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(Journal of Leukocyte Biology. 2002;71:255-261.)
© 2002 by Society for Leukocyte Biology

Activation of phagocytic cell NADPH oxidase by norfloxacin: a potential mechanism to explain its bactericidal action

Rajaa El Bekay*, Moisés Álvarez*, Modesto Carballo*, José Martín-Nieto{dagger}, Javier Monteseirín{ddagger}, Elizabeth Pintado*, Francisco J. Bedoya* and Francisco Sobrino*,{ddagger}

* Departamento de Bioquímica Médica y Biología Molecular, and
{dagger} Servicio de Inmunología y Alergia, Hospital Universitario Virgen Macarena, Universidad de Sevilla, Spain; and
{ddagger} Division de Genética, Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, Spain

Correspondence: Francisco Sobrino, Departamento de Bioquímica Médica y Biología Molecular, Facultad de Medicina, Av. Sánchez Pizjuán 4, E-41009 Sevilla, Spain. E-mail: fsobrino{at}cica.es


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ABSTRACT
 
The mechanisms underlying the bactericidal power of fluoroquinolones against intracellular parasites in host macrophages remain poorly understood. We have analyzed the effect of norfloxacin, a fluoroquinolone antibiotic, on the production of reactive oxygen intermediates (O2•- and H2O2) and NADPH oxidase activity in mouse macrophages. The generation of anion superoxide (O2•-) was found to be significantly greater in macrophages incubated with norfloxacin than in untreated controls. This enhancing effect of norfloxacin was dose-dependent and reached maximal values within 10 min after its addition. The O2•- generated was mainly intracellular, as determined by the use of specific dyes, such as lucigenin and luminol, and able to diffuse freely through the cell membrane. Also, the production of H2O2 was increased in macrophages in response to norfloxacin. The positive effect of norfloxacin was associated to an enhanced mobilization of NADPH oxidase subunits p47phox and p67phox from the cytosol to the plasma membrane in phagocytic cells. The effect of the antibiotic persisted in vivo for several hours. These data support the notion that norfloxacin inhibits mycobacterial growth within phagocytic cells by enhancing intracellular production of O2•- and other reactive oxygen species.

Key Words: macrophages • reactive oxygen species • O2 • - • H2O2


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INTRODUCTION
 
New therapeutic strategies have been assayed against bacteria that grow intracellulary, such as Mycobacteria and those of the genera Salmonella, Listeria, and Legionella [1 2 3 ]. However, their effective killing results were hampered by the difficulty in achieving sufficiently high concentrations of antibiotics within animal cells [4 5 6 ]. This is attributable to a reduced transport of antibiotic into the cells and in some cases, to antibiotic efflux carried out by specific membrane transporters [7 ]. It has been shown that the fluoroquinolone antibiotic norfloxacin (NFX) is a substrate for organic-anion transporters in J774 macrophages [8 ] and that gemfibrozil, an inhibitor of anion transport [9 ], enhances the intracellular accumulation of NFX [6 ]. Although the microbiological data raised by Rudin et al. [6 ] clearly demonstrate that NFX attenuates the cytotoxic effect of Lysteria monocytogenes, the molecular mechanism accounting for the bactericidal effect of NFX has not been addressed.

It is known that the pathogens referred to above are able to grow intracellulary within macrophages [1 2 3 ] and that these cells exhibit a potent reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity responsible for the synthesis of superoxide anion (O2•-) as a bactericidal molecule [10 , 11 ], in addition to other reactive species derived from reactive oxygen species (ROS) or nitrogen [12 ]. Recently, data have been described suggesting that pathogens growing intracellularly can depress the respiratory burst as well as other macrophage functions [13 14 15 ], in contrast with previous studies showing opposite effects [16 , 17 ].

