Evaluation of assays for the measurement of bovine neutrophil reactive oxygen species

Veterinary Immunology and Immunopathology 115 (2007) 107–125 www.elsevier.com/locate/vetimm

Evaluation of assays for the measurement of bovine neutrophil reactive oxygen species Manuela Rinaldi a, Paolo Moroni a, Max J. Paape b, Douglas D. Bannerman b,* a b

Department of Veterinary Pathology, Hygiene and Public Health, University of Milan, Milan 20133, Italy Bovine Functional Genomics Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, United States Received 10 July 2006; received in revised form 18 September 2006; accepted 21 September 2006

Abstract During mastitis and other bacterial-mediated diseases of cattle, neutrophils play a critical role in the host innate immune response to infection. Neutrophils are among the earliest leukocytes recruited to the site of infection and contribute to host innate immune defenses through their ability to phagocytose and kill bacteria. The bactericidal activity of neutrophils is mediated, in part, through the generation of reactive oxygen species (ROS). Extracellular release of ROS can induce injury to host tissue as well, and aberrant release of ROS has been implicated in the pathogenesis of certain inflammatory-mediated diseases. Due to their essential role in bacterial clearance and implicated involvement in the pathogenesis of other diseases, there is much interest in the study of neutrophil-generated ROS. Several assays have been developed to measure ROS production, however, many of these have not been evaluated with bovine neutrophils. The objectives of the current study were to evaluate different assays capable of measuring bovine neutrophil ROS, and to compare the results of assays never previously tested with bovine neutrophils to those obtained from more well-established assays frequently used with these cells. Eight different assays were evaluated, including: luminol, isoluminol, and methyl cypridina luciferin analog (MCLA) chemiluminescence assays; Amplex Red, dihydroethidium (DHE), dichlorodihydrofluorescein diacetate (CM-H2DCFDA), and dihydrorhodamine 123 fluorescence assays; and the cytochrome c absorbance assay. The assays were evaluated in the context of their abilities to detect ROS produced in response to two agonists commonly used to induce neutrophil activation, phorbol 12-myristate, 13-acetate (PMA) and opsonized zymosan. Diphenyleneiodonium chloride, a NADPH oxidase inhibitor, was used to assess the specificity of the assays to detect ROS. The ability of these assays to discriminate between intra- and extracellular ROS and to specifically detect distinct ROS was evaluated using superoxide dismutase and catalase, which scavenge extracellular superoxide and hydrogen peroxide, respectively. With the exception of the DHE assay, all assays detected bovine neutrophil ROS generation elicited by PMA and zymosan. PMA, but not zymosan, was able to stimulate neutrophil generation of ROS at levels that were detectable with DHE. The MCLA chemiluminescence assay was the only assay that detected ROS produced in response to each of the lowest concentrations of PMA and zymosan tested. To our

Abbreviations: BLAD, bovine leukocyte adhesion deficiency; CMF-HBSS, calcium- and magnesium-free Hanks’ balanced salt solution; CMH2DCFDA, 5-(6)-chloromethyl-20 ; 70 -dichlorodihydrofluorescein diacetate, acetyl ester; DHR, dihydrorhodamine 123; DMSO, dimethyl sulphoxide; DHE, dihydroethidium; DPI, diphenyleneiodonium chloride; HRP, horseradish peroxidase; MCLA, methyl cypridina luciferin analog; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate, 13-acetate; PMN, polymorphonuclear neutrophil; RFI, relative fluorescence index; ROS, reactive oxygen species; SOD, superoxide dismutase * Corresponding author at: Bovine Functional Genomics Laboratory, USDA-Agricultural Research Service, BARC-East, Building 1040, Room #2, Beltsville, MD 20705-2350, United States. Tel.: +1 301 504 5066; fax: +1 301 504 5306. E-mail address: [email protected] (D.D. Bannerman). 0165-2427/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2006.09.009

108

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125

knowledge, this is the first study to evaluate DHE-, MCLA-, Amplex Red-, and isoluminol-based assays for the measurement of bovine neutrophil ROS, and the most comprehensive comparative study of ROS assays under similar experimental conditions. # 2006 Elsevier B.V. All rights reserved. Keywords: Bovine; Cattle; Leukocyte; Neutrophil; Reactive oxygen species; Respiratory burst

1. Introduction During mastitis and other bacterial-mediated diseases of cattle, polymorphonuclear neutrophils (PMN’s) are one of the first innate immune effector cells to be recruited to the site of infection. Impaired recruitment or functioning of PMN’s is negatively correlated with host clearance of bacteria (Schalm et al., 1976; Roth and Kaeberle, 1981; Cai et al., 1994; Tkalcevic et al., 2000; Nagahata, 2004). During mastitis, for example, experimentally induced neutropenia results in uncontrollable infection and the development of gangrenous mastitis (Schalm et al., 1976). Conversely, enhanced recruitment of PMN’s to the infected mammary glands of cows with normal levels of circulating PMN’s is associated with more rapid clearance of intramammary infection (Burvenich et al., 2003; Paape et al., 2003). Perhaps the strongest evidence for the essential role of PMN’s in contributing to host defense mechanisms in cattle comes from studies with Holstein cattle with bovine leukocyte adhesion deficiency (BLAD) (Nagahata, 2004). The molecular basis of BLAD is a single point mutation in the gene for the PMN adhesion molecule, CD18, which results in impaired PMN recruitment to sites of infection. Corresponding with impaired PMN recruitment, this condition is characterized by recurrent bacterial infections and often premature death due to complications arising from these infections. The microbicidal activity of PMN’s governs their ability to control and eradicate invading bacterial pathogens. PMN’s produce a variety of bactericidal molecules including defensins, bactericidal permeability-increasing protein, lysozyme, and reactive oxygen species (ROS) (Gudmundsson and Agerberth, 1999; Burg and Pillinger, 2001). Generation of the latter, which is also referred to as respiratory or oxidative burst activity, is a characteristic property of phagocytes such as the PMN. PMN generation of ROS is critical to their role in host defense as impaired production is associated with increased susceptibility to infection (Baehner, 1990; Dinauer, 1993). Generation of ROS is mediated by a membraneassociated, multi-component protein enzyme complex referred to as either NADPH oxidase or phagocyte

oxidase (phox) (Hampton et al., 1998; Babior, 1999). This enzyme complex is inactive in unstimulated PMN’s and is only assembled upon cell activation. NADPH oxidase assembly and activation is elicited in response to both receptor-dependent and -independent events. A variety of factors can induce NADPH oxidase activation in bovine PMN’s including opsonized bacteria or zymosan, aggregated IgG, and protein kinase C activators such as phorbol myristate acetate (PMA) (Czuprynski and Hamilton, 1985; Leino and Paape, 1993, 1996; Rambeaud et al., 2006). Upon assembly and activation, NADPH oxidase facilitates the shuttling of electrons from cytosolic NADPH to oxygen present in the phagosome or in the proximal extracellular environment (Babior, 1984; Weiss, 1989; El-Benna et al., 2005). Superoxide anion (O2) is the most proximally generated ROS by NADPH oxidase and forms as a result of one molecule of oxygen acting as an acceptor for a single donated electron (Weiss, 1989; Burg and Pillinger, 2001). Superoxide anion, in turn, serves as the precursor for the formation of other ROS (Weiss, 1989; Hampton et al., 1998). Hydrogen peroxide (H2O2) is rapidly formed from superoxide anion by spontaneous dismutation or enzymatic dismutation by superoxide dismutase. Transition metals can catalyze the formation of the powerful oxidant, hydroxyl radical (OH), from superoxide anion and hydrogen peroxide. In the presence of halogens (e.g., Cl), myeloperoxidase, which is a major component of the PMN azurophilic granules, catalyzes the conversion of hydrogen peroxide into highly toxic hypohalous acids [e.g., hypochlorous acid (HOCl)]. Hypochlorous acid, for example, can then react with amines or hydrogen peroxide to form chloramines or singlet oxygen (1O2), respectively. Thus, the production of superoxide anion by NADPH oxidase serves to provide a substrate for the generation of an array of ROS that mediate PMN bactericidal activity. ROS are detected both intracellulary and extracellularly (Hampton et al., 1998). This is due, in part, to the localization of NADPH oxidase to the cellular membrane. Internalization of the cell membrane and intracellular trafficking of the formed phagosome,

