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Systematic study on ROS production induced by oleic, linoleic, and γ-linolenic acids in human and rat neutrophils
Free Radical Biology & Medicine 41 (2006) 1124 – 1132 www.elsevier.com/locate/freeradbiomed
Original Contribution
Systematic study on ROS production induced by oleic, linoleic, and γ-linolenic acids in human and rat neutrophils Elaine Hatanaka a,⁎, Adriana Cristina Levada-Pires a , Tania Cristina Pithon-Curi a,b , Rui Curi a a
Institute of Biomedical Sciences, Department of Physiology and Biophysics, University of São Paulo, Avenida Prof. Lineu Prestes, 1524, 05508-900 Butantã, São Paulo, SP, Brazil b Cruzeiro do Sul University, São Paulo, Brazil Received 12 February 2006; revised 23 June 2006; accepted 25 June 2006 Available online 4 July 2006
Abstract The effects of oleic, linoleic, and γ-linolenic acids on the production of ROS by unstimulated and PMA-stimulated neutrophils were investigated by using five techniques: luminol- and lucigenin-amplified chemiluminescence, cytochrome c, hydroethidine, and phenol red reduction. Using lucigenin-amplified chemiluminescence, an increase in extracellular superoxide levels was observed by the treatment of neutrophils with the fatty acids. There was also an increase in intracellular ROS levels under similar conditions as measured by the hydroethidine technique. An increment in the intra- and extracellular levels of H2O2 was also observed in neutrophils treated with oleic acid as measured by phenol red reduction assay. In the luminol technique, peroxidase activity is required in the reaction of luminol with ROS for light generation. Oleic, linoleic, and γ-linolenic acids inhibited the myeloperoxidase activity in stimulated neutrophils. So, these fatty acids jeopardize the results of ROS content measured by this technique. Oleic, linoleic, and γ-linolenic acids per se led to cytochrome c reduction and so this method also cannot be used to measure ROS production induced by fatty acids. Oleic, linoleic, and γ-linolenic acids do stimulate ROS production by neutrophils; however, measurements using the luminol-amplified chemiluminescence and cytochrome c reduction techniques require further analysis. © 2006 Elsevier Inc. All rights reserved. Keywords: Fatty acids; ROS; Luminol; Lucigenin; Cytochrome c; Hydroethidine; Neutrophils; Free radicals
Neutrophils are the first cells that migrate to tissues in response to invading pathogens. The antimicrobial function of these phagocytes depends on the release of lytic enzymes stored in cytoplasmatic granules and on generation of S superoxide (O2 − ). In phagocytes, superoxide is mainly generated by the reaction of oxygen and NADPH through the NADPH oxidase complex [1]. This enzyme complex is formed by subunits found in intracellular granules and plasma membrane. After activation, NADPH oxidase components containing granules fuse to phagocytic vacuoles and generate superoxide. Also, granules may migrate to the cell surface and release superoxide into the extracellular space [2]. Superoxide anion and hydrogen peroxide (H2O2) generated
⁎ Corresponding author. Fax: +55 11 3091 7285. E-mail address: [email protected] (E. Hatanaka). 0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2006.06.014
by the NADPH oxidase give rise to other reactive oxygen species (ROS) that are strong cytolytic agents, such as hypochlorous acid (formed from H2O2 and chloride ions by the action of myeloperoxidase (MPO) released from neutrophil granules) and hydroxyl radical [1–3]. Superoxide can also be generated through the mitochondrial electron transport chain, xanthine–xanthine oxidase, and cytochrome P450. Mitochondria generate superoxide mostly by the univalent reduction of oxygen in complexes I and III of the electron transport chain [4]. Changes in mitochondrial generation of superoxide can be assessed by the addition of mitochondria uncouplers that are depolarizing agents and inhibitors of the respiratory chain [5]. ROS generated by neutrophils play an important role in the inflammatory response by regulating other immune reactive cells [6,7]. Fatty acids have been used for the treatment of various diseases that involve oxidative stress, such as coronary
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heart disease [8] and rheumatic arthritis [9]. However, the effects of long-chain fatty acids on ROS production by neutrophils remain to be fully established. Some authors showed an increase [10–12], whereas others found a decrease, in ROS production by neutrophils in response to fatty acid treatment [13–16]. This discrepancy may be due to the methods used to measure intra- and extracellular ROS levels. In this study intra- and extracellular levels of ROS were measured in neutrophils treated with oleic, linoleic, and γlinolenic acids. The luminol technique was used to measure intra- and extracellular ROS levels. Hydroethidine reduction was employed to measure intracellular ROS production. Cytochrome c reduction and lucigenin-amplified chemiluminescence were used to determine extracellular superoxide anion levels. Phenol red reduction was employed to measure intra- and extracellular H2O2 levels. Material and methods RPMI 1640 medium, Hepes, penicillin, and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Fatty acids, glutamine, luminol, lucigenin, cytochrome c, hydroethidine, peroxidase type II, hydrogen peroxide, phenol red, Histopaque, oyster glycogen, and trypan blue were supplied by Sigma Chemical Co (St. Louis, MO, USA). Fatty acids were dissolved in ethanol. The final concentration of ethanol in the assay medium did not exceed 0.05%. A preliminary experiment showed that ethanol at this concentration is not toxic to neutrophils and does not interfere with the results obtained. Reagents, water, and plastic ware used in the experiments were all endotoxin-free.
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Human and rat neutrophil treatment Lucigenin (1 mM), luminol (1 mM), phenol red (0.28 mM), hydroethidine (1 μM), or cytochrome c (0.1 mM) was added to neutrophil (2.5 × 106 cells/ml) incubation medium when required. Immediately afterward, cells were treated with various concentrations (0, 10, 25, 50, 100, and 200 μM) of oleic, linoleic, or γ-linolenic acid and phorbol myristate acetate (PMA) (54 ng/ml). ROS release was monitored for 20 min. The assays were run in PBS buffer supplemented with CaCl2 (1 mM), MgCl2 (1.5 mM), and glucose (10 mM), at 37°C, in a final volume of 0.3 ml. Lucigenin-enhanced chemiluminescence assay Lucigenin is extensively used to measure the production of reactive oxygen species by chemiluminescence. After being excited by superoxide anion, lucigenin releases energy in the form of light. Lucigenin-amplified chemiluminescence is a specific method for studying the kinetics of superoxide production by neutrophils. In this method, the response to xanthine–xanthine oxidase presented a positive correlation with light measurement and did not show augmentation of chemiluminescence when MPO was added to the assay medium [19]. Also, neutrophil chemiluminescence induced by fMetLeu-Phe (fMLP) is dose-dependently inhibited by scavengers of superoxide anions but not by azide, catalase, mannitol, or taurine; so this is a specific method to measure superoxide anion production [20]. The chemiluminescence response was monitored for 20 min, at 37°C, in a microplate luminometer (EG&G Berthold LB96V). Luminol-enhanced chemiluminescence assay
Rat neutrophil preparation Male Wistar rats weighing 180 ± 20 g were obtained from the Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Brazil. The rats were maintained at 23°C under a light:dark cycle of 12:12 h. Food and water were given ad libitum. The Animal Care Committee of the Institute of Biomedical Sciences approved the experimental procedure of this study. Rats were killed by decapitation without anesthesia. Neutrophils were obtained by intraperitoneal (ip) lavage using 40 ml sterile PBS, 4 h after the ip injection of 10 ml sterile oyster glycogen solution (Sigma; Type II) at 1% in PBS [17]. The number of viable cells (> 98%) was counted in a Neubauer chamber using a light microscope (Nikkon, Japan) and trypan blue solution at 1%. Human neutrophil preparation Human neutrophils were isolated from blood of healthy volunteers, as previously described [18], using a commercial gradient of Ficoll–Hypaque (Histopaque). The Ethical Committee of the Institute of Biomedical Sciences approved the experimental procedure of this study.