We have chosen peritoneal macrophages with the aim of testing the working hypothesis that NFX treatment could restore the synthesis of O2•- through the activation of NADPH oxidase. Conflicting data have been described as well on the response to fluoroquinolone treatment by phagocytic cells. Whereas some authors found no change in the ROS-dependent chemiluminescence promoted by fluoroquinolones on polymorphonuclear cells [18 , 19 ], these antibiotics have been shown to exhibit photosensitizing properties when administered in vivo regarding the generation of ROS [20 ]. In the present work, an analysis of the type of released ROS and their enzymatic source were addressed. The NFX-stimulated ROS production process was analyzed by four methods, each with a different specificity for the type and intra- or extracellular location of the detected ROS. We show that NFX clearly promotes in macrophages and neutrophils an increase of the intracellular production of O2•- and hydrogen peroxide (H2O2), which involve the mobilization of NADPH oxidase cytosolic subunits p47phox and p67phox to the plasma membrane and subsequent enzyme activation [21 22 23 ]. This NFX effect was also observed upon in vivo treatment of mice with the antibiotic and was persistent during several hours as evidenced by in vitro assays of NADPH oxidase activity in particulate and cytosolic macrophage fractions.


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MATERIALS AND METHODS
 
Chemicals and reagents
Dextran T-500 was obtained from Amersham Pharmacia Biotech (Barcelona, Spain). Lymphocyte separation medium and Hanks’ balanced salt solution (HBSS) were obtained from Bio-Whittaker (Verviers, Belgium). Norfloxacin, phorbol 12-myristate 13-acetate (PMA), dibutiryl-cyclic AMP, gemfibrozil, cytochrome c, horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG), lucigenin, HRP, and diphenyleneiodonium (DPI) were obtained from Sigma Chemical Co. (Madrid, Spain). 2,7-Dichlorohydrofluorescein diacetate (DCFDA) was purchased from Molecular Probes (Junction City, OR). Rabbit polyclonal antibodies raised against p47phox and p67phox were kindly donated by professor O. T. G. Jones (Department of Biochemistry, University of Bristol, U.K.). Luminol (5-amino-2,3-dihydrophthalazine-1,4-dione) was purchased from Serva Feinbiochemica GmbH (Madrid, Spain), and 4-iodophenol was from Aldrich Chemical Co. (Madrid, Spain). Blotting nitrocellulose membranes were purchased from Bio-Rad (Hercules, CA), and NADPH was from Boehringer Mannheim GmbH (Mannheim, Germany).

Preparation of peritoneal macrophages and human neutrophils
Peritoneal macrophages were isolated from mice kept under standard laboratory diet with free access to food and water. The animals (20–25 g) were killed by decapitation, and macrophages were obtained by peritoneal lavage as previously described [24 ]. The cells were pelleted by centrifugation, suspended in Hepes-buffered Krebs-Ringer (KR-Hepes), composed of 118 mM NaCl, 4.75 mM KCl, 1.18 mM H2PO4, 1.18 mM MgSO4, 1.25 mM CaCl2, 10 mM glucose, and 25 mM Hepes, pH 7.4, and immediately used for experiments. For in vivo experiments, macrophages were obtained from mice treated with NFX (20 mg/kg weight, dissolved in 0.9% NaCl plus 5 mM NaOH), injected intraperitoneally (i.p.) daily during 4 days. Untreated control mice were injected with the solvent alone and analyzed in parallel.

Human peripheral neutrophils were obtained from healthy blood donors, following informed consent. Neutrophils were isolated from fresh heparinized blood as indicated [25 ] and further purified by dextran sedimentation, followed by Ficoll-Paque gradient centrifugation and hypotonic lysis of residual erythrocytes. Neutrophils were washed twice in HBSS and then suspended at a density of 107 cells/ml in KR-Hepes buffer.

O2•- production
O2•- release was measured using three different methods: For the (i) lucigenin- and (ii) luminol-luminescence methods, macrophages were suspended at a density of 106 cells/ml in KR-Hepes buffer and preincubated at 37°C for 5 min. Then 15 µM lucigenin or 15 µM luminol was added, and the assay was carried out as indicated previously [26 ], except that HRP (8 mU/ml) was included when luminol was used. PMA or NFX was added to the cellular suspension, and chemiluminescence emission was recorded at different times using a 1250 BioOrbit luminometer. (iii) When the cytochrome c reduction method [27 ] was used, macrophages (106 cells/ml) were preincubated for 5–10 min with NFX in KR-Hepes buffer, then cytochrome c (80 µM) was added, and the absorbance at 550 nm was recorded using a 8452A Diode Array Hewlett Packard spectrophotometer [24 ].

