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Superoxide production by NADPH oxidases plays an important role in the development and progression of cardiovascular disease (CVD). However, measurement of superoxide (O2•−), a marker of oxidative stress, remains a challenging task in clinical and translational studies. In this study we analyzed O2•− production in cultured human lymphoblast cell lines by three different methods: a) superoxide dismutase (SOD)-inhibitable cytochrome C reduction, b) spin trapping of superoxide with 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO) and 5-diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), and c) using electron spin resonance (ESR) with the cell-permeable spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH). Lymphocytes were isolated and immortalized by an Epstein-Barr Virus (EBV)-transformation procedure. Superoxide was measured in cultured lymphoblast cell lines at baseline and upon stimulation with phorbol 12-myristate 13-acetate (PMA). Cytochrome C and the spin traps EMPO and DEPMPO detected 2 to 5 times less superoxide compared to CMH. Thus, CMH provided the most quantitative measurement of superoxide generation in human lymphoblast cell lines. Superoxide detection with CMH was linear dependent on cell concentration and was inhibited by SOD but not by catalase. Both cell-permeable polyethylene glycol (PEG)-SOD and extracellular Cu,Zn-SOD inhibited O2•− detection by 90% in PMA-stimulated cells, suggesting a predominantly extracellular O2•− generation in human lymphoblasts. Our study describes a new technique for O2•− measurement in cultured human lymphoblasts using ESR and CMH. A highly sensitive in vitro measurement of O2•− in human cell lines would allow investigators to study genotype/phenotype interactions in translational studies.
Superoxide (O2•−) production plays an important role in redox cell signaling and development of pathophysiological conditions, such as hypertension, ischemia-reperfusion injury, inflammation and atherosclerosis . However, detection of O2•− is still a challenging problem. One of the most sensitive and definitive methods of O2•− detection is electron spin resonance (ESR) [2, 3]. The ESR spin-trapping technique has been used to detect O2•− radicals induced by inflammation via neutrophil NADPH oxidase in cellular systems in vitro . However, the commonly used nitrone spin traps have a very low efficacy for trapping of O2•− radicals (Figure 1) . Thus, formation of the radical adduct is limited by slow kinetics of O2•− trapping and obstruction by antioxidants. Furthermore, superoxide radical adducts suffer from decomposition to hydroxyl (•OH)-radical adducts by glutathione (GSH) peroxidase . Finally, both O2•− and •OH-radical adducts can be reduced to ESR silent hydroxylamines by ascorbate, transition metals, or flavin-enzymes (Figure 1) .
Recently, cyclic hydroxylamines were found to be effective scavengers of O2•− radicals [8, 9]. Hydroxylamine probes 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethyl-piperidine (PPH) and 1-hydroxy-3-carboxy-pyrrolidine (CPH) have been previously used for quantitative detection of extracellular O2•−. The advantage of hydroxylamine probes is that they are effective scavengers of O2•− and produce a stable nitroxide radical . Previously, we reported the activity of the phagocytic NADPH oxidase in neutrophils from healthy subjects using CPH, and measuring O2•− as SOD-inhibitable formation of 3-carboxyproxyl .
In the current investigation, we studied superoxide production in cultured lymphoblast cell lines at baseline and upon stimulation with phorbol 12-myristate 13-acetate (PMA) by three methods: a) superoxide dismutase (SOD)-inhibitable cytochrome C reduction, b) spin trapping of superoxide with 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO) and 5-diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), and c) using ESR with the cell-permeable spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) (Figure 1) . Reaction of O2•− with CMH is much faster (1.2*104 M−1s−1) than with nitrone spin traps, thereby enabling the hydroxylamines to compete with cellular antioxidants and react with both extra- and intracellular O2•−.
Our study describes a new technique for O2•− measurement in cultured human lymphoblasts using ESR and CMH.
Spin traps 5-diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO) and spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) were purchased from Alexis Corporation (San Diego, USA). Polyethylene-glycol-conjugated superoxide dismutase (PEG-SOD), and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma-Aldrich (St. Louis, MO). All other reagents were obtained from Sigma-Aldrich.
