured by trypan blue assay and MTT assay for each size ZnO particles at different concentrations (12, 120, 240, 600, 1200 µmol/L). The zinc probe, Fluozin-3, was used to detect the intracellular free zinc. Transmission electron microscopy was adopted to observe the cellular ultrastructure and the uptake of ZnO.

RESULTSAll four kinds of ZnO were rod shape, with a purity of > 99.9 wt%, and they were classified as zincite phase crystal and their surface areas were in accordance with the sizes. The viability (ZnO-n10: (97 ± 19)%, (91 ± 4)%, (24 ± 4)%, (15 ± 2)%; ZnO-n30: (111 ± 4)%, (81 ± 3)%, (24 ± 2)%, (27 ± 8)%; ZnO-n60: (105 ± 11)%, (73 ± 20)%, (43 ± 11)%, (28 ± 14)%; ZnO-µm: (88 ± 16)%, (62 ± 7)%, (22 ± 4)%, (13 ± 5)%) of cells exposed to ZnO decreased with the increasing of the concentration of ZnO from 12 to 600 µmol/L (r values were 0.965, 0.979, 0.998, 0.992, and the t values were 19.8, 25.3, 76.3, 40.9, respectively, P < 0.05). The liability (ZnO-n10: (98 ± 1)%, (67 ± 2)%, (59 ± 7)%, (13 ± 13)%, (5 ± 4)%; ZnO-n30: (98 ± 1)%, (97 ± 2)%, (50 ± 3)%, (20 ± 14)%, (7 ± 2)%; ZnO-n60: (97 ± 2)%, (88 ± 5)%, (48 ± 10)%, (12 ± 5)%, (4 ± 1)%; ZnO-µm: (96 ± 1)%, (76 ± 3)%, (58 ± 3)%, (19 ± 5)%, (20 ± 10)%) of cells exposed to ZnO decreased with the increasing of the concentration of ZnO from 12 to 600 µmol/L (r valued at 0.982, 0.956, 0.972, 0.980, and the t valued at 19.3, 12.1, 15.6, 18.5, respectively, P < 0.05). The increase of the zinc concentration showed by the zinc fluorescence probe was 121 ± 11, which was similar to the fluorescence of cells treated with ZnAc(2) (132 ± 14, F = 0.6, P > 0.05) at the Zn-equivalent concentration. There was no statistic difference for the percents of high zinc content cells in total cells exposed to ZnO-n30 (87.6 ± 2.6)% and these exposed to ZnAc(2) (86.9 ± 3.2)% (F = 1.5, P > 0.05).

CONCLUSIONZnO nanoparticles are highly cytotoxic to U937 cells and the solubilization of ZnO is the main toxicological mechanism.

Cell Survival ; Humans ; Monocytes ; drug effects ; ultrastructure ; Nanoparticles ; toxicity ; U937 Cells ; Zinc Oxide ; toxicity

Adhesion events of monocytes represent an important step in inflammatory responses induced by chemokines. The β1-integrin CD29 is a major adhesion molecule regulating leukocyte migration and extravasation. Although several adhesion molecules have been known as regulators of CD29, the molecular interactions between CD29 and its regulatory adhesion molecules (such as CD98 and CD147) have not been fully elucidated. Therefore, in this study, we examined whether these molecules are functionally, biochemically, and cell-biologically associated using monocytic U937 cells treated with aggregation-stimulating and blocking antibodies, as well as enzyme inhibitors. The surface levels of CD29, CD98, and CD147 (but not CD43, CD44, and CD82) were increased. The activation of CD29, CD98, and CD147 by ligation of them with aggregation-activating antibodies triggered the induction of cell-cell adhesion, and sensitivity to various enzyme inhibitors and aggregation-blocking antibodies was similar for CD29-, CD98-, and CD147-induced U937 cell aggregation. Molecular association between these molecules and the actin cytoskeleton was confirmed by confocal microscopy and immunoprecipitation. These results strongly suggest that CD29 might be modulated by its biochemical and cellular regulators, including CD98 and CD147, via the actin cytoskeleton.
Actin Cytoskeleton ; Antibodies ; Antibodies, Blocking ; Chemokines ; Enzyme Inhibitors ; Immunoprecipitation ; Leukocytes ; Ligation ; Microscopy, Confocal ; Monocytes ; U937 Cells*

