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1.
A human-derived monocytic cell line (U937) was induced to phagocytose Mycobacterium phlei by the addition of phorbol myristate acetate (PMA) to the culture medium for 50-60 h. Cells not treated with PMA were unable to phagocytose M. phlei. Magnetic beads enabled a rapid and highly efficient separation of phagocytosed and free bacteria to be achieved, an approach which is particularly useful if colony plating is used to enumerate bacterial survival within phagocytic cells. Fluorescence-activated cell sorting (FACS) analysis showed that 98% of U937 cells contained viable bacteria after 3 h.  相似文献   

2.
The U937 human monocytic cell line was studied to determine its ability to generate a respiratory burst after stimulation with phorbol myristate acetate (PMA) or opsonized zymosan. U937 cells cultured in normal medium produced virtually no superoxide anion or chemiluminescence in response to either stimulus. In contrast, U937 cells cultured in medium containing soluble factors from activated lymphocytes produced significant O2- and chemiluminescence when stimulated with PMA or opsonized zymosan. The chemiluminescence in response to PMA was maximal in U937 cells precultured with these soluble factors for 3 days, whereas maximal responsiveness to opsonized zymosan was not observed until 5 to 6 days of lymphokine exposure. Although this ability to generate a respiratory burst persisted for a number of days in U937 cells that were subsequently recultured in normal medium, this responsiveness was gradually lost in the continued absence of these factors. The data indicate that the U937 monocytic cell line can be activated or induced to differentiate by soluble factors released by activated lymphocytes. In the process, these cells acquire the ability to generate a respiratory burst. The U937 cell line may serve as a useful model for the study of the ontogeny and regulation of the respiratory burst during human monocytic differentiation.  相似文献   

3.
4.
Abstract The human histiocytic lymphoma cell line U937 can be induced to differentiate along the monocyte/macrophage pathway by either phorbol myristate acetate (PMA) or by the combination of retinoïc acid (RA) and 1,25-dihydroxyvitamin D3 (VD). U937 cells treated with either PMA or RA/VD were able to phagocytose Salmonella typhimurium in the presence of non-immune human serum. However, only cells differentiated by RA/VD were capable of developing an oxidative metabolic burst in response to infection. Since the oxidative burst is considered to be a potent antimicrobial mechanism, we investigated its effect on S. typhimurium . The oxidative burst failed to affect either the viability or the multiplication of S. typhimurium suggesting that if plays only a minor role in the host defence against S. typhimurium .  相似文献   

5.
6.
The proliferation and differentiation signals of myelogeneous U937 cells are provided by extracellular stimuli, such as lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA). In a DNA-native-polyacrylamide gel assay system, we demonstrated that a particular nuclease activity is expressed in PMA-stimulated U937 cells and secreted into the culture medium. The nuclease activity was induced in U937 cells by LPS treatment, while the secretion of the enzyme was undetected in the culture medium. Therefore, it is likely that the expression and secretion of the particular nuclease in U937 cells are controlled by extracellular stimulations, such as PMA and LPS treatment.  相似文献   

7.
The activation of phospholipase D by platelet-activating factor (PAF) in the human promonocytic cell line U937 has been investigated. In cells prelabeled with [3H]palmitic acid, addition of PAF or phorbol 12-myristate 13-acetate (PMA) induced the synthesis of [3H]phosphatidylethanol, indicating phospholipase D activation. When U937 cells were preincubated for 5 min with PMA, and then stimulated with PAF, formation of phosphatidylethanol was greatly enhanced. In contrast, under the same experimental conditions PMA treatment blocked completely the PAF-induced inositol phosphates formation in cells prelabeled with [3H]inositol. Thus, PMA treatment demonstrates that phospholipase D activation can occur independently from phosphoinositide-specific phospholipase C activation during PAF stimulation in U937 cells. On the other hand, the data herein presented suggest that influx of external calcium is required for phospholipase D activation by PAF, as assessed by complete inhibition of the enzyme activity by chelation of extracellular calcium or by treatment with the calcium channel blocker verapamil. Based on these findings, a hypothetical model for phospholipase D activation is discussed.  相似文献   

