Monocyte-like Cell Line U937 Research Articles

Differential induction of VCAM-1 on human iliac venous and arterial endothelial cells and its role in adhesion.

Venous and arterial large vessel endothelial cells (EC) were compared for their constitutive and TNF-alpha-induced expression of the cell-surface adhesion molecules ICAM-1 and -2, VCAM-1 and ELAM-1 by FACS analysis. Human iliac venous and arterial EC (HIVEC and HIAEC) constitutively express ICAM-1 and ICAM-2. TNF-alpha increases the expression of ICAM-1, but not ICAM-2, and induces the expression of ELAM-1 on both EC types. However, TNF-alpha induces VCAM-1 cell-surface expression and mRNA only in venous, but not in arterial EC. We next investigated the function of these adhesion molecules and their ligands, LFA-1, very late activation Ag (WLA-L) and sialylated Lewis x glycoprotein (sLe(x)), in adhesion assays with the monocyte-like cell line U937. Untreated U937 cells do not adhere to untreated HIVEC or HIAEC and adhesion is much lower to TNF-alpha-treated arterial than to TNF-alpha-treated venous EC. In adhesion-inhibition assays we demonstrate that U937 cell adhesion to TNF-alpha-treated HIVEC is mediated by VCAM-1/VLA-4 and ELAM-1/sLe(x) interaction, whereas the lower adhesion to TNF-alpha-treated HIAEC is only mediated by ELAM-1/sLe(x) interaction. U937 cells treated with the phorbol ester PMA for 3 days adhere to both HIVEC and HIAEC; this adhesion is mediated by LFA-1 interaction with ICAM-1 and/or -2. Adhesion of PMA-treated U937 cells is increased by TNF-alpha treatment of EC. This increased adhesion is mediated in part by the TNF-alpha-induced VCAM-1 expression on venous EC. Therefore, the cell-surface adhesion molecule VCAM-1 is differentially induced on these two EC types and the differential expression is functionally important in U937 cell adhesion.

Modulation of cell surface antigens and regulation of phagocytic activity mediated by CD11b in the monocyte-like cell line U937 in response to lipopolysaccharide.

Modulation of the cellular antigens and regulation of the phagocytic activity of the monocyte-like cell line U937 after culture with lipopolysaccharide (LPS) were investigated. CD14 expression was induced on the surface of the U937 cells after 48 h of culture with LPS and then they became adhesive with numerous filamentous filopodia extruded on the cell surface, exhibiting the enhanced expression of CD16 and CD23, the activation cell surface markers for differentiation into macrophage. However, no induction or enhancement of the cell surface expression was observed with respect to CD11b, CD18, HLA-A, B, C, HLA-DR, DQ, DP or CD57. These U937 cells also acquired the ability to produce superoxide anions and to phagocytose the Salmonella enteritidis strain, 116-54. This phagocytosis was inhibited by the anti-CD11b monoclonal antibodies, but not by the anti-CD14, anti-CD16, anti-CD18, anti-CD23, anti-HLA-A, B, C or anti-HLA-DR monoclonal antibodies. These findings indicate that the phagocytic activity against Salmonella enteritidis 116-54 induced by LPS is mediated mainly via the CD11b molecule, but is not associated with the increased expression of CD11b. Puromycin and cycloheximide, inhibitors of protein synthesis, or a divalent cation-chelating agent, EDTA completely inhibited this phagocytic activity. Interestingly, EDTA was found to suppress specifically the CD11b expression on the U937 cells cultured with LPS. No phagocytic activity was induced when the U937 cells cultured with LPS were incubated at 4 degrees C, but restored to the control level when shifted up to 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of cell-surface galactosyltransferase with immunoglobulins