Leucocyte extravasation has been known to play an important role in inflammatory reactions including contact dermatitis. Previous studies suggested that CD99 regulates β1 integrin activity and may be a novel therapeutic target molecule for inflammatory diseases. In this study, the effects of CD99-derived peptide, CD99CRIII3, on inflammatory reactions in contact dermatitis mouse model were investigated. CD99CRIII3 decreased β1-integrin activity in human monocytic U937 cells. CD99CRIII3 inhibited the adhesion of U937 monocytes to human umbilical vein endothelial cells and their extravasation through human umbilical vein endothelial cells. CD99CRIII3 reduced inflammation in the phorbol myristate acetate-induced contact dermatitis mice in a dose-dependent manner. These results indicate that CD99CRIII3 suppresses the extravasation of monocytes and inflammatory reactions in the animal model of the contact dermatitis, suggesting that CD99CRIII3 could be a new drug candidate against inflammatory skin diseases.
Animals ; Dermatitis, Contact* ; Human Umbilical Vein Endothelial Cells ; Humans ; Inflammation ; Mice* ; Models, Animal ; Monocytes* ; Myristic Acid ; Skin Diseases ; U937 Cells

Although Mycobacterium marinum is closely related to M. tuberculosis H37Rv (M. tbc) genomically, clinical outcome of human infection is quite different. The role of the host macrophage in determining differential pathologic responses was analyzed using an in vitro model of macrophage infection. By using subtractive hybridization, thirty-two differentially expressed genes were identified in the monocytic cell line U937 infected with M. tbc or M. marinum. Among them, IL-8 mRNA expression was more prominent in the M. tbc-infected U937 cells by Northern hybridization than in those infected with M. marinum. The IL-8 production was significantly lower in M. marinum-infected U937 or monocytes when compared with those infected by other strains of mycobacteria, such as M. tuberculosis H37Ra, M. bovis BCG or M. smegmatis. To identify possible mechanisms underlying these differences, changes in the expression of molecules such as nuclear factor-kappaB (NF-kappaB) involved in the signaling pathway activated by mycobacteria were assessed. U937 cells infected with M. tbc showed a significant degradation of IkBa proteins compared with M. marinum-infected U937 cells. Collectively, these results implicate distinct differences in IL-8 production in human macrophages infected with M. tbc or M. marinum, and suggest important role of IL-8 in the immunopathogenesis of tuberculosis.
Cell Line* ; Humans ; Interleukin-8* ; Macrophages ; Monocytes ; Mycobacterium bovis ; Mycobacterium marinum* ; Mycobacterium tuberculosis* ; Mycobacterium* ; RNA, Messenger ; Tuberculosis ; U937 Cells

BACKGROUND: Epidemiological studies have shown correlation between low incidence of coronary heart disease and high consumption of fish products. It has been suggested that this may be due to the high content of polyunsaturated fatty acids of the n-3 fatty acid group in fish oil. In animal studies eicosapentaenoic acid (EPA) inhibited attachment of monocytes to the arterial endothelium. METHOD: Adhesion assay was performed on the endothelial cells of the human umbilical vein with 25, 50, 100, 200 micrometer EPA-treated U937 cells. The endothelial cells were activated with lipopoysaccharide (LPS). The adhesion assay was repeated with oxidized EPA. EPA was oxidized with CuSO4 and ascorbic acid. RESULT:Viability of U937 cells were not affected by concentrations up to 200 micrometer of EPA and oxidized EPA. LPS treatment of endothelium notably increased the number of U937 cells attached to endothelial cells on the adhesion assay. However, treatment of EPA, native or oxidized, to U937 cells did not affect the number of U937 cells attached to LPS activated endothelial cells. CONCLUSION: EPA treatment, native or oxidized, of U937 cells did not affect U937 cell-endothelial cell adhesion. This suggests that inhibition of monocyte-endothelial cells attachment by EPA is not due to the effects of EPA on monocytes.
Animals ; Ascorbic Acid ; Cell Adhesion* ; Coronary Disease ; Eicosapentaenoic Acid* ; Endothelial Cells ; Endothelium ; Fatty Acids, Unsaturated ; Fish Products ; Humans ; Incidence ; Monocytes ; U937 Cells ; Umbilical Veins