8.
Human monocytes synthesize large amounts of the second complement component (C2) after incubation with a T-lymphocyte product called monocyte complement stimulator (MCS). The human monocyte-like cell line, U937, also synthesizes C2 and can be stimulated to increase this synthesis by lymphokine-rich culture supernates. Additionally, phorbol myristate acetate (PMA), an agent which induces maturational changes in other macrophage-like cell lines, also stimulates C2 synthesis by U937 cells. Lymphokine and PMA stimulation of C2 secretion by U937 are both reversibly inhibitable by cycloheximide. At optimal concentrations for stimulation of C2 synthesis, PMA inhibits [3H]thymidine incorporation by U937 indicating that increased C2 is not due to increased numbers of U937 cells.  相似文献   

9.
Membrane-associated interleukin 1 (IL 1) activity was induced on the human macrophage tumor cell line, U937, by pretreatment with phorbol myristic acid (PMA). Incubation of PMA-treated, paraformaldehyde-fixed U937 cells with the murine cell line D10.G4.1 in the presence of concanavalin A caused an increase in DNA synthesis as measured by the uptake of tritiated thymidine. Paraformaldehyde-fixed U937, not pretreated with PMA, showed little or no activity. A rabbit polyclonal antibody directed against human IL 1 neutralized all membrane-associated IL 1-like activity, as measured by the inhibition of D10.G4.1 cell proliferation. PMA-treated U937 caused a pronounced enhancement of PGE2 production from a human chondrosarcoma cell line, SW-1353. Membrane-associated IL 1 induced a more potent PGE2 response than did a maximal concentration of soluble IL 1. Rabbit antihuman IL 1 neutralized membrane-bound IL 1 induction of PGE2. The data presented here raise the possibility that membrane-bound IL 1 may play a primary role in the pathophysiology of the inflammatory disease process.  相似文献   

10.
Human monocytes are known to metabolize arachidonic acid (AA) and to release prostaglandins upon stimulation. Previous data indicate that in vitro maturation and differentiation of monocytes result in alteration of this property with greatly diminished response to stimulators of release of prostaglandin E (PGE) and thromboxane B2 (TxB2) occurring after cells have been cultured. To further study the effects of differentiation on human monocyte AA metabolism, a model system was established based upon the human histiocytic cell line U937. Among tested stimulants, which included opsonized zymosan, complement fragment C3b, phorbol myristate acetate (PMA), calcium ionophore A23187, and concanavalin A, it was found that Escherichia coli lipopolysaccharide (LPS) was unique in that it stimulated increased release of TxB2 from U937 cells. The effect of the phorbol ester PMA, a compound commonly used to induce differentiation of U937, on the ability of U937 to respond to LPS was examined. Following 48 hr of treatment with PMA, U937 became capable of releasing both PGE and TxB2 in response to small doses of LPS. As previously observed for human monocytes, the release of PGE was delayed for several hours following stimulation and failed to reach maximal cumulative levels in culture until 24-48 hr following stimulation. In contrast to human monocytes, PMA-induced U937 were capable of maintaining their responsiveness to LPS for several days. Thus, the U937 cell line provides a useful model for study of the effects of differentiation of human mononuclear phagocytes on their ability to metabolize AA, and for the effects of LPS on histiocytic tumor cell prostaglandin release.  相似文献   

