Flow cytometry and spectral imaging multiphoton microscopy analysis of CD36 expression with quantum dots 605 of untreated and 7-ketocholesterol-treated human monocytic cells

OBJECTIVE: To evaluate CD36 expression with quantum dots 605 (QDs 605) on untreated and 7-ketocholesterol (7KC)-treated monocytic U937 cells by flow cytometry (FCM) and confocal and multiphoton laser scanning microscopy (CLSM). STUDY DESIGN: Cells were analyzed by CLSM, following flow cytometric quantification of CD36 expression and 7KC uptake. Image sequences were obtained by spectral analysis in monophoton and multiphoton CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm to differentiate emission spectra. In CLSM analysis, cell deposits were screened in ultraviolet excitation modes to optimize the possibilities of QDs 605 and have the benefit of nuclei counterstaining by DAPI. RESULTS: FCM and CLSM reveal the expression of CD36 by means of QDs 605. FCM provides information on 7KC uptake. CLSM provides the localization of 7KC vs. DAPI. As factor curves and images show the red, narrow emission of QDs 605 vs. violet and blue emissions of 7KC and DAPI, respectively, a reliable identification of CD36 is obtained. CONCLUSION: QDs 605 are useful tools to perform antigenic expression in FCM and CLSM. Moreover, CLSM and subsequent spectral analysis provide a more specific characterization of QDs 605 fluorescent emission in the UV excitation mode and a simultaneous identification of 7KC.     

Localization of protein-protein interactions in live cells using confocal and spectral imaging FRET microscopy

Microscopy has become an essential tool for cellular protein investigations. The development of new fluorescent markers such as green fluorescent proteins generated substantial opportunities to monitor protein-protein interactions qualitatively and quantitatively using advanced fluorescence microscope techniques including wide-field, confocal, multiphoton, spectral imaging, lifetime, and correlation spectroscopy. The specific aims of the investigation of protein dynamics in live specimens dictate the selection of the microscope methodology. In this article confocal and spectral imaging methods to monitor the dimerization of alpha enhancer binding protein (C/EBPalpha) in the pituitary GHFT1-5 living cell nucleus have been described. Also outline are issues involved in protein imaging using light microscopy techniques and the advantages of lifetime imaging of protein-protein interactions.     

7-Ketocholesterol favors lipid accumulation and colocalizes with Nile Red positive cytoplasmic structures formed during 7-ketocholesterol-induced apoptosis: analysis by flow cytometry, FRET biphoton spectral imaging microscopy, and subcellular fractionation.

BACKGROUND: Oxidized low-density lipoproteins play key roles in atherosclerosis. Their toxicity is at least in part due to 7-ketocholesterol (7KC), which is a potent inducer of apoptosis. In this study on human promonocytic U937 cells, we determined the effects and the interactions of 7KC with cellular lipids during 7KC-induced apoptosis. METHODS: Morphologic and functional changes were investigated by microscopic and flow cytometric methods after staining with propidium iodide, 3,3'-dihexyloxacarbocyanine iodide, and Hoechst 33342. Cellular lipid content was identified by using filipin to quantify free cholesterol and Nile Red (NR), which emit a yellow or orange-red fluorescence in the presence of neutral and polar lipids, respectively. After staining with NR, interactions of 7KC with cellular lipids were identified by fluorescence resonance energy transfer biphoton spectral imaging confocal microscopy and by subcellular fractionation, gas chromatography, and mass spectrometry. RESULTS: During 7KC-induced apoptosis the fluorescence from filipin and the ratio of measured (orange-red vs. yellow) fluorescence of NR were enhanced. Spectral analysis of images obtained in biphoton mode and resulting factor images demonstrated the occurrence of fluorescence resonance energy transfer between 7KC and NR and the subsequent colocalization of 7KC and NR. These data were in agreement with biochemical characterization and demonstrated that 7KC and neutral and polar lipids accumulate in NR-stained cytoplasmic structures. CONCLUSIONS: During 7KC-induced apoptosis, 7KC modifies the cellular content of neutral and polar lipids, favors free cholesterol accumulation, and colocalizes with neutral and polar lipids that are inside NR-stained cytoplasmic structures.     