Luminol (5-amino-2,3-dihydro-1,4-phthalazindione) is a chemical light amplifier. One important point to be considered in this technique is that MPO-derived metabolites are responsible for the excitation of luminol [21]. Therefore, neutrophil degranulation can influence the results by releasing MPO found in the azurophil granules during the respiratory burst. Neutrophils were treated as described above in the presence of luminol (1 mM). Chemiluminescence was measured as described above. Flow-cytometric measurement of reactive oxygen metabolites using hydroethidine Hydroethidine has been widely used for the flow-cytometric measurement of intracellular ROS production. Hydroethidine, a reduced derivative of ethidium bromide, easily penetrates into the cells and shows weak fluorescence when excited by light at 480-nm wavelength. Hydroethidine is intracellularly oxidized by oxygen radicals, being converted into ethidium bromide that tightly binds to DNA and shows a strong red fluorescence [22,23]. One advantage of this method is the possibility of evaluating the response of individual cells. It provides statistically reliable distribution of cells according to the
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following activating states: dormant, primed, active, or resting [24]. Neutrophils were treated as described above in the presence of hydroethidine (1 μM). The fluorescence was measured using the FL3 channel in a FACSCalibur flow cytometer (Becton–Dickinson, San Jose, CA, USA). Ten thousand events were analyzed per experiment. Cytochrome c reduction assay The reduction of cytochrome c by reactive oxygen species can be measured by spectrophotometry. A control reaction was carried out with 50 and 100 μM oleic, linoleic, and γlinolenic acids and cytochrome c (0.1 and 1.0 mM) without cells. The reaction was run for 30 min in PBS in a final volume of 0.3 ml. After that, changes in absorbance were monitored at 550 nm. Hydrogen peroxide determination For the measurement of hydrogen peroxide levels, a single, rapid, and inexpensive method described by Pick et al. [25] was used. The assay is based on horseradish peroxidase (HRP)mediated oxidation of phenol red by H2O2. The reaction leads to formation of a colored compound that shows absorbance at 610 nm [25]. The phenol red assay allows the detection of reactive oxygen species both inside and outside the cells. Neutrophils were treated as described above in the presence of phenol red (0.28 mM) and 1 U/ml HRP type II. The reaction was stopped by the addition of 10 μl of 1 N sodium hydroxide aqueous solution. Measurement of MPO release from neutrophils Neutrophils (2 × 106 cells/ml) were exposed for 30 min, at 37°C, to oleic, linoleic, and γ-linolenic acids (0, 10, 25, 50, 100, and 200 μM) in the presence or absence of PMA. After incubation, the medium was immersed into ice and centrifuged at 500 g for 10 min, at 4°C, to separate the supernatant from the cells. The supernatant was used to measure MPO activity. The reaction was run in PBS, H2O2 (0.1 mM) and luminol (1 mM), at 37°C, in a final volume of 0.3 ml. Chemiluminescence was determined as described above [26].
acids in the reaction of ROS with the reagents, peroxide was added to the phenol red assay, and xanthine and xanthine oxidase were added to the lucigenin and luminol assays, without cells. There was no effect of the fatty acids on the lucigenin, luminol, and phenol red ROS-detecting systems. Neutrophils treated with oleic, linoleic, and γ-linolenic acids showed an increase in superoxide levels as shown by cytochrome c reduction (data not shown). However, the control reactions, containing 100 μM oleic, linoleic, or γ-linolenic acids with cytochrome c (0.1 mM), without cells, also showed an increase in absorbance (Fig. 1). The same result was obtained with 1.0 mM cytochrome c and 50 μM oleic, linoleic, or γlinolenic acid (data not shown). Oleic, linoleic, and γ-linolenic acids per se led to cytochrome c reduction. Therefore, this method cannot be recommended as a reliable measurement of ROS production induced by fatty acids at the concentration used in this study. As indicated in the Fig. 2, oleic and linoleic acids strongly inhibited basal and PMA-stimulated ROS production by rat neutrophils in a dose-dependent manner. The inhibitory effect of oleic acid on ROS release by unstimulated neutrophils was 15% for 5 μM and varied from 68 to 86% for 10, 25, 50, 100, and 200 μM. In PMA-stimulated cells, ROS release was decreased by 56–93% for the same fatty acid concentrations. The inhibitory effect of linoleic acid on neutrophil ROS release was 25% for 5 μM, 41% for 10 μM, and approximately 90% for 25, 50, 100, and 200 μM. In PMA-stimulated neutrophils, the inhibition induced by linoleic acid was 39% for 5 μM, 43% for 10 μM, and about 95% for 25, 50, 100, and 200 μM (Fig. 2). The inhibitory effect of γ-linolenic acid on PMA-induced neutrophil ROS release was less pronounced than that of oleic and linoleic acids. γ-Linolenic acid caused a significant effect at the 200 μM concentration only (64.5% reduction compared to control). Oleic, linoleic, and γ-linolenic acids inhibited MPO activity in the incubation medium of PMA-stimulated neutrophils (Fig. 3). Taking into consideration that the lightgenerating reaction is peroxidase-dependent [21], ROS production by neutrophils is underestimated by using the luminol-amplified chemiluminescence technique in neutrophils treated with fatty acids. The increase in superoxide production induced by fatty acids in neutrophils using the lucigenin-amplified chemiluminescence
Statistical analysis Comparisons were performed using one-way ANOVA and the Dunnett test. The significance was set at p < 0.05. Results were obtained from three to five separate experiments and are expressed as means ± SEM. Results Appropriate controls were carried out using 10, 25, 50, 100, and 200 μM oleic, linoleic, and γ-linolenic acids in the assays (luminol, lucigenin, and phenol red) without cells. The three fatty acids did not directly affect the luminol, lucigenin, and phenol red assays. To test for possible interference by the fatty
Fig. 1. Cytochrome c reduction by oleic, linoleic, and γ-linolenic acids (100 μM) in assay medium without cells. Results are presented as means ± SEM of two experiments carried out in triplicate. *p < 0.05 for comparison between control condition and treatment with fatty acids.