Intracellular levels of H2O2
DCFDA was used as an indicator of the quantity of intracellular H2O2 [28 ]. Briefly, 2 x 106 macrophages or neutrophils were incubated at 37°C for 1 h in the dark in the presence of 2.5 µM DCFDA (dissolved in ethanol). The cells were then rinsed twice with KR-Hepes buffer, and the fluorescence intensity was measured at different times in an LS-5 Perkin-Elmer spectrofluorometer, using excitation and emission wavelengths of 503 nm and 529 nm, respectively.

NADPH oxidase activity in a cell-free system
Mice were killed by decapitation, and cytosolic and membrane fractions were prepared as described above for the analysis of p47phox and p67phox mobilization. NADPH oxidase activity was quantified on the basis of O2•- production by measuring the superoxide dismutase-inhibitable reduction of cytochrome c at 37°C [27 ]. Enzyme activity was assayed in both fractions using 80 µM cytochrome c, and the reaction was started by addition of 1 mM NADPH. The absorbance changes at 550 nm were recorded, and the O2•- released was calculated using an extinction coefficient of 21.1 mM-1 • cm-1.

p47phox and p67phox Mobilization
Human neutrophils (107 cells/ml) were lysed on ice for 30 min in a 60 µl buffer containing 100 mM Hepes, pH 7.3, 100 mM KCl, 3 mM NaCl, 3 mM MgCl2, 1.25 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA), and the protease inhibitors phenylmethylsulfonyl fluoride (PMSF; 1 mM), leupeptin, aprotinin (10 µg/ml each), and benzamidine (150 µg/ml). The cells were disrupted by sonication on ice (20W, three bursts of 5 s each separated by 30-s intervals). Unbroken cells and debris were removed by centrifugation at 10,000 g for 5 min at 4°C. The supernatant obtained after further ultracentrifugation at 100,000 g for 5 min at 4°C constituted the cytosolic fraction. The pellet obtained was resuspended in a buffer containing 120 mM NaH2PO4, pH 7.4, 1 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 20% (v/v) glycerol, 40 mM octylglucoside, and the protease inhibitors indicated above and then recentrifuged at 20,000 g for 40 min at 4°C. The supernatant obtained, containing solubilized membranes, was used for the analysis of p47phox and p67phox mobilization [29 , 30 ]. With this purpose, the cytosolic and solubilized-membrane fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5% (w/v) polyacrylamide gels and electroblotted onto nitrocellulose membranes using a semidry device (Bio-Rad). The membranes were blocked for 1 h in TBS (50 mM Tris/HCl, pH 7.5, 150 mM NaCl) containing 3% (w/v) bovine serum albumin (BSA), then rinsed twice with TBS containing 0.1% (v/v) Tween 20 (TBST), and probed overnight with rabbit anti-p67phox IgG or anti-p47phox diluted 1:1000 in TBST. After extensive washing with TBST, the filters were incubated for 60 min with HRP-conjugated goat anti-rabbit IgG (1:5000 dilution), and bound secondary antibody was detected by an enhanced chemiluminescence assay [25 ]. Briefly, the membranes were incubated in 1 ml fresh luminescence-reagent solution, composed of 10 mM Tris/HCl pH 8.5, 2.25 mM luminol, 0.015% (v/v) H2O2, and 0.45 mM 4-iodophenol. Luminol (predissolved in 50 µl 1 M NaOH) and 4-iodophenol were freshly prepared in 10 ml Tris/HCl, pH 8.5. After 1 min of incubation, the membranes were placed on filter paper, covered with Saran Wrap, and exposed to X-ray films in the dark for 1–5 min. The protein concentration in the lysate was determined by the Bradford procedure [31 ] using BSA as standard.


