Regulation of the growth and differentiation of a human monocytic cell line by lymphokines. I. Induction of superoxide anion production and chemiluminescence

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.     

Superoxide anion participation in human monocyte-mediated oxidation of low-density lipoprotein and conversion of low-density lipoprotein to a cytotoxin

Human monocytes, upon activation with opsonized zymosan, altered low-density lipoprotein (LDL) during a 24-h co-incubation, resulting in its oxidation and acquisition of cytotoxic activity against target fibroblast cell lines. Both the oxidation of LDL and its conversion to a cytotoxin were enhanced with time of incubation, with the most substantial changes occurring after 6 h of culture of LDL with activated monocytes. Unactivated monocytes did not mediate either alteration. Superoxide anion (O2-) participated in both the oxidation of LDL and its conversion to a cytotoxin since addition of superoxide dismutase (SOD) at the beginning of the co-incubation inhibited, in a concentration dependent fashion, both the monocyte-mediated oxidation and the monocyte-mediated conversion of LDL to a cytotoxin. As expected, the rate of superoxide anion release was greatest during the respiratory burst, very early in the 24-h incubation (0 to 2 h); however, exposure of LDL to monocytes during the respiratory burst was not required for LDL oxidation. The lower levels of O2- released by the cells hours after the respiratory burst had subsided were sufficient to lead to the initiation of LDL oxidation. Three results indicated that the oxidative modification of LDL into a cytotoxin required O2(-)-independent free radical propagation after O2(-)-dependent initiation. First, oxidation of LDL exposed to the activated, superoxide anion-releasing monocytes for 6 h could be almost completely blocked by the addition at 6 h of the general free radical scavenger butylated hydroxytoluene, but not by SOD. Second, LDL oxidation proceeded even after removal of LDL from the superoxide anion-producing, activated cells after various durations of exposure. Third, the development of substantial levels of lipid peroxidation products and the development of greater cytotoxicity occurred after 6 h of exposure of LDL to activated cells, long after peak O2- release had subsided. These results lead us to conclude that monocyte-mediated oxidation of LDL, leading to its transformation into a cytotoxin, requires release of O2- occurring as a result of activation but not necessarily during the respiratory burst, and also requires O2(-)-independent free radical propagation. The modification of LDL into a potent toxin by activated monocytes may explain the tissue damage in atherosclerotic lesions and other pathologic sites in which inflammatory cells congregate.     

Infection of differentiated U937 cells by Salmonella typhimurium: Absence of correlation between oxidative burst and antimicrobial defence

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 .     

Cellular Thiol Production and Oxidation of Low-Density Lipoprotein

Compelling evidence suggests that low-density lipoprotein (LDL) is oxidized by cells within the arterial intima and that, once oxidized, it is profoundly atherogenic. The precise mechanism(s) by which cells promote the oxidation of LDL in vivo are not known; in vitro, however, oxidation of LDL can be enhanced by a number of differing mechanisms, including reaction with free and protein-bound metal ions, thiols, reactive oxygen species, lipoxygenase, myeloperoxidase and peroxynitrite. This review is concerned with the mechanisms by which cells enhance the oxidation of LDL in the presence of transition metals; in particular, the regulation, pro- and anti-oxidant consequences, and mechanism of action of cellular thiol production are examined, and contrasted with thiol-independent oxidation of LDL in the presence of transition metals.     

Induction of oxidative DNA damage in u937 cells by TNF or anti-Fas stimulation

TNF and Fas signaling pathways are reported to induce mitochondrial damage associated with production of oxygen radicals. We examined whether such radical production elicited detectable nuclear DNA damage in U937 cells following treatment with TNF or with anti-Fas antibodies. Using GC-mass spectroscopy for analysing base oxidation, several oxidized species increased significantly following TNF treatment, whereas anti-Fas resulted in less detectable oxidative damage using this assay. Cytogenetic analysis showed that, in the presence of aphidicolin, which blocks several types of DNA repair, TNF induced extensive chromosomal damage. Aphidicolin also synergized with TNF and anti-Fas in inducing cell death which was prevented by reducing atmospheric oxygen or addition of n -acetyl cysteine, a scavenger of oxygen radicals. Thus, several lines of evidence point to the TNF and Fas pathways inducing extensive oxidative DNA damage and repair, and suggest potential roles for these pathways in mutagenesis and aging.     