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An additive effect on fatty acid-induced superoxide production by neutrophils was observed when PMA was added to the assay medium (Fig. 5). The additive effect of the fatty acids and PMA on superoxide production was more pronounced in human than in rat neutrophils and occurred mainly by the treatment of the cells with γ-linolenic acid. Human neutrophils treated with PMA plus oleic (50 and 100 μM), linoleic (50 and 100 μM), or γ-linolenic (10, 50, 100, and 200 μM) acid showed an additive increase in superoxide production. For oleic acid, this increment was of 3.2 and 4.3 times higher compared with control
Fig. 2. Intra- and extracellular ROS levels in rat neutrophils (2.5 × 106 cells/ml) as measured by the luminol-amplified chemiluminescence method, in the absence and in the presence of various concentrations of oleic, linoleic, and γlinolenic acids (0, 10, 50, 100, and 200 μM) with or without PMA. Results are presented as means ± SEM of at least three experiments carried out in triplicate.
technique is a reproductive finding. There was no significant interference or cross-reaction of fatty acids with lucigenin itself. Kinetic studies showed that induction of superoxide production in human neutrophils is a fast event that occurs within minutes after neutrophil treatment with oleic, linoleic, and γ-linolenic acids (50 μM) (Fig. 4). By using the lucigenin-amplified chemiluminescence assay, S an increase in extracellular O2 − levels was observed by treatment of neutrophils with the three fatty acids. Oleic, linoleic, and γ-linolenic acids (in human neutrophils) and oleic and linoleic acids (in rat neutrophils) raised the chemiluminescent signal to a magnitude expected for classical stimuli, such as PMA or opsonized particles, 3 to 4 orders of magnitude higher than in unstimulated cells (Fig. 5) [27].
Fig. 3. Changes in MPO activity in the incubation medium of PMA-stimulated rat neutrophils treated with oleic, linoleic, and γ-linolenic acids. Results are presented as means ± SEM of at least three experiments carried out in triplicate. *p < 0.05 for comparison between control condition and treatment with PMA and #p < 0.05, ##p < 0.01, for comparison between PMA and fatty acids.
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Fig. 4. Kinetics of light emission by unstimulated neutrophils (2.5 × 106 cells/ ml) treated with oleic, linoleic, and γ-linolenic acids (50 μM) in the presence of lucigenin (1 mM).
(neutrophils plus PMA), respectively, for 50 and 100 μM. For linoleic acid at 50 and 100 μM, the increment was of 3.6 times higher compared to control, whereas for γ-linolenic acid the increase was 1.5-, 2.6-, 3.8-, and 2.4-fold for 10, 50, 100, and 200 μM, respectively. Rat neutrophils treated with γ-linolenic acid did not show any change in superoxide production; however, when PMA was added to the assay medium an additive effect on superoxide anion production was observed, 2.2 times higher for 10 and 50 μM compared to control. The treatment of rat neutrophils with oleic, linoleic, and γlinolenic acids increased the intracellular levels of ROS as indicated by the reduction of hydroethidine (Fig. 6). For oleic acid (50 and 100 μM), the increment in ROS production was 3.6-fold higher compared to control. For linoleic acid, the increment was over three times higher compared to control for
Fig. 5. Superoxide anion levels in the incubation medium of (A–C) rat and (D–F) human neutrophils (2.5 × 106 cells/ml) as measured by the lucigenin assay in the absence and in the presence of various concentrations of oleic, linoleic, and γ-linolenic acids (0, 10, 50, 100, and 200 μM) with or without PMA. Results are presented as means ± SEM of at least three experiments carried out in triplicate. *p < 0.05, **p < 0.01, and ***p < 0.001, due the effects of the fatty acids compared with control and #p < 0.05, ###p < 0.001, for comparison between columns of the same concentration of fatty acids, in the presence or absence of PMA.
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Fig. 6. Intracellular ROS production by rat neutrophils (2.5 × 106cells/ml), as measured by the hydroethidine technique in the absence and in the presence of various concentrations of oleic, linoleic, and γ-linolenic acids (0, 10, 50, 100, and 200 μM) with or without PMA. Results are presented as means ± SEM of at least three experiments carried out in triplicate. *p < 0.05, **p < 0.01, for comparison between the treatment with oleic, linoleic, and γ-linolenic acids and control.
both 50 and 100 μM. For γ-linolenic acid the increment was 2.0-fold for the 200 μM concentration. Oleic, linoleic, and γ-linolenic acids did not significantly increase the intra- and extracellular basal levels of H2O2 in human and rat neutrophils as showed by the phenol red reduction assay. However, there was a positive dose–response correlation between fatty acid concentrations and H2O2 production. The Pearson correlation found was r = 0.97 and p = 0.004 for oleic acid and r = 0.92 and p = 0.02 for linoleic and γ-linolenic acids (Fig. 7). An additive effect on H2O2 production was observed when PMA was added to the assay medium in both rat and human neutrophils treated with oleic acid (Fig. 7). Discussion ROS production by neutrophils is primarily associated with phagocyte defense against foreign organisms and occurs
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mainly through the NADPH oxidase complex. NADPH oxidase is assembled and activated either in the plasma membrane or in the membrane of internalized phagosomes. The reactive oxygen species generated will then either be released from the cells (activation in the plasma membrane) or be retained inside the phagocyte (activation in the phagosomal membrane) [1,2,28,29]. Mitochondria are also considered to be an important intracellular site for superoxide generation, which occurs mostly by the univalent reduction of oxygen in complexes I and III of the electron transport chain [4,5]. Evidence is presented herein that oleic, linoleic, and γlinolenic acids cause a marked increase in intra- and extracellular ROS levels in incubated rat and human neutrophils. One important point observed is that these fatty acids interfered with cytochrome c reduction and luminol-amplified chemiluminescence assays. Thus, the contradictory findings of the fatty acid effects on ROS production found in the literature can be in part due to the methods used. The fatty acids tested caused a direct reduction of cytochrome c. Therefore, the effects of fatty acids on superoxide production cannot be measured by cytochrome c reduction assay under the conditions herein used. A similar effect was observed by Hardy et al. [30]. Another important point of this technique is that H2O2 may also interfere with the assay. Accumulation of H2O2 in the measuring system can result in a reoxidation of Fe2+ cytochrome c back to the Fe3+ form, thus giving underestimated results [29]. In addition to the direct effect on cytochrome c reduction, fatty acids can also jeopardize the light emission techniques (luminol/isoluminol and lucigenin) when the concentration used affects the turbidity of the medium. Measurements of intracellular ROS levels can also be underestimated because neutrophils rapidly release oxygen species to the extracellular medium. An increase in ROS production induced by fatty acids in neutrophils was found by using lucigenin-amplified chemiluminescence, hydroethidine, and phenol red reduction techniques, whereas a decrease was observed with luminol-enhanced chemiluminescence assay. To address this discrepancy, the effects of oleic, linoleic, and γ-linolenic acids on MPO activity in the neutrophil incubation medium were examined. Oleic, linoleic, and γ-linolenic acids reduced MPO activity in the incubation medium of PMA-stimulated neutrophils. Our results agree with those of Naccache et al. [31]. These authors reported that oleic, linoleic, and linolenic acids inhibit neutrophil granule secretion in response to addition of fMLP [31]. Taking into consideration that the light-generating reaction is peroxidasedependent [21], ROS production by neutrophils is underestimated by using the luminol-amplified chemiluminescence technique in fatty acid-treated neutrophils. The influence of neutrophil degranulation on ROS production through the respiratory burst by using the luminol technique was also estimated by Dahlgren and Stendahl [32]. In this study, NADPH oxidase activity was measured in cytoplasts and in normal cells. The cytoplasts lacking all granules were then deficient in MPO activity. Under this condition, there was a decrease in chemiluminescence [32]. Therefore, luminol-enhanced chemi-
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Fig. 7. Hydrogen peroxide levels in incubation medium of (A–C) rat and (D–F) human neutrophils (2.5 × 106 cells/ml) as measured by the phenol red method in the absence and in the presence of various concentrations of oleic, linoleic, and γ-linolenic acids (0, 10, 50, 100, and 200 μM) with or without PMA. Results are presented as means ± SEM of at least three experiments carried out in triplicate. *p < 0.05, **p < 0.01, for comparison between the treatment with the oleic, linoleic, and γ-linolenic acids and the control and #p < 0.05, ###p < 0.001, for comparison between columns of the same concentration of the fatty acids in the presence and absence of PMA.
