Supra-physiological doses of testosterone affect membrane oxidation of human neutrophils monitored by the fluorescent probe C11-BODIPY581/591
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- de Souza-Junior, T.P., Yamada, A.K., Simão, R. et al. Eur J Appl Physiol (2013) 113: 1241. doi:10.1007/s00421-012-2538-y
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The purpose of this study was to determine the effects of supra-physiological doses of testosterone (TES) on membrane oxidation of activated human neutrophils in vitro using an innovative and sensitive technique: the real-time detection with the fluorescence probe C11-BODIPY581/591. Methodological controls were performed with the lipid-soluble and powerful antioxidant astaxanthin at different neutrophil density cultures. Neutrophils from nine healthy young men (23.4 ± 2.5 years, 174.4 ± 7.0 cm height, and 78.3 ± 7.0 kg weight) were isolated and treated with 0.1 or 10 μM TES for 24 h and subsequently labeled with the free radical-sensitive probe C11-BODIPY581/591 for monitoring membrane oxidation after neutrophil activation with phorbol-12-myristate-13-acetate (PMA). First-order exponential decay kinetic indicated that both 0.1 and 10 μM TES severely increased baseline membrane oxidation in non-activated human neutrophils (compared to control). However, similar kinetics of membrane oxidation were observed in control and 0.1 μM TES-treated neutrophils after PMA activation, whereas chemical activation did not alter the baseline higher rates of membrane oxidation in 10 μM TES-treated neutrophils. The data presented here support the hypothesis that TES exerts distinct effects on the membrane oxidation of human neutrophils, depending on its dose (here, 102 to 104-fold higher than physiological levels in men) and on PMA activation of the oxidative burst. Furthermore, this paper also presents an innovative application of the free radical-sensitive probe C11-BODIPY581/591 for monitoring (auto-induced) membrane oxidation as an important parameter of viability and, thus, responsiveness of immune cells in inflammatory processes.
Reactive oxygen/nitrogen species
Thiobarbituric acid reactive substances
Vascular smooth muscle cells
Anabolic-androgenic steroids (AAS) have been long used in the treatment of chronic debilitating illnesses, trauma, burns, surgery, and radiation therapy (Shehzad et al. 2001). The desire for improved body aesthetics, physical function, and sport performance has also fueled anabolic drug abuse over the past 50 years (Hoffman et al. 2009). Since aging is regularly associated with a gradual decline in circulating testosterone concentrations and decreased musculature in men, AAS replacement therapies have been currently suggested to circumvent the age-related morbidity and cognitive impairment in men (Dillon et al. 2010). Accordingly, supra-physiologic (50 nM = 50-fold normal) dosing of testosterone for eight continuous weeks has been shown protective to muscle mass and fatigue resistance in orchiectomized mice compared to sham operated animals (Axell et al. 2006). The protective action of testosterone on skeletal muscle and cognitive capacities may in part be due to its anti-inflammatory actions or through direct modulation of anti-catabolic pathways (Thompson et al. 2006; Liva and Voskuhl 2001; Malkin et al. 2004). On the other hand, concerns about the potential adverse effects of higher doses of testosterone have encouraged the development of strategies to define a possible “pharmacological window” for beneficial AAS effects in men.
Regarding inflammatory responses, increased levels of pro-inflammatory cytokines (e.g., IL-8, TNFα) stimulates the migration of circulating neutrophils to the inflammatory site, followed by activation of the membrane enzyme NADPH oxidase and massive production of reactive oxygen/nitrogen species (ROS/RNS) (Dong et al. 2011; Krüeger et al. 2009; Zembron-Lacny et al. 2010). Although the oxidative burst of neutrophils represents a key-event in eliciting efficient immune responses, the massive production of ROS/RNS can also negatively: (a) affect the structural integrity of the neutrophil membrane; (b) limit the microbicidal and phagocytic activities of neutrophils due to oxidative modification of membrane proteins; (c) induce apoptosis prematurely leading to cell degradation; (d) shorten the half-life of active neutrophils in inflammatory sites; and, as a general effect; (e) compromise general immune responsiveness (Ottonello et al. 2011). Lipid/membrane oxidation is notoriously recognized as a harmful event related to cell dysfunction and, thus, possibly related here to loss of neutrophil viability during inflammatory processes. Thus, by strategically pre-attaching the free radical-sensitive fluorescent probe C11-BODIPY581/591 (Drummen et al. 2002) to neutrophil membranes, we aim here to investigate the effects of supra-physiological doses (0.1 and 10 μM) of testosterone on the membrane oxidation kinetics of activated human neutrophils in vitro, as an important marker of immune efficiency. Furthermore, this paper also presents an innovative application of C11-BODIPY581/591 for monitoring the progression of the auto-induced membrane oxidation in immune cells. Proper methodological controls were performed with the powerful lipid-soluble antioxidant astaxanthin (Barros et al. 2001; Naguib 2000) in order to check the susceptibility of neutrophil membranes to ROS/RNS—especially peroxyl and alkoxyl radicals, main propagating agents of lipid oxidation—and the effects of cell density to the fluorescence signal.