1 Acyl-2 acetyl-sn-glycero-3 phosphocholine decreases the susceptibility of low-density lipoprotein to oxidative modification by copper ions,monocytes or endothelial cells

The effects of platelet-activating factor (PAF) and its analogue, 1 acyl-2 acetyl-sn-glycero-3 phosphocholine (1 acyl-2 acetyl-GPC), were investigated on the oxidative modification of low-density lipoprotein (LDL) by copper ions, U937 monocyte-like cells or endothelial cells, by determination of the lipid peroxidation end products (TBARS) content and measurement of the electrophoretic mobility of the particle. 1 Acyl-2 acetyl-GPC, in the concentration range 1–5 μg/ml, inhibited LDL oxidation in a dose-dependent manner in the three systems, whereas PAF had no effect. The protective effect of 1 acyl-2 acetyl-GPC was markedly more important when oxidative modification was performed with endothelial cells, leading to total inhibition at 5 μg/ml. At the same concentration, the TBARS production was inhibited by 60% and 20% with monocytes and copper ions, respectively. The degradation by J774 macrophage-like cells of LDL modified by copper ions, U937 monocyte-like cells or endothelial cells was also inhibited when modification was performed in the presence of 1 acyl-2 acetyl-GPC. Furthermore, preincubation of the LDL particle with 1 acyl-2 acetyl-GPC before modification protected the lipoprotein against oxidation, whereas preincubation of the cultured cells with the phospholipid had no effect. Thus 1 acyl-2 acetyl-GPC decreases the susceptibility of the LDL particle to oxidative modification, possibly by intercalation within the lipid phase of the particle. Since LDL oxidation is believed to play an important role in the initiation and progression of atherosclerosis, this inhibitory effect of 1 acyl-2 acetyl-GPC might be of importance in view of the fact that this phospholipid is produced concomitantly with PAF in some inflammatory cells.     

Oxidative modification of low density lipoprotein (LDL) by activated human monocytes and the cell lines U937 and HL60

Summary Human peripheral blood monocytes, upon activation, have the capacity to oxidize low density lipoprotein (LDL) and render the LDL toxic to cultured cells. Previous studies by our laboratory indicate that this process is mediated by free radicals in that it can be prevented by addition of free radical scavengers and antioxidants during the incubation of monocytes with LDL. Here we report that optimal modification of LDL by monocytes was influenced by media composition. In the absence of added metal ions, oxidation was distinctly dependent on the concentration of monocytes as well as LDL concentration. Exposure of monocytes to lipopolysaccharide or stimulation of phagocytosis by opsonized zymosan resulted in marked enhancement of LDL oxidation compared to other activating agents. After exposure to activated monocytes, lipid oxidation products in the supernatant were found both in a high molecular weight fraction containing LDL (>30 000 Daltons) and in a lipoprotein-free, low molecular weight fraction (<30 000 Daltons), yet only the high molecular weight, LDL-containing fraction was toxic to target cells. In addition, human myelomonocytic cell lines U937 and HL60 were shown to mediate oxidation of LDL. As with monocytes, exposing these cells to opsonized zymosan caused the level of LDL oxidation to be significantly enhanced. These findings offer further insight into the mechanisms of monocyte-mediated oxidation of lipoproteins and will facilitate studies investigating the role of monocyte-modified LDL in tissue injury. This project was funded by grants form the American Heart Association-Northeast Ohio Affiliate and the National Institutes of Health, Bethesda, MD (HL-29582).     

Protein oxidation and proteolysis in RAW264.7 macrophages: effects of PMA activation

Macrophages are stimulable cells able to increase the production of reactive oxygen and nitrogen species dramatically for a short period of time. Free radicals and other oxidants are able to oxidize the intracellular protein pool. These oxidized proteins are selectively recognized and degraded by the intracellular proteasomal system. We used the mouse macrophage-like cell line RAW264.7 to test whether macrophagial cells are able to increase their protein turnover after oxidative stress and whether this is accompanied by an increased protein oxidation. Macrophagial cells are particularly susceptible to bolus additions of hydrogen peroxide and peroxynitrite. In further experiments we activated RAW264.7 cells with PMA to test whether the production of endogenous oxidants has analogous effects. A clear dependence of the protein turnover and protein oxidation on the oxidative burst could be measured. In further experiments the role of the proteasomal system in the selective removal of oxidized proteins could be revealed exploring the proteasome specific inhibitor lactacystin. Therefore, although oxidants are able to attack the intracellular protein pool in macrophages, these cells are able to remove oxidized proteins selectively and protect the intracellular protein pool from oxidation.     