Detection of the activity of β-1,4-galactosyltransferase (β-1,4-GT) in suspensions of viable mouse hepatocytes, the human hepatoma cell line Hep G2, the human colonic adenocarcinoma cell line HT-29, the monocyte-like cell line U937, and human splenic B and T lymphocytes suggested the presence of β-1,4-GT, in an enzymatically active form, on plasma membranes. The presence of β-1,4-GT on cell surfaces was also indicated from the effect of trypsinization of live cells, which significantly reduced cell surface β-1,4-GT activity, but did not affect the activity associated with cytoplasmic membranes. Furthermore, the presence of β-1,4-GT on the cell surface was demonstrated by indirect immunofluorescence staining of cells with anti-β-1,4-GT antibody. The detection of radioactivity in immunoglobulins (Ig) and their component chains after incubation with suspensions of intact cells in the presence of Mn 2+ and UDP-[ 3H]-galactose, indicated that Ig molecules were galactosylated. In the absence of UDP-[ 3H]-galactose, β-1,4-GT on cell surfaces, or immobilized on Sepharose-4B, formed stable complexes with galactose acceptors, including Ig. The efficiency of binding decreased in the order: J chain > α chain > μ chain > polymeric IgA2 > monomeric/polymeric IgA1 > IgM > IgG. Thus, β-1,4-GT could act as a cell-surface receptor for Ig through a cation-dependent, lectin-like association of the β-1,4-GT with the carbohydrate moieties of the Ig. This was confirmed by indirect surface immunofluorescence and radiolabeled ligand binding assays. The binding was inhibitable by EDTA, α-lactalbumin (in the presence of glucose), GlcNAc, or uridine 3',5'dialdehyde. At 37°C, the apparent affinity constants and association rate constants of interaction between cell surface β-1,4-GT on glutaraldehyde-fixed HT-29 and U937 cells and α2 chain or monomeric IgA1 were in the range from 7.1 × 10 7 to 4.6 × 10 8 M −1 and from 1 × 10 5 to 3 × 10 6 M −1 s −1, respectively. The dissociation rate constants and half time of dissociation calculated from these data were in the range from 2.1 × 10 −2 to 5.0 × 10 −4 s −1 and from 33 to 1380 s, respectively. The number of α2 or IgA1 molecules bound per HT-29 and U937 cell were in the range from 1.9 × 10 5 to 1.3 × 10 6. The binding of IgA by the cell surface β-1,4-GT was not associated with internalization or the catabolic degradation of the ligand.

Modulation of the transcriptional rate of Fcγ receptor mRNA in human mononuclear phagocytes

Human monocytes and macrophages bear three classes of cell surface receptors for the Fc portion of IgG (FcγRI, FcγRII, and FCγRIII). These receptors mediate phagocytosis and other effector functions and are important in the pathophysiology of hematologic disease, inflammation, and host defense. We have previously shown that interferon-γ (IFN-γ) and dexamethasone modulate total FcγRII mRNA levels as well as FcγRI and FcγRII protein expression on monocytes and the monocyte-like cell line U937. In this study, we investigated the modulation of FcγRI mRNA. Additionally, we utilized mRNA stability and nuclear run-on assays to study the mechanism of the modulation of Fcγ receptor transcripts in the monocyte/macrophage cell line U937. In U937 cells, IFN-γ increased FcγRI mRNA levels 7.5-fold. Treatment with dexamethasone reduced FcγRI mRNA levels to 0.6-fold of baseline and inhibited by 20–60% the increase in mRNA observed after treatment of the U937 cells with IFN-γ. On monocytes, treatment with IFN-γ increased monocyte FcγRI mRNA 6.7-fold. Cotreatment of the IFN-γ-stimulated monocytes with dexamethasone resulted in a 160% further increase in FcγRI expression. FCγRI and FCγRII mRNA half-lives were then determined in U937 cells by incubation with dexamethasone and/or IFN-γ for 16 hr and then arresting mRNA transcription with actinomycin-D (10 μg/ml). The mRNA half-lives in untreated U937 cells were 3.3 ± 0.3 hr (FcγRI) and 3.1 ± 0.3 hr (FcγRII). For either FcγRI and FcγRII, the effect of dexamethasone and/or IFN-γ on mRNA half-life was not significant ( P > 0.5). We also performed nuclear run-on experiments with U937 cells which indicated that IFN-γ increased the transcription of FcγRI 4.2-fold and FcγRII 1.7-fold. Our data suggest that these changes in FcγRI and FcγRII protein are likely due, at least in part, to increases in mRNA levels secondary to alteration in gene transcription.