Materials and methods
Highly purified chemicals for usual solutions, including specific buffers, were purchased from Labsynth (Diadema, SP, Brazil). Other chemicals like Histopaque-1077, dimethyl sulfoxide (DMSO), Triton X-100, astaxanthin, and phorbol-12-myristate 13-acetate (PMA) were obtained from Sigma-Aldrich Chemical Co (St Louis, MO, USA). The fluorescent probe 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY581/591) was purchased from Molecular Probes (Ontario, Canada).
Nine healthy, untrained young men (undergraduate students from University Metropolitan of Santos, Brazil) volunteered to participate in this research. Subject (mean ± SD) age, height, and body mass were (23.4 ± 2.5) years (174.4 ± 7.0) cm, and (78.3 ± 7.0) kg, respectively. Blood collecting protocol and all other experimental procedures were approved by the Ethics Committee in Research of University Cruzeiro do Sul (Ethical Protocol CEP-002-2011), which conforms to the Standards for Research Using Human Subjects, Resolution 196/96 of the USA National Health Council of 10/10/1996.
Human neutrophils in culture
Blood samples (10 mL) were drawn from the forearm cubital vein of volunteers using EDTA-containing Vacutainer® kits. Heparinized tubes were avoided, since eventual iron contamination could accelerate oxidative degradation of samples (Freitas et al. 2008). Blood samples were diluted in the same volume of phosphate buffered saline solution (0.137 M NaCl, 2.7 mM KCl, 8.0 mM Na2HPO4, pH 7.4) and neutrophils were separated using Histopaque 1077 according to the manufacturer’s instructions (Sigma-Aldrich, MO, USA). The plasma and intermediary layer were removed and both neutrophils and erythrocytes were collected from sediment. Erythrocytes were lysed with a hemolysis solution (150 mM NH4Cl, 10 mM NaHCO3, 0.1 mM EDTA, pH 7.4) and immediately centrifuged for 10 min (600g at 4 °C). This procedure was repeated twice for full red blood cell lyse. Thereafter, neutrophils were washed with PBS followed by centrifugation for 10 min (600g at 4 °C). After centrifugation, isolated neutrophils were maintained in RPMI-1640 medium supplemented with 2 mM glutamine, 20 mM HEPES, 10 % fetal calf serum, 10 U/mL penicillin, and 10 mg/mL streptomycin. The number of viable cells (>95 %) was determined in a Neubauer chamber under an optical microscope by Trypan blue exclusion (Marin et al. 2010). Alternatively, a second set of human neutrophils were isolated from blood samples by including a pre-cleaning treatment with Dextran 500. Blood samples (5 mL) were mixed with 6.7 mL of 6 % non-pyrogenic Dextran 500 (w/v) in sterile 0.9 % NaCl solution and the upper cell layer was transferred to a 50-mL polypropylene tube for a brief centrifugation (740g for 10 min, room temperature). Afterwards, supernatant was discarded and the total cell pellet was resuspended in sterile 0.9 % NaCl solution. Next, the diluted white blood cell suspension was carefully applied on top of 7 mL of Histopaque 1077 (in a 50-mL polypropylene tube), as previously described. All materials used for blood collection (syringes, needles, bottles) were disposable and handled by health/medical professionals of the ICAFE/Universidade Cruzeiro do Sul to prevent any potential physical complication.