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RESULTS
 
Mouse peritoneal macrophages and blood human neutrophils were used as biological systems to study the potential ability of NFX to promote the production by these cells of ROS. The levels of O2•-, H2O2, and other ROS were analyzed by four separate methods, each with a different specificity toward the location and type of ROS produced: (i) The lucigenin-based luminescence method was specific for O2•- [32 ], whereas (ii) luminol-based luminescence correlated well with the total ROS produced by the cells [33 , 34 ]. Because luminol and lucigenin permeate freely through the cell membrane, their luminescence was an indication of intra- and extracellular ROS [33 , 34 ]. Conversely, (iii) the assay based on cytochrome c reduction [28 ] was highly specific for O2•- released to the extracellular medium, because the cytochrome was not able to access the cell interior [35 ]. Finally, (iv) the DCFDA method was used as an indicator of intracellular or intra- plus extracellular H2O2 levels [28 ], depending on whether the dye was removed from the medium, respectively.

As shown in Figure 1 A , when macrophages were incubated with NFX at the indicated concentrations (10–100 µM), and luminol-based chemiluminescence was recorded, a dose-dependent increase of ROS production was obtained, which was maximal at a 100 µM dose of NFX. To analyze whether O2•- was present among the ROS produced in response to NFX, lucigenin-based luminescence was also recorded (Fig. 1B) . A clear stimulatory effect was also found under similar experimental conditions, with a maximal O2•- production being attained at 50 µM NFX. The effect of 100 nM PMA, a well-known activator of NADPH oxidase [36 ], was also analyzed to ascertain the functional responsiveness of macrophages. The amount of O2•- produced in response to 50 µM NFX was half the level elicited by PMA under the same conditions (Fig. 1B) , thus indicating a relatively high activation of NADPH oxidase from macrophages by NFX. The somewhat different dose effect observed when comparing lucigenin- with luminol-based luminescence suggests that several types of ROS in addition to O2•- were released by macrophages in response to NFX. In separate experiments, it was verified that NFX by itself did not elicit lucigenin- or luminol-based chemiluminescence production in a cell-free system (unpublished results).



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  		Figure 1. Kinetics and dose-effect of NFX on O2•- and ROS production by mouse macrophages. Macrophages (106 cells/ml) were suspended in KR-Hepes buffer and preincubated at 37°C for 5 min. NFX was then added (arrow) at the following doses: 10 µM (•), 25 µM ({blacksquare}), 50 µM ({blacktriangleup}), and 100 µM ({blacklozenge}). Also, the effect of 200 nM PMA ({circ}) was analyzed. Luminol (A) or lucigenin (B), both at a 15 µM concentration, was added prior to NFX or PMA, and the chemiluminescence response was recorded for the times shown. Control experiments without additions were carried out in parallel, giving negligible levels of luminescence (not shown). Plotted values are the mean ± SE from three separate experiments in which each measurement was performed in triplicate.

Previously, it has been shown that gemfibrozil enhances the intracellular accumulation of NFX and hence improves the efficiency of fluoroquinolones against bacterial pathogens [6 ]. We found that the production of O2•- was not affected upon addition of 50 µM gemfibrozil to the lucigenin-based assay (unpublished results). Hence, it may be concluded that the described cooperative effect between gemfibrozil and NFX possibly results from the capacity of the latter to act as an inhibitor of anion transport through the membrane [9 ] rather than as an enhancer of NFX-dependent O2•- production. Extracellular O2•- production by macrophages was measured using the cytochrome c method. NFX by itself was unable to elicit any O2•- extracellular production (unpublished results). However, as shown in Figure 2 , the addition of NFX, 15 min prior to phorbol ester treatment, greatly potentiated the PMA-dependent O2•- production. This effect suggested that NFX and PMA could act on some common regulatory step, such as activation of protein kinase C. In this context, it was found that NFX, when added directly on a cell-free system, did not affect cytochrome c reduction (unpublished results).