Oxalomalate,a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase,enhances lipid peroxidation-mediated oxidative damage in U937 cells

Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. Cytosolic NADP+-dependent isocitrate dehydrogenase (ICDH) in U937 cells produces NADPH, an essential reducing equivalent for the antioxidant system. The protective role of ICDH against lipid peroxidation-mediated oxidative damage in U937 cells was investigated in control cells pre-treated with oxalomalate, a competitive inhibitor of ICDH. Upon exposure to 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) to U937 cells, which induces lipid peroxidation in membranes, the viability was lower and the protein oxidation, lipid peroxidation, and oxidative DNA damage, reflected by an increase in 8-hydroxy-2'-deoxyguanosine, were higher in oxalomalate-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species, as measured by the oxidation of 2',7'-dichlorodihydrofluorescin, as well as the significant decrease in the intracellular GSH level in oxalomalate-treated U937 cells upon exposure to AAPH. These results suggest that ICDH plays an important role as an antioxidant enzyme in cellular defense against lipid peroxidation-mediated oxidative damage through the removal of reactive oxygen species.     

Prooxidant and antioxidant properties of Trolox C, analogue of vitamin E, in oxidation of low-density lipoprotein

Trolox C (Trolox), a water-soluble analogue of vitamin E lacking the phytyl chain, was investigated with respect to its effect on the oxidation of low-density lipoprotein (LDL). Trolox was added at different time points of LDL oxidation induced by Cu2+ and aqueous peroxyl radicals. In the case of Cu2+ -induced LDL oxidation, the effect of Trolox changed from antioxidant to prooxidant when added at later time points during oxidation; this transition occurred whenever alpha-tocopherol was just consumed in oxidizing LDL. Thus, in the case of Cu2+ -dependent LDL oxidation, the presence of lipophilic antioxidants in the LDL particle is likely to be a prerequisite for the antioxidant activity of Trolox. When oxidation was induced by peroxyl radicals, as a model of metal-independent oxidation, the effect of Trolox was always antioxidant, suggesting the importance of Cu2+ /Cu+ redox-cycling in the prooxidant mechanism of Trolox. Our data suggest that, in the absence of significant amounts of lipophilic antioxidants, LDL becomes highly susceptible to oxidation induced by transition metals in the presence of aqueous reductants.     

p-Hydroxyphenylacetaldehyde, the major product of tyrosine oxidation by the activated myeloperoxidase system can act as an antioxidant in LDL

The oxidative modification of low density lipoprotein (LDL) may play a significant role in atherogenesis. HOCl generated by the myeloperoxidase/H2O2/Cl- system of activated neutrophils may be operative in vivo making LDL atherogenic. Tyrosine has been found to be oxidized by HOCl to p-hydroxyphenylacetaldehyde (p-HA) capable of modifying phospholipid amino groups in LDL. As an amphiphatic phenolic compound, p-HA may have the potential to act as an antioxidant in the lipid phase of LDL. The present results show that (a) tyrosine exerts a protective effect on LDL modification by HOCl, (b) p-HA could act as antioxidant associated with the lipoprotein preventing cell- and transition metal ion-mediated LDL oxidation and (c) p-HA was able to scavenge free radicals.     

The cellular location and specificity of bacterial cytochrome c peroxidases.

We have found that an anti-CD11c monoclonal antibody (MAb) inhibits the respiratory burst induced in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells as well as in human peripheral blood monocytes and neutrophils upon cell stimulation with concanavalin A. The MAb had no effect, however, when the added stimulus was fMet-Leu-Phe or PMA. Flow cytometry analyses indicated that concanavalin A was able to interact with CD11c. The anti-CD11c MAb inhibited significantly concanavalin A binding to differentiated U937 cells, and concanavalin A blocked binding of anti-CD11c MAb to the cells. Binding of labelled concanavalin A to membrane proteins which were separated by PAGE and transferred to nitrocellulose paper indicated that proteins with apparent molecular masses similar to those of CD11c (150 kDa) and CD18 (95 kDa) molecules were the main concanavalin A-binding proteins in differentiated U937 cells as well as in mature neutrophils. Similar experiments carried out in the presence of the anti-CD11c MAb showed a specific and significant inhibition of concanavalin A binding to the CD11c molecule. These results indicate that concanavalin A binds to the CD11c molecule and this binding is responsible for the concanavalin A-induced respiratory burst in PMA-differentiated U937 cells as well as in human mature monocytes and neutrophils.     

Salicylate promotes myeloperoxidase-initiated LDL oxidation: antagonization by its metabolite gentisic acid.