Neutrophils (2 × 106 cell/mL), in a RPMI-1640 medium, were treated with 0.1 and 10 μM testosterone (1.0 mg/mL in 1,2-dimethoxyethane, T5411-1ML, Sigma-Aldrich), and incubated in a humidified atmosphere of 5 % CO2 at 37 °C for 24 h, in accordance with preliminary studies (Marin et al. 2010). Control cultures (treated with same volumes of the drug vehicle, 1,2-dimethoxyethane or DMSO) were simultaneously incubated under the same experimental conditions. Neither 1,2-dimethoxyethane nor DMSO (vehicles for TES administration) showed significant toxic effects to isolated neutrophils at concentrations applied here (lower than 2 % of total medium volumes).
Control assays with astaxanthin (AST)
In order to examine the susceptibility of neutrophil membranes to (auto-induced) oxidation, human neutrophils in culture were pre-loaded with 5 or 10 μM astaxanthin (AST), a powerful antioxidant carotenoid recognized by its highly specific scavenging activity against the main propagating agents of lipid oxidation processes: peroxyl and alkoxyl radicals (ROO· and RO·, respectively). Thus, it is expected that AST could intercept both ROO· and RO· radicals and prevent neutrophil membrane from oxidation in active neutrophils. Noteworthy, AST does not affect sensitivity or the electronic configuration/properties (thus, fluorescence) when concomitantly incorporated with C11-BODIPY581/591 in biological membranes (Naguib 2000). The effects of cell density (5 × 105, 1 × 106, and 2 × 106 cell/well) were also checked in control experiments.
C11-BODIPY581/591 loading in membranes
Cultured neutrophils were concentrated by centrifugation (740g for 10 min at room temperature), resuspended in PBS (0.137 M NaCl, 2.7 mM KCl, 8.0 mM Na2HPO4, pH 7.4), and seeded in 96-well fluorescence microplates (1 × 105/well). Subsequently, neutrophils were incubated with 3.2 μM C11-BODIPY581/591 (in DMSO), in the dark, at 37 °C and smoothly shaken for 1 h for adequate labeling, modifying the original protocol described by Makrigiorgos (1997). The probe C11-BODIPY581/591 is very sensitive to oxidation initiated by ROO· and RO· radicals (Drummen et al. 2002). The fluorescent probe C11-BODIPY581/591: (a) anchors with high stability to biological membranes by strong hydrophobic interaction of its undecanoic acid moiety with lipid bilayers; (b) shows good spectral separation of the non-oxidized (595 nm) and oxidized (520 nm) forms; (c) has a good photo-stability and displays very few fluorescence artifacts; (d) is virtually insensitive to microenvironmental changes, such as pH or solvent polarity; (e) once oxidized, C11-BODIPY581/591 remains lipophilic and does not spontaneously leave the lipid bilayer; (f) is not cytotoxic to most model mammal cells; and (g) is comparably sensitive to oxidation of common unsaturated fatty acids of biological membranes (Papa et al. 1999).
where y is relative fluorescence intensity, y0 is relative fluorescence intensity at time zero (equals 1.0), A1 is attenuation factor, k is rate constant and t is time (min).
Statistical analysis on C11-BODIPY581/591 kinetics was performed by paired student t test of all 56 data points, followed by the Tukey’s post-test. Origin Pro8 8SR0 software was used for statistical analyses and graph preparation (v8.0725/B725; OriginLab Corporation, Northampton, MA, USA).
First-order decay parameters of C11-BODIPY581/591 fluorescence in resting and chemically activated (100 ng PMA/105 cells) human neutrophils pre-treated in vitro with testosterone (TES) 0.1 or 10 μM for 24 h
0.910 ± 0.275
0.090 ± 0.027
18.45 ± 0.26
Control + PMA*,a,g,h
0.911 ± 0.301
0.058 ± 0.003
9.90 ± 2.63
TES 0.1 μMb,d,e
0.935 ± 0.005
0.065 ± 0.005
2.49 ± 0.37
TES 0.1 μM + PMAd,g,i
0.910 ± 0.046
0.087 ± 0.045
6.82 ± 0.46
TES 10 μMc,e,f
0.911 ± 0.019
0.094 ± 0.018
4.31 ± 1.26
TES 10 μM + PMAf,h,i
0.964 ± 0.019
0.030 ± 0.019
4.45 ± 0.41
As expected, PMA addition to control neutrophils significantly activated the oxidative burst, as shown by comparing open circles in Figs. 2 and 3, and based on the calculated k values in Table 1 (k was 1.86-fold lower in the presence of PMA than in control neutrophils). On the other hand, PMA activation surprisingly diminished the degree of membrane oxidation in 0.1 μM TES-treated neutrophils compared to non-activated cells (k was 2.74-fold higher in PMA-activated neutrophils than in basal conditions), to reach identical scores as control neutrophils (Table 1). Contrarily, PMA addition did not alter the high rate of oxidation (k) in membranes of human neutrophils pre-treated with 10 μM TES for 24 h.