Extracellular-superoxide dismutase expression during monocytic differentiation of U937 cells

Leukemic cell lines, such as U937, THP-1, and HL60 cells, can differentiate into macrophages following exposure to various agents including 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro. It is well known that TPA enhances reactive oxygen species (ROS) generation through the activation of NADPH oxidase (NOX), and ROS act as mediators in TPA signaling. Extracellular-superoxide dismutase (EC-SOD) is a major anti-oxidative enzyme that protects the cells from damaging effects of superoxide. Recently, the reduction of Cu/Zn-SOD and the induction of Mn-SOD by TPA in leukemic cells have been reported; however, the regulation of EC-SOD by TPA remains poorly understood. Here, we explored the regulation of EC-SOD during the monocytic differentiation of U937 cells by TPA. We observed the reduction of EC-SOD and Cu/Zn-SOD, whereas the induction of Mn-SOD during the differentiation of U937 cells. The reduction of EC-SOD and Cu/Zn-SOD was attenuated by pretreatments with GF109203X (an inhibitor of protein kinase C, PKC), diphenyleneiodonium (an inhibitor of NOX), and U0126 (an inhibitor of mitogen-activated protein kinase kinase, MEK/extracellular-signal regulated kinase, ERK). Interestingly, pretreatment with BAY11-7082 (an inhibitor of nuclear factor-κB, NF-κB) suppressed the reduction of Cu/Zn-SOD, but not of EC-SOD. Furthermore, we also determined the involvement of newly synthesized protein and the instability of mRNA in the reduction of EC-SOD. Overall, our results suggest that the expression of EC-SOD is decreased by TPA through intracellular signaling consisting of PKC, NOX-derived ROS and MEK/ERK, but not of NF-κB signaling.     

Serglycin expression during monocytic differentiation of U937-1 cells

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.      

Differentiation of monocytic U937 cells under static magnetic field exposure

We present here a morphological, cytochemical and biochemical study of the macrophagic differentiation of human pro-monocytic U937 cells exposed to moderate intensity (6 mT) static magnetic fields (MF). It was found that the following substances induced differentiation in U937 cells to a progressively lower degree: 50 ng/mL 12-0-tetradecanoyl-13-phorbol acetate (TPA), low concentration of glutamine (0,05 mM/L), 10% dimethyl sulfoxide (DMSO) and 100 mM/L Zn++. Differentiated U937 cells shift from a round shape to a macrophage-like morphology, from suspension to adhesion growth and acquire phagocytotic activity, the cytoskeleton adapting accordingly. Exposure to static MF at 6 mT of intensity decreases the degree of differentiation for all differentiating molecules with a consequent fall in cell adhesion and increased polarization of pseudopodia and cytoplasmic protrusions. Differentiation alone, or in combination with exposure to static MFs, affects the distribution and quantity of cell surface sugar residues, the surface expression of markers of macrophage differentiation, and phagocytotic capability. Our results indicate that moderate-intensity static MFs exert a considerable effect on the process of macrophage differentiation of pro-monocytic U937 cells and suggest the need for further studies to investigate the in vivo possible harmful consequences of this.     

Cholesterol modulates PAF-stimulated Ca2(+)-mobilization in monocytic U937 cells

We investigated the effect of cellular cholesterol content on platelet activating factor (PAF)-stimulated Ca2+ mobilization in the human monocytic cell line U937. When cholesterol auxotroph U937 cells were depleted of cellular cholesterol by a 48-h incubation in delipidated medium, a 40% reduction in the PAF (100 nM)-stimulated increase in cytosolic Ca2+ concentration was seen. Ca2+ mobilization following stimulation with LTD4 (10 nM) or ATP (10 microM) was not affected. Addition of LDL (100 micrograms/ml, 24 h) to the delipidated medium completely recovered cellular cholesterol content and PAF-induced Ca2+ mobilization. These two LDL effects had very similar time- and dose-dependences. Partial recoveries of PAF-induced Ca2+ mobilization, seen after addition of pure cholesterol dissolved in ethanol (30 micrograms/ml, 24 h) or acetyl-LDL (100 micrograms/ml, 24 h), were associated with partial recoveries of cellular cholesterol content. Our results indicate that cellular cholesterol influences PAF-stimulated events in monocytic cells.     