Oxysterols in cultured bovine aortic smooth muscle cells and in the monocyte-like cell line U937.

Oxysterols in cultured bovine aortic smooth muscle cells and in the monocyte-like cell line U937 were analyzed by gas-liquid chromatography. The following products were detected: 7 alpha- and 7 beta-hydroxycholesterol, 7-ketocholesterol, cholesterol-alpha-epoxide, cholesterol-beta-epoxide, cholestanetriol, and 20- and 25-hydroxycholesterol. The proportions of oxidized cholesterol varied from 1 to 3% of total cholesterol. The concentrations of oxysterols were lower when the smooth muscle cells were maintained in a vitamin E-enriched medium, and were higher in U937 cells when the cells were activated with phorbol myristate acetate. The cell oxysterol content appears to be regulated by factors that inhibit or enhance free radical generation. The concentrations of oxysterols found in cells may serve as an indicator of the extent of lipid peroxidation.

Plasma membrane and intracellular pools of transferrin receptors decline during in vitro cultivation of U937 cells

Transferrin receptor expression in the monocyte-like cell line U937 was investigated during in vitro cultivation. U937 cells expressed a single class of high affinity surface transferrin receptors (KD approximately 4 nM), with apparent subunit Mr of 90-95,000 Da as determined by SDS-reducing PAGE. [125I]-transferrin binding studies on detergent-solubilized cells revealed that half to two-thirds of the total functional binding sites were located intracellularly. Radioligand binding, immunofluorescence and flow cytometry studies were performed on intact, detergent-solubilized, or saponin-permeabilized cells, using either transferrin or the anti-transferrin receptor monoclonal antibody OKT9 IgG. These studies demonstrated that functional and antigenic transferrin receptor levels were maximal on cells 24 h after subculture at low density and declined during the culture period. Scatchard analysis of radioligand binding data suggested that the decline in functional transferrin binding sites resulted from a decline in the number of available receptors. These results demonstrate that in U937 cells there is a density-dependent regulation of transferrin receptor expression, resulting in a loss of functional and antigenic receptors from both plasma membrane and intracellular locations.

Induction of CD 14 antigen on the surface of U937 cells by an interleukin‐6 autocrine mechanism after culture with formalin‐killed Gram‐negative bacteria

In order to better understand the regulation of CD14 antigen on the surface of the monocyte-like cell line U937 in response to bacteria, the expression and regulation of CD14 antigen on these cells when cultured with formalin-killed bacteria were determined using the monoclonal antibody MY-4 and analyzed by means of the indirect immunofluorescence method. CD14 expression was induced on the U937 cells after about 48 hours of culture with all of the formalin-killed Gram-negative bacteria used in this study but with none of the Gram-positive bacteria. Maximum expression was obtained after culture with formalin-killed Salmonella enteritidis strain 116-54. Various cytokines such as interleukin-1 beta, interleukin-2, interleukin-6, interferon-gamma and tumor-necrosis factor-alpha were assayed in the culture supernatant of U937 cells cultured with or without formalin-killed Salmonella enteritidis 116-54 using an enzyme-immunoassay or radioimmunoassay system. The U937 cells were found to produce a large amount of interleukin-6 in response to formalin-killed Salmonella enteritidis 116-54. On the other hand, culture supernatant (referred to as conditioned medium) obtained from the U937 cells after 72 h of culture with formalin-killed Salmonella enteritidis 116-54 also induced strong expression of CD14 antigen 48 to 72 h later, and this was blocked by the addition of anti-human interleukin-6 antibody. These findings suggest that the expression of CD14 antigen on the surface of U937 cells cultured with formalin-killed Gram-negative bacteria is induced by interleukin-6 and can be explained on the basis of the autocrine mechanism of interleukin-6.