Independent to which protocol for neutrophil isolation was used (including pre-treatment with 6 % Dextran or not), noisy signals of fluorescence were detected during the first 5–10 min after PMA activation of the oxidative burst (Figs. 1, 2, 3). This effect was observed either when neutrophils were pre-loaded with the powerful antioxidant AST or by TES addition, which suggests that structural changes in cell membrane following PMA activation are the most probable molecular event involved. It is also plausible that the observed structural changes were caused by the organic solvent DMSO, which solubilizes the activation factor PMA. Nevertheless, after the stabilization period, the fluorescence signal of C11-BODIPY581/591 was only stable and apparently responsive to free radicals when neutrophil density was kept <1 × 106 cell/well (Fig. 1a–d). Undesirable iron contamination in samples (probably from hemolysis) should also be kept in mind when evaluating quantitative parameters of membrane oxidation of neutrophils and other immune cells (Haag-Weber et al. 1989).
Unexpectedly, Fig. 1b, c (experiments performed neutrophil densities >1 × 106 cell/well) revealed that C11-BODIPY581/591 fluorescence did not adequately respond to AST incorporation in neutrophil membranes. These results clearly demonstrate that the free radical-sensitive probe is not stoichiometrically exposed to ROS/RNS produced by active neutrophils. Physicochemical changes in neutrophil membranes due to cell-cell interactions (especially here, in high cell density cultures) might explain the unexpected pattern of fluorescence kinetics observed in Fig. 1b, c. Fluorescent probes, including C11-BODIPY581/591, normally change their excitation and emission spectra when in aggregate forms or upon strong cell-cell interactions, changes in membrane fluidity, loss of lipid raft integrity, and/or mechanical stress (de la Haba et al. 2012; Verstraeten et al. 2010). Nevertheless, by limiting cell-cell interactions in 5 × 105 cell/well cultures, C11-BODIPY581/591 (at a final concentration of 3.2 μM) was properly challenged by ROS/RNS generated by activated neutrophils, and the dose-dependent effect of AST (a powerful antioxidant) was adequately evidenced (Fig. 1d). Based on these results, further studies—as those with TES—were performed with 1 × 105 neutrophil/well cultures, which accordingly provided more stable fluorescent signals (Figs. 2, 3). As shown for other cell types, the novel approach presented here (the real-time monitoring of membrane oxidation in human neutrophils by C11-BODIPY581/591) seems to adequately reproduce the oxidative challenges faced by activated neutrophils, since PMA addition accelerated membrane oxidation (and permeability, Shasby et al. 1983) in control neutrophils due to ROS/RNS production. However, other chemical stimuli, e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF), lipopolysaccharide (LPS) or LPS in combination with interferon-gamma (IFN-γ), can also activate the oxidative burst in neutrophils through different signaling pathways (Atzeni et al. 2002). Therefore, further studies evaluating specific receptor-mediated activation of neutrophils are required, since PMA is a non-specific polymorphonuclear cell activator (Paik et al. 2005).