In collaboration with the Emory University General Clinical Research Center human immortalized lymphoblast cell lines were developed from peripheral blood mononuclear cells of subjects with and without CVD at the Atlanta Veterans Affairs Medical Center (AVAMC). The study was approved by the Institutional Review Board of Emory University and the AVAMC’s Research and Development Committee. All subjects provided informed consent.
Lymphocytes were isolated from whole blood by Ficoll density gradient centrifugation . After low speed centrifugation of freshly sampled venous blood (10 ml), the buffy coat and red blood cells (4 ml) were diluted with 4 ml phosphate buffered saline (PBS). The diluted blood was layered on 5 ml Ficoll paque PLUS solution. After centrifugation at 500 X g at 20° C for 30 min, the buffy coat was gently removed and diluted with 10 ml PBS. After low centrifugation at 200 X g for 7 min at room temperature, the supernatant was discarded. All subsequent steps were performed at 4°C. The remaining red pellet underwent hypotonic lysis by the addition of 9 ml H2O. After 20 sec, osmolarity was restored with 2 ml of 10 times concentrated PIPES (piperazine-N,N’-bis-[2-ethano-sulfonic-acid]) buffer, followed by centrifugation at 200 X g for 7 min. The remaining pellet was resuspended in 5 ml RPMI and 10% fetal bovine serum (FBS) and incubated for 45 min in 5% CO2 at 37°C. The non-adherent cells were gently washed and collected. Finally, lymphocytes were pelleted after room temperature low speed centrifugation at 200 X g for 7 min. This procedure yielded 5–20 X 106 lymphocytes for each 10 ml of blood.
To initiate B lymphocyte cultures, lymphocytes were infected with the B95-8 strain of Epstein-Barr Virus (EBV) . After EBV-transformation, B lymphocytes were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine,100 U/ml penicillin, and 100 mg/ml streptomycin, at 37°C in a humid atmosphere saturated with 5% CO2. The medium was changed twice weekly. Cell counts and viability of >95% (trypan blue stain) were monitored on a daily basis for 2 weeks until stored in liquid nitrogen for later in vitro phenotyping experiments.
Superoxide radical was measured at room temperature by ESR using a Bruker EMX spectrometer (Bruker Biospin) using spin traps EMPO and DEPMPO, or spin probe CMH (CMH) . Briefly, cells were washed with PBS, and resuspended in Krebs-Hepes buffer (KHB). Subsequently, 0.25 x 106 EBV-transformed lymphoblasts were placed in 0.1 ml KHB with 100 _M diethylenetriaminepentaacetic acid (DTPA) and incubated with PMA, polyethylene glycol (PEG-SOD), and PMA plus SOD. Results were expressed as pmoles of O2•− _ released per 106 cells per minute.
Superoxide formation was also determined by monitoring the SOD-inhibitable reduction of cytochrome C [13, 14]. Cells were harvested by centrifugation and washed once in Hank’s balanced salt solution (HBSS). Cells (1.25 x 107) were finally resuspended in 1 mL of HBSS and preincubated at 37°C for 10 min. The reaction was started by mixing 80 μL of the cell suspension (106 cells in HBSS) with 20 μL of the substrate solution containing cytochrome C (160 μM), catalase (40 μg·mL−1) and PMA in HBSS; cytochrome C reduction was recorded at 37°C for 10 min using a dual-wavelength ELISA-reader (at 550–540 nm) in the presence or absence of SOD. The amount of O2•− released was calculated using an extinction coefficient of 21 mM 1 cm 1 and expressed as the mean determination of multiple experiments ± SE.
Incubation of human lymphoblasts with the spin probe CMH resulted in the generation of the ESR signal of 3-methoxycarbonyl-proxyl nitroxide (CM•) (Figure 2A). Time scan of the low-field component of the ESR signal showed linear accumulation of CM• in unstimulated cells. Stimulation of cells by PMA (10 μM) led to several-fold increase in the slope of CM• kinetics (Figure 2B).