Effects of caspase inhibitors (z-VAD-fmk, z-VDVAD-fmk) on Nile Red fluorescence pattern in 7-ketocholesterol-treated cells: investigation by flow cytometry and spectral imaging microscopy.

BACKGROUND: The 7-ketocholesterol (7KC)-induced cell death has some characteristics of apoptosis and is associated with polar lipid accumulation. So, we investigated the effects of the broad-spectrum caspase inhibitor z-VAD-fmk and of the caspase-2 inhibitor z-VDVAD-fmk on lipid profile evaluated by staining with Nile Red (NR). METHODS: The 7KC-treated human monocytic U937 cells were cultured in the absence or in the presence of the caspase inhibitors z-VAD-fmk or z-VDVAD-fmk. When staining with NR is performed, neutral and polar lipids have yellow and orange/red emission, respectively, and fluorescence was then analyzed by flow cytometry (FCM) and by confocal laser scanning microscopy (CLSM) combined with subsequent image processing. The 3D-image sequences were obtained by means of CLSM using spectral analysis, and were analyzed by the factor analysis of medical image sequences algorithm to differentiate spectra inside mixed fluorescence emission and get corresponding specific images. RESULTS: By FCM, comparatively to untreated cells, higher percentages of red fluorescent cells were identified in 7KC-treated cells. Factor curves and images reveal orange and red fluorescence emissions in 7KC-treated cells and show yellow, orange, and red fluorescence emissions in 7KC-treated cells cultured in the presence of z-VAD-fmk or z-VDVAD-fmk. CONCLUSIONS: Our data support that investigation by FCM and by spectral analysis in CLSM associated with subsequent image processing provides useful tools to determine the effect of caspase inhibitors on lipid content evaluated with NR. They also favor the hypothesis of relationships between caspase activity and polar lipid accumulation.     

Prosaposin: a new player in cell death prevention of U937 monocytic cells

We report that prosaposin binds to U937 and is active as a protective factor on tumor necrosis factor alpha (TNFalpha)-induced cell death. The prosaposin-derived saposin C binds to U937 cells in a concentration-dependent manner, suggesting that prosaposin behaves similarly. Prosaposin binding induces U937 cell death prevention, reducing both necrosis and apoptosis. This effect was inhibited by mitogen-activated protein ERK kinase (MEK) and sphingosine kinase (SK) inhibitors, indicating that prosaposin prevents cell apoptosis by activation of extracellular signal-regulated kinases (ERKs) and sphingosine kinase. Prosaposin led to rapid ERK phosphorylation in U937 cells as detected by anti-phospho-p44/42 mitogen-activated protein (MAP) kinase and anti-phosphotyrosine reactivity on ERK immunoprecipitates. It was partially prevented by apo B-100 and pertussis toxin (PT), suggesting that both lipoprotein receptor-related protein (LRP) receptor and Go-coupled receptor may play a role in the prosaposin-triggered pathway. Moreover, sphingosine kinase activity was increased by prosaposin treatment as demonstrated by the enhanced intracellular formation of sphingosine-1-phosphate (S-1-P). The observation that the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin prevented the prosaposin effect on cell apoptosis suggests that sphingosine kinase exerts its anti-apoptotic activity by the PI3K-Akt pathway. Thus, cell apoptosis prevention by prosaposin occurs through ERK phosphorylation and sphingosine kinase. The biological effect triggered by prosaposin might be extended to primary cells because it triggers Erk phosphorylation in peripheral blood mononuclear cells (PBMCs). This is the first evidence of a biological effect consequent to a signal transduction pathway triggered by prosaposin in cells of non-neurological origin.     