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125

which contains the membrane-associated complex, results in intracellular generation of ROS within the phagosomal compartment. Generation of extracellular ROS is attributed to direct production from cell membrane-associated NADPH oxidase and/or escape of ROS from forming phagosomes that have not completely sealed and internalized during the process of phagocytosis (Babior, 1984; Dahlgren and Karlsson, 1999). Although generation of ROS is essential for optimal PMN bactericidal activity, there is debate about whether these molecules are directly toxic to bacteria under the physiological conditions that exist within the phagosome. Recent evidence suggests an indirect role in which NADPH oxidase pumps electrons into the phagosome, resulting in the induction of a charge across the membrane (Segal, 2005). The compensating influx of ions, in turn, provides an optimized environment for the enzymes released from cytoplasmic granules to exert their bactericidal activity. In addition to their direct or indirect role in bacterial killing, extracellular release of these molecules can induce injury to the host itself. In fact, there is increasing evidence that ROS are among the most injurious substances released from cells and that dysregulated release of these molecules is involved in the pathogenesis of a variety of inflammatory diseases (Fantone and Ward, 1985; Weiss, 1989; Ricevuti, 1997). For example, there are several reports suggesting that in the setting of mastitis, where PMN concentrations can approach 50 million cells/ml of milk (Bannerman et al., 2004), that activated PMN’s induce direct injury to the mammary epithelium (Capuco et al., 1986; Ledbetter et al., 2001; Long et al., 2001; Burvenich et al., 2004; Lauzon et al., 2005). Thus, despite their critical role in contributing to PMN host defense capabilities, ROS also contribute to the deleterious injury associated with the inflammation elicited in response to bacterial infection. Because of their prominent role in facilitating PMNmediated clearance of bacterial pathogens and their implicated role in contributing to the pathogenesis of inflammatory-mediated diseases, there is strong interest in the study of PMN-generated ROS. Although there have been several assays developed to measure human PMN production of ROS, many of these have never been evaluated with bovine PMN’s. The objective of the current study was to evaluate different assays for measuring bovine PMN ROS production and to compare the differential stimulatory activity of two distinct agonists, PMA and opsonized zymosan, which are commonly utilized to induce PMN respiratory burst activity.

109

2. Materials and methods 2.1. Reagents Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and isoluminol (6-amino-2,3-dihydro-1,4-phthalazinedione) (both from Sigma Chemical Co., St. Louis, MO) were prepared as 1 M stock solutions in dimethyl sulphoxide (DMSO). Phorbol 12-myristate, 13-acetate (PMA) (Sigma Chemical Co.), dihydroethidium (DHE) (Molecular Probes, Inc., Eugene, OR), and diphenyleneiodonium chloride (DPI) (Calbiochem–Novabiochem Corp., San Diego, CA) were prepared as 10 mM stock solutions in DMSO. Methyl cypridina luciferin analog (MCLA: 2-methyl-6-(4-methoxyphenyl)-3,7dihydroimidazol[1,2-a]pyrazin-3-one, hydrochloride) and 5-(6)-chloromethyl-20 ,70 -dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) (both from Molecular Probes, Inc.) were prepared as 1 mM stock solutions in DMSO. Dihydrorhodamine 123 (DHR) was obtained as a 5 mM solution in DMSO (Molecular Probes, Inc.). Cytochrome c from bovine heart and superoxide dismutase (SOD) from bovine erythrocytes (both from Sigma Chemical Co.) were prepared as 1 mM and 1000 units/ml stock solutions, respectively, in calcium- and magnesium-free Hanks’ balanced salt solution (CMF-HBSS). Catalase, isolated from bovine liver, was obtained as a prepared suspension in water (Sigma Chemical Co.). Peroxidase type VI from horseradish (Sigma Chemical Co.) was prepared as a stock solution of 500 U/ml in endotoxin-free water. Zymosan was activated by suspending 500 mg of zymosan A from Saccharomyces cerevisiae (Sigma Chemical Co.) in 50 ml of HBSS. The suspension was boiled for 20 min, mixed in a blender for 30 s, and then centrifuged at 250  g for 10 min. The pellet was washed twice by resuspension in HBSS and recentrifugation as above. After the final wash, zymosan was resuspended in 50 ml of a 65% solution of pooled bovine serum diluted in phosphate-buffered saline (PBS). Following a 1 h incubation at 37 8C, the suspension was centrifuged and the resulting pellet washed twice as above. After the final wash, activated zymosan was resuspended in HBSS at a final concentration of 10 mg/ml, aliquotted, and stored at 70 8C. 2.2. Isolation of bovine blood PMN’s Clinically healthy lactating Holstein cows from the USDA-ARS Beltsville dairy herd were used as blood donors for all experiments. The use and care of all

110

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125

animals in this study were approved by the Beltsville Agricultural Research Center’s Animal Care and Use Committee. Blood was obtained from the coccygeal vein and collected into Vacutainer1 glass tubes containing acid-citrate-dextrose (Becton-Dickinson Corp, Franklin, Lakes, NJ). The tubes were immediately inverted five times and stored on ice. PMN’s were isolated using a Percoll1-gradient as previously described (Weber et al., 2001). Briefly, 20 ml of blood were transferred to 50 ml polypropylene conical tubes and centrifuged (1000  g) for 20 min at 4 8C. The plasma and buffy coat were aseptically aspirated and discarded. The remaining cells were suspended in 34 ml of ice-cold PBS and the suspension slowly pipetted down the side of a clean 50 ml polypropylene conical tube containing 10 ml of 1.084 g/ml Percoll1 (Sigma Chemical Co.). The tubes were centrifuged (400  g) for 40 min at 22 8C. The supernatant, mononuclear cell layer, and Percoll1 were aseptically aspirated and a pellet composed of PMN’s and erythrocytes was retained. Erythrocytes were lysed by mixing 1 volume of cells with 2 volumes of an ice cold 0.2% NaCl solution and inverting the tube for 1 min. Tonicity was restored by the addition of one-half volume of a 3.7% NaCl solution. The tubes were centrifuged (500  g) for 2 min at 4 8C. The cell pellet was washed twice by resuspension in CMF-HBSS and recentrifugation for 1 min at 4 8C. Cells were enumerated using an electronic particle counter (Coulter Electronics, Inc., Hialeah, FL). Cell viability and differential cell counts were determined by trypan blue (Sigma Chemical Co.) exclusion and Wright staining, respectively. PMN purity was >95% and viability >90%. PMN concentrations were adjusted with CMF-HBSS and maintained on ice until used in the various assays described below. 2.3. Cytochrome c reduction assay The cytochrome c reduction assay was performed as previously described (Sartorelli et al., 2000; Teufelhofer et al., 2003). For studies evaluating dose-dependent changes in cytochrome c reduction, 50 ml of HBSS or varying concentrations of PMA or zymosan, and 10 ml of cytochrome c (1 mM) were added to 2  105 PMN’s suspended in 20 ml of CMF-HBSS in wells of a 96-well plate. For studies evaluating the effect of specific ROS inhibitors on cytochrome c reduction, PMN’s were first incubated for 10 min at 37 8C with 20 ml of DPI (80 mM) or 2 ml of SOD (1000 U/ml). Cytochrome c and a fixed concentration of either PMA (160 mM) or zymosan (1 mg/ml) were then added as above. All reactions were adjusted to a final volume of 200 ml with

HBSS. Absorbance was measured on a Synergy HTTM multi-modal plate reader (Bio-Tec Instruments, Inc., Winooski, VT). Optical density (OD) was measured at 10 min intervals at a wavelength of 550 nm and a reference wavelength of 670 nm, and the difference of the two OD’s recorded. Background values, calculated from wells with cytochrome c diluted in HBSS, were subtracted from all values. 2.4. MCLA chemiluminescence assay The MCLA chemiluminescence assay was performed as previously described (Nishida et al., 1989; Pronai et al., 1992) with slight modification. For studies evaluating dose-dependent changes in MCLA-derived chemiluminescence, 37.5 ml of HBSS or varying concentrations of PMA or zymosan, and 7.5 ml of MCLA (2 mM) were added to 4  105 PMN’s suspended in 50 ml of CMF-HBSS in wells of a 96-well plate. For studies evaluating the effect of specific ROS inhibitors on MCLA-dependent chemiluminescence, PMN’s were first incubated for 10 min at 37 8C with 20 ml of DPI (60 mM) or 1.5 ml of SOD (1000 U/ml). MCLA and a fixed concentration of either PMA (160 nM) or zymosan (1 mg/ml) were then added as above. All reactions were adjusted to a final volume of 150 ml with HBSS. Chemiluminescence was measured every 10 min with a VeritasTM microplate luminometer (Turner Biosystems Inc., Sunnyvale, CA). Because MCLA is rapidly consumed (Pronai et al., 1992), 7.5 ml of MCLA (2 mM) was added to all wells between each time point at which the plate was read throughout the course of chemiluminescent measurements. Background values, defined as the mean chemiluminescent values of MCLA diluted in HBSS, were subtracted from all readings. 2.5. Amplex1 Red (10-acetyl-3,7 dihydroxyphenoxazine) fluorescence assay The Amplex Red-dependent fluorescence assay was performed according to the manufacturer’s instructions (Molecular Probes, Inc.) and previously published methods (Mohanty et al., 1997; Zhou et al., 1997). For studies evaluating dose-dependent changes in Amplex Red-derived fluorescence, 20 ml of HBSS or varying concentrations of PMA or zymosan, and 80 ml of the Amplex Red reaction mixture [75 ml of Amplex Red (10 mM) and 150 ml of horseradish peroxidase (10 U/ml) diluted in 11.8 ml of Krebs Ringer phosphate glucose solution (145 mM NaCl, 5.7 mM Na2HPO4, 4.86 mM KCl, 0.54 mM CaCl2, 1.22 mM MgSO4, 5.5 mM glucose)] were added to 1.5  104 PMN’s suspended