Urokinase receptor mRNA level and gene transcription are strongly and rapidly increased by phorbol myristate acetate in human monocyte-like U937 cells.

We have studied the effect of the tumor promotor phorbol myristate acetate (PMA) on the level of mRNA for the receptor for urokinase-type plasminogen activator (u-PAR) in the human monocyte-like cell line U937. PMA causes an early increase in the u-PAR mRNA level which reaches a maximal 50-fold enhancement after 24 h of treatment. Half-maximal stimulation occurs at approximately 5 nM PMA. The effect is observed only with phorbol esters that also act as tumor promotors. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) also increases the level of u-PAR mRNA. Nuclear run-on experiments show a time-dependent increase in the u-PAR gene transcription rate after exposure of the cells to PMA. The PMA-induced increase in u-PAR mRNA is paralleled by a time-dependent increase in u-PAR protein as detected by cross-linking studies with radiolabeled ligand. We conclude that PMA stimulates transcription of the u-PAR gene in U937 cells, and this is responsible at least in part for the accumulation of the u-PAR mRNA and for the subsequent increase in urokinase-binding capacity.

β-Endorphin Binding to Naloxone-Insensitive Sites on a Human Mononuclear Cell Line (U937): Effects of Cations and Guanosine Triphosphate*

Some of the functional effects of beta-endorphin on immune cells are resistant to inhibition by naloxone. To further characterize the beta-[125I]endorphin-binding site mediating these effects and its response to cations and GTP, the human monocyte-like cell line U937 was used. Incubation of intact cells and beta-[125I]endorphin for 60 min at 4 C demonstrated a saturable, high affinity binding site [Kd = 1.2 +/- 0.5 X 10(-8) M (mean +/- SE; n = 4] competed by equimolar beta-endorphin and N-acetyl (Ac)-beta-endorphin but not by naloxone, morphine, or selective opiate receptor agonists. Competition studies showed that beta-endorphin-(6-31) and beta-endorphin-(28-31) were approximately 5- and 100-fold less potent, respectively, whereas beta-endorphin-(1-16) or -(1-27) was ineffective. Covalent cross-linking of beta-[125I]endorphin to intact cells and resolution by gel electrophoresis showed dominant bands at 59K and 44K and a minor band at 66K. The bands at 44K and 66K were completely displaced by increasing equivalent concentrations of beta-endorphin and N-Ac-beta-endorphin. Increasing concentrations of mono (Na+, K+)- and divalent (Ca2+, Mg2+, Mn2+) cations reduced the binding of beta-[125I]endorphin to U937 membrane; beta-[125I]endorphin binding to rat brain membrane showed similar cation sensitivity. GTP gamma-sulfate (GTP gamma S; 10(-4) M) alone reduced binding to U937 membrane by 25%. In the presence of Na+ (100 or 150 mM) or Mg2+ (10 mM), GTP gamma S reduced binding by an additional 50%. Moreover, GTP gamma S (10(-8)-10(-4) M) in the presence of Na+ (100 mM) reduced binding in a dose-dependent manner, whereas GMP was ineffective. In conclusion, beta-endorphin binds to sites on human U937 cells similar to those observed on normal murine splenocytes. Although naloxone insensitive, these sites exhibit properties, such as size, salt sensitivity, and coupling to a GTP-binding protein, that are similar to those observed for agonist binding to brain opiate receptors.

Cell surface antigens and function of monocytes and a monocyte-like cell line before and after infection with HIV

The human immunodeficiency virus (HIV-1) preferentially infects cells that express the CD4 molecule, including monocytes and cells of the monocyte lineage. The monocyte-like cell line U937 and monocytes isolated from peripheral blood lymphocytes (PBL) were infected with HIV-1. Cell surface antigen expression was determined in infected and noninfected cells as was the ability to stimulate in mixed lymphocyte reaction. The CD4 antigen decreased in infected cells U937 and PBL monocytes. MHC class II antigens HLA-DR, HLA-DQ, and HLA-DP increased in HIV-1 infected U937 cells. In infected PBL-derived monocytes, HLA-DR increased, HLA-DQ decreased, and HLA-DP was unchanged. Infected U937 and PBL monocytes were capable of stimulating allogeneic lymphocytes, thus demonstrating retention of the alloantigen presentation function of HIV-1-infected monocytes.