luminescence is not an appropriate technique to measure ROS release by neutrophils treated with fatty acids under the conditions herein used. As mentioned above, oleic, linoleic, and linolenic acids inhibit neutrophil granule secretion by fMLP- and PMAstimulated neutrophils. However, in the absence of stimuli, Bates et al. [33] showed that fatty acids increase neutrophil degranulation as follows: linolenic > linoleic > oleic. In general, as the number of double bonds in the 18-carbon fatty acid molecule increases, so does its ability to stimulate degranulation and oxidative burst in unstimulated neutrophils [33,34]. This may explain the fact that the inhibitory effect of γ-linolenic acid on PMA-induced ROS production as measured by the luminol technique is less pronounced than that caused by linoleic and oleic acids.
PMA addition to the assay medium caused an additive effect on superoxide and hydrogen peroxide production induced by oleic, linoleic, and γ-linolenic acids. These results support the proposal of a possible priming effect of fatty acids on neutrophil oxidative burst. Neutrophils exist in various states of activation such as primed, activated, and spent. Priming and activation are biochemically integrated events. Priming is a state of preactivation of dormant neutrophils that enables a prompt response of the cells to a microbicidal activity [35]. Small biochemical changes may trigger priming and large changes may lead to full activation including degranulation. Priming agents such as cytokines (TNF-α, IL-8) [36,37], phospholipase A2 [38], platelet-activating factor [39], and serum amyloid A [27] cause an increment in oxygen consumption when a second stimulus occurs [40].
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Primed oxygenation activity may result from the activation of one or more of the components of the neutrophil transduction pathways induced by fatty acids. This may include fluxes of free cations (Na+, K+, and Ca2+), changes in membrane potential, activation of intracellular proteases, increases in arachidonic acid and phospholipid metabolism, phosphorylation of specific proteins (oxidase components), and increase in the intracellular concentrations of cyclic nucleotides [40,41]. Fatty acids support the structural basis of the modulation of the membrane lipid organization and the subsequent regulation of G-protein-coupled receptor signaling [42]. Hardy et al. demonstrated that pretreating neutrophils with arachidonic, eicosapentaenoic, and docosahexanoic acids enhances their capacity to respond to either fMLP or PMA, thereby producing more superoxide than when challenged with the stimulators only [34,43]. In the present study, oleic, linoleic, and γ-linolenic acids showed a modulatory effect on inflammation by stimulating ROS production. However, ROS measurement can be jeopardized by using luminol-amplified chemiluminescence and cytochrome c reduction techniques. Acknowledgments The authors are indebted to the Fundação de Amparo à Pesquisa do Estado de São Paulo and to the Conselho Nacional de Desenvolvimento Científico e Tecnológico for financial support. References [1] Babior, B. M. NADPH oxidase: an update. Blood 93:1464–1476; 1999. [2] Johnson, J. L.; Park, J. W.; Benna, J. E.; Faust, L. P.; Inanami, O.; Babior, B. M. Activation of p47 (PHOX), a cytosolic subunit of the leukocyte NADPH oxidase—Phosphorylation of Ser-359 or Ser-370 precedes phosphorylation at other sites and is required for activity. J. Biol. Chem. 273:35147–35152; 1999. [3] Dahlgren, C.; Karlsson, A. Respiratory burst in human neutrophils. J. Immunol. Methods 232:3–14; 1999. [4] Andreyev, A. Y.; Kushnareva, Y. E.; Starkov, A. A. Mitochondrial metabolism of reactive oxygen species. Biochemistry 70:200–214; 2005. [5] Kovacic, P.; Pozos, R. S.; Somanathan, R.; Shangari, N.; O'Brien, P. J. Mechanism of mitochondrial uncouplers, inhibitors, and toxins: focus on electron transfer, free radicals, and structure–activity relationships. Curr. Med. Chem. 12:2601–2623; 2005. [6] Forman, H. J.; Torres, M. Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling. Am. J. Respir. Crit. Care Med. 15:4–8; 2002. [7] Williams, M. S.; Kwon, J. T cell receptor stimulation, reactive oxygen species, and cell signaling. Free Radic. Biol. Med. 15:1144–1151; 2004. [8] Harper, C. R.; Jacobson, T. A. Usefulness of omega-3 fatty acids and the prevention of coronary heart disease. Am. J. Cardiol. 96:1521–1529; 2005. [9] Stamp, L. K.; James, M. J.; Cleland, L. G. Diet and rheumatoid arthritis: a review of the literature. Semin. Arthritis Rheum. 35:77–94; 2005. [10] Moriuchi, H.; Zaha, M.; Fukumoto, T.; Yuizono, T. Activation of polymorphonuclear leukocytes in oleic acid-induced lung injury. Intens. Care Med. 24:709–715; 1998. [11] Yamaguchi, T.; Kaneda, M.; Kakinuma, K. Effect of saturated and unsaturated fatty acids on the oxidative metabolism of human neutrophils:
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[32] Dahlgren, C.; Stendahl, O. Role of myeloperoxidase in luminol-dependent chemiluminescence of polymorphonuclear leukocytes. Infect. Immun. 39:736–741; 1983. [33] Bates, E. J.; Ferrante, A.; Smithers, L.; Poulos, A.; Robinson, B. S. Effect of fatty acid structure on neutrophil adhesion, degranulation and damage to endothelial cells. Atherosclerosis 116:247–259; 1995. [34] Ferrante, A.; Hii, C. S.; Costabile, M. Regulation of neutrophil functions by long chain fatty acids. In: Gabrilovich, D.I. (Ed.), The Neutrophils: New Outlook for Old Cells. Imperial College Press, London; 2005. [35] Sheppard, F. R.; Kelher, M. R.; Moore, E. E.; McLaughlin, N. J.; Banerjee, A.; Silliman, C. C. Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation. J. Leukocyte Biol. 78:1025–1042; 2005. [36] Dewas, C.; Dang, P. M.; Gougerot-Pocidalo, M. A.; El-Benna, J. TNF-alpha induces phosphorylation of p47 (phox) in human neutrophils: partial phosphorylation of p47phox is a common event of priming of human neutrophils by TNF-alpha and granulocyte– macrophage colony-stimulating factor. Immunology 171:4392–4398; 2003. [37] Guichard, C.; Pedruzzi, E.; Dewas, C.; Fay, M.; Pouzet, C.; Bens, M.; Vandewalle, A.; Ogier-Denis, E.; Gougerot-Pocidalo, M. A.; Elbim, C. Interleukin-8-induced priming of neutrophil oxidative burst requires