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125

in 10 ml of Krebs Ringer phosphate glucose solution in wells of a 96-well plate. For studies evaluating the effect of specific ROS inhibitors on Amplex Red-dependent fluorescence, PMN’s were first incubated for 10 min at 37 8C with 10 ml of either DPI (96 mM) or catalase (12,000 U/ml), or 1.2 ml of SOD (1000 U/ml). Amplex Red and a fixed concentration of either PMA (240 nM) or zymosan (1.5 mg/ml) were then added as above. All reactions were adjusted to a final volume of 120 ml with HBSS. Fluorescence was measured every 15 min on a Synergy HTTM multi-modal plate reader (Bio-Tec Instruments, Inc.) at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Background values, defined as the mean fluorescent values of Amplex Red reaction mixture diluted in HBSS, were subtracted from all readings. 2.6. CM-H2DCFDA and DHR fluorescence assays The CM-H2DCFDA and DHR fluorescence assays were performed in accordance with previously established methods (Wang and Joseph, 1999; Lee et al., 2003; Li et al., 2004; Becker et al., 2005). For studies evaluating dose-dependent changes in CM-H2DCFDAfluorescence, 30 or 37.5 ml of varying concentrations of PMA or zymosan, respectively, and 7.5 ml of CMH2DCFDA (200 mM), were added to 4  105 PMN’s suspended in 50 ml of CMF-HBSS in wells of a 96-well plate. For studies evaluating the effect of specific ROS inhibitors on CM-H2DCFDA-dependent fluorescence, PMN’s were first incubated for 10 min at 37 8C with 20 ml of either DPI (60 mM) or catalase (7500 U/ml), or 1.5 ml of SOD (1000 U/ml). CM-H2DCFDA and a fixed concentration of either PMA (200 nM) or zymosan (1 mg/ml) were then added as above. All reactions were adjusted to a final volume of 150 ml with HBSS. Studies with DHR were performed as described for CMH2DCFDA with the exception that PMN’s were preincubated with 7.5 ml of DHR (20 mM) for 15 min and then subsequently stimulated as described. Fluorescence was measured every 15 min on a Synergy HTTM multi-modal plate reader (Bio-Tec Instruments, Inc.) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Background values, defined as the mean fluorescent values of CM-H2DCFDA or DHR diluted in HBSS, were subtracted from all readings. 2.7. DHE flow cytometric assay The DHE flow cytometric assay was performed based upon previously published methods (Walrand et al., 2003). For all studies with this assay, 100 ml of

111

DHE (200 mM) were added to 1  106 PMN’s suspended in 500 ml of CMF-HBSS. For those studies evaluating the effect of specific ROS inhibitors on DHEdependent fluorescence, 160 ml of DPI (100 mM) or catalase (12,500 U/ml), or 20 ml of SOD (1000 units/ ml) were also added. For all studies with DHE, reaction volumes were adjusted to 1.8 ml with HBSS and the cell suspension and reaction mixture incubated for 15 min at 37 8C in a shaking (30 rpm) water bath. Two hundred microlitres of HBSS or varying concentrations of PMA or zymosan were subsequently added, and the incubation allowed to proceed. At varying time points after addition of each agonist, cellular fluorescence was measured with a Coulter1 Epics1 XL-MCL flow cytometer (Beckman Coulter, Inc., Fullerton, CA). Four hundred microlitres of 1% methylene blue were added to each sample immediately prior to assaying on the flow cytometer to quench extracellular fluorescence (Jain et al., 1991). A relative fluorescence index (RFI) was calculated for each sample by multiplying the percentage of cells fluorescing by the mean channel fluorescence and dividing the resulting product by 100 (Salgar et al., 1991). 2.8. Luminol and isoluminol chemiluminescence assays The luminol and isoluminol chemiluminescence assays were performed with slight modifications of previously established methods (Leino and Paape, 1993; Lundqvist and Dahlgren, 1996). For studies evaluating dose-dependent changes in luminol-derived chemiluminescence, 60 ml of HBSS or varying concentrations of PMA or zymosan, and 20 ml of luminol (5 mM) were added to 2  105 PMN’s suspended in 100 ml of CMF-HBSS in wells of a 96-well plate. For studies evaluating the effect of specific ROS inhibitors on luminol-dependent chemiluminescence, PMN’s were first incubated for 10 min at 37 8C with 20 ml of either DPI (80 mM) or catalase (10,000 U/ml), 2 ml of SOD (1000 U/ml), or the combination of both catalase and SOD. Luminol and a fixed concentration of either PMA (133 nM) or zymosan (833 mg/ml) were then added as above. All reactions were adjusted to a final volume of 200 ml with HBSS. Reactions with isoluminol were set up exactly as described above with the exception that luminol was substituted with 20 ml of isoluminol (500 mM) and 5 ml of horseradish peroxidase (HRP: 160 U/ml) (Sigma Chemical Co.), and that these reagents were added to 2  105 PMN’s suspended in 95 ml of CMF-HBSS. Chemiluminescence was measured at varying intervals with a VeritasTM

112

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125

microplate luminometer (Turner Biosystems Inc.). Background values, defined as the mean chemiluminescent values of luminol or isoluminol diluted in HBSS, were subtracted from all readings.

obtained from all experimental conditions to those obtained from unstimulated PMN’s or PMN’s stimulated with PMA or zymosan, respectively. A P-value of <0.05 was considered significant.

2.9. Determination of intraassay and interassay variability

3. Results

To assess intraassay variability, assays were performed as described with PMN’s isolated from a single cow plated in quintuplicate and exposed to PMA (40 mM for evaluation of cytochrome c reduction and 40 nM for all other assays) or HBSS (unactivated). The area under the curve was calculated from plotted data points for each experimental condition and the foldincrease in values over unactivated PMN’s calculated for each of the five wells containing activated PMN’s. The coefficient of variation (CV) was determined from the calculated fold-increase values in each well of the quintuplicately plated PMN’s. The entire experiment was repeated two more times on different days and the mean CV of the three independent experiments reported. To assess interassay variability, PMN’s isolated from three cows were exposed to PMA or HBSS (unactivated), and the mean fold-increase in ROS generation of activated versus unactivated PMN’s calculated for all three cows from the area under the curve of plotted data points. The assay was then repeated two more times on different days with PMN’s re-isolated from the same cows. The CV was determined from the mean fold-increase in ROS generation calculated for each experiment performed on the three separate days. 2.10. Statistical methods With the exception of the DHE flow cytometric assay, measurements (i.e., absorbance, chemiluminescence, fluorescence) for all other assays were continuously collected at regular intervals up to 120 min post-stimulation. To enable calculation of cumulative ROS production over the entire period in which measurements were performed, the area under the curve was calculated from plotted data points for each experimental condition using GraphPad Prism Version 4.00 for Windows (GraphPad Software, Inc., San Diego, CA). A one-way analysis of variance (ANOVA) (GraphPad Software, Inc.) was used to compare the mean responses between experimental groups. For studies evaluating dose-dependent responses or inhibitor-dependent effects on ROS generation, the Dunnett post hoc comparison test was used to compare values

3.1. Evaluation of assays measuring extracellular ROS production The measurement of cytochrome c reduction is a commonly used technique for the measurement of extracellular superoxide (Munzel et al., 2002; Tarpey et al., 2004). To assess the ability of this compound to measure bovine PMN ROS, blood PMN’s were stimulated with increasing concentrations of PMA (Fig. 1A) or zymosan (Fig. 1B) and absorbance was measured. Bovine PMN’s exposed to either agonist demonstrated a dose-dependent increase in ROS that could reduce cytochrome c as evidenced by increases in absorbance. Relative to unstimulated PMN’s, PMA and zymosan elicited maximal fold-increases in absorbance of 11 and 5, respectively. To determine the specificity of this assay to measure ROS, PMN’s were pre-treated for 10 min with either the NADPH oxidase inhibitor, DPI (8 mM), or SOD (10 U/ml), the latter of which is an extracellular scavenger of superoxide (Dahlgren and Karlsson, 1999). PMN’s were subsequently stimulated with PMA (40 mM) (Fig. 1C) or zymosan (250 mg/ml) (Fig. 1D), and absorbance was measured. DPI and SOD, respectively, inhibited >70 and 90% of the PMA- and zymosan-induced changes in cytochrome c reduction. MCLA-derived chemiluminescence has been reported to be a highly specific indicator of extracellular superoxide production (Nishida et al., 1989; Pronai et al., 1992). To evaluate the ability of this compound to measure bovine PMN ROS, blood PMN’s were stimulated with increasing concentrations of PMA (Fig. 2A) or zymosan (Fig. 2B) and MCLA-dependent chemiluminescence measured. With the highest concentrations of agonists assayed, increases in MCLAdependent chemiluminescence were apparent within 10 min of PMN stimulation. Both PMA and zymosan induced dose-dependent increases in chemiluminescence and even the lowest concentrations tested elicited detectable increases that were significantly (P < 0.01) elevated relative to unstimulated PMN’s. Relative to unstimulated PMN’s, maximal increases in chemiluminescence of 37- and 15-fold were evoked by PMA and zymosan, respectively. DPI inhibited 82 and 95% of the PMA (40 nM)- and zymosan (250 mg/ml)-evoked chemiluminescence, respectively (Fig. 2C and D),