Regulation of the Expression of the Low-Affinity IgE Receptor (FcεRII) 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.

Mononuclear cells adhering to placenta involved in fetal alloantigen presentation and immunosuppression

The human immunodeficiency virus (HIV-1) preferentially infects cells that express the CD4 molecule, including monocytes and cells of the monocyte lineage. The monocyte-like cell line U937 and monocytes isolated from peripheral blood lymphocytes (PBL) were infected with HIV-1. Cell surface antigen expression was determined in infected and noninfected cells as was the ability to stimulate in mixed lymphocyte reaction. The CD4 antigen decreased in infected cells U937 and PBL monocytes. MHC class II antigens HLA-DR, HLA-DQ, and HLA-DP increased in HIV-1 infected U937 cells. In infected PBL-derived monocytes, HLA-DR increased, HLA-DQ decreased, and HLA-DP was unchanged. Infected U937 and PBL monocytes were capable of stimulating allogeneic lymphocytes, thus demonstrating retention of the alloantigen presentation function of HIV-1-infected monocytes.

Effect of low-density lipoprotein on the expression of high affinity Fcγ receptors

A substrain of the human monocyte-like cell line U937, which is a cholesterol auxotroph, was used to study the effect of cellular cholesterol depletion on the expression of the type I Fc receptor for IgG (FcγRI). Measurement of FcγRI expression was performed by immunofluorescence and flow cytometry using the monoclonal antibody (mAb) 32.2, which is specific for an epitope on FcγRI, and monomeric IgG 2a, which binds to the ligand binding site of FcγRI. Incubation of these cells for 24 h in growth medium containing delipidated fetal calf serum depletes cellular cholesterol without affecting growth or viability. While incubation of U937 cells with human interferon-γ (IFN-γ) increased FcγRI expression, cholesterol depletion after cell growth in media containing delipidated serum and IFN-γ resulted in reduced binding of both mAb 32.2 and IgG 2a. A significant decrease in the number of cell surface binding sites, as measured by mean fluorescence intensity, was observed after cholesterol depletion. Supplementation of the delipidated serum medium with pure cholesterol in an ethanol/bovine serum albumin mixture, which replenished cellular cholesterol and supported growth, failed to restore antibody binding significantly. In contrast, low-density lipoprotein (LDL) which also delivered cholesterol to the cells restored binding both in terms of the number of the reactive cells and cell surface receptor density. High-density lipoprotein (HDL 3), which does not deliver cholesterol to the cells, showed results similar to those obtained with pure cholesterol. This indicates that either LDL cholesterol is better utilized for membrane synthesis than pure cholesterol or that LDL provides another component, in addition to cholesterol, which is required for expression of FcγRI, but not for growth. These studies indicate a role for LDL in regulating the expression of FcγRI on the cell surface.

Elastase inhibitor. Characterization of the human elastase inhibitor molecule associated with monocytes, macrophages, and neutrophils.

A fast-acting inhibitor of serine elastase has been detected at high levels in human neutrophils, fresh monocytes, matured monocytes, and macrophages. The elastase inhibitor was isolated from large scale cultures of the monocyte-like cell line U937 by DNase chromatography, disulfide exchange, Phenyl-Sepharose, Red A-agarose, and DEAE HPLC chromatography with an average yield of 480 micrograms from 1.8 x 10(10) cells. The isolated polypeptide was verified as elastase inhibitor by its ability to (a) form a covalent complex with elastase; and (b) inhibit the elastinolytic activity of elastase. The purified elastase inhibitor molecule is unique, i.e., physiochemical and/or functional properties distinguish it from all other serine proteinase inhibitors. Treatment with iodoacetamide abrogates the ability of the molecule to form a complex with elastase, thereby providing evidence for the presence of an essential cysteine residue. Based on functional criteria, this elastase inhibitor has been grouped with the proteinase inhibitors of the serpin superfamily. The purified elastase inhibitor is a single polypeptide of Mr approximately 42,000. The NH2 terminus appears to be blocked. Compositional analyses indicates five cysteine residues per molecule of approximately 360 amino acid residues. Negligible levels of carbohydrate were detected on gas-liquid chromatography. This finding and the insensitivity of the molecule to peptide N-glycosidase F treatment strongly indicate that the elastase inhibitor is a nonglycosylated protein.