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Original Contribution
Systematic study on ROS production induced by oleic, linoleic, and γ-linolenic acids in human and rat neutrophils Elaine Hatanaka a,⁎, Adriana Cristina Levada-Pires a , Tania Cristina Pithon-Curi a,b , Rui Curi a a
Institute of Biomedical Sciences, Department of Physiology and Biophysics, University of São Paulo, Avenida Prof. Lineu Prestes, 1524, 05508-900 Butantã, São Paulo, SP, Brazil b Cruzeiro do Sul University, São Paulo, Brazil Received 12 February 2006; revised 23 June 2006; accepted 25 June 2006 Available online 4 July 2006
Abstract The effects of oleic, linoleic, and γ-linolenic acids on the production of ROS by unstimulated and PMA-stimulated neutrophils were investigated by using five techniques: luminol- and lucigenin-amplified chemiluminescence, cytochrome c, hydroethidine, and phenol red reduction. Using lucigenin-amplified chemiluminescence, an increase in extracellular superoxide levels was observed by the treatment of neutrophils with the fatty acids. There was also an increase in intracellular ROS levels under similar conditions as measured by the hydroethidine technique. An increment in the intra- and extracellular levels of H2O2 was also observed in neutrophils treated with oleic acid as measured by phenol red reduction assay. In the luminol technique, peroxidase activity is required in the reaction of luminol with ROS for light generation. Oleic, linoleic, and γ-linolenic acids inhibited the myeloperoxidase activity in stimulated neutrophils. So, these fatty acids jeopardize the results of ROS content measured by this technique. Oleic, linoleic, and γ-linolenic acids per se led to cytochrome c reduction and so this method also cannot be used to measure ROS production induced by fatty acids. Oleic, linoleic, and γ-linolenic acids do stimulate ROS production by neutrophils; however, measurements using the luminol-amplified chemiluminescence and cytochrome c reduction techniques require further analysis. © 2006 Elsevier Inc. All rights reserved. Keywords: Fatty acids; ROS; Luminol; Lucigenin; Cytochrome c; Hydroethidine; Neutrophils; Free radicals
Neutrophils are the first cells that migrate to tissues in response to invading pathogens. The antimicrobial function of these phagocytes depends on the release of lytic enzymes stored in cytoplasmatic granules and on generation of S superoxide (O2 − ). In phagocytes, superoxide is mainly generated by the reaction of oxygen and NADPH through the NADPH oxidase complex [1]. This enzyme complex is formed by subunits found in intracellular granules and plasma membrane. After activation, NADPH oxidase components containing granules fuse to phagocytic vacuoles and generate superoxide. Also, granules may migrate to the cell surface and release superoxide into the extracellular space [2]. Superoxide anion and hydrogen peroxide (H2O2) generated
⁎ Corresponding author. Fax: +55 11 3091 7285. E-mail address: [email protected] (E. Hatanaka). 0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2006.06.014
by the NADPH oxidase give rise to other reactive oxygen species (ROS) that are strong cytolytic agents, such as hypochlorous acid (formed from H2O2 and chloride ions by the action of myeloperoxidase (MPO) released from neutrophil granules) and hydroxyl radical [1–3]. Superoxide can also be generated through the mitochondrial electron transport chain, xanthine–xanthine oxidase, and cytochrome P450. Mitochondria generate superoxide mostly by the univalent reduction of oxygen in complexes I and III of the electron transport chain [4]. Changes in mitochondrial generation of superoxide can be assessed by the addition of mitochondria uncouplers that are depolarizing agents and inhibitors of the respiratory chain [5]. ROS generated by neutrophils play an important role in the inflammatory response by regulating other immune reactive cells [6,7]. Fatty acids have been used for the treatment of various diseases that involve oxidative stress, such as coronary
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heart disease [8] and rheumatic arthritis [9]. However, the effects of long-chain fatty acids on ROS production by neutrophils remain to be fully established. Some authors showed an increase [10–12], whereas others found a decrease, in ROS production by neutrophils in response to fatty acid treatment [13–16]. This discrepancy may be due to the methods used to measure intra- and extracellular ROS levels. In this study intra- and extracellular levels of ROS were measured in neutrophils treated with oleic, linoleic, and γlinolenic acids. The luminol technique was used to measure intra- and extracellular ROS levels. Hydroethidine reduction was employed to measure intracellular ROS production. Cytochrome c reduction and lucigenin-amplified chemiluminescence were used to determine extracellular superoxide anion levels. Phenol red reduction was employed to measure intra- and extracellular H2O2 levels. Material and methods RPMI 1640 medium, Hepes, penicillin, and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Fatty acids, glutamine, luminol, lucigenin, cytochrome c, hydroethidine, peroxidase type II, hydrogen peroxide, phenol red, Histopaque, oyster glycogen, and trypan blue were supplied by Sigma Chemical Co (St. Louis, MO, USA). Fatty acids were dissolved in ethanol. The final concentration of ethanol in the assay medium did not exceed 0.05%. A preliminary experiment showed that ethanol at this concentration is not toxic to neutrophils and does not interfere with the results obtained. Reagents, water, and plastic ware used in the experiments were all endotoxin-free.