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125

113

Fig. 1. Cytochrome c reduction assay for the measurement of extracellular reactive oxygen species (ROS). Blood neutrophils isolated from five cows were exposed to increasing concentrations of PMA (A) or zymosan (Zym) (B) in the presence of cytochrome c, and absorbance was measured every 10 min (left y-axis). To analyze concentration-dependent generation of ROS, the area under the curve was calculated from plotted data points for each concentration (right y-axis). *, **Significantly increased (P <0.05 and <0.01, respectively) relative to unstimulated neutrophils. In other studies, cytochrome c was used to assay ROS production (left y-axis) in blood neutrophils (n = 5) pre-treated for 10 min with superoxide dismutase (SOD: 10 U/ml) or diphenyleneiodonium chloride (DPI: 8 mM) and subsequently exposed to PMA (40 mM) (C) or Zym (250 mg/ml) (D). The area under the curve for each experimental condition was calculated (right y-axis). **Significantly decreased (P < 0.01) relative to neutrophils stimulated with either PMA or zymosan alone.

whereas, SOD completely abrogated the response elicited by either agonist. Measurement of fluorescence resulting from the enzyme-catalyzed oxidation of Amplex Red to the highly fluorescent molecule, resorufin, has been reported to be a highly specific and sensitive method for the detection of extracellular hydrogen peroxide (Zhou et al., 1997; Forteza et al., 2005). To assess the ability of this compound to measure bovine PMN ROS, blood PMN’s were stimulated with increasing concentrations of PMA (Fig. 3A) or zymosan (Fig. 3B) and fluorescence was measured. All concentrations of PMA used to activate PMN’s, and zymosan concentrations 250 mg/ml, elicited an increase in Amplex Reddependent fluorescence. Maximal increases in the generation of ROS that oxidized Amplex Red in

response to PMA or zymosan were reflected by relative fluorescent values that were 158- and 14-fold higher, respectively, than unactivated PMN’s. Regardless of the agonist, DPI completely inhibited this response (Fig. 3C and D). SOD enhanced Amplex Red-dependent fluorescence in wells containing PMA-stimulated PMN’s, whereas it had no effect on those containing PMN’s activated with zymosan. To further evaluate the specificity of this assay, PMN’s were pre-treated for 10 min with catalase (1000 U/ml), a cell impermeable enzyme that scavenges extracellular hydrogen peroxide (Dahlgren and Karlsson, 1999), and subsequently stimulated with PMA (40 nM) or zymosan (250 mg/ ml) (Fig. 3E and F). Catalase completely blocked the Amplex Red-dependent fluorescence in wells containing PMN’s stimulated with PMA or zymosan.

114

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125

Fig. 2. Assay of extracellular production of reactive oxygen species (ROS) with MCLA. Blood neutrophils isolated from five cows were exposed to increasing concentrations of PMA (A) or zymosan (Zym) (B) in the presence of MCLA, and chemiluminescence measured every 10 min (left yaxis). To analyze concentration-dependent generation of ROS, the area under the curve was calculated from plotted data points for each concentration (right y-axis). **Significantly increased (P < 0.01) relative to unstimulated neutrophils. In other studies, MCLA-dependent chemiluminescence (left y-axis) was assayed in blood neutrophils (n = 5) pre-treated for 10 min with superoxide dismutase (SOD: 10 U/ml) or diphenyleneiodonium chloride (DPI: 8 mM) and subsequently exposed to PMA (40 nM) (C) or Zym (250 mg/ml) (D). The area under the curve for each experimental condition was calculated (right y-axis). **Significantly decreased (P < 0.01) relative to neutrophils stimulated with either PMA or zymosan alone.

3.2. Evaluation of assays measuring intracellular ROS production CM-H2DCFDA is a membrane-permeable fluorescent compound that is trapped intracellularly following esterase cleavage and fluoresces when oxidized by hydrogen peroxide and other ROS, such as peroxynitrite anion (Rothe and Valet, 1990; LeBel et al., 1992; Crow, 1997; Possel et al., 1997). To evaluate the ability of this compound to measure bovine PMN ROS, blood PMN’s were stimulated with increasing concentrations of PMA (Fig. 4A) or zymosan (Fig. 4B) and H2DCFDAdependent fluorescence was measured. Both PMA and zymosan, respectively, elicited dose-dependent increases in fluorescence at concentrations 0.04 mM and 125 mg/ml, respectively. Relative to unactivated PMN’s, the maximal concentrations of PMA and zymosan tested elicited an 11- and 6.5 fold-increase in fluorescence, respectively. DPI significantly inhibited

(P < 0.01) both PMA- and zymosan-induced H2DCFDA-dependent fluorescence by 85% and 69%, respectively (Fig. 4C and D). In contrast, pre-treatment with either SOD (Fig. 4C and D) or catalase (Fig. 4E and F) had no effect on PMA- or zymosan-induced H2DCFDA-dependent fluorescence. Similar to CM-H2DCFDA, DHR is a membranepermeable compound that fluoresces upon oxidation by peroxynitrite and hydrogen peroxide (Henderson and Chappell, 1993; Kooy et al., 1994; Walrand et al., 2003). The ability of this compound to measure bovine PMN ROS production was evaluated by stimulating PMN’s with increasing concentrations of PMA (Fig. 5A) or zymosan (Fig. 5B) and measuring DHRmediated fluorescence. PMA and zymosan evoked measurable dose-dependent increases in DHRmediated fluorescence at concentrations 0.004 mM and 125 mg/ml, respectively. Maximal increases in fluorescence relative to unstimulated cells were 30- and

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125

115

Fig. 3. Assay of extracellular production of hydrogen peroxide with Amplex Red. Blood neutrophils isolated from five cows were exposed to increasing concentrations of PMA (A) or zymosan (Zym) (B) in the presence of Amplex Red, and fluorescence was measured every 15 min (left yaxis). To analyze concentration-dependent generation of ROS, the area under the curve was calculated from plotted data points for each concentration (right y-axis). **Significantly increased (P < 0.01) relative to unstimulated neutrophils. In other studies, Amplex Red-dependent fluorescence (left y-axis) was assayed in blood neutrophils (n = 5) pre-treated for 10 min with superoxide dismutase (SOD: 10 U/ml) (C and D), diphenyleneiodonium chloride (DPI: 8 mM) (C and D), or catalase (Cat: 1000 U/ml) (E and F), and subsequently exposed to PMA (40 nM) (C and E) or Zym (250 mg/ml) (D and F). The area under the curve for each experimental condition was calculated (right y-axis). **Significantly different (P < 0.01) relative to neutrophils stimulated with either PMA or zymosan alone.

13-fold higher following respective exposure to PMA or zymosan. PMA- and zymosan-induced DHR-dependent fluorescence was significantly inhibited (P < 0.01) by 98 and 83%, respectively, in the presence of DPI, whereas SOD had no inhibitory effect on either agonist (Fig. 5C and D). Catalase partially inhibited the DHR-mediated

fluorescence response evoked by PMA (42% inhibition) and zymosan (21% inhibition) (Fig. 5E and F). DHE is a cell permeable compound that is readily oxidized by superoxide to form ethidium bromide, a highly fluorescent compound that intercalates with DNA (Rothe and Valet, 1990; Filatov et al., 1995; Walrand

116

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125

Fig. 4. Assay of intracellular production of reactive oxygen species (ROS) with CM-H2DCFDA. Blood neutrophils isolated from five cows were exposed to increasing concentrations of PMA (A) or zymosan (Zym) (B) in the presence of CM-H2DCFDA, and fluorescence was measured every 15 min (left y-axis). To analyze concentration-dependent generation of ROS, the area under the curve was calculated from plotted data points for each concentration (right y-axis). *, **Significantly increased (P <0.05 and <0.01, respectively) relative to unstimulated neutrophils. In other studies, CM-H2DCFDA-dependent fluorescence (left y-axis) was assayed in blood neutrophils (n = 5) pre-treated for 10 min with superoxide dismutase (SOD: 10 U/ml) (C and D), diphenyleneiodonium chloride (DPI: 8 mM) (C and D), or catalase (Cat: 1000 U/ml) (E and F), and subsequently exposed to PMA (40 nM) (C and E) or Zym (250 mg/ml) (D and F). The area under the curve for each experimental condition was calculated (right y-axis). *, ** Significantly decreased (P <0.05 and <0.01, respectively) relative to neutrophils stimulated with either PMA or zymosan alone.

et al., 2003). To assess the ability of this compound to measure bovine PMN ROS, blood PMN’s were stimulated for 30 min with increasing concentrations of PMA or zymosan (Fig. 6A) and fluorescence was measured on a flow cytometer at varying time points.