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  		Figure 2. NFX enhances PMA-dependent extracellular O2•- production by macrophages. Macrophages were suspended in KR-Hepes buffer at 106 cells/ml and preincubated without or with 25 µM NFX at 37°C for 15 min, followed by the addition of 80 µM cytochrome c and 200 nM-PMA (arrow). The change of cytochrome c absorbance at 550 nm was recorded for the indicated times. The data illustrated are representative of four separate experiments giving similar results.

Collectively, present data can be interpreted as the result of NADPH oxidase activity of macrophages being sensitive to NFX-promoted activation. To verify that NFX administered in vivo exerted the same effect on intracellular O2•- production observed in vitro, mice were i.p. injected daily with 20 mg NFX during 4 consecutive days, and intracellular O2•- production was measured using the lucigenin method in macrophages isolated from NFX-treated and untreated control mice. Figure 3 shows that macrophages obtained from NFX-treated mice produced ninefold more O2•- than those extracted from nontreated mice in the absence of any other stimulus. A clearly potentiating effect of NFX was also detected when macrophages from NFX-treated and untreated mice were incubated with 100 nM PMA. DPI, an inhibitor of NADPH oxidase activity [37 ], was used to analyze whether the enhanced O2•- production could be ascribed to this enzyme. When macrophages from NFX-treated and untreated mice were preincubated with DPI, a clear inhibition of PMA-dependent O2•- production was observed in both cases (Fig. 3) . This indicated indirectly that NFX administered in vivo elicited a stimulation of NADPH oxidase activity in mouse macrophages. In a different set of experiments, staurosporin, a known inhibitor of protein kinases, was added at the same time as NFX. It was found that a dose of 500 nM staurosporin canceled NFX-dependent O2•- production completely when added in vitro (unpublished results). Dibutiryl cyclic AMP (at 1.5 mM) added simultaneously to NFX also reduced its stimulating effect on O2•- production by macrophages (unpublished results). To measure intracellular H2O2 levels in macrophages under culture conditions, the oxidation of DCFDA was monitored by fluorescence measurements carried out for 70 min in macrophages that had been preincubated with this compound and then exposed to NFX. Table 1 illustrates that NFX induced an increase in H2O2 production by macrophages. A maximal effect was observed at 50 µM NFX, which declined at higher doses (unpublished results). It was verified that NFX did not exert any direct effect on the hydrolysis of DCFDA (unpublished results), this ruling out an artifactual effect of NFX on DCFDA oxidation.



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  		Figure 3. Effect of NFX in vivo treatment on O2•- production by macrophages. Mice were i.p. injected for 4 days with NFX (20 mg/kg weight; solid bars) or with the solvent alone (open bars). Peritoneal macrophages (106 cells/ml) were suspended in KR-Hepes buffer and preincubated at 37°C for 5 min with or without 50 nM DPI. Thereafter, the cells were incubated with or without 100 nM PMA for 15 min. Chemiluminescence was measured in the presence of 15 µM lucigenin. Plotted values are the mean ± SE from three separate experiments in which each measurement was performed in triplicate.


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  		Table 1. Effect of Norfloxacin on H2O2 Production by Macrophages

To test whether other phagocytic cells could also be targets for NFX, the production of H2O2 in response to this compound was also evaluated in human neutrophils. Figure 4A and B, illustrates that results similar to those obtained for macrophages regarding H2O2 production were obtained upon incubation of neutrophils with NFX. Two different experimental approaches were used in this case. First, the cells were preincubated with DCFDA for 30 min and then without being washed, treated with different doses of NFX (Fig. 4A) . The total fluorescence (from the cells plus the medium) was recorded during 2 h, and NFX was found to increase intra- and extracellular H2O2 production also in human neutrophils. Second, the cells were washed to remove DCFDA from the medium and subsequently were treated with NFX (Fig. 4B) . Similarly, the presence of NFX induced an increase in the intracellular levels of H2O2. The comparison of Fig. 4A with B indicated that most of the H2O2 produced was released to the medium (ca. eightfold extracellular-to-intracellular ratio).