Plasminogen Activation Initiated by Single-chain Urokinase-type Plasminogen Activator: Potentiation by U937 Monocytes

The binding of urokinase-type plasminogen activators (u-PA) to receptors on various cell types has been proposed to be an important feature of many cellular processes requiring extracellular proteolysis. We have investigated the effect of single-chain u-PA binding to the monocyte-like cell line U937 on plasminogen activation. A 16-fold acceleration of the activation of plasminogen was observed at optimal concentrations of single-chain u-PA. This potentiation was abolished by the addition of either 6-aminohexanoic acid or the amino-terminal fragment of u-PA, thus demonstrating the requirement for specific binding of both single-chain u-PA and plasminogen to the cells. The mechanism of the enhancement of plasmin generation appears to be due primarily to an increase in the rate of feedback activation of single-chain u-PA to the more active two-chain u-PA by cell-bound plasmin, initially generated by single-chain u-PA. This increased activity of the plasminogen activation system in the presence of U937 cells provides a mechanism whereby u-PAs may exert their influence in a variety of cell-associated proteolytic events.

Expression of functional cell surface [formula omitted]-inactivator by U937 cells

We have previously shown that the human monocyte-like cell line U937 synthesizes C 1-INA and expresses cell surface C 1-INA . In this report we provide evidence that this surface-expressed C 1-INA is functionally active. Intact U937 cells demonstrated functional C 1-INA activity in a hemolytic assay. This activity was blocked when the cells were incubated with monospecific antibody to C 1-INA , and was not detectable in cell-free supernatants of U937 cells. SDS-PAGE analysis of radiolabeled U937 cell surface proteins purified by anti-C 1-INA affinity chromatography revealed two distinct bands. One protein had a M r of 105 kDa identical to plasma C 1-INA , and the second had a M r of 200 kDa. We were unable to determine the identity of the 200 kDa protein by Western blotting with anti-C 1-INA . However, the possibility exists that this 200 kDa molecule may represent a C 1-INA receptor, a dimeric form of C 1-INA , or an unrelated cell surface protein with affinity for C 1-INA . Furthermore, we show that treatment of U937 cells with phorbol ester resulted in an increase in the percentage of cells expressing surface C 1-INA . These results suggest that U937 cells express functional cell surface C 1-INA , which could function in vivo to protect these human tumor cells from lysis by host complement.

Cultured human endothelial cells stimulated with cytokines or endotoxin produce an inhibitor of leukocyte adhesion.

Activation of cultured human endothelial cells (HEC) by inflammatory stimuli, such as interleukin 1 (IL-1), tumor necrosis factor (TNF), and bacterial endotoxin (lipopolysaccharide, LPS), increases their surface adhesiveness for blood leukocytes and related cell lines. We now report that activated HEC also generate a soluble leukocyte adhesion inhibitor (LAI), which accumulates in conditioned media from IL-1-, TNF-, or LPS-treated, but not sham-treated, HEC cultures. LAI significantly inhibits the adhesion of PMN and monocytes to activated, but not unactivated, HEC. In contrast, LAI has no effect on the adhesion of lymphocytes, the promyelocytic cell line HL-60 or the monocyte-like cell line U937 to HEC monolayers. LAI appears to act directly on the leukocyte, but does not inhibit either agonist-induced responses in PMN (membrane depolarization, changes in cytosolic calcium concentration, superoxide production) or PMN attachment to serum-coated plastic surfaces. Endothelial generation of LAI is blocked by actinomycin D but not by aspirin or indomethacin. Preliminary biochemical characterization indicates that LAI is a soluble, protein-containing molecule that is heat- and acid-stable. Fractionation by HPLC gel filtration yields a single peak of LAI activity (14,000 less than Mr greater than 24,000). Thus, in addition to proadhesive cell surface changes, the endothelium may also actively contribute to the regulation of endothelial-leukocyte interactions at sites of inflammation in vivo through the production of soluble adhesion inhibitors such as LAI.