Porphyromonas gingivalis fimbriae induce adhesion of monocytic cell line U937 to endothelial cells

This study used the human monocytic cell line U937 to examine whether or not Porphyromonas gingivalis fimbriae could induce the adhesion of monocytes to endothelial cells. An in vitro adhesion assay was used to investigate the effects of the fimbriae on U937 cell adhesion to human umbilical vein endothelial cells (HUVEC). The fimbriae enhanced U937 cell adhesion to HUVEC in a dose-dependent manner. U937 cells adhered better to HUVEC pretreated with the fimbriae for a minimum of 2 hr than to untreated HUVEC. The enhanced adhesion was inhibited by a monoclonal antibody against P. gingivalis 381 fimbriae. Pretreatment of U937 cells with the fimbriae for 24 hr enhanced U937 cell adhesion to HUVEC approximately 4-fold. This phenomenon was inhibited by an anti-CD11b antibody, suggesting the involvement of CD11b. These results indicate that P. gingivalis fimbriae can induce monocyte adhesion to the endothelial cell surface. They also suggest that the fimbriae may be involved in the initial event for infiltration of monocytes into the periodontal tissues of individuals with adult periodontitis.     

Oxidized LDL immune complexes induce release of sphingosine kinase in human U937 monocytic cells

The transformation of macrophages into foam cells is a critical event in the development of atherosclerosis. The most studied aspect of this process is the uptake of modified LDL through the scavenger receptors. Another salient aspect is the effect of modified LDL immune complexes on macrophages activation and foam cell formation. Macrophages internalize oxidized LDL immune complexes (oxLDL-IC) via the Fc-gamma receptor and transform into activated foam cells. In this study we examined the effect of oxLDL-IC on sphingosine kinase 1 (SK1), an enzyme implicated in mediating pro-survival and inflammatory responses through the generation of the signaling molecule sphingosine-1-phosphate (S1P). Intriguingly, oxLDL-IC, but not oxLDL alone, induced an immediate translocation and release of SK1 into the conditioned medium as evidenced by fluorescence confocal microscopy. Immunoblot analysis of cell lysates and conditioned medium revealed a decrease in intracellular SK1 protein levels accompanied by a concomitant increase in extracellular SK1 levels. Furthermore, measurement of S1P formation showed that the activity of cell-associated SK decreased in response to oxLDL-IC compared to oxLDL alone, whereas the activity of SK increased extracellularly. Blocking oxLDL-IC binding to Fc-gamma receptors resulted in decreased levels of extracellular S1P. The data also show that cell survival of human U937 cells exposed to oxLDL-IC increased compared to oxLDL alone. Exogenously added S1P further increased cell survival induced by oxLDL-IC. Taken together, these findings indicate that S1P may be generated extracellularly in response to modified LDL immune complexes and may therefore promote cell survival and prolong cytokine release by activated macrophages.     

Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis.

BACKGROUND: Specific signal detection has been a fundamental issue in fluorescence microscopy. In the context of tissue samples, this problem has been even more pronounced, with respect to spectral overlap and autofluorescence. METHODS: Recent improvements in confocal laser scanning microscopy combine sophisticated hardware to obtain fluorescence emission spectra on a single-pixel basis and a mathematical procedure called "linear unmixing" of fluorescence signals. By improving both the specificity of fluorescence acquisition and the number of simultaneously detectable fluorochromes, this technique of spectral imaging (SI) allows complex interrelations in cells and tissues to be addressed. RESULTS: In a comparative approach, SI microscopy on a quantitative basis was compared to conventional bandpass (BP) filter detection, demonstrating substantial superiority of SI with respect to detection accuracy and dye combination. An eight-color immunofluorescence protocol for tissue sections was successfully established. Moreover, advanced use of SI in fluorescence resonance energy transfer (FRET) applications using enhanced green fluorescence protein (EGFP) and enhanced yellow fluorescence protein (EYFP) in a confocal set up could be demonstrated. CONCLUSIONS: This novel technology will help to perform complex multiparameter investigations at the cellular level by increasing the detection specificity and permitting simultaneous use of more fluorochromes than with classical techniques based on emission filters. Moreover, SI significantly extends the possibilities for specialized microscopy applications, such as the visualization of macromolecular interactions or conformational changes, by detecting FRET.     