The oxidative modification of low density lipoprotein (LDL) may play a significant role in atherogenesis. Tyrosyl radicals generated by myeloperoxidase (MPO) can act as prooxidants of LDL oxidation. Taking into consideration, that monophenolic compounds are able to form phenoxyl radicals in presence of peroxidases, we have tested salicylate, in its ability to act as a prooxidant in the MPO system. Measurement of conjugated dienes and lipid hydroperoxides were taken as indicators of lipid oxidation. Exposure of LDL preparations to MPO in presence of salicylate revealed that the drug could act as a catalyst of lipid oxidation in LDL. The radical scavenger ascorbic acid as well as heme poisons (cyanide, azide) and catalase were inhibitory. The main metabolite of salicylic acid, gentisic acid, showed inhibitory action in the MPO system. Even when lipid oxidation was maximally stimulated by salicylate the LDL oxidation was efficaciously counteracted in presence of gentisic acid at salicylate/gentisic acid ratios that could be reached in plasma of patients receiving aspirin medication. Gentisic acid was also able to impair the tyrosyl radical catalyzed LDL peroxidation. The results suggest that salicylate could act like tyrosine via a phenoxyl radical as a catalyst of LDL oxidative modification by MPO. But the prooxidant activity of this radical species is effectively counteracted by the salicylate metabolite gentisic acid.     

Induction of the oxidative response and of concanavalin A-binding capacity in maturing human U937 cells

Differentiation of U937 cells with phorbol myristate acetate (PMA) induces high stimulation by concanavalin A of the respiratory burst as well as an increase in concanavalin A-binding cell capacity. New concanavalin A-binding proteins are detected as differentiated U937 cells acquire their capacity to be activated by concanavalin A. We identified several concanavalin A-binding proteins, of molecular mass 30-200 kDa, in PMA-differentiated cells, but only some of them seem to be directly related to the concanavalin A effect on the respiratory burst. One of these candidates could be a glycoprotein with an apparent molecular mass of 140 kDa which behaved as a major concanavalin A-binding protein and is expressed on differentiated cells at the time these cells respond maximally to concanavalin A.     

Singlet oxygen mediated DNA degradation by copper nanoparticles: potential towards cytotoxic effect on cancer cells

The DNA degradation potential and anti-cancer activities of copper nanoparticles of 4-5 nm size are reported. A dose dependent degradation of isolated DNA molecules by copper nanoparticles through generation of singlet oxygen was observed. Singlet oxygen scavengers such as sodium azide and Tris [hydroxyl methyl] amino methane were able to prevent the DNA degradation action of copper nanoparticles confirming the involvement of activated oxygen species in the degradation process. Additionally, it was observed that the copper nanoparticles are able to exert cytotoxic effect towards U937 and Hela cells of human histiocytic lymphoma and human cervical cancer origins, respectively by inducing apoptosis. The growth characteristics of U937 and Hela cells were studied applying various concentrations of the copper nanoparticles.     

Low-density lipoprotein modification by normal,myeloperoxidase-deficient and NADPH oxidase-deficient granulocytes and the impact of redox active transition metal ions

The modification of low-density lipoprotein (LDL) by normal, myeloperoxidase (MPO)-deficient and NADPH oxidase-deficient granulocytes was investigated using the monoclonal antibody (mAb) OB/04, which was originally generated against copper-oxidized LDL. Incubation of LDL with normal granulocytes increased the reactivity of LDL with mAb OB/04. These effects were even more pronounced using MPO-deficient granulocytes. Inhibitors of oxidative reactions (the NADPH oxidase inhibitor diphenyleneiodonium chloride [DPI], catalase, superoxide dismutase [SOD]) did not significantly reduce LDL oxidation by normal granulocytes. Furthermore, granulocytes of a patient with NADPH oxidase deficiency were almost equally effective as normal granulocytes, indicating that oxidative burst-derived reactive oxygen species are of only minor importance in the generation of mAb OB/04-detectable new epitopes on LDL in vitro. In contrast, incubation of LDL with iron and copper prior to and during incubation with normal granulocytes markedly enhanced the generation of OB/04-detectable epitopes. It is supposed that, besides superoxide (in normal and MPO-deficient granulocytes) or instead of superoxide (in NADPH oxidase-deficient granulocytes), lytic enzymes released by activated granulocytes may enhance the availability of transition metals for oxidation of LDL. Our results support the concept that transition-metal-dependent pathways of LDL oxidation in combination with degranulation products of granulocytes are important.     

