Granulocyte-macrophage colony stimulating factor modulates the production of TNF alpha by differentiated U937 cells infected with Leishmania major

In this work, the production of tumor necrosis factor alpha (TNF alpha) during interaction of human phagocytes with the intracellular parasite Leishmania major was further investigated. The human monocytic cell line U937, differentiated with a combination of 1 alpha, 25 dihydroxyvitamin D3 (VD) and retinoic acid (RA), or with granulocyte macrophage colony stimulating factor (GM-CSF) was used. Differentiated U937 cells were infected with Leishmania major promastigotes, and TNF alpha was assayed in cell culture supernatants. It was found that the cytokine was produced only by U937 cells differentiated with VD/RA and further incubated with GM-CSF and LPS or interferon gamma (IFN gamma). L. major induced TNF alpha production only in the presence of GM-CSF. No direct relationship was found, however, between production of TNF alpha and resistance of differentiated U937 cells to infection with L. major.     

LPS-stimulated release of prostaglandin E and thromboxane B2 from the U937 cell line

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.     

Induction of NF-KB during monocyte differentiation by HIV type 1 infection

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.     

Characterization of a factor from the U937 cell line that enhances the toxicity of human eosinophils to Schistosoma mansoni larvae

A factor produced by lipopolysaccharide-stimulated human monocytes, monocyte-derived eosinophil cytotoxicity-enhancing factor (M-ECEF), increases the ability of human eosinophils to kill larvae of Schistosoma mansoni. In order to purify this monokine, a continuous cell line was sought as a generator of source material. It was found that high titers of an ECEF-like activity could be obtained from the U937 cell line cultured in serum-free medium. Production of this activity was optimal when cells were cultured with PMA for 2 days and were further treated with LPS for 2 days. PMA and LPS alone did not enhance eosinophil cytotoxicity and could be separated completely from U937-ECEF activity by reversed-phase HPLC. Thus, the activity was not due to carry-over of these two stimuli. U937-ECEF was compared with M-ECEF by a number of analytical methods. ECEF from both sources was resistant to several denaturing treatments but was sensitive to proteases or to reduction and alkylation. U937-ECEF exhibited activity profiles similar, if not identical, to those of M-ECEF when subjected to molecular sizing HPLC in the presence of 8 M urea, isoelectric focusing, and reversed-phase HPLC. The activity has apparent m.w. of 17,000 and 32,000, isoelectric points ranging from 3.8 to 5.1, and one or more reversed-phase HPLC retention times, depending on the method of sample preparation. These results demonstrate certain physical characteristics of M-ECEF, show that the U937 cell line is an appropriate source for the purification of M-ECEF, and provide information that will allow the design of a purification strategy. Although it appears that tumor necrosis factor (TNF) or a TNF-like molecule is a component of M-ECEF, a major component of M-ECEF is different from TNF as judged by the 1) physical characteristics of M-ECEF, 2) low direct toxicity of M-ECEF to L929 cells, 3) comparative stability of M-ECEF to heat treatment, and 4) inability of an anti-TNF monoclonal antibody to remove M-ECEF activity.     

Inhibition of p38 kinase reveals a TNF-alpha-mediated, caspase-dependent, apoptotic death pathway in a human myelomonocyte cell line

TNF-alpha transduces signals of survival or death via its two receptors, R1/p55/p60 and RII/p80/p75. The role of caspases as effectors of cell death is universally accepted, although caspase inhibitors may potentiate TNF cytotoxicity in some instances. In conditions when macromolecular synthesis is blocked, caspases are part of the machinery that executes TNF-triggered apoptotic death in U937, a human myelomonocyte cell line, and in the Jurkat T cell line. However, inhibition of p38 mitogen-activated protein kinase (p38 MAPK) triggered TNF cytotoxicity in U937 cells and murine splenic macrophages, but not the Jurkat cell line. TNF induced expression of the antiapoptotic protein c-IAP2 (cytoplasmic inhibitor of apoptosis protein 2), and was blocked in the presence of a p38 MAPK inhibitor, which also induced caspase-dependent, TNF-mediated apoptosis in U937 cells. Thus, inhibition of p38 MAPK resulted in the activation of caspase 9 and cleavage of the adaptor molecule BH3 interacting domain death agonist, and blocked NF-kappaB-mediated transactivation, without affecting the nuclear translocation of NF-kappaB. Collectively, these data show that activation of p38 MAPK is critical to cell survival by TNF in U937 cells, and demonstrate lineage-specific regulation of TNF-triggered signals of activation or apoptosis.     