11.
The bacterial neomycin phosphotransferase gene driven by the Moloney mouse leukemia virus long terminal repeat (LTR) or SV40 early region promoter was introduced into the human promonocyte-macrophage cell line, U937, and into the pluripotential human embryonic teratocarcinoma cell line, NT2/D1. Clonally derived cell lines capable of growing in 2-4 mg/ml of the aminoglycoside antibiotic, G418 (Geneticin), were established and transfected with pHIVCat, a plasmid expressing the bacterial chloramphenicol acetyl transferase (CAT) activity under the control of the human immunodeficiency virus (HIV-1) LTR. All of the G418 resistant (neo(r)) U937 cell lines and 10 of 14 neo(r) NT2/D1 cell lines exhibited reduced basal levels of CAT expression or impaired responses to activation of the HIV-1 LTR by phorbol 12-myristate 13-acetate (PMA) when compared to the parental lines. Other differences included inhibition of tat activation of the HIV-1 LTR and increased sensitivity of U937 cells to human tumor necrosis factor alpha. The expression of other eukaryotic promoters including the HTLV-1 LTR, SV40 ori sequences, and the human beta-actin gene promoter was similarly affected. However, differentiation of the neo(r) U937 cells into macrophages was neither delayed nor impaired. Because PMA is an activator of protein kinase C (PKC) and a potent inducer of HIV-1 directed gene expression, the amounts, sensitivity to G418, and cytosol to membrane translocation of this enzyme were determined in the wild type and neo(r) U937 cells. G418 at concentrations too low to affect cell growth (12-150 micrograms/ml) inhibited PMA-induced transactivation responses in wild type cells but did not inhibit PKC-dependent protein phosphorylation in vitro. PKC activities in the wild type and neo(r) cells were similar in absolute amounts and in the cytosol-membrane distribution of the enzyme. In contrast with wild type cells, however, all of the cytosolic Ca(2+)-phospholipid-dependent form of PKC disappeared from the neo(r) cells within 30 min after PMA induction. The results suggested that, depending upon the cell type, gene cotransfer using aminoglycoside resistance as a selectable marker may seriously perturb important cellular control mechanisms such as the PKC pathway leading to activation of gene expression.  相似文献   

12.
Monocytes are a part of the innate immune system. Their differentiation into macrophages changes their cellular proteome and secretome. Particularly secretome components such as cytokines are crucial for immune response and inflammation in many diseases. Differentiation of human lymphoma cell line U937 can be triggered by phorbol 12‐myristate 13‐acetate (PMA). Screening of the cytokine release in U937 upon PMA stimulation by cytometric bead array almost exclusively showed interleukin‐8 (IL‐8). Next, a label‐free nanoLC‐ESI‐MS/MS‐sSRM method for quantification of IL‐8 in the cell secretome was established and applied to monitor the time kinetics of PMA treatment in different concentrations. Targeted secretome analysis was achieved by scheduled SRM‐MS using one proteotypic peptide as precursor ion and four mass transitions. Label‐free quantification was performed by external calibration using IL‐8 standard. Validation results indicated that the method was suited for the quantification of IL‐8 in the secretome. The maximal IL‐8 release of 62.4 ng/mL was observed after incubating cells treated by 50 ng/mL PMA for 48 h. The method can now be used for quantification of IL‐8 release from different cells under various conditions. Furthermore, it can be easily expanded to other secreted proteins detected by untargeted screening methods.  相似文献   

13.
The effects of several cytokines and phorbol myristate acetate (PMA) on LFA-1 and ICAM-1 expression on a human eosinophilic leukemia cell line, EoL-3, were investigated and compared with those of a human monocytic leukemia cell line, U937. EoL-3 cells expressed large amounts of LFA-1 and small amounts of ICAM-1, and their expression was regulated similarly in EoL-3 cells and U937 cells. Interferon-gamma (IFN-gamma) enhanced ICAM-1 expression but not LFA-1 expression, and PMA augmented both LFA-1 and ICAM-1 expression. IFN-gamma and PMA showed an additive effect on ICAM-1 expression. These results collectively suggest that expression of LFA-1 and ICAM-1 is regulated differently and that IFN-gamma and PMA regulate the expression through different mechanisms. PMA but not IFN-gamma induced homotypic adhesion of EoL-3 and U937 cells, suggesting that PMA but not IFN-gamma activated the adhesive function of these cells. Staurosporin, an inhibitor of protein kinases (PKs), partly suppressed IFN-gamma- and PMA-augmented expression of ICAM-1 on EoL-3 and U937 cells, but did not affect PMA-augmented LFA-1 expression, suggesting that staurosporin-sensitive PKs are involved in IFN-gamma- and PMA-augmented ICAM-1 expression but not in PMA-augmented LFA-1 expression. The role of protein kinase C (PK-C) in these mechanisms was not revealed because a PK-C inhibitor, H-7, did not show any definitive effect on IFN-gamma- and PMA-induced expression of LFA-1 and ICAM-1. Moreover, cyclic AMP (cAMP)- and cGMP-dependent pathways were not shown to be involved in the augmentation of the expression of these molecules.  相似文献   