Sensitization of U937 cells to heat shock by oxalomalate, a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase

Heat shock may increase oxidative stress due to increased production of reactive oxygen species (ROS) and/or the promotion of cellular oxidation events. Cytosolic NADP

+

-dependent isocitrate dehydrogenase (ICDH) in U937 cells produces NADPH, an essential reducing equivalent for the antioxidant system. The protective role of ICDH against heat shock in U937 cells was investigated in control and cells treated with oxlalomalate, a competitive inhibitor of ICDH. Upon exposure to heat shock, the viability was lower and the protein oxidation, lipid peroxidation and oxidative DNA damage were higher in oxalomalate-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of ROS, as measured by the oxidation of 2'7'-dichlorodihydrofluorescin in U937 cells treated with oxalomalate. These results suggest that ICDH plays an important role as an antioxidant defense enzyme in cellular defense against heat shock through the removal of ROS.     

Lipophilic beta-blockers inhibit monocyte and endothelial cell-mediated modification of low density lipoproteins.

The effects of propranolol, pindolol and metoprolol on the modification of low density lipoprotein (LDL) by U937 monocyte-like cells, endothelial cells and copper ions were studied by determination of the lipid peroxidation product content and measurement of the relative electrophoretic mobility of the particle. Propranolol and pindolol inhibited LDL oxidation by U937 cells in a dose-dependent manner from 10 to 100 microM, whereas metoprolol had no effect. In the case of LDL modification by endothelial cells, all the three beta-blockers were efficient within the same range of concentrations, and the order of potency was propranolol greater than pindolol greater than metoprolol. In vitro oxidation of LDL in the presence of copper ions was also inhibited by propranolol; pindolol and metoprolol had no significant protective effect in this system. These results concerning the inhibitory action of beta-blockers were confirmed by testing the degradation of modified LDL by J774 macrophages. Although the concentrations of the drugs utilized in this study are relatively high, in long-term treatment beta-blockers might accumulate in target tissues, and the protective effect of propranolol against LDL oxidation might be involved in its inhibitory action on atherosclerosis previously reported in animal models.     

Prooxidant and antioxidant properties of Trolox C,analogue of vitamin E,in oxidation of low-density lipoprotein

Trolox C (Trolox), a water-soluble analogue of vitamin E lacking the phytyl chain, was investigated with respect to its effect on the oxidation of low-density lipoprotein (LDL). Trolox was added at different time points of LDL oxidation induced by Cu²⁺ and aqueous peroxyl radicals. In the case of Cu²⁺ -induced LDL oxidation, the effect of Trolox changed from antioxidant to prooxidant when added at later time points during oxidation; this transition occurred whenever α-tocopherol was just consumed in oxidizing LDL. Thus, in the case of Cu²⁺-dependent LDL oxidation, the presence of lipophilic antioxidants in the LDL particle is likely to be a prerequisite for the antioxidant activity of Trolox.

When oxidation was induced by peroxyl radicals, as a model of metal-independent oxidation, the effect of Trolox was always antioxidant, suggesting the importance of Cu²⁺/Cu⁺ redox-cycling in the prooxidant mechanism of Trolox. Our data suggest that, in the absence of significant amounts of lipophilic antioxidants, LDL becomes highly susceptible to oxidation induced by transition metals in the presence of aqueous reductants.     

Differentiation of human U937 promonocytic cells is impaired by moderate copper deficiency

Copper (Cu) deficiency suppresses macrophage activities in animals and humans. Our previous studies indicated that the induction of Cu deficiency in differentiated U937 monocytic cells impairs respiratory burst and bactericidal activities and lipopolysaccharide-mediated secretion of inflammatory mediators. The current investigation examined the roles of Cu in the monocytic differentiation process. Human U937 promonocytic cells were exposed to a high affinity Cu chelator (5 microM 2,3,2-tetraamine [tet]) for 24 hr before inducing differentiation by treatment with 1,25-dihydroxyvitamin D3 plus interferon-gamma (DI). This procedure decreased cell Cu by 55% without compromising cellular Zn, Fe, or general metabolic activities. Lower Cu status significantly attenuated the expression of maturation markers Mac-1 (CD11b), ICAM-1 (CD54), and LPS-R (CD14). This change was associated with a marked suppression in respiratory burst activity and killing of Salmonella. To examine if the adverse effect of inadequate Cu on the DI-induced differentiation represented a more general defect, U937 cells were treated with phorbol 12-myristate 13-acetate (PMA). Lower Cu status also suppressed PMA-mediated differentiation of U937 cells. Supplemental Cu, but not Zn or Fe, blocked the tet-induced declines in cell Cu, expression of maturation markers, and respiratory burst and bactericidal activities. These results demonstrate that Cu is essential for the monocytic differentiation process that contributes to the competency of the host's defense system.