PMA evoked dose-dependent increases in relative fluorescence, whereas, measurable increases in response to zymosan were not detected. PMN stimulation with zymosan for up to 120 min failed to elicit a significant increase in fluorescence relative to unactivated PMN’s

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125

117

Fig. 5. Assay of intracellular production of reactive oxygen species (ROS) with DHR. Blood neutrophils isolated from five cows were exposed to increasing concentrations of PMA (A) or zymosan (Zym) (B) in the presence of DHR, and fluorescence was measured every 15 min (left y-axis). To analyze concentration-dependent generation of ROS, the area under the curve was calculated from plotted data points for each concentration (right yaxis). **Significantly increased (P < 0.01) relative to unstimulated neutrophils. In other studies, DHR-dependent fluorescence (left y-axis) was assayed in blood neutrophils (n = 5) pre-treated for 10 min with superoxide dismutase (SOD: 10 U/ml) (C and D), diphenyleneiodonium chloride (DPI: 8 mM) (C and D), or catalase (Cat: 1000 U/ml) (E and F), and subsequently exposed to PMA (40 nM) (C and E) or Zym (250 mg/ml) (D and F). The area under the curve for each experimental condition was calculated (right y-axis). *, **Significantly decreased (P <0.05 and <0.01, respectively) relative to neutrophils stimulated with either PMA or zymosan alone.

(data not shown). Maximal increases in the generation of ROS that oxidized DHE in response to PMA were reflected by relative fluorescent values that were 39-fold higher than unactivated PMN’s. DPI inhibited >96% of PMA-induced DHE oxidation, whereas, catalase and SOD, which are restricted to the extracellular compartment, had no inhibitory effect (Fig. 6B).

3.3. Evaluation of luminol-based assays for the measurement of ROS production Luminol-derived chemiluminescence has been reported to be elicited in response to the generation of an array of intracellular and extracellular ROS, including superoxide anion, hydroxyl radicals, hydrogen peroxide,

118

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125

Fig. 6. Assay of intracellular generation of reactive oxygen species (ROS) with DHE. Blood neutrophils isolated from five cows were exposed to increasing concentrations of PMA or zymosan (Zym) in the presence of DHE, and fluorescence was measured 30 min following stimulation (A). **Significantly increased (P < 0.01) relative to unstimulated neutrophils. In other studies, DHE-dependent fluorescence was assayed in blood neutrophils (n = 5) pre-treated for 15 min with superoxide dismutase (SOD: 10 U/ml), diphenyleneiodonium chloride (DPI: 8 mM), or catalase (Cat: 1000 U/ml), and subsequently exposed to PMA (40 nM) (B). **Significantly decreased (P < 0.01) relative to neutrophils stimulated with PMA alone.

peroxynitrite, and hypochlorous acid (Saez et al., 2000; Munzel et al., 2002). To evaluate the ability of this compound to measure bovine PMN ROS, blood PMN’s were stimulated with increasing concentrations of PMA (Fig. 7A) or zymosan (Fig. 7B) and chemiluminescence measured. Both PMA and zymosan elicited dosedependent increases in luminol-dependent chemiluminescence at concentrations 0.04 mM and 125 mg/ml, respectively. Relative to unstimulated PMN’s, maximal increases in chemiluminescence of 330- and 440-fold were evoked by PMA and zymosan, respectively. DPI inhibited >90% of the luminol-dependent chemiluminescence in wells containing PMN’s stimulated with PMA or zymosan (Fig. 7C and D). SOD inhibited

PMA- and zymosan-evoked luminol-dependent chemiluminescence by 41 and 25%, although the latter inhibition did not reach a level that was statistically significant. Pre-incubation with catalase did not significantly alter chemiluminescence in PMN’s activated with either agonist (Fig. 7E and F). Pre-treatment with both SOD and catalase blocked 65% of the increase in PMA-elicited chemiluminescence, whereas, the combination of inhibitors had no effect on zymosan-evoked responses. Isoluminol-dependent chemiluminescence has been reported to be a specific indicator of extracellular superoxide (Lundqvist and Dahlgren, 1996; Dahlgren and Karlsson, 1999). To assess the ability of this compound to measure bovine PMN ROS, blood PMN’s were stimulated with increasing concentrations of PMA (Fig. 8A) or zymosan (Fig. 8B) and chemiluminescence measured. Both PMA and zymosan elicited dosedependent increases in isoluminol-dependent chemiluminescence at concentrations 0.004 mM and 125 mg/ ml, respectively. Relative to unstimulated PMN’s, maximal increases in chemiluminescence of 159and 33-fold were induced by PMA and zymosan, respectively. DPI inhibited >95% of the isoluminoldependent chemiluminescence in wells containing PMN’s stimulated with PMA or zymosan, whereas, SOD inhibited these responses by >70% (Fig. 8C and D). In contrast to the lack of effect on zymosan-induced responses, pre-incubation with catalase inhibited 63% of the chemiluminescence response of PMA-activated PMN’s (Fig. 8E and F). Pre-treatment with both catalase and SOD blocked 97 and 70% of the chemiluminescence elicited by PMA and zymosan, respectively. 3.4. Intraassay and interassay variability of evaluated ROS assays The intraassay and interassay variability was determined for each assay as described in Section 2. The intraassay coefficient of variation for all assays was <7%, whereas, those values for interassay variation ranged from a low of 7.66% for the luminol assay to a high of 62.81% for the DHE flow cytometric assay (Table 1). 4. Discussion The current study compared a total of eight different assays for measuring ROS produced by bovine PMN’s stimulated with two common agonists used to elicit oxidative burst, PMA and opsonized zymosan. PMA evoked bovine PMN production of ROS at levels that were detectable by all assays, and with the exception of

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125

119

Fig. 7. Luminol-dependent chemiluminescence assay for reactive oxygen species (ROS) production. Blood neutrophils isolated from five cows were exposed to increasing concentrations of PMA (A) or zymosan (Zym) (B) in the presence of luminol, and chemiluminescence measured every 5 min (left y-axis). To analyze concentration-dependent generation of ROS, the area under the curve was calculated from plotted data points for each concentration (right y-axis). **Significantly increased (P < 0.01) relative to unstimulated neutrophils. In other studies, luminol-dependent chemiluminescence (left y-axis) was assayed in blood neutrophils (n = 5) pre-treated for 10 min with superoxide dismutase (SOD: 10 U/ml) (C and D), diphenyleneiodonium chloride (DPI: 8 mM) (C and D), catalase (Cat: 1000 U/ml) (E and F) or the combination of Cat and SOD (E and F), and subsequently exposed to PMA (40 nM) (C and E) or Zym (250 mg/ml) (D and F). The area under the curve for each experimental condition was calculated (right y-axis). **Significantly decreased (P < 0.01) relative to neutrophils stimulated with either PMA or zymosan alone.

the DHE assay, so did zymosan. The respective changes in fluorescence, chemiluminescence, and absorbance of the various assays were confirmed to be reflective of increased ROS production, as the majority of the increase in these responses was inhibited by the NADPH oxidase inhibitor, DPI (Table 2). SOD, which catalyzes the dismutation of superoxide to hydrogen peroxide, and catalase, which reduces

hydrogen peroxide to water, are cell-impermeable enzymes capable of scavenging extracellularly released ROS (Dahlgren and Karlsson, 1999). The properties of these enzymes enabled their use to evaluate the ability of the assays to discriminate between intracellular versus extracellular ROS, and to specifically identify the extracellular ROS generated (Table 2).

120

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125

Fig. 8. Isoluminol-dependent chemiluminescence assay for reactive oxygen species (ROS) production. Blood neutrophils isolated from five cows were exposed to increasing concentrations of PMA (A) or zymosan (Zym) (B) in the presence of isoluminol, and chemiluminescence measured (left y-axis). To analyze concentration-dependent generation of ROS, the area under the curve was calculated from plotted data points for each concentration (right y-axis). **Significantly increased (P < 0.01) relative to unstimulated neutrophils. In other studies, isoluminol-dependent chemiluminescence (left y-axis) was assayed in blood neutrophils (n = 5) pre-treated for 10 min with superoxide dismutase (SOD: 10 U/ml) (C and D), diphenyleneiodonium chloride (DPI: 8 mM) (C and D), catalase (Cat: 1000 U/ml) (E and F) or the combination of Cat and SOD (E and F), and subsequently exposed to PMA (40 nM) (C and E) or Zym (250 mg/ml) (D and F). The area under the curve for each experimental condition was calculated (right y-axis). **Significantly decreased (P < 0.01) relative to neutrophils stimulated with either PMA or zymosan alone.