Tumor necrosis factor-alpha activation by adenovirus E1A 13S CR3 occurs in a cell-dependent and cell-independent manner

The adenovirus (Ad) early gene product 13S transactivates the tumor necrosis factor (TNF)-alpha promoter in inflammatory cells. We examined both the subdomains of E1A and the upstream TNF promoter elements involved. In both Jurkat and U-937 cells, zinc finger or carboxyl region mutation of Ad E1A 13S conserved region 3 resulted in a significant loss of transactivation of the TNF promoter (> or =69%). For both cell types there was a TNF-negative regulatory element in the -242 to -199 region and a positive regulatory element between -199 and -118. In contrast, an upstream positive regulatory element was detected in different regions in both cell types. In U-937 cells the positive regulatory unit was between -600 and -576, whereas in Jurkat cells it was between -576 and -242. The U-937 upstream element was dependent on a site previously designated epsilon in cooperation with an adjacent nuclear factor-kappaB-2a site. Therefore, transactivation of the TNF promoter by Ad 13S in lymphocyte and monocyte cell types involves similar subdomains of the E1A protein, but cell-specific TNF promoter elements.     

Prosomes (proteasomes) changes during differentiation are related to the type of inducer

The core of the 26S proteasome, the 20S prosome, is a highly organized multi-protein complex found in large amount in malignant cells. Differentiation of several cell lines, including the monoblastic U937 and the lymphoblastoid CCRF-CEM, is accompanied by a general decrease in the prosome concentration when phorbol-myrirtic-acetate (PMA) and retinoic acid plus dihydroxyvitamine D3 (RA+VD) are used. Incubation of U937 cells for three days with PMA or RA+VD causes differentiation, but the resulting patterns of prosome labeling in the cell and on the plasma membrane are not the same. In contrast, the same kind of prosome changes occur in U937 and CCRF-CEM cells when PMA is used as inducer. The intracellular distribution of prosomes is also linked to malignancy and differentiation. Prosomes are found in the nucleus and the cytoplasm of cancer cells; and treatment with RA+VD decreases the prosomes in the nucleus whereas PMA causes various prosome proteins changes. These results indicate that prosomes are important in cell regulation and in the expression of malignancy.     

Induction of collagenase production in U937 cells by phorbol ester and partial purification of the induced enzyme

The U937 cell line is a monoblast-like cell line that can be induced to differentiate when treated with phorbol ester or a variety of other agents. Collagenase was detected in the media of U937 cell cultures after treatment with phorbol myristate acetate (PMA) at concentrations of 5 ng/mL or greater. In general, no collagenase was detected in the media of untreated cells. The induced collagenase cleaved native type I collagen into the 3/4 and 1/4-length fragments and showed the inhibition by ethylenediaminetetraacetic acid characteristic of the action of mammalian collagenases. Collagenase activity could be detected in the media of treated cells 12-18 h after the addition of PMA. Secretion of collagenase continued for 2-3 days after PMA addition. The production of collagenase by PMA-treated U937 cells was inhibited by actinomycin D and cycloheximide, suggesting that the induction of the enzyme is the result of de novo synthesis. The collagenase secreted by U937 cells induced with PMA has been purified 12-fold by using DEAE-Sephacel followed by wheat germ agglutinin-agarose chromatography. The apparent molecular mass of this U937 collagenase, determined by gel filtration chromatography on the partially purified enzyme, was 29-36 kilodaltons.     