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Human and rat neutrophil treatment Lucigenin (1 mM), luminol (1 mM), phenol red (0.28 mM), hydroethidine (1 μM), or cytochrome c (0.1 mM) was added to neutrophil (2.5 × 106 cells/ml) incubation medium when required. Immediately afterward, cells were treated with various concentrations (0, 10, 25, 50, 100, and 200 μM) of oleic, linoleic, or γ-linolenic acid and phorbol myristate acetate (PMA) (54 ng/ml). ROS release was monitored for 20 min. The assays were run in PBS buffer supplemented with CaCl2 (1 mM), MgCl2 (1.5 mM), and glucose (10 mM), at 37°C, in a final volume of 0.3 ml. Lucigenin-enhanced chemiluminescence assay Lucigenin is extensively used to measure the production of reactive oxygen species by chemiluminescence. After being excited by superoxide anion, lucigenin releases energy in the form of light. Lucigenin-amplified chemiluminescence is a specific method for studying the kinetics of superoxide production by neutrophils. In this method, the response to xanthine–xanthine oxidase presented a positive correlation with light measurement and did not show augmentation of chemiluminescence when MPO was added to the assay medium [19]. Also, neutrophil chemiluminescence induced by fMetLeu-Phe (fMLP) is dose-dependently inhibited by scavengers of superoxide anions but not by azide, catalase, mannitol, or taurine; so this is a specific method to measure superoxide anion production [20]. The chemiluminescence response was monitored for 20 min, at 37°C, in a microplate luminometer (EG&G Berthold LB96V). Luminol-enhanced chemiluminescence assay
Rat neutrophil preparation Male Wistar rats weighing 180 ± 20 g were obtained from the Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Brazil. The rats were maintained at 23°C under a light:dark cycle of 12:12 h. Food and water were given ad libitum. The Animal Care Committee of the Institute of Biomedical Sciences approved the experimental procedure of this study. Rats were killed by decapitation without anesthesia. Neutrophils were obtained by intraperitoneal (ip) lavage using 40 ml sterile PBS, 4 h after the ip injection of 10 ml sterile oyster glycogen solution (Sigma; Type II) at 1% in PBS [17]. The number of viable cells (> 98%) was counted in a Neubauer chamber using a light microscope (Nikkon, Japan) and trypan blue solution at 1%. Human neutrophil preparation Human neutrophils were isolated from blood of healthy volunteers, as previously described [18], using a commercial gradient of Ficoll–Hypaque (Histopaque). The Ethical Committee of the Institute of Biomedical Sciences approved the experimental procedure of this study.
Luminol (5-amino-2,3-dihydro-1,4-phthalazindione) is a chemical light amplifier. One important point to be considered in this technique is that MPO-derived metabolites are responsible for the excitation of luminol [21]. Therefore, neutrophil degranulation can influence the results by releasing MPO found in the azurophil granules during the respiratory burst. Neutrophils were treated as described above in the presence of luminol (1 mM). Chemiluminescence was measured as described above. Flow-cytometric measurement of reactive oxygen metabolites using hydroethidine Hydroethidine has been widely used for the flow-cytometric measurement of intracellular ROS production. Hydroethidine, a reduced derivative of ethidium bromide, easily penetrates into the cells and shows weak fluorescence when excited by light at 480-nm wavelength. Hydroethidine is intracellularly oxidized by oxygen radicals, being converted into ethidium bromide that tightly binds to DNA and shows a strong red fluorescence [22,23]. One advantage of this method is the possibility of evaluating the response of individual cells. It provides statistically reliable distribution of cells according to the
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following activating states: dormant, primed, active, or resting [24]. Neutrophils were treated as described above in the presence of hydroethidine (1 μM). The fluorescence was measured using the FL3 channel in a FACSCalibur flow cytometer (Becton–Dickinson, San Jose, CA, USA). Ten thousand events were analyzed per experiment. Cytochrome c reduction assay The reduction of cytochrome c by reactive oxygen species can be measured by spectrophotometry. A control reaction was carried out with 50 and 100 μM oleic, linoleic, and γlinolenic acids and cytochrome c (0.1 and 1.0 mM) without cells. The reaction was run for 30 min in PBS in a final volume of 0.3 ml. After that, changes in absorbance were monitored at 550 nm. Hydrogen peroxide determination For the measurement of hydrogen peroxide levels, a single, rapid, and inexpensive method described by Pick et al. [25] was used. The assay is based on horseradish peroxidase (HRP)mediated oxidation of phenol red by H2O2. The reaction leads to formation of a colored compound that shows absorbance at 610 nm [25]. The phenol red assay allows the detection of reactive oxygen species both inside and outside the cells. Neutrophils were treated as described above in the presence of phenol red (0.28 mM) and 1 U/ml HRP type II. The reaction was stopped by the addition of 10 μl of 1 N sodium hydroxide aqueous solution. Measurement of MPO release from neutrophils Neutrophils (2 × 106 cells/ml) were exposed for 30 min, at 37°C, to oleic, linoleic, and γ-linolenic acids (0, 10, 25, 50, 100, and 200 μM) in the presence or absence of PMA. After incubation, the medium was immersed into ice and centrifuged at 500 g for 10 min, at 4°C, to separate the supernatant from the cells. The supernatant was used to measure MPO activity. The reaction was run in PBS, H2O2 (0.1 mM) and luminol (1 mM), at 37°C, in a final volume of 0.3 ml. Chemiluminescence was determined as described above [26].
acids in the reaction of ROS with the reagents, peroxide was added to the phenol red assay, and xanthine and xanthine oxidase were added to the lucigenin and luminol assays, without cells. There was no effect of the fatty acids on the lucigenin, luminol, and phenol red ROS-detecting systems. Neutrophils treated with oleic, linoleic, and γ-linolenic acids showed an increase in superoxide levels as shown by cytochrome c reduction (data not shown). However, the control reactions, containing 100 μM oleic, linoleic, or γ-linolenic acids with cytochrome c (0.1 mM), without cells, also showed an increase in absorbance (Fig. 1). The same result was obtained with 1.0 mM cytochrome c and 50 μM oleic, linoleic, or γlinolenic acid (data not shown). Oleic, linoleic, and γ-linolenic acids per se led to cytochrome c reduction. Therefore, this method cannot be recommended as a reliable measurement of ROS production induced by fatty acids at the concentration used in this study. As indicated in the Fig. 2, oleic and linoleic acids strongly inhibited basal and PMA-stimulated ROS production by rat neutrophils in a dose-dependent manner. The inhibitory effect of oleic acid on ROS release by unstimulated neutrophils was 15% for 5 μM and varied from 68 to 86% for 10, 25, 50, 100, and 200 μM. In PMA-stimulated cells, ROS release was decreased by 56–93% for the same fatty acid concentrations. The inhibitory effect of linoleic acid on neutrophil ROS release was 25% for 5 μM, 41% for 10 μM, and approximately 90% for 25, 50, 100, and 200 μM. In PMA-stimulated neutrophils, the inhibition induced by linoleic acid was 39% for 5 μM, 43% for 10 μM, and about 95% for 25, 50, 100, and 200 μM (Fig. 2). The inhibitory effect of γ-linolenic acid on PMA-induced neutrophil ROS release was less pronounced than that of oleic and linoleic acids. γ-Linolenic acid caused a significant effect at the 200 μM concentration only (64.5% reduction compared to control). Oleic, linoleic, and γ-linolenic acids inhibited MPO activity in the incubation medium of PMA-stimulated neutrophils (Fig. 3). Taking into consideration that the lightgenerating reaction is peroxidase-dependent [21], ROS production by neutrophils is underestimated by using the luminol-amplified chemiluminescence technique in neutrophils treated with fatty acids. The increase in superoxide production induced by fatty acids in neutrophils using the lucigenin-amplified chemiluminescence
Statistical analysis Comparisons were performed using one-way ANOVA and the Dunnett test. The significance was set at p < 0.05. Results were obtained from three to five separate experiments and are expressed as means ± SEM. Results Appropriate controls were carried out using 10, 25, 50, 100, and 200 μM oleic, linoleic, and γ-linolenic acids in the assays (luminol, lucigenin, and phenol red) without cells. The three fatty acids did not directly affect the luminol, lucigenin, and phenol red assays. To test for possible interference by the fatty
Fig. 1. Cytochrome c reduction by oleic, linoleic, and γ-linolenic acids (100 μM) in assay medium without cells. Results are presented as means ± SEM of two experiments carried out in triplicate. *p < 0.05 for comparison between control condition and treatment with fatty acids.