Differential effects of unsaturated fatty acids on phospholipid synthesis in human leukemia monocytic U937 cells

The biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in monocyte-like leukemia U937 cells was monitored by adding [³H]choline, [¹⁴C]ethanolamine or [¹⁴C]glycerol to the culture media; incorporation into phospholipid (PL) increased with time. The effect of unsaturated fatty acids (UFA) on PC and PE synthesis was investigated by pretreating U937 cells for 72h with 10 μM 18:1 (n –9), 18:2 (n –6), 18:3 (n –3), 20:4 (n –6) and 20:5 (n –3). The UFA caused no alteration in cell growth, as evidenced by light microscopy and the incorporation of [³H]thymidine and [³H]leucine. Total cellular uptake of radioactive precursors remained unaffected by all the treatments. Pretreatment with 20:5 resulted in approximately 25 per cent reduction in the incorporation of [³H]choline into PL, while no significant effect was detected with the other UFAs. 18:3, 20:4 and 20:5 depressed the incorporation of [¹⁴C]ethanolamine into PL by 34 per cent, 28 per cent and 49 per cent respectively. However, there was no redistribution of label with any of the treatments. 18:3, 20:4 and 20:5 also antagonized the stimulatory effect of endotoxin (LPS) on PC and PE synthesis. In addition, the incorporation from [¹⁴C]glycerol into PC and PE was reduced by 18:3, 20:4 and 20:5. Although the PL composition of the cells remained essentially unaffected, our study shows that chronic treatment of U937 cells with n –3 PUFA (20:5) depressed PC and PE synthesis, and 18:3 and 20:4 also caused inhibition of PE synthesis.     

Nitric oxide augments release of chemokines from monocytic U937 cells: modulation by anti-inflammatory pathways

Nitric oxide (NO) appears to act as an inflammatory mediator on monocytic cells. Exogenous NO augmented release of chemokines from human promonocytic U937 cells and peripheral blood mononuclear cells. Pharmacological strategies aiming at modulation of NO-induced release of interleukin-8 (IL-8) were investigated in U937 cells in detail. Release of IL-8 was down-regulated by transforming growth factor beta2 (TGF-beta2), by the protein tyrosine-kinase inhibitor genistein, and via rises in intracellular cyclic AMP, generated by prostaglandin E(2), rolipram, pentoxifylline, forskolin, or dibutyryl-cyclic AMP. In addition, incubation with the synthetic glucocorticoid dexamethasone or suppression of activity of p38 mitogen-activated protein (MAP) kinases by SB-203580 modulated release of IL-8. Activation of p38 MAP kinases was confirmed by the demonstration of an augmented appearance of phosphorylated p38 in the presence of NO. The present data suggest that exposure to exogenous NO resembles activation of U937 cells by proinflammatory stimuli. The anti-inflammatory cytokine TGF-beta2, as well as anti-inflammatory or immunosuppressive agents such as genistein, pentoxifylline, rolipram, dexamethasone, and SB-203580 modulate inflammatory, chemokine-inducing actions of NO.     

MiR-424 regulates monocytic differentiation of human leukemia U937 cells by directly targeting CDX2

MiR-424 plays an important role via promoting the monocytic differentiation in many human leukemia cell lines. Here, we report that miR-424 decreased miR-125b expression to 36 % by directly targeting caudal type homeobox 2. However, miR-424 also decreased expression of Fes, PU.1 and colony-stimulating factor receptor (MCSFR). As Fes, PU.1 and MCSFR were down-regulated by over-expression of miR-125b (unpublished work), a similar effect of miR-424 and Fes siRNA on CD64, Egr-1, Egr-2 and CEBPA indicates that Fes may be an important downstream target of miR-424. We hypothesize that miR-424 promotes monocytic differentiation by regulating other critical factors and miR-424 has high affinity for these factors. For the first time, the molecular mechanism of miR-424 during monocytic differentiation of U937 cells has been elucidated in this study.     

Long time incubation of monocytic U 937 cells with LDL increases specific paf-acether binding and the cellular acetylhydrolase activity.