Tumor necrosis factor signal transduction. Cell-type-specific activation and translocation of protein kinase C

We have investigated the changes in protein kinase C (PKC) activity after treatment of several cell lines with TNF. Binding studies with [3H]phorbol dibutyrate (PBt2) on whole cells revealed rapid and transient activation of PKC in Jurkat, K562, and U937 cells with a maximum of phorbol ester binding at 6 min after TNF treatment. As shown by Scatchard analysis, the TNF-induced increase of [3H]PBt2 binding reflected increments of phorbol ester binding site numbers rather than greater binding affinities. Upon subfractionation of TNF-treated U937 cells a transient increase of PBt2 binding in the membrane fraction was accompanied by a long term loss of PBt2-binding in the cytosol, indicating a TNF-induced translocation of PKC from the cytosol to the cell membrane. With histone III-S as a substrate, the determination of specific PKC activity revealed similar kinetics of PKC translocation in U937 cells. TNF also induced PKC translocation in K562 and Jurkat cells. However, although TNF caused long term down-regulation of cytosolic PKC activity in U937 cells, the cytosolic PKC activity only transiently decreased in both Jurkat and K562 cells and then recovered to near basal levels. In the human nonmalignant fibroblast cell line CCD18, PKC was not activated by TNF. Our data suggest that PKC activation may play a major role in TNF signal transduction in some, but not all target cells.     

The THP-1 cell line is a urokinase-secreting mononuclear phagocyte with a novel defect in the production of plasminogen activator inhibitor-2

Mononuclear phagocytes regulate the generation of plasmin by secreting urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-2 (PAI-2). We investigated the production of plasminogen activator (PA) and PA inhibitor by the human monocytic leukemia cell line, THP-1. Similar to U937 monoblast-like cells and peripheral blood monocytes (PBM), THP-1 cells produce a PA that is specifically neutralized by anti-uPA antibody and comigrates with human high molecular mass uPA (54 kDa) on casein-plasminogen zymogaphy. PA activity could be dissociated from intact THP-1 cells by brief treatment with a weak acid-glycine buffer, indicating that the uPA is secreted and bound to receptors on the plasma membrane. Regulation of uPA proceeds normally in THP-1 cells, with cell-associated PA activity increasing from 77 +/- 20 to 163 +/- 26 and 325 +/- 30 mPU/10(6) cells in response to PMA and LPS, respectively; parallel increases in steady state levels of uPA mRNA were observed. In contrast to normal expression of uPA activity, functional PAI-2 could not be demonstrated in either the conditioned media or cell lysates of THP-1 under basal or stimulated conditions. Both U937 and PBM secrete low levels of PA inhibitor activity that increase substantially in response to stimulation with PMA and LPS. Immunoreactive PAI-2, measured by ELISA, was undetectable in THP-1 lysates or conditioned medium, but was consistently present in U937 and PBM, paralleling the presence of PA inhibitor activity. THP-1 cells express low levels of an abnormally sized mRNA for PAI-2 and demonstrate a regulatory defect whereby steady state levels of PAI-2 mRNA are markedly reduced upon stimulation with PMA or LPS. By contrast, U937 and PBM respond to identical stimulation with increases in PAI-2 mRNA. We conclude that THP-1 cells express a structurally abnormal species of PAI-2 mRNA, with complete loss of inhibitory activity as well as altered function of PMA- and LPS-responsive regulatory elements.     

Ectopic expression of CXCR5/BLR1 accelerates retinoic acid- and vitamin D(3)-induced monocytic differentiation of U937 cells

The product of the blr1 gene is a CXC chemokine receptor (CXCR5) that regulates B lymphocyte migration and has been implicated in myelomonocytic differentiation. The U937 human leukemia cell line was used to study the role of blr1 in retinoic acid-regulated monocytic leukemia cell growth and differentiation. blr1 mRNA expression was induced within 12 hr by retinoic acid in U937 cells. To determine whether the early induction of blr1 might regulate inducible monocytic cell differentiation, U937 cells were stably transfected with blr1 (U937/blr1 cells). Ectopic expression of blr1 caused no significant cell cycle or differentiation changes, but caused the U937/blr1 cells to differentiate faster when treated with either retinoic acid or 1alpha,25-dihydroxyvitamin D(3). Treated with retinoic acid, U937/blr1 cells showed a greater increase in the percentage of CD11b expressing cells than vector control cells. Retinoic acid also induced a higher percentage of functionally differentiated blr1 transfectants as assessed by nitroblue tetrazolium reduction. U937/blr1 cells underwent moderate growth inhibition on treatment with retinoic acid. Similar results occurred with 1alpha,25-dihydroxyvitamin D(3). Because blr1 was induced early during cell differentiation and because its overexpression accelerated monocytic differentiation, it may be important for signals controlling cell differentiation.