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An additive effect on fatty acid-induced superoxide production by neutrophils was observed when PMA was added to the assay medium (Fig. 5). The additive effect of the fatty acids and PMA on superoxide production was more pronounced in human than in rat neutrophils and occurred mainly by the treatment of the cells with γ-linolenic acid. Human neutrophils treated with PMA plus oleic (50 and 100 μM), linoleic (50 and 100 μM), or γ-linolenic (10, 50, 100, and 200 μM) acid showed an additive increase in superoxide production. For oleic acid, this increment was of 3.2 and 4.3 times higher compared with control
Fig. 2. Intra- and extracellular ROS levels in rat neutrophils (2.5 × 106 cells/ml) as measured by the luminol-amplified chemiluminescence method, in the absence and in the presence of various concentrations of oleic, linoleic, and γlinolenic acids (0, 10, 50, 100, and 200 μM) with or without PMA. Results are presented as means ± SEM of at least three experiments carried out in triplicate.
technique is a reproductive finding. There was no significant interference or cross-reaction of fatty acids with lucigenin itself. Kinetic studies showed that induction of superoxide production in human neutrophils is a fast event that occurs within minutes after neutrophil treatment with oleic, linoleic, and γ-linolenic acids (50 μM) (Fig. 4). By using the lucigenin-amplified chemiluminescence assay, S an increase in extracellular O2 − levels was observed by treatment of neutrophils with the three fatty acids. Oleic, linoleic, and γ-linolenic acids (in human neutrophils) and oleic and linoleic acids (in rat neutrophils) raised the chemiluminescent signal to a magnitude expected for classical stimuli, such as PMA or opsonized particles, 3 to 4 orders of magnitude higher than in unstimulated cells (Fig. 5) [27].
Fig. 3. Changes in MPO activity in the incubation medium of PMA-stimulated rat neutrophils treated with oleic, linoleic, and γ-linolenic acids. Results are presented as means ± SEM of at least three experiments carried out in triplicate. *p < 0.05 for comparison between control condition and treatment with PMA and #p < 0.05, ##p < 0.01, for comparison between PMA and fatty acids.
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Fig. 4. Kinetics of light emission by unstimulated neutrophils (2.5 × 106 cells/ ml) treated with oleic, linoleic, and γ-linolenic acids (50 μM) in the presence of lucigenin (1 mM).
(neutrophils plus PMA), respectively, for 50 and 100 μM. For linoleic acid at 50 and 100 μM, the increment was of 3.6 times higher compared to control, whereas for γ-linolenic acid the increase was 1.5-, 2.6-, 3.8-, and 2.4-fold for 10, 50, 100, and 200 μM, respectively. Rat neutrophils treated with γ-linolenic acid did not show any change in superoxide production; however, when PMA was added to the assay medium an additive effect on superoxide anion production was observed, 2.2 times higher for 10 and 50 μM compared to control. The treatment of rat neutrophils with oleic, linoleic, and γlinolenic acids increased the intracellular levels of ROS as indicated by the reduction of hydroethidine (Fig. 6). For oleic acid (50 and 100 μM), the increment in ROS production was 3.6-fold higher compared to control. For linoleic acid, the increment was over three times higher compared to control for
Fig. 5. Superoxide anion levels in the incubation medium of (A–C) rat and (D–F) human neutrophils (2.5 × 106 cells/ml) as measured by the lucigenin assay in the absence and in the presence of various concentrations of oleic, linoleic, and γ-linolenic acids (0, 10, 50, 100, and 200 μM) with or without PMA. Results are presented as means ± SEM of at least three experiments carried out in triplicate. *p < 0.05, **p < 0.01, and ***p < 0.001, due the effects of the fatty acids compared with control and #p < 0.05, ###p < 0.001, for comparison between columns of the same concentration of fatty acids, in the presence or absence of PMA.
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Fig. 6. Intracellular ROS production by rat neutrophils (2.5 × 106cells/ml), as measured by the hydroethidine technique in the absence and in the presence of various concentrations of oleic, linoleic, and γ-linolenic acids (0, 10, 50, 100, and 200 μM) with or without PMA. Results are presented as means ± SEM of at least three experiments carried out in triplicate. *p < 0.05, **p < 0.01, for comparison between the treatment with oleic, linoleic, and γ-linolenic acids and control.
both 50 and 100 μM. For γ-linolenic acid the increment was 2.0-fold for the 200 μM concentration. Oleic, linoleic, and γ-linolenic acids did not significantly increase the intra- and extracellular basal levels of H2O2 in human and rat neutrophils as showed by the phenol red reduction assay. However, there was a positive dose–response correlation between fatty acid concentrations and H2O2 production. The Pearson correlation found was r = 0.97 and p = 0.004 for oleic acid and r = 0.92 and p = 0.02 for linoleic and γ-linolenic acids (Fig. 7). An additive effect on H2O2 production was observed when PMA was added to the assay medium in both rat and human neutrophils treated with oleic acid (Fig. 7). Discussion ROS production by neutrophils is primarily associated with phagocyte defense against foreign organisms and occurs
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mainly through the NADPH oxidase complex. NADPH oxidase is assembled and activated either in the plasma membrane or in the membrane of internalized phagosomes. The reactive oxygen species generated will then either be released from the cells (activation in the plasma membrane) or be retained inside the phagocyte (activation in the phagosomal membrane) [1,2,28,29]. Mitochondria are also considered to be an important intracellular site for superoxide generation, which occurs mostly by the univalent reduction of oxygen in complexes I and III of the electron transport chain [4,5]. Evidence is presented herein that oleic, linoleic, and γlinolenic acids cause a marked increase in intra- and extracellular ROS levels in incubated rat and human neutrophils. One important point observed is that these fatty acids interfered with cytochrome c reduction and luminol-amplified chemiluminescence assays. Thus, the contradictory findings of the fatty acid effects on ROS production found in the literature can be in part due to the methods used. The fatty acids tested caused a direct reduction of cytochrome c. Therefore, the effects of fatty acids on superoxide production cannot be measured by cytochrome c reduction assay under the conditions herein used. A similar effect was observed by Hardy et al. [30]. Another important point of this technique is that H2O2 may also interfere with the assay. Accumulation of H2O2 in the measuring system can result in a reoxidation of Fe2+ cytochrome c back to the Fe3+ form, thus giving underestimated results [29]. In addition to the direct effect on cytochrome c reduction, fatty acids can also jeopardize the light emission techniques (luminol/isoluminol and lucigenin) when the concentration used affects the turbidity of the