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  		Figure 4. Time course of DCFDA oxidation by human neutrophils stimulated by NFX. Neutrophils (2x106 cells/ml) were incubated with 2.5 µM DCFDA at 37°C for 1 h. Thereafter, the cells were left unwashed (A) or washed three times (B) prior to the addition of NFX at the indicated doses, and the fluorescence was recorded. (A) DCFDA oxidation produced by total (intracellular plus extracellular) H2O2; (B) intracellular oxidation of DCFDA. Plotted values are the mean ± SE from three separate experiments in which each measurement was performed in triplicate.

The translocation of subunits p47phox and p67phox from the cytosol to the plasma membrane is an essential step for the activation of NADPH oxidase [29 , 30 ]. Thus, experiments were carried out on neutrophils aimed at analyzing whether such translocation was involved in an NFX-dependent O2•- increase. With this purpose, cytosolic and membrane fractions from neutrophils, which had been treated with NFX, were subjected to immunoblotting analysis using antibodies against the p67phox and p47phox subunits of NADPH oxidase. Results shown in Figure 5 A illustrate that the translocation of p67phox from the cytosol to the plasma membrane was dependent on the dose of NFX, following a time course that paralleled its disappearance from the cytosol. In Figure 5B , the dependence on NFX of p47phox translocation to the membrane is illustrated. Similar experiments could not be carried out on macrophages because the available antibodies did not recognize macrophage p47phox or p67phox.



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  		Figure 5. Effect of norfloxacin on the translocation from the cytosol to the plasma membrane of p47phox and p67phox subunits of human neutrophil NADPH oxidase. Neutrophils (107 cells/ml) were incubated with 10, 25, or 50 µM NFX for 15 min at 37°C. Cytosolic and plasma-membrane fractions were separated electrophoretically and transferred to nitrocellulose as indicated in Materials and Methods. The nitrocellulose membrane was then probed with anti-p67phox (A) and anti-p47phox (B) antibodies, and the corresponding bands were detected by luminol-enhanced chemiluminescence. The figure shown is representative of three independent experiments.

To analyze whether the NFX action persisted after its removal from the cell milieu, a set of mice were i.p. injected with NFX for 4 days, and NADPH-oxidase activity was assayed in vitro in cytosolic and plasma membrane fractions isolated from peritoneal macrophages. We observed that the presence of both cellular fractions was necessary to achieve maximal NADPH-oxidase activity (Fig. 6 ), in agreement with previous studies [10 , 11 ]. Moreover, the mixture of cytosolic and membrane fractions from macrophages isolated from NFX-treated mice contained 2.5-fold more NADPH oxidase activity as compared with levels found in macrophages from untreated mice. This suggests that NFX given in vivo could stimulate the recruitment and translocation of some undefined component(s) of the NADPH oxidase complex to the membrane, by its phosphorylation or dephosphorylation or by lipid released by activation of phospholipases A2, C, or D. Further work is required to understand the molecular mechanisms that control this process.



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  		Figure 6. Cell-free NADPH oxidase activity in macrophage cytosol and plasma membrane. Cytosolic (Cyt) and plasma-membrane (Mem) fractions were obtained from macrophages isolated from mice treated with NFX (20 mg/kg) during 4 days (hatched bars) or treated with the solvent alone (0.9% NaCl plus 5 mM NaOH; open bars). NADPH oxidase activity was measured in both fractions (30 µg/ml protein in each assay), separated or as a mixture of both fractions. The reaction was started by the simultaneous addition of 1 mM NADPH and 80 µM cytochrome c, and the absorbance at 550 nm was recorded for 10 min. The activity values shown are the mean ± SD from three separate experiments.

To analyze whether the present observations were specific for NFX, experiments with ofloxacin, another fluoroquinolone antibiotic, were also performed. Ofloxacin increased O2•- and ROS production as well in mouse macrophages and human neutrophils in a time- and dose-dependent manner (unpublished results), suggesting that the effects described above for NFX could be a general property of this family of antibiotics.