Besides the well established role of low density lipoproteins (LDL), the phospholipid PAF-acether (paf) seems to be involved in atherogenesis. The effect of LDL (10 micrograms/ml for 24 h, n = 3) on paf binding characteristics of monocyte/macrophage-like U 937 cells was investigated using the radioligand [3H]paf, unlabeled paf and the paf receptor antagonist WEB 2086. The specific [3H]paf binding significantly increased at 1.4 nM (P less than 0.02) and 2.8 nM (P less than 0.01) added [3H]paf with an increased number of paf binding sites in the Scatchard plot analysis of the data. Specific paf binding was functionally active since paf mediated a cellular [Ca2+]i rise. The protein kinase C (PKC) activator PMA (1 nM, 37 degrees C) expressed specific [3H]paf binding already after a 15-min incubation period, indicating a PKC activation as the decisive step of paf receptor expression. LDL also stimulated the paf degrading cellular acetylhydrolase significantly by increasing both Km (9.4 +/- 1.9 vs. 2.0 +/- 0.5 microM, P less than 0.02) and vmax (0.5 +/- 0.2 vs. 0.2 +/- 0.0 nmol/min per mg cell protein, P less than 0.02). The data demonstrate that LDL increases the number of paf receptors on monocyte/macrophage-like U 937 cells and interferes with the dynamics and/or synthesis of the cellular acetyl hydrolase. These effects could be of importance in the pathogenesis of atherosclerosis.     

Escherichia coli induces apoptosis in human monocytic U937 cells through the Fas/FasL signaling pathway

Apoptosis is a genetically regulated cellular suicide mechanism that plays an essential role in development and in defense of multicellular organism. Escherichia coli (E. coli) can induce monocyte apoptosis; however, the mechanism is not clear. This study determines if Fas/FasL regulates E. coli-induced human monocyte line U937 cell apoptosis. We found that infection of U937 cells with E. coli induced rapid cell death in a dose- and time-dependent manner displaying the characteristic features of apoptosis. Moreover, opsonized E. coli induced U937 apoptosis with a higher apoptotic rate (53.29 ± 5.83%) than non-opsonized E. coli (19.37 ± 2.56%). Studying the underlying mechanisms we found that the E. coli-induced apoptosis was associated with a more prominent induction expression of Fas/FasL in a time- and dose-dependent manner. Furthermore, E. coli treatment resulted in a significant increase in the levels of DR5, TRAIL, and FADD, but exerted no statistically significant effects on the levels of DR4. The activity of caspase-8 enzyme increased in infection groups, positively correlated with apoptosis rate. Taken together, these results clearly indicate that receptor-mediated phagocytosis of E. coli induces apoptosis. Moreover, our findings suggest a possible regulatory role of Fas/FasL in the pathway of E. coli infection.     

Vitamin C recycling and function in human monocytic U-937 cells

The uptake, recycling, and function of ascorbic acid was evaluated in cultured U-937 monocytic cells. Dehydroascorbic acid, the two-electron oxidized form of the vitamin, was taken up on the glucose transporter and reduced to ascorbate to a much greater extent than ascorbate itself was accumulated by the cells. In contrast to dehydroascorbic acid, ascorbate entered the cells on a sodium- and energy-dependent transporter. Intracellular ascorbate enhanced the transfer of electrons across the cell membrane to extracellular ferricyanide. Rates of ascorbate-dependent ferricyanide reduction were saturable, fivefold greater than basal rates, and facilitated by intracellular recycling of ascorbate. Whereas reduction of dehydroascorbic acid concentrations above 400 microM consumed reduced glutathione (GSH), even severe GSH depletion by 1-chloro-2,4-dinitrobenzene was without effect on the ability of the cells to reduce concentrations of dehydroascorbic acid likely to be in the physiologic range (< 200 microM). Dialyzed cytosolic fractions from U-937 cells reduced dehydroascorbic acid to ascorbate in an NADPH-dependent manner that appeared due to thioredoxin reductase. However, thioredoxin reductase did not account for the bulk of dehydroascorbic acid reduction, since its activity was also decreased by treatment of intact cells with 1-chloro-2,4-dinitrobenzene. Thus, U-937 cells loaded with dehydroascorbic acid accumulate ascorbate against a concentration gradient via a mechanism that is not dependent on GSH or NADPH, and this ascorbate can serve as the major source of electrons for transfer across the plasma membrane to extracellular ferricyanide.