The reduction of cytochrome c and MCLA-derived chemiluminescence have both been reported to be specific indicators of extracellular superoxide production (Nishida et al., 1989; Nakano, 1990; Pronai et al., 1992; Munzel et al., 2002). In the current study, the finding that SOD almost completely inhibited both

PMA- and zymosan-evoked responses detected by cytochrome c and MCLA establishes the specificity of these assays to solely detect extracellular superoxide produced by activated bovine PMN’s. In human leukocytes, changes in Amplex Redmediated fluorescence have been shown to specifically

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125 Table 1 Intraassay and interassay variability

Cytochrome c reduction assay MCLA chemiluminescence assay Amplex Red fluorescence assay CM-H2DCFDA fluorescence assay DHR fluorescence assay DHE flow cytometric assay Luminol chemiluminescence assay Isoluminol chemiluminescence assay a

Intraassay (CV %)a

Interassay (CV %)a

5.88 4.67 6.77 5.03 3.63 3.03 6.86 3.15

47.55 14.78 45.74 14.69 33.32 62.81 7.66 9.44

121

Joseph, 1999; Myhre et al., 2003; Walrand et al., 2003). That neither SOD nor catalase had a statistically significant inhibitory effect on PMA- or zymosanevoked increases in fluorescence in the CM-H2DCFDA and DHE assays suggests that these assays solely detect intracellular ROS generated by bovine PMN’s. Interestingly, although SOD demonstrated no inhibitory effect on DHR-mediated fluorescence generated from bovine PMN’s stimulated with either agonist, extracellularly-restricted catalase partially blocked these responses. This latter finding may seem inconsistent with reports that the oxidation of DHR by hydrogen peroxide and peroxynitrite anion is restricted to intracellular sources of these ROS (Henderson and Chappell, 1993; Kooy et al., 1994; Walrand et al., 2003). However, hydrogen peroxide crosses cellmembranes, and it has been proposed that scavenging of extracellular hydrogen peroxide may lead to the establishment of a concentration gradient favoring extracellular diffusion and, thus, corresponding decreases in its intracellular concentration (Crow, 1997). Consistent with the current findings with bovine PMN’s, catalase has been reported to block DHRmediated fluorescence in response to PMA in other cell types (Henderson and Chappell, 1993). Chemiluminescence generated by luminol is indicative of the production of intracellular and/or extracellular ROS (Briheim et al., 1984; Rest, 1994). There are conflicting findings as to the identity of the ROS that directly evoke luminol-dependent chemiluminescence and luminol has been reported to measure superoxide (Faulkner and Fridovich, 1993; Lundqvist and Dahlgren, 1996), hydroxyl radical (Yildiz and Demiryurek, 1998; Nemeth et al., 2002), hydrogen peroxide (Castro

Coefficient of variation (CV) values expressed as percentages.

reflect increases in extracellular hydrogen peroxide (Mohanty et al., 1997). The findings in the current study that catalase inhibited 98% of the PMA- and zymosan-evoked increases in Amplex Red-mediated fluorescence establishes the specificity of this assay to solely detect bovine PMN-generated extracellular hydrogen peroxide. Interestingly, Amplex Red fluorescence was higher among PMA-activated PMN’s incubated with SOD than those stimulated in the absence of SOD (Fig. 3C). Based on SOD’s ability to catalyze the conversion of superoxide to hydrogen peroxide, the corresponding increase in Amplex Red fluorescence is not surprising. There was also a tendency towards slightly increased fluorescence in zymosan-stimulated PMN’s exposed to SOD, but the increase did not reach a statistically significant level. Three of the assays evaluated in the present study, CM-H2DCFDA, DHE, and DHR, have been established to measure intracellular, but not extracellular, ROS from PMN’s derived from non-bovine sources (Wang and

Table 2 Summarized comparisons of assays used to measure bovine neutrophil reactive oxygen species (ROS) Assay

Cytochrome c MCLA Amplex Red H2DCFDA DHR DHE Luminol Isoluminol a b c d e

ROS



O2 O2 H2O2 H2O2, ONOO H2O2, ONOO O2 HOCl, O2, H2O2, ONOO, OH O2

Location a

E E E I I I E, I E, I

Stimulation indexb (PMA (ZYM)) 11 38 158 11 30 39 331 159

(5) (15) (14) (6) (13) (1) (448) (33)

ROS inhibitorsc DPI 70 (73) 82 (95) 100 (100) 85 (69) 98 (84) 96 (nd) 91 (100) 95 (99)

SOD 100 (95) 100 (100) –e(–) – (–) – (–) – (nd) 41 (–) 70 (75)

CAT d

nd (nd) nd (nd) 98 (100) – (–) 42 (21) – (nd) – (–) 63 (–)

CAT + SOD nd nd nd nd nd nd 66 97

(nd) (nd) (nd) (nd) (nd) (nd) (–) (70)

I: intracellular; E: extracellular. Maximal fold-increase relative to unstimulated neutrophils as determined in the dose–response studies. Data presented for each inhibitor is expressed as the percent inhibition of PMA- or zymosan-induced fold-increase in bovine neutrophil ROS. nd: not determined. –: not significantly inhibited.

122

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125

et al., 1996; Yildiz and Demiryurek, 1998), peroxynitrite (Radi et al., 1993), and hypochlorous acid (Brestel, 1985; Myhre et al., 2003). The finding in the present study that SOD partially blocked PMA-induced luminol chemiluminescence suggests the ability of this assay to detect extracellularly-generated bovine PMN superoxide. There was also a trend towards decreased fluorescence among zymosan-stimulated PMN’s exposed to SOD, but this difference did not reach a statistically significant level. Because zymosan was demonstrated to be a less potent inducer of superoxide production in both the cytochrome c and MCLA assays, the superoxide component responsible for the luminoldependent increases in chemiluminescence may be too small to be detectably inhibited by SOD. Catalase had no inhibitory effect on PMA- or zymosan-evoked luminol-responses despite the ability of these two agonists to induce PMN generation of extracellular hydrogen peroxide, as determined by increased Amplex Red-mediated fluorescence. This finding suggests that increases in luminol-dependent chemiluminescence do not reflect changes in extracellular hydrogen peroxide production. The combined findings with these two inhibitors suggests that the majority of the measurable change in bovine PMN luminol-dependent chemiluminescence reflects either intracellular increases in ROS production and/or extracellular increases in ROS other than superoxide and hydrogen peroxide. In contrast to luminol, isoluminol does not readily diffuse across cell membranes and, thus, preferentially detects extracellular ROS (Lundqvist et al., 1995). The finding in the current study that SOD inhibited the majority of isoluminol-mediated chemiluminescence in bovine PMN’s stimulated with either PMA or zymosan is consistent with previous findings with human PMN’s (Lundqvist and Dahlgren, 1996). Interestingly, catalase also partially inhibited the response evoked by PMA, but not zymosan. As demonstrated with the Amplex Red assay, hydrogen peroxide generation is stimulated to a much lower extent by zymosan than by PMA (Fig. 3). Thus, the ability of the isoluminol assay to detect catalase-mediated inhibition of extracellularly generated hydrogen peroxide may be beyond the sensitivity of the isoluminol assay due to the lower production of this ROS in response to zymosan. Exposure to both SOD and catalase completely inhibited PMA-induced changes, suggesting an additive effect of these two enzymes to block superoxide- and hydrogen peroxide-evoked increases in isoluminol chemiluminescence. The combination of catalase and SOD inhibited zymosan-induced changes to an equivalent extent as that of SOD alone consistent with the

inability of catalase to inhibit zymosan-induced increases in isoluminol-mediated chemiluminescence. Compared to classical assays such as ELISA’s, where identical samples can be compared on different days, the interassay variation among the current assays could not be evaluated with identical samples due to the short lifespan of PMN’s (Savill et al., 1989). Thus, the interassay variability reflected not only variation within the assays themselves on different days, but also variation in the PMN isolation and handling procedures. The individual interassay variability of unactivated PMN’s was substantially lower than that of activated PMN’s (data not shown) in the three assays with the highest interassay variability calculated for the foldincreases in stimulation (i.e., cytochrome c, Amplex Red, and DHE) (Table 1). This suggests that an additional source of variation may have included differential PMN responsiveness to stimulation with PMA on each day. Factors influencing PMN responsiveness could include differential environmental and/ or physiological influences on the repeatedly sampled animals. For example, heat stress due to daily fluctuations in environmental temperature may have altered PMN responsiveness similar to that reported by others investigating the chemotactic responses of bovine PMN’s (Elvinger et al., 1992). Veterinary manipulations, such as bleeding, can affect circulating corticosteroid levels (Alam and Dobson, 1986), which in turn, can downregulate PMN function (Murata and Hirose, 1991; Burton et al., 1995). Thus, variation in sampling time needed to draw blood samples from each cow, which can be influenced by animal cooperativity, could result in differential exposure of PMN to inflammatory regulating mediators prior to isolation from the cow. The results of studies investigating the kinetics of ROS production with PMN’s from other species using the cytochrome c (Sartorelli et al., 2000), MCLA (Pronai et al., 1992), Amplex Red (Mohanty et al., 1997), luminol, and isoluminol (Lundqvist and Dahlgren, 1996) assays are comparable to those in the present study with bovine PMN’s. Although we are not aware of published studies evaluating the detailed kinetics of PMN production of ROS as measured with the DHE, DHR, and CM-H2DCFDA assays, the initial detection of ROS at 30 min post-stimulation with these assays in the present study is consistent with times at which others have detected increases in these ROS using these probes (Rothe and Valet, 1990; Bassoe et al., 2003; Lee et al., 2003). The differential sensitivity of the assays to detect PMA- or zymosan-evoked responses in the current study is presumably due to the differential