medium. Measurements of intracellular ROS levels can also be underestimated because neutrophils rapidly release oxygen species to the extracellular medium. An increase in ROS production induced by fatty acids in neutrophils was found by using lucigenin-amplified chemiluminescence, hydroethidine, and phenol red reduction techniques, whereas a decrease was observed with luminol-enhanced chemiluminescence assay. To address this discrepancy, the effects of oleic, linoleic, and γ-linolenic acids on MPO activity in the neutrophil incubation medium were examined. Oleic, linoleic, and γ-linolenic acids reduced MPO activity in the incubation medium of PMA-stimulated neutrophils. Our results agree with those of Naccache et al. [31]. These authors reported that oleic, linoleic, and linolenic acids inhibit neutrophil granule secretion in response to addition of fMLP [31]. Taking into consideration that the light-generating reaction is peroxidasedependent [21], ROS production by neutrophils is underestimated by using the luminol-amplified chemiluminescence technique in fatty acid-treated neutrophils. The influence of neutrophil degranulation on ROS production through the respiratory burst by using the luminol technique was also estimated by Dahlgren and Stendahl [32]. In this study, NADPH oxidase activity was measured in cytoplasts and in normal cells. The cytoplasts lacking all granules were then deficient in MPO activity. Under this condition, there was a decrease in chemiluminescence [32]. Therefore, luminol-enhanced chemi-
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Fig. 7. Hydrogen peroxide levels in incubation medium of (A–C) rat and (D–F) human neutrophils (2.5 × 106 cells/ml) as measured by the phenol red method in the absence and in the presence of various concentrations of oleic, linoleic, and γ-linolenic acids (0, 10, 50, 100, and 200 μM) with or without PMA. Results are presented as means ± SEM of at least three experiments carried out in triplicate. *p < 0.05, **p < 0.01, for comparison between the treatment with the oleic, linoleic, and γ-linolenic acids and the control and #p < 0.05, ###p < 0.001, for comparison between columns of the same concentration of the fatty acids in the presence and absence of PMA.
luminescence is not an appropriate technique to measure ROS release by neutrophils treated with fatty acids under the conditions herein used. As mentioned above, oleic, linoleic, and linolenic acids inhibit neutrophil granule secretion by fMLP- and PMAstimulated neutrophils. However, in the absence of stimuli, Bates et al. [33] showed that fatty acids increase neutrophil degranulation as follows: linolenic > linoleic > oleic. In general, as the number of double bonds in the 18-carbon fatty acid molecule increases, so does its ability to stimulate degranulation and oxidative burst in unstimulated neutrophils [33,34]. This may explain the fact that the inhibitory effect of γ-linolenic acid on PMA-induced ROS production as measured by the luminol technique is less pronounced than that caused by linoleic and oleic acids.
PMA addition to the assay medium caused an additive effect on superoxide and hydrogen peroxide production induced by oleic, linoleic, and γ-linolenic acids. These results support the proposal of a possible priming effect of fatty acids on neutrophil oxidative burst. Neutrophils exist in various states of activation such as primed, activated, and spent. Priming and activation are biochemically integrated events. Priming is a state of preactivation of dormant neutrophils that enables a prompt response of the cells to a microbicidal activity [35]. Small biochemical changes may trigger priming and large changes may lead to full activation including degranulation. Priming agents such as cytokines (TNF-α, IL-8) [36,37], phospholipase A2 [38], platelet-activating factor [39], and serum amyloid A [27] cause an increment in oxygen consumption when a second stimulus occurs [40].
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Primed oxygenation activity may result from the activation of one or more of the components of the neutrophil transduction pathways induced by fatty acids. This may include fluxes of free cations (Na+, K+, and Ca2+), changes in membrane potential, activation of intracellular proteases, increases in arachidonic acid and phospholipid metabolism, phosphorylation of specific proteins (oxidase components), and increase in the intracellular concentrations of cyclic nucleotides [40,41]. Fatty acids support the structural basis of the modulation of the membrane lipid organization and the subsequent regulation of G-protein-coupled receptor signaling [42]. Hardy et al. demonstrated that pretreating neutrophils with arachidonic, eicosapentaenoic, and docosahexanoic acids enhances their capacity to respond to either fMLP or PMA, thereby producing more superoxide than when challenged with the stimulators only [34,43]. In the present study, oleic, linoleic, and γ-linolenic acids showed a modulatory effect on inflammation by stimulating ROS production. However, ROS measurement can be jeopardized by using luminol-amplified chemiluminescence and cytochrome c reduction techniques. Acknowledgments The authors are indebted to the Fundação de Amparo à Pesquisa do Estado de São Paulo and to the Conselho Nacional de Desenvolvimento Científico e Tecnológico for financial support. References [1] Babior, B. M. NADPH oxidase: an update. Blood 93:1464–1476; 1999. [2] Johnson, J. L.; Park, J. W.; Benna, J. E.; Faust, L. P.; Inanami, O.; Babior, B. M. Activation of p47 (PHOX), a cytosolic subunit of the leukocyte NADPH oxidase—Phosphorylation of Ser-359 or Ser-370 precedes phosphorylation at other sites and is required for activity. J. Biol. Chem. 273:35147–35152; 1999. [3] Dahlgren, C.; Karlsson, A. Respiratory burst in human neutrophils. J. Immunol. Methods 232:3–14; 1999. [4] Andreyev, A. Y.; Kushnareva, Y. E.; Starkov, A. A. Mitochondrial metabolism of reactive oxygen species. Biochemistry 70:200–214; 2005. [5] Kovacic, P.; Pozos, R. S.; Somanathan, R.; Shangari, N.; O'Brien, P. J. Mechanism of mitochondrial uncouplers, inhibitors, and toxins: focus on electron transfer, free radicals, and structure–activity relationships. Curr. Med. Chem. 12:2601–2623; 2005. [6] Forman, H. J.; Torres, M. Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling. Am. J. Respir. Crit. Care Med. 15:4–8; 2002. [7] Williams, M. S.; Kwon, J. T cell receptor stimulation, reactive oxygen species, and cell signaling. Free Radic. Biol. Med. 15:1144–1151; 2004. [8] Harper, C. R.; Jacobson, T. A. Usefulness of omega-3 fatty acids and the prevention of coronary heart disease. Am. J. Cardiol. 96:1521–1529; 2005. [9] Stamp, L. K.; James, M. J.; Cleland, L. G. Diet and rheumatoid arthritis: a review of the literature. Semin. Arthritis Rheum. 35:77–94; 2005. [10] Moriuchi, H.; Zaha, M.; Fukumoto, T.; Yuizono, T. Activation of polymorphonuclear leukocytes in oleic acid-induced lung injury. Intens. Care Med. 24:709–715; 1998. [11] Yamaguchi, T.; Kaneda, M.; Kakinuma, K. Effect of saturated and unsaturated fatty acids on the oxidative metabolism of human neutrophils:
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