CMH detects both extra- and intracellular O2•− [11, 15]. Extracellular O2•− can be quantified by inhibition of the ESR signal by Cu,Zn-SOD, while supplementation of cells with cell-permeable PEG-SOD will inhibit detection of both extra- and intracellular O2•−. Addition of Cu,Zn-SOD (50 U/ml) strongly inhibited accumulation of CM• both in PMA-stimulated and unstimulated cells to similar levels (Figure 2B). These results confirm specific detection of O2•− by CMH. Furthermore, supplementation of cells with cell-permeable PEG-SOD showed inhibition of the ESR signal similar to extracellular Cu,Zn-SOD (Figure 3). Both cell-permeable PEG-SOD and extracellular Cu,Zn-SOD inhibited O2•− production by 90% in PMA-stimulated cells (Figure 3). The amount of O2•− generated was calculated as a difference between the cellular sample and buffer. These data imply that both PMA-stimulated and unstimulated cells produce predominantly extracellular O2•−.
It has been previously shown that cyclic hydroxylamines such as CMH do not directly react with hydrogen peroxide (H2O2) [16, 17]. However, transition metals and heme proteins may stimulate oxidation of CMH in the presence of H2O2 . Therefore, we treated human lymphoblasts with cell-permeable PEG-catalase (100 U/ml) in order to test the role of H2O2 in CMH oxidation in lymphoblast suspensions. Treatment of lymphoblasts with PEG-catalase did not affect basal O2•− production but slightly decreased O2•− generation in PMA-stimulated cells (Figures 4). It is important to note that in the presence of SOD, CMH is exposed to H2O2. Meanwhile, addition of catalase to cells and SOD did not change CM• formation (Figure 4C). Inhibition of O2•− generation by catalase in PMA-stimulated cells (30.8 pmol/106/min vs 19.8 pmol/106/min in the presence of catalase) can be explained by redox regulation of phagocytic NADPH oxidase in PMA-stimulated lymphoblasts rather than presence of SOD activity because catalase did not affect O2•− detection in the xanthine oxidase system (data not shown). Thus, the CMH signal was not dependent on cellular H2O2, but scavenging of H2O2 attenuated PMA-mediated stimulation of NADPH oxidase.
Superoxide production was proportional to PMA concentration, and duration of PMA treatment (Figures 5). Five minute incubation with 10 μM PMA provided optimal condition for cell stimulation.
Superoxide detection with CMH was linear dependent on cell concentration, and was inhibited by SOD but not by catalase (Figure 6A). In order to have sufficient amount of cells and linear dependence of ESR signal on cell number we selected 2.5 x106 cells/ml as the optimal cell concentration. Furthermore, analysis of O2•− production at various concentrations of CMH did not show significant increase in O2•− detection at concentrations of CMH higher than 1 mM (Figure 6B). Therefore, 1 mM CMH scavenges most of the cellular O2•−. It was previously shown that CMH allows high sensitivity detection of both extra- and intracellular O2•−. The fact that further increase in CMH concentration did not result in substantial increase in detected O2•− supports our observation that human lymphoblasts generate predominantly extracellular O2•−.
Despite unambiguous detection of O2•− by the spin trap DEPMPO its application to cells is limited by bioreduction of the DEPMPO/O2•− radical adduct. Indeed, in human lymphoblasts treated with DEPMPO (Figure 7, DEPMPO: cells) we did not observe a spectrum for the O2•− radical adduct. The 8-line ESR spectra correspond to DEPMPO/•OH radical adduct produced by GSH peroxidase-mediated decomposition of DEPMPO/•OOH adduct (Figure 1). Formation of DEPMPO/•OH was significantly inhibited by Cu,Zn-SOD. Spin trapping of O2•− in cells stimulated with PMA resulted in formation of a mixture of DEPMPO/•OH and DEPMPO/•OOH adducts (Figure 7). Cu,Zn-SOD inhibited formation of both radical adducts, which supports the detection of extracellular O2•− followed by cell-mediated decomposition of DEPMPO/•OOH to DEPMPO/•OH. Meanwhile, accumulation of radical adducts (followed by intensity of low-field ESR component) was not linear and flattened after a few minutes, suggesting bioreduction of the radical adducts.