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125

generation of specific ROS elicited by the two different agonists and the corresponding differential sensitivity of the respective assays to detect the individual ROS generated. 5. Conclusions The comparative findings of the current study clearly demonstrate that the assays differ in their applicability to the measurement of bovine PMN ROS. CMH2DCFDA, DHR, and DHE fluorescence assays are useful for the measurement of intracellular bovine PMN ROS, whereas, cytochrome c reduction, MCLA chemiluminescence, and Amplex Red fluorescence assays are applicable to the measurement of extracellular ROS. The specificity of the assays measuring extracellular bovine PMN ROS under the stated conditions was also defined and cytochrome c- and MCLA-based assays were established to be highly specific for measuring superoxide, whereas, Amplex Red was highly specific for the detection of hydrogen peroxide. In terms of sensitivity, the MCLA chemiluminescence assay was the only one that could detect measurable ROS in response to both of the lowest concentrations of PMA and zymosan tested. Acknowledgements The authors would like to acknowledge Glenn R. Welch for his technical assistance with the flow cytometer. Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. References Alam, M.G., Dobson, H., 1986. Effect of various veterinary procedures on plasma concentrations of cortisol, luteinising hormone and prostaglandin F2 alpha metabolite in the cow. Vet. Rec. 118, 7–10. Babior, B.M., 1984. Oxidants from phagocytes: agents of defense and destruction. Blood 64, 959–966. Babior, B.M., 1999. NADPH oxidase: an update. Blood 93, 1464– 1476. Baehner, R.L., 1990. Chronic granulomatous disease of childhood: clinical, pathological, biochemical, molecular, and genetic aspects of the disease. Pediatr. Pathol. 10, 143–153. Bannerman, D.D., Paape, M.J., Lee, J.W., Zhao, X., Hope, J.C., Rainard, P., 2004. Escherichia coli and Staphylococcus aureus elicit differential innate immune responses following intramammary infection. Clin. Diagn. Lab. Immunol. 11, 463–472. Bassoe, C.F., Li, N., Ragheb, K., Lawler, G., Sturgis, J., Robinson, J.P., 2003. Investigations of phagosomes, mitochondria, and acidic

123

granules in human neutrophils using fluorescent probes. Cytometry B Clin. Cytom. 51, 21–29. Becker, S., Dailey, L.A., Soukup, J.M., Grambow, S.C., Devlin, R.B., Huang, Y.C., 2005. Seasonal variations in air pollution particleinduced inflammatory mediator release and oxidative stress. Environ. Health Perspect. 113, 1032–1038. Brestel, E.P., 1985. Co-oxidation of luminol by hypochlorite and hydrogen peroxide implications for neutrophil chemiluminescence. Biochem. Biophys. Res. Commun. 126, 482–488. Briheim, G., Stendahl, O., Dahlgren, C., 1984. Intra- and extracellular events in luminol-dependent chemiluminescence of polymorphonuclear leukocytes. Infect. Immun. 45, 1–5. Burg, N.D., Pillinger, M.H., 2001. The neutrophil: function and regulation in innate and humoral immunity. Clin. Immunol. 99, 7–17. Burton, J.L., Kehrli Jr., M.E., Kapil, S., Horst, R.L., 1995. Regulation of L-selectin and CD18 on bovine neutrophils by glucocorticoids: Effects of cortisol and dexamethasone. J. Leukoc. Biol. 57, 317–325. Burvenich, C., Monfardini, E., Mehrzad, J., Capuco, A.V., Paape, M.J., 2004. Role of neutrophil polymorphonuclear leukocytes during bovine coliform mastitis: Physiology or pathology? Verh. K. Acad. Geneeskd. Belg. 66, 97–150. Burvenich, C., Van Merris, V., Mehrzad, J., Diez-Fraile, A., Duchateau, L., 2003. Severity of E. coli mastitis is mainly determined by cow factors. Vet. Res. 34, 521–564. Cai, T.Q., Weston, P.G., Lund, L.A., Brodie, B., McKenna, D.J., Wagner, W.C., 1994. Association between neutrophil functions and periparturient disorders in cows. Am. J. Vet. Res. 55, 934–943. Capuco, A.V., Paape, M.J., Nickerson, S.C., 1986. In vitro study of polymorphonuclear leukocyte damage to mammary tissues of lactating cows. Am. J. Vet. Res. 47, 663–668. Castro, L., Alvarez, M.N., Radi, R., 1996. Modulatory role of nitric oxide on superoxide-dependent luminol chemiluminescence. Arch. Biochem. Biophys. 333, 179–188. Crow, J.P., 1997. Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: Implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide 1, 145–157. Czuprynski, C.J., Hamilton, H.L., 1985. Bovine neutrophils ingest but do not kill Haemophilus somnus in vitro. Infect. Immun. 50, 431–436. Dahlgren, C., Karlsson, A., 1999. Respiratory burst in human neutrophils. J. Immunol. Meth. 232, 3–14. Dinauer, M.C., 1993. The respiratory burst oxidase and the molecular genetics of chronic granulomatous disease. Crit. Rev. Clin. Lab. Sci. 30, 329–369. El-Benna, J., Dang, P.M., Gougerot-Pocidalo, M.A., Elbim, C., 2005. Phagocyte NADPH oxidase: a multicomponent enzyme essential for host defenses. Arch. Immunol. Ther. Exp. (Warsz). 53, 199–206. Elvinger, F., Natzke, R.P., Hansen, P.J., 1992. Interactions of heat stress and bovine somatotropin affecting physiology and immunology of lactating cows. J. Dairy Sci. 75, 449–462. Fantone, J.C., Ward, P.A., 1985. Polymorphonuclear leukocytemediated cell and tissue injury: oxygen metabolites and their relations to human disease. Hum. Pathol. 16, 973–978. Faulkner, K., Fridovich, I., 1993. Luminol and lucigenin as detectors for O2. Free Radic. Biol. Med. 15, 447–451. Filatov, M.V., Varfolomeeva, E.Y., Ivanov, E.I., 1995. Flow cytofluorometric detection of inflammatory processes by measuring respiratory burst reaction of peripheral blood neutrophils. Biochem. Mol. Med. 55, 116–121.