14.
Induction of NF-KB during monocyte differentiation by HIV type 1 infection   总被引:11,自引:0,他引:11  
The production of human immunodeficiency virus type 1 (HIV-1) progeny was followed in the U937 promonocytic cell line after stimulation either with retinoic acid or PMA, and in purified human monocytes and macrophages. Electrophoretic mobility shift assays and Southwestern blotting experiments were used to detect the binding of cellular transactivation factor NF-KB to the double repeat-KB enhancer sequence located in the long terminal repeat. PMA treatment, and not retinoic acid treatment of the U937 cells acts in inducing NF-KB expression in the nuclei. In nuclear extracts from monocytes or macrophages, induction of NF-KB occurred only if the cells were previously infected with HIV-1. When U937 cells were infected with HIV-1, no induction of NF-KB factor was detected, whereas high level of progeny virions was produced, suggesting that this factor was not required for viral replication. These results indicate that in monocytic cell lineage, HIV-1 could mimic some differentiation/activation stimuli allowing nuclear NF-KB expression.  相似文献   

15.
Kaneko YS  Ikeda K  Nakanishi M 《Life sciences》1999,65(21):2251-2258
The effects of phorbol 12-myristate 13-acetate (PMA) on DNA damage-induced apoptosis were examined in promyelocytic leukemia cells, U937, in comparison with other differentiation-inducing agents to clarify the role of protein kinase C (PKC) vis-a-vis cellular differentiation in apoptosis. The apoptosis of U937 cells was observed at as early as 1-1.5 h following UV irradiation, with most cells being in apoptotic state at 3 h. Pretreatment with PMA for as short as 5 min was sufficient to inhibit apoptosis induced by UV irradiation, whereas apparent changes in cell cycle distributions and expression of differentiation markers by PMA were not observed until 12 h and 48 h, respectively. The inhibition of apoptosis by PMA was completely abolished by the pretreatment with calphostin C, a PKC inhibitor, and 4 alpha-phorbor 12,13-didecanoate, which is unable to activate PKC, did not protect U937 cells against apoptosis induced by UV irradiation. Other differentiation inducers, such as cyclic AMP and active vitamin D3, did not affect the UV-induced apoptosis of U937 cells. Taken together, it was suggested that PMA inhibits DNA damage-induced apoptosis through the activation of PKC rather than as a result of differentiation of U937 cells.  相似文献   

16.
The p21 (cip1/waf1) protein induces cell cycle arrest through inhibition of the activity of cdk (cyclin dependent kinase)/cyclin complexes. Expression of p21 is induced in a p53-dependent manner by DNA damage. p21 can also be induced independently of p53 by phorbol ester or okadaic acid. In this study, we have addressed the role of the PKC (protein kinase C) signaling pathway in the induction of p21 in response to PMA (phorbol myristate acetate) and okadaic acid. Levels of p21 (protein and mRNA) rapidly increased (within approximately 4 h) in U937 cells treated with PMA. The PKC-specific inhibitors RO 31-8220 and GF109203X down-regulated PMA or okadaic acid-induced p21 expression. Following persistent PKC activation, p21 mRNA levels remained elevated, indicating an enhanced stability of the mRNA. Using actinomycin D to measure mRNA stability and p21 promoter luciferase assays to measure activity, we provide evidence to support a role for the PKC signaling pathway in p21 mRNA stability. Thus, PKC regulates the amount of p21 in U937 cells at the level of mRNA accumulation and translation.  相似文献   