In comparison, ESR spectra of CMH plus O2•− is linear and consistent (Figure 7). All samples containing CMH showed less noise in their ESR spectra due to higher intensity of the 3-line spectrum compared to the 12-line spectrum of DEPMPO radical adducts. Finally, accumulation of ESR signal in CMH containing samples was linear in time and the amount of detected O2•− with CMH was 5-fold higher than with the spin trap DEPMPO (Figure 7). Of note, the presence of small initial ESR spectra in CMH samples did not interfere with the measurements because time scans show only the increase in ESR amplitude.
Spin trapping of O2•− with EMPO produced only the EMPO/•OH adduct, which was inhibited by Cu,Zn-SOD (Figure 8A). These data suggest that cells transformed EMPO/•OOH into EMPO/•OH much faster than DEPMPO/•OOH into DEPMPO/•OH. Incubation of cells with the spin probe CMH resulted in accumulation of CM• nitroxide, which was also inhibited by Cu,Zn-SOD (Figure 8B). In addition, ESR spectra of CMH containing samples did have much better signal to noise ratio compared to EMPO samples. Interestingly, accumulation of EMPO/•OH radical adducts in unstimulated cells was not linear, while ESR kinetics in CMH containing samples was linear and the amount of detected O2•− with CMH was 3-fold higher than with EMPO (Figure 8C, D).
It has been previously reported that cyclic hydroxylamine probes, such as CPH and CMH, detect more cellular O2•− than cytochrome C . This difference was particularly larger in PMA-stimulated neutrophils, potentially due to oxidation of the ferrous cytochrome by H2O2. Indeed, SOD-inhibitable reduction of cytochrome C was twice lower than the amount of O2•− detected by CMH in unstimulated cells (Table 1). Moreover, the amount of O2•− detected by cytochrome C was 4.3-times lower than the amount of O2•− detected by CMH in PMA-stimulated cells (Table 1).
We have analyzed O2•− production in cultured lymphoblast cell lines obtained from subjects with and without CVD. We found that basal O2•− production in lymphoblast cell lines of CVD patients (n=8) compared to non-CVD subjects (n=8) was 10.9 ± 1.5 vs 7.6 ± 0.9 pmol O2•−/106 cells/min, respectively (p=0.04). In addition, PMA-stimulated O2•− production in lymphoblast cell lines of CVD patients compared to non-CVD subjects were not statistically significantly different (data not shown). This preliminary data demonstrate that CMH was successfully used for O2•− measurements in cultured lymphoblast cell lines obtained from subjects with and without CVD. Larger clinical association studies with more characterized phenotypes are required to confirm these observations.
In the present study, we observed that the cell-permeable spin probe CMH provided the most quantitative measurement of O2•− generation in human lymphoblast cell lines. As shown in table 1, cytochrome C and the spin traps EMPO and DEPMPO detected 2 to 4 times less O2•− compared to CMH. Higher reactivity of CMH with O2•− and the stability of the CM nitroxide (product of CMH and O2•− reaction) are likely responsible for higher O2•− detection by CMH. In addition, we show that human lymphoblasts predominantly produce extracellular O2•−, confirming the phagocytic NADPH oxidase as the main source of O2•− in human lymphoblasts. Recently, a new cell-impermeable spin probe CAT1H has been suggested for quantification of extracellular O2•− . Our preliminary data (data not shown) suggest that CAT1H could be used for measurements of NADPH oxidase activity in human lymphoblasts due to specific detection of extracellular O2•−.