The Role of Fatty Acid Metabolites in the Differentiation of the Human Monocyte-Like Cell Line U937

The human monocyte cell line, U937, can be induced to terminally differentiate into macrophage-like cells when treated with gamma-interferon. However, if these cells were treated with gamma-interferon and esculetin, an inhibitor of the lipoxygenase pathway, or BW755C, an inhibitor of both the lipoxygenase and the cyclooxygenase pathways, a marked inhibition in cellular differentiation occurred. In contrast, inhibitors of only the cyclooxygenase pathway had no effect on differentiation. These studies suggest a role for lipoxygenase products of arachidonic acid in the differentiation of the human U937 cell line. Arachidonic acid utilized in the production of eicosanoids is derived from phospholipids by the action of phospholipase A2 and phospholipase C. When U937 cells were cultured in medium supplemented with gamma-interferon, there was a striking increase in the level of phosphatidylcholine and phosphatidylethanolamine-specific phospholipase A2 activities and phosphatidylinositol-specific phospholipase C activity as compared to control cells. More ever, although there was not a significant difference in the incorporation of labeled arachidonic acid or linoleic acid into the major phospholipids of differentiated U937 cells as compared to undifferentiated control cells, there was a marked increase in the relative amount of the labeled arachidonic acid released from the differentiated cells as lipoxygenase products compared to cyclooxygenase products. These data suggest that lipoxygenase products may be essential in the differentiation process of U937 cells and that enhanced phospholipase enzyme activities that occur during differentiation help explain how arachidonic acid becomes available to form lipoxygenase products.

Determination of intermediates, products and cleavage site in the reaction between plasminogen activator inhibitor type-2 and urokinases

Several specific inhibitors for plasminogen activators have been isolated from various organs and cell lines, those from human placenta and the human monocyte-like cell line U-937 being virtually identical. The reaction between this type of inhibitor, designated as type-2, and high- M r and low- M r urokinase-type plasminogen activators was followed by reversed-phase high-performance liquid chromatography and gel electrophoresis. The components, their stable complexes and their dissociation and cleavage products could be clearly identified in both systems. The amino acid sequence of the inhibitor at the cleavage site was determined to be -Met-Thr-Gly-Arg↓Thr-Gly-His-Gly-. A 35-residue carboxy-terminal fragment was found to be released.

Secreted proteins of human monocytes. Analysis by two-dimensional gel electrophoresis and effect of lipopolysaccharide.

A monocyte-rich preparation from the adherent cell fraction of human peripheral blood leukocytes was incubated for 1-8 h with [35S]methionine or [3H]leucine in the presence and absence of bacterial lipopolysaccharide (LPS). The macromolecules released into the supernatant were analysed by two-dimensional gel electrophoresis and radioautography. A complex labelling pattern involving at least 20 easily demonstrable and apparently distinct products with a broad range of molecular masses and isoelectric points was observed. LPS or LPS plus actinomycin in combination markedly stimulated the labelling and release of at least twelve different macromolecules ranging in apparent Mr from 12,000 to 46,000. Studies with monocytes that had been additionally purified by centrifugal elutriation and with the monocyte-like human cell line U-937 indicated that monocytes rather than contaminating cells were the source of these products. The majority of the secreted products were unique and did not cross-react with antibodies to interleukin 1 or tumour necrosis factor. The high resolving capacity of two-dimensional gel electrophoresis may be useful to define further the diverse biological activities and potential monokines released from monocytes at various stages of their differentiation and activation.