17.
Intracellular Ca2+ mobilization in U937 cells was studied. Stimulation of immature U937 cells with leukotriene B4 (LTB4) increased intracellular Ca2+ levels, whereas stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) failed to increase intracellular Ca2+ levels. U937 cells cultured with 1.5% dimethyl sulfoxide (DMSO) for 4 days (DMSO-U937 cells) responded to LTB4 and possessed the ability to respond to fMLP. U937 cells cultured with 1 ng/ml phorbol myristate acetate (PMA) for 4 days (PMA-U937 cells) lost the ability to respond to LTB4, although they responded to fMLP. Treatment of DMSO-U937 cells with 100 ng/ml PMA for 3 min suppressed intracellular Ca2+ increase induced by LTB4 and fMLP. The fMLP-induced Ca2+ rise in PMA-U937 cells was not suppressed by a further treatment with 100 ng/ml PMA. DMSO-U937 cells responded to inositol 1,4,5-trisphosphate (IP3), indicating that IP3 functions as a messenger of intracellular Ca2+ mobilization from endoplasmic reticulum in U937. The magnitude and duration of the rise in Ca2+ induced by IP3 in DMSO-U937 cells treated with 100 ng/ml PMA for 3 min were similar to those of the controls. When DMSO-U937 cells were Ca2+-depleted, addition of Ca2+ resulted in a transient overshoot of Ca2+ influx. However, the transient overshoot was not observed, when PMA-U937 cells were tested. These results indicate that Ca2+ efflux in PMA-U937 cells is increased by an activated exit pump, which may be directly or indirectly related to the functional state of PMA-U937 cells.  相似文献   

18.
Serglycin is the major proteoglycan in most hematopoietic cells, including monocytes and macrophages. The monoblastic cell line U937-1 was used to study the expression of serglycin during proliferation and differentiation. In unstimulated proliferating U937-1 cells serglycin mRNA is nonconstitutively expressed. The level of serglycin mRNA was found to correlate with the synthesis of chondroitin sulfate proteoglycan (CSPG). The U937-1 cells were induced to differentiate into different types of macrophage-like cells by exposing the cells to PMA, RA, or VitD3. These inducers of differentiation affected the expression of serglycin mRNA in three different ways. The initial upregulation seen in the normally proliferating cells was not observed in PMA treated cells. In contrast, RA increased the initial upregulation, giving a reproducible six times increase in serglycin mRNA level from 4 to 24 h of incubation, compared to a four times increase in the control cells. VitD3 had no effect on the expression of serglycin mRNA. The incorporation of (35S)sulfate into CSPG decreased approximately 50% in all three differentiated cell types. Further, the (35S)CSPGs expressed were of larger size in PMA treated cells than controls, but smaller after RA treatment. This was due to the expression of CSPGs, with CS-chains of 25 and 5 kDa in PMA and RA treated cells, respectively, compared to 11 kDa in the controls. VitD3 had no significant effect on the size of CSPG produced. PMA treated cells secreted 75% of the (35S)PGs expressed, but the major portion was retained in cells treated with VitD3 or RA. The differences seen in serglycin mRNA levels, the macromolecular properties of serglycin and in the PG secretion patterns, suggest that serglycin may have different functions in different types of macrophages.   相似文献   

19.
The binding of human urokinase-type plasminogen activator (u-PA) to the surface of the human monocytic cell line U937 was studied by immunological detection of bound u-PA or binding of biotinylated diisopropyl fluorophosphate-inactivated human u-PA visualized by light or electron microscopy. Untreated U937 cells showed a characteristic binding pattern, with the majority of the u-PA bound to the microvillar-containing protruding pole of the cells. After treatment with the phorbol ester PMA, the resulting adherent cell population was very heterogeneous with respect to both cellular morphology and u-PA binding. The bound u-PA was distributed on both the dorsal and the substrate side of the cells, and the patches of bound u-PA could not be correlated to any typical membrane conformations or cell-cell or cell-substratum contacts. When a monoclonal antibody directed against the amino-terminal fragment (ATF) of u-PA was used, the results were identical regardless of whether intact u-PA or ATF was used for binding to the cells. In contrast, when a monoclonal antibody recognizing the non-receptor-binding protease domain of u-PA was used, bound ATF showed no staining, while bound intact u-PA was stained as efficiently as above. The alteration of u-PA receptor distribution following treatment with PMA could be related to the changes in glycosylation and ligand affinity of the purified u-PA receptor previously described following PMA treatment of U937 cells.  相似文献   

20.
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.  相似文献   

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