Atherosclerosis is considered as a chronic inflammatory process. However, the nature of the oxidant signaling that regulates monocyte adhesion and its underlying mechanism is poorly understood. We investigated the role of reactive oxygen species on the vascular cell adhesion molecule-1 (VCAM-1) and monocyte adhesion in the cultured endothelial cells. TNF-alpha at a range of 1~30 ng/ml induced VCAM-1 expression dose-dependently. BCECF-AM-labeled U937 cells firmly adhered on the surface of endothelial cells when the endothelial cells were incubated with TNF-alpha (15 ng/ml). Ten micromol/L of SB203580, an inhibitor of p38 MAPK, significantly reduced TNF-alpha-induced VCAM-1 expression, compared to the JNK inhibitor (40micromol/L of SP60015) or ERK inhibitor (40micrommol/L of U0126). Also, SB203580 significantly inhibited TNF-alpha-induced monocyte adhesion in HUVEC. Superoxide production was minimal in the basal condition, however, treatment of TNF-alpha induced superoxide production in the dihydroethidine-loaded endothelial cells. Diphenyleneiodonium (DPI, 10micromol/L), an inhibitor of NADPH oxidase, and rotenone (1micromol/L), an inhibitor of mitochondrial complex I inhibited TNF-alpha-induced superoxide production, VCAM-1 expression and monocyte adhesion in the endothelial cells. Taken together, our data suggest that NADPH oxidase and mitochondrial ROS were involved in TNF-alpha-induced VCAM-1 and monocyte adhesion in the endothelial cells.
Atherosclerosis ; Endothelial Cells* ; Monocytes* ; NADP* ; NADPH Oxidase* ; p38 Mitogen-Activated Protein Kinases ; Reactive Oxygen Species ; Rotenone ; Superoxides ; Tumor Necrosis Factor-alpha ; U937 Cells ; Vascular Cell Adhesion Molecule-1*

OBJECTIVE: To purify and identify HMGB1 secreted by liver cells HepG2 and immune cells U937. METHODS: We cultured the liver cell lines HepG2 and immune cell lines U937, and stimulated them with HMGB1 (400 ng/mL) for 20 h. Then the supernatant was collected. Ultrafiltration centrifugation, CM-Sepharose cation, DEAE-Sepharose anion exchange chromatography, Sephadex G75-gel filtration chromatography, and immunoprecipitation were used for purification. The molecular weight and identity of HMGB1 was confirmed by SDS-PAGE and Western blot. RESULTS: A sharp stained protein band with a molecular weight of about 26 kD was obtained by SDS-PAGE analysis and shown to be HMGB1 confirmed by Western blot. CONCLUSION High purified HMGB1 can be separated from these two cell lines.
Cell Culture Techniques ; Electrophoresis, Polyacrylamide Gel ; methods ; HMGB1 Protein ; isolation & purification ; metabolism ; Hep G2 Cells ; Hepatocytes ; metabolism ; Humans ; Monocytes ; metabolism ; U937 Cells