Measurements of O2•− by reduction of cytochrome C is a reproducible and cost-efficient assay. However, the cytochrome C assay has several limitations: (a) it detects only extracellular O2•−; (b) has low sensitivity; and (c) is not specific and can be reduced by ascorbate and flavin enzymes [2, 3]. In addition, it has been previously suggested that the cytochrome C assay underestimates O2•− values due to oxidation of cytochrome C by cellular H2O2, which may account for significant differences between CMH and cytochrome C in PMA-stimulated O2•− generation (Table 1).
Recently, it has been shown that spin trapping detects mainly extracellular but not intracellular O2•−  due to low efficacy for O2•− trapping . This issue, however, is not the major limiting factor here due to the extracellular O2•− production by human lymphoblasts. By examining the ESR spectra of DEPMPO and EMPO radical adducts, we show that biodegradation and bioreduction of the radical adducts are the major problems regarding spin trapping of O2•− produced by human lymphoblasts. Non-linear kinetics is likely associated with decay of the EMPO and DEPMPO superoxide adducts. In comparison, linear CMH kinetics supports resistance of the CM-nitroxide to bioreduction. Meanwhile, new analogs of EMPO may be more resistant to bioreduction .
The fact that CMH detected more O2•− than spin traps or cytochrome C may suggest that CMH is the source of additional O2•− production. Our data does not support such a conclusion because a) amount of O2•− was proportional to the cell number (Figure 6A); b) amount of detected O2•− was not increased above 1mM CMH (Figure 6B); c) amount of O2•− detected in the xanthine oxidase system was 25% of oxidized xanthine determined spectrophotometrically, which corresponds to normal O2•−/H2O2 ratio for xanthine oxidase; d) the background oxidation of CMH was rather minimal (Figure 2B: Buffer); and e) PMA-stimulation of O2•− production was similar for CMH (8.8 fold) and spin trap EMPO (6 fold). Thus, our data support a higher efficiency of O2•− detection by CMH compared with spin traps.
The phagocytic NADPH oxidase of neutrophils and lymphoblasts has been extensively studied in chronic granulomatous disease (CGD), where defects caused by genetic variations such as deletions, insertions, and single nucleotide polymorphisms (SNP) in various enzyme subunits lead to impairment in respiratory burst and O2•− production [21–25]. More recently, lymphoblasts have been used in studies of NADPH oxidase in relation to hypertension and pre-eclampsia. An increase in p22phox expression and O2•− production was reported in hypertensive patients, and there was an enhanced agonist-stimulated NADPH oxidase–mediated O2•− production in pre-eclampsia, which may be important in mediating the endothelial dysfunction seen in this disease [26, 27]. More recently, we have shown that the presence of the CC genotype of the CYBA C242T SNP is associated with significantly increased basal NADPH oxidase activity in human lymphoblasts of patients with CVD, independent of risk factors for atherosclerosis .
Previously, we showed that NADPH oxidase activity was genetically determined as measured in freshly isolated neutrophils using CPH . In addition, environmental factors such as dietary supplementation with antioxidants, i.e. ascorbate, are of great interest in the prevention and management of many chronic diseases. However, antioxidants may interfere with detection of O2•− by cytochrome C or spin traps because of direct reduction of ferricytochrome and reduction of radical adducts, but not with probes such as CPH and CMH due to their resistance to bioreduction . Thus, detection of O2•− by CMH may be advantageous in the assessment of antioxidant drugs and compounds.
In conclusion, we describe a new technique for O2•− measurement in cultured human lymphoblast cell lines using ESR and the spin probe CMH. We observed that cultured human lymphoblasts generate predominantly extracellular O2•−. Finally, we believe that this highly sensitive and quantitative O2•− measurement in human cell lines will be useful to investigators who study genotype/phenotype interactions in translational studies of diseases related to oxidative stress.
This research was supported by National Institutes of Health grants PO-1 HL058000-05 and PO-1 HL075209, the Emory University Research Committee, the Emory University General Clinical Research Center (M01-RR00039), Atlanta, Georgia, and by National Institutes of Health cardiovascular training grant T-32 HL07745 to P. M. and S. S. W.
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