124

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125

Forteza, R., Salathe, M., Miot, F., Forteza, R., Conner, G.E., 2005. Regulated hydrogen peroxide production by Duox in human airway epithelial cells. Am. J. Respir. Cell Mol. Biol. 32, 462–469. Gudmundsson, G.H., Agerberth, B., 1999. Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system. J. Immunol. Meth. 232, 45–54. Hampton, M.B., Kettle, A.J., Winterbourn, C.C., 1998. Inside the neutrophil phagosome: Oxidants, myeloperoxidase, and bacterial killing. Blood 92, 3007–3017. Henderson, L.M., Chappell, J.B., 1993. Dihydrorhodamine 123: a fluorescent probe for superoxide generation? Eur. J. Biochem. 217, 973–980. Jain, N.C., Paape, M.J., Berning, L., Salgar, S.K., Worku, M., 1991. Functional competence and monoclonal antibody reactivity of neutrophils from cows injected with Escherichia coli endotoxin. Comp. Haematol. Int. 1, 10–20. Kooy, N.W., Royall, J.A., Ischiropoulos, H., Beckman, J.S., 1994. Peroxynitrite-mediated oxidation of dihydrorhodamine 123. Free Radic. Biol. Med. 16, 149–156. Lauzon, K., Zhao, X., Bouetard, A., Delbecchi, L., Paquette, B., Lacasse, P., 2005. Antioxidants to prevent bovine neutrophilinduced mammary epithelial cell damage. J. Dairy Sci. 88, 4295–4303. LeBel, C.P., Ischiropoulos, H., Bondy, S.C., 1992. Evaluation of the probe 20 ,70 -dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem. Res. Toxicol. 5, 227–231. Ledbetter, T.K., Paape, M.J., Douglass, L.W., 2001. Cytotoxic effects of peroxynitrite, polymorphonuclear neutrophils, free-radical scavengers, inhibitors of myeloperoxidase, and inhibitors of nitric oxide synthase on bovine mammary secretory epithelial cells. Am. J. Vet. Res. 62, 286–293. Lee, V.M., Quinn, P.A., Jennings, S.C., Ng, L.L., 2003. NADPH oxidase activity in preeclampsia with immortalized lymphoblasts used as models. Hypertension 41, 925–931. Leino, L., Paape, M.J., 1993. Comparison of the chemiluminescence responses of bovine neutrophils to differently opsonized zymosan particles. Am. J. Vet. Res. 54, 1055–1059. Leino, L., Paape, M.J., 1996. Regulation of activation of bovine neutrophils by aggregated immunoglobulin G. Am. J. Vet. Res. 57, 1312–1316. Li, S.Y., Gomelsky, M., Duan, J., Zhang, Z., Gomelsky, L., Zhang, X., Epstein, P.N., Ren, J., 2004. Overexpression of aldehyde dehydrogenase-2 (ALDH2) transgene prevents acetaldehyde-induced cell injury in human umbilical vein endothelial cells: Role of ERK and p38 mitogen-activated protein kinase. J. Biol. Chem. 279, 11244–11252. Long, E., Capuco, A.V., Wood, D.L., Sonstegard, T., Tomita, G., Paape, M.J., Zhao, X., 2001. Escherichia coli induces apoptosis and proliferation of mammary cells. Cell Death Differ. 8, 808–816. Lundqvist, H., Dahlgren, C., 1996. Isoluminol-enhanced chemiluminescence: a sensitive method to study the release of superoxide anion from human neutrophils. Free Radic. Biol. Med. 20, 785–792. Lundqvist, H., Kricka, L.J., Stott, R.A., Thorpe, G.H., Dahlgren, C., 1995. Influence of different luminols on the characteristics of the chemiluminescence reaction in human neutrophils. J. Biolumin. Chemilumin. 10, 353–359. Mohanty, J.G., Jaffe, J.S., Schulman, E.S., Raible, D.G., 1997. A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative. J. Immunol. Meth. 202, 133–141.

Munzel, T., Afanas’ev, I.B., Kleschyov, A.L., Harrison, D.G., 2002. Detection of superoxide in vascular tissue. Arterioscler. Thromb. Vasc. Biol. 22, 1761–1768. Murata, H., Hirose, H., 1991. Suppression of bovine neutrophil function by sera from cortisol-treated calves. Br. Vet. J. 147, 63–70. Myhre, O., Andersen, J.M., Aarnes, H., Fonnum, F., 2003. Evaluation of the probes 2’,7’-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem. Pharmacol. 65, 1575–1582. Nagahata, H., 2004. Bovine leukocyte adhesion deficiency (BLAD): a review. J. Vet. Med. Sci. 66, 1475–1482. Nakano, M., 1990. Determination of superoxide radical and singlet oxygen based on chemiluminescence of luciferin analogs. Meth. Enzymol. 186, 585–591. Nemeth, K., Furesz, J., Csikor, K., Schweitzer, K., Lakatos, S., 2002. Luminol-dependent chemiluminescence is related to the extracellularly released reactive oxygen intermediates in the case of rat neutrophils activated by formyl-methionyl-leucyl-phenylalanine. Haematologia (Budap) 31, 277–285. Nishida, A., Kimura, H., Nakano, M., Goto, T., 1989. A sensitive and specific chemiluminescence method for estimating the ability of human granulocytes and monocytes to generate O2. Clin. Chim. Acta 179, 177–181. Paape, M.J., Bannerman, D.D., Zhao, X., Lee, J.W., 2003. The bovine neutrophil: structure and function in blood and milk. Vet. Res. 34, 597–627. Possel, H., Noack, H., Augustin, W., Keilhoff, G., Wolf, G., 1997. 2,7Dihydrodichlorofluorescein diacetate as a fluorescent marker for peroxynitrite formation. FEBS Lett. 416, 175–178. Pronai, L., Nakazawa, H., Ichimori, K., Saigusa, Y., Ohkubo, T., Hiramatsu, K., Arimori, S., Feher, J., 1992. Time course of superoxide generation by leukocytes—the MCLA chemiluminescence system. Inflammation 16, 437–450. Radi, R., Cosgrove, T.P., Beckman, J.S., Freeman, B.A., 1993. Peroxynitrite-induced luminol chemiluminescence. Biochem. J. 290 (Pt 1), 51–57. Rambeaud, M., Clift, R., Pighetti, G.M., 2006. Association of a bovine CXCR2 gene polymorphism with neutrophil survival and killing ability. Vet. Immunol. Immunopathol. 111, 231–238. Rest, R.F., 1994. Measurement of human neutrophil respiratory burst activity during phagocytosis of bacteria. Meth. Enzymol. 236, 119–136. Ricevuti, G., 1997. Host tissue damage by phagocytes. Ann. N.Y. Acad. Sci. 832, 426–448. Roth, J.A., Kaeberle, M.L., 1981. Effects of in vivo dexamethasone administration on in vitro bovine polymorphonuclear leukocyte function. Infect. Immun. 33, 434–441. Rothe, G., Valet, G., 1990. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 20 ,70 -dichlorofluorescin. J. Leukoc. Biol. 47, 440–448. Saez, F., Motta, C., Boucher, D., Grizard, G., 2000. Prostasomes inhibit the NADPH oxidase activity of human neutrophils. Mol. Hum. Reprod. 6, 883–891. Salgar, S.K., Paape, M.J., Alston-Mills, B., Miller, R.H., 1991. Flow cytometric study of oxidative burst activity in bovine neutrophils. Am. J. Vet. Res. 52, 1201–1207. Sartorelli, P., Paltrinieri, S., Comazzi, S., 2000. Non-specific immunity and ketone bodies. II. In vitro studies on adherence and superoxide anion production in ovine neutrophils. J. Vet. Med. A Physiol. Pathol. Clin. Med. 47, 1–8. Savill, J.S., Wyllie, A.H., Henson, J.E., Walport, M.J., Henson, P.M., Haslett, C., 1989. Macrophage phagocytosis of aging neutrophils

M. Rinaldi et al. / Veterinary Immunology and Immunopathology 115 (2007) 107–125 in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages. J. Clin. Invest. 83, 865–875. Schalm, O.W., Lasmanis, J., Jain, N.C., 1976. Conversion of chronic staphylococcal mastitis to acute gangrenous mastitis after neutropenia in blood and bone marrow produced by an equine antibovine leukocyte serum. Am. J. Vet. Res. 37, 885–890. Segal, A.W., 2005. How neutrophils kill microbes. Annu. Rev. Immunol. 23, 197–223. Tarpey, M.M., Wink, D.A., Grisham, M.B., 2004. Methods for detection of reactive metabolites of oxygen and nitrogen: In vitro and in vivo considerations. Am. J. Physiol. Regul. Integr. Comp. Physiol. 286, R431–R444. Teufelhofer, O., Weiss, R.M., Parzefall, W., Schulte-Hermann, R., Micksche, M., Berger, W., Elbling, L., 2003. Promyelocytic HL60 cells express NADPH oxidase and are excellent targets in a rapid spectrophotometric microplate assay for extracellular superoxide. Toxicol. Sci. 76, 376–383. Tkalcevic, J., Novelli, M., Phylactides, M., Iredale, J.P., Segal, A.W., Roes, J., 2000. Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity 12, 201–210.

125

Walrand, S., Valeix, S., Rodriguez, C., Ligot, P., Chassagne, J., Vasson, M.P., 2003. Flow cytometry study of polymorphonuclear neutrophil oxidative burst: a comparison of three fluorescent probes. Clin. Chim. Acta 331, 103–110. Wang, H., Joseph, J.A., 1999. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic. Biol. Med. 27, 612–616. Weber, P.S., Madsen, S.A., Smith, G.W., Ireland, J.J., Burton, J.L., 2001. Pre-translational regulation of neutrophil L-selectin in glucocorticoid-challenged cattle. Vet. Immunol. Immunopathol. 83, 213–240. Weiss, S.J., 1989. Tissue destruction by neutrophils. N. Engl. J. Med. 320, 365–376. Yildiz, G., Demiryurek, A.T., 1998. Ferrous iron-induced luminol chemiluminescence: a method for hydroxyl radical study. J. Pharmacol. Toxicol. Meth. 39, 179–184. Zhou, M., Diwu, Z., Panchuk-Voloshina, N., Haugland, R.P., 1997. A stable non-fluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal. Biochem. 253, 162–168.