Effects of TES on membrane oxidation
Testosterone treatment in vitro increased baseline membrane oxidation of non-activated neutrophils in a dose-independent matter, as shown in Fig. 1 and (k values) in Table 1. However, we cannot confirm if this effect was caused by baseline overproduction of ROS/RNS in resting neutrophils and/or by TES-mediated increase of antioxidant defenses. Marin et al. (2010) elegantly reported that pre-incubation with 10 nM, 0.1 and 10 μM TES clearly inhibited superoxide radical (O2·−) production in of human neutrophils after PMA activation (albeit, at a fivefold lower concentration). Under these circumstances, the authors reported lower indexes of lipid oxidation (using the thiobarbituric-acid reactive substances assay; TBARS) in 10 nM and 0.1 μM TES-treated cells, but not after 10 μM TES incubation. Although the TBARS assay is less sensitive than the C11-BODIPY581/591 method applied here (Aldini et al. 2001), these data sustain the hypothesis that 0.1 μM TES mediates suppressive effects on ROS production in PMA-activated neutrophils. Accordingly, Juliet et al. (2004) have already reported the suppressive effects of 0.01 μM TES treatment (tenfold lower concentration than in our study) on NADPH oxidase assemblage (both p67phox and p47phox expression). Regarding cellular mechanisms, Chignalia et al. (2012) demonstrated that 0.1 μM TES induces vascular smooth muscle cells (VSMCs) migration via NADPH oxidase-derived ROS and c-Src (a regulator of redox-sensitive migration) dependent pathways in hypertensive animals. The observed effect was promoted by both genomic and non-genomic mechanisms: TES increased Nox1 and Nox4 mRNA levels and p47phox protein expression, which are differentially regulated in VSMCs from Wistar-Kyoto rats and hypertensive rats (Chignalia et al. 2012). Thus, depending on the supra-physiological doses of TES, specific mechanisms are apparently activated to influence redox metabolism in neutrophils. Strong evidence points towards the assembling process and/or migration of NADPH oxidase sub-particles to neutrophil membrane (Juliet et al. 2004; Chignalia et al. 2012).
Based on a well-accepted hormesis principle, too low ROS/RNS production would limit crucial redox-dependent processes such as cell signaling, H2O2-mediated proliferation, and oxidative burst responses in polymorphonuclear cells, whereas excessive ROS/RNS concentrations would cause undesirable oxidative modifications to important cellular biomolecules (Valko et al. 2007). Other authors have also reported dose-dependent ambiguous effects of TES in immune cells, at the same micromolar range (Greabu et al. 2003; Juliet et al. 2004). Although TES concentrations reported here represent 102 to 104-fold higher doses than those physiologically found in average men (Shehzad et al. 2001), our study demonstrates that supra-physiological doses of TES can distinctively affect the progression of membrane oxidation in activated neutrophils, a molecular event that is truthfully related to cell viability, neutrophil turnover scores, and immune responsiveness.
Altogether, our results show that supra-physiological doses of TES impose higher oxidative conditions on circulating (non-activated) neutrophils. This may enhance the general antioxidant capacity of circulating neutrophils as a response to the TES-induced baseline oxidation of membranes. On the other hand, 0.1 μM TES treatment appropriately restored membrane oxidation rates back to those found in PMA-activated control neutrophils, whereas the pre-treatment with 10 μM TES (100-fold higher concentration) sustained the high susceptibility of neutrophils to auto-induced membrane oxidation. Thus, the tricky aspect of obtaining maximal benefits from medical TES treatments (e.g., hormone reposition, post-surgery recovery, etc.) is to apply an optimized TES concentration—matching its “pharmacological window”—that appropriately balances its required anabolic and/or androgenic effects with related immunosuppressive effects due to increased membrane oxidation in activated neutrophils. In a much worse scenario involving circulating neutrophils, it is worthwhile mentioning that TES benefits are normally obtained at nano/micro molar concentrations in plasma, which, thus, require sensitive methods to investigate such relevant physiological processes. The technique presented here represents a useful tool to investigate that hypothesis.
The authors are indebted to Dr. Rosemari Otton, and M.Sc. Douglas Marin Popp for providing neutrophil samples and resources during preliminary studies at Laboratório de Fisiologia Celular, campus Analia Franco, Universidade Cruzeiro do Sul, São Paulo, Brazil, and the assistance of Dr. Douglas Ganini for neutrophil isolation in further experiments. Dr. Marcelo Paes de Barros is also indebted to Prof. Rui Curi, at Department of Biophysics and Physiology, University of São Paulo, Brazil, for chemicals and laboratory facilities during neutrophil isolation. This research is supported by Fundação de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP 2009/12342-8), Programa de Suporte à Pós-graduação de Instituições de Ensino Particulares, da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (PROSUP/CAPES, Brazil), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (Bolsas Produtividade em Pesquisa, Nivel 2, #312404/2009-3, CNPq, Brazil).
Conflict of interest
All authors of the present manuscript declare that there is no any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations that could inappropriately influence, or be perceived to influence our work.