No abstract available.
Cytokines ; Monocytes*

We tried to construct and identify the recombinant replication-deficient adenovirus vector coding for human tissue factor pathway inhibitor 2 (hTFPI-2) gene by AdMax system in HEK293 cells. Firstly, we obtained hTFPI-2 gene from the recombinant plasmid pIRES2-EGFP-TFPI-2 by PCR using primers with restriction endonuclease site of EcoRI or SacI. After digesting the hTFPI-2 gene and plasmid PDC316-IRES-EGFP shuttle vector, we ligated them with T4 ligase and formed the recombinant shuttle vector PDC316-IRES-EGFP-hTFPI-2. It was confirmed that the ligation product was inserted the gene of hTFPI-2 correctly by sequencing. Then we took cotransfection of HEK293 cells with the recombinant shuttle vector and genomic plasmid pBHGloxdeltaE1,3Cre by liposome lipofectamine2000, and finished the package of recombinant adenovirus Ad-hTFPI-2. The results of the PCR test and restriction endonuclease digestion confirmed the successful construction of the recombinants Ad-hTFPI-2. Furthermore, we measured the titre of Ad-hTFPI-2 with the aid of green fluorescence protein expression after multiplication and purification. The titre was 0.931 x 10(12) pfu/ml. Finally, we infected U937 monocytes by purified Ad-hTFPI-2, and determined the infection efficiency and the TFPI-2's level and activity. The efficiency of Ad-hTFPI-2 infection in U937 cells was 89.33%. After infected by Ad-hTFPI-2, the TFPI-2's level in supernatant increased about 7 fold. Also the TFPI-2 in supernatant had activities of inhibiting trypsin and plasmin. The recombinant adenovirus with the hTFPI-2 gene was constructed successfully. It will be helpful for the further investigation of its potentiality to be applied in antiatherosclerosis.
Adenoviridae ; genetics ; metabolism ; Defective Viruses ; genetics ; metabolism ; Genetic Vectors ; genetics ; Glycoproteins ; biosynthesis ; genetics ; Humans ; Monocytes ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; U937 Cells

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine fulfilling a broad variety of immunoregulatory functions. Monocytes and macrophages play a pivotal role in inflammation and immune regulation. NF-kappaB and HIF-1 are known to increase expression of the TNF-alpha gene in a separate way. METHODS: Human monocytic leukemia, U937 cells, were transfected using the standard electroporation method for intracellular expression of NF-kappaB and HIF-1. We performed analysis using the mammalian two-hybrid assay and co-immunoprecipitation assay for detection of protein interaction of both proteins. In addition, chromatin immunoprecipitation analysis was performed for examination of NF-kappaB and HIF-1 binding on the TNF-alpha gene promoter. RESULTS: Here we show that NF-kappaB and HIF-1 cooperatively induced an increase in expression of the TNF-alpha gene dependent on promoter activity by the direct protein interaction of these two transcription factors. Hypoxia signaling induced marked enhancement of the transactivation of TNF-alpha promoter by HIF-1 and NF-kappaB. A tandem NF-kappaB/HIF-1 binding site was identified within the TNF-alpha promoter, which acted as a strong enhancer element. Physical association of the Rel domain of NF-kappaB and the N-TD domain of HIF-1 was required. Hypoxia treatment also resulted in a significant increase in the protein interaction of NF-kappaB and HIF-1 in vivo. Both transcription factors were recruited on the chromatin TNF-alpha promoter dependent on hypoxia stimuli. CONCLUSION: The results of this study indicate that a variety of extracellular signals for activation of TNF-alpha gene expression might converge on the transcriptional regulation through the NF-kappaB/HIF-1 signaling pathway.
Anoxia ; Binding Sites ; Chromatin ; Chromatin Immunoprecipitation ; Electroporation ; Enhancer Elements, Genetic ; Gene Expression ; Humans ; Immunoprecipitation ; Inflammation ; Leukemia ; Macrophages ; Monocytes ; NF-kappa B ; Proteins ; Transcription Factors ; Transcriptional Activation ; Tumor Necrosis Factor-alpha ; Two-Hybrid System Techniques ; U937 Cells