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DISCUSSION
 
Although the bactericidal properties of norfloxacin are well established, its mechanism of action has long remained a controversial issue. Present data illustrate that NFX acts directly on macrophages and neutrophils to elicit an increased production of intracellular O2•- and other ROS, such as H2O2. This NFX-enhanced production of ROS fulfills the necessary criteria to implicate the classical NADPH oxidase from phagocytic cells as the causal factor, according to the following considerations: (i) The NFX-dependent O2•- and ROS production was monitored following a set of well-standardized methods, all of which gave concordant results. (ii) The activation of NADPH oxidase and O2•- production was accompanied by the translocation of its p47phox and p67phox subunits from the cytosol to the plasma membrane. (iii) The NFX-dependent O2•- production was canceled by DPI, a compound known to act as an inhibitor of NADPH- and other flavin-containing oxidases [37 ]. Similarly, ROS production was inhibited partially by staurosporin, an inhibitor of several protein kinase(s). Furthermore, the presence of dibutiryl cyclic AMP reduced by about 50% the NFX-dependent O2•- production. A similar inhibitory effect has been described previously to occur in PMA-stimulated neutrophils [38 ]. (iv) In a cell-free system, macrophages harvested from NFX-treated mice showed that O2•- production, measured on the basis of cytochrome c reduction, was specific for NADPH as the substrate. It is interesting that the effect of NFX given in vivo persisted through the time elapsed during preparation of the cytosol and plasma-membrane fractions, suggesting that the NFX-promoted changes in the protein composition of NADPH oxidase are rather stable. (v) The stimulating effect of NFX on ROS production required the presence of cellular components, thus discarding an artifactual action by NFX on some of the reagents used in the measurements. Similarly, it has been shown that fluoroquinolone antibiotics exhibit photosensitizing properties in relation to the generation of ROS by granulocytes cells [20 ]. However, other studies have failed in detecting changes in the levels of O2•- production upon incubation of granulocytes with different fluoroquinolones [19 ], possibly because of methodological differences, such as the prior stimulation of neutrophils with PMA. It is noteworthy that NFX, in contrast to other well-established stimulators of NADPH oxidase (such as PMA), triggers O2•- synthesis mainly inside the cells. This conclusion was reached with the use of different dyes that freely permeate through the plasma membrane (such as lucigenin and luminol) and that are unable to diffuse across (such as cytochrome c).

Early studies have shown that the functionality of macrophages becomes profoundly affected upon infection by pathogens [13 14 15 ]. In addition, an impairment of macrophage functions after their ingestion of Plasmodium falciparum-infected erythrocytes [39 ] or the decreasing activity of macrophages found upon repeated infections with this parasite [40 ] may indicate a damaged function in phagocytes. Also the zymosan-stimulated release of O2•- did not increase in peritoneal macrophages from Listeria-infected mice as compared with normal mice [41 ]. In another case, a correlation between an increased metabolic activity of macrophages from animals previously infected with bacillus Calmette-Guerin or L. monocytogenes and inhibition of bacterial growth has been described [42 ]. In this context, the enhancer effect of NFX on ROS production could help to restore the intracellular machinery of phagocytic cells responsible for killing the ingested microorganisms. In summary, the present study allows us to define a mechanism to interpret NFX action on macrophages, which links ROS production in the cytosol to its bactericidal power [43 ].


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ACKNOWLEDGEMENTS
 
This work was financed by Grants from Ministerio de Educación y Cultura (SAF/2000-117) and the Fondo de Investigaciones Sanitarias (FIS), Spain, No. 97/1289, awarded to F. S.; a grant from Ministerio de Educación y Cultura (SAF 2000-161) given to F. J. B.; and a grant from Fundation of SEIAC, Spain, Bial-Arístegui, and Hycor Biomedical (Garden Grove, CA) awarded to J. M.

Received August 12, 2001; revised September 19, 2001; accepted September 24, 2001.


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