Journal of Dairy Science
Volume 89, Issue 11 , Pages 4188-4201, November 2006

Effect of Cis-Urocanic Acid on Bovine Neutrophil Generation of Reactive Oxygen Species

  • M. Rinaldi

      Affiliations

    • Department of Veterinary Pathology, Hygiene and Public Health, University of Milan, Italy 20133
    • ,
  • P. Moroni

      Affiliations

    • Department of Veterinary Pathology, Hygiene and Public Health, University of Milan, Italy 20133
    • ,
  • L. Leino

      Affiliations

    • BioCis Pharma, Ltd., Turku, Finland 20520
    • ,
  • J. Laihia

      Affiliations

    • BioCis Pharma, Ltd., Turku, Finland 20520
    • ,
  • M.J. Paape

      Affiliations

    • Bovine Functional Genomics Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705
    • ,
  • D.D. Bannerman

      Affiliations

    • Bovine Functional Genomics Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705
    • Corresponding Author InformationCorresponding author.

Received 22 March 2006; accepted 25 April 2006.

Article Outline

Abstract 

Neutrophils play a fundamental role in the host innate immune response during mastitis and other bacterial-mediated diseases of cattle. One of the critical mechanisms by which neutrophils contribute to host innate immune defenses is through their ability to phagocytose and kill bacteria. The ability of neutrophils to kill bacteria is mediated through the generation of reactive oxygen species (ROS). However, the extracellular release of ROS can be deleterious to the host because ROS induce tissue injury. Thus, in diseases such as mastitis that are accompanied by the influx of neutrophils, the generation of large quantities of ROS may result in significant injury to the mammary epithelium. cis-Urocanic acid (cis-UCA), which is formed from the UV photoisomerization of the trans isoform found naturally in human and animal skin, is an immunosuppressive molecule with anti-inflammatory properties. Little is known about the effect of cis-UCA on neutrophils, although one report demonstrated that it inhibits human neutrophil respiratory burst activity. However, the nature of this inhibition remains unknown. Because of the potential therapeutic use that a molecule such as cis-UCA may have in blocking excessive respiratory burst activity that may be deleterious to the host, the ability of cis-UCA to inhibit bovine neutrophil production of ROS was studied. Further, because neutrophil generation of ROS is necessary for optimal neutrophil bactericidal activity, a response which is critical for the host innate immune defense against infection, the effects of cis-UCA on bovine neutrophil phagocytosis and bacterial killing were assayed. cis-Urocanic acid dose-dependently inhibited the respiratory burst activity of bovine neutrophils as measured by luminol chemiluminescence. Subsequently, the effect of cis-UCA on the production of specific oxygen radicals was investigated using more selective assays. Using 2 distinct assays, we established that cis-UCA inhibited the generation of extracellular superoxide. In contrast, cis-UCA had no effect on the generation of intracellular levels of superoxide or other ROS. At concentrations that inhibited generation of extracellular superoxide, bovine neutrophil phagocytosis and bacterial activity remained intact. Together, these data suggest that cis-UCA inhibits the tissue-damaging generation of extracellular ROS while preserving neutrophil bactericidal activity.

Key words: dairy cow, neutrophil, reactive oxygen species, respiratory burst

 

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Introduction 

Polymorphonuclear neutrophils (PMN) play an essential role in defending the host against invading microbial pathogens. Defects in PMN recruitment to the site of infection (Schalm et al., 1976; Nagahata, 2004) or reduced PMN functioning (Roth and Kaeberle, 1981; Cai et al., 1994; Tkalcevic et al., 2000) or both are associated with impaired host clearance of bacteria. Recruitment of PMN to the site of infection is mediated by chemokines, complement components, and arachidonate metabolites, and the rapidity with which PMN are recruited influences the outcome of infection (Burvenich et al., 2003; Paape et al., 2003). Once at the site of infection, activated PMN recognize, engulf, and kill bacterial pathogens, the latter of which is dependent upon the production of microbicidal peptides, proteases, and reactive oxygen species (ROS; Gudmundsson and Agerberth, 1999; Burg and Pillinger, 2001).

The generation of ROS, also referred to as respiratory or oxidative burst activity, is a characteristic property of phagocytes such as the PMN. Oxidative burst activity plays a critical role in PMN-mediated defenses against bacteria, because defects in the ability to generate ROS are associated with increased susceptibility to infection (Baehner, 1990; Dinauer, 1993). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a membrane-associated, multicomponent protein enzyme complex that participates directly in the generation of ROS (Hampton et al., 1998; Babior, 1999). In unstimulated PMN, this enzyme complex remains dormant. Upon activation, the enzyme complex triggers the shuttling of electrons from cytosolic NADPH to oxygen present in the phagosomal compartment or in the extracellular milieu. In general, one molecule of oxygen acts as an acceptor for a single donated electron, leading to the generation of superoxide anion () (Weiss, 1989). Superoxide anion is the most proximally generated ROS by NADPH oxidase, and, in turn, serves as a precursor to the formation of other powerful oxidants (Weiss, 1989; Hampton et al., 1998). Superoxide anions can rapidly form hydrogen peroxide (H2O2) by spontaneous dismutation or enzymatic dismutation by superoxide dismutase. The interaction of superoxide anion and hydrogen peroxide in the presence of a transition metal catalyst can give rise to the formation of hydroxyl radical (OH·), a powerful oxidant. Myeloperoxidase (MPO), a major component of the azurophilic granules, catalyzes the transformation of hydrogen peroxide in the presence of halogens (e.g., Cl−) into highly toxic molecules such as hypochlorous acid (HOCl−). Hypochlorous acid can, in turn, react with hydrogen peroxide or amines to form singlet oxygen (1O2) and chloramines, respectively. There is some controversy regarding the actual direct bactericidal activity of the individual ROS in vivo (Hampton et al., 1998; Segal, 2005); however, it is clear that NADPH oxidase-dependent generation of superoxide anion initiates a cascade resulting in the production of an array of ROS that mediate PMN bactericidal activity.

Although the ability to generate ROS is essential for optimal PMN bactericidal activity, ROS do not discriminate against pathogens and host tissue and induce injury to both. Increasing evidence suggests that ROS comprise some of the most injurious substances released from cells and that they exert their deleterious effect through a variety of mechanisms, including lipid peroxidation and the modification of DNA, which can include the induction of strand breaks (Henson and Johnston, 1987; Weiss, 1989; Hampton et al., 1998). In addition, there is evidence that the oxidizing environment created by the generation of ROS can enhance PMN-derived protease activity, the latter of which is also injurious to host tissues. In diseases such as mastitis, where PMN concentrations approach 50million cells/mL of milk following IMI (Bannerman et al., 2004), the potential for tissue damage is great. In fact, several reports suggest that activated PMN induce direct injury to the mammary epithelium both in vitro and in vivo (Capuco et al., 1986; Ledbetter et al., 2001; Long et al., 2001; Burvenich et al., 2004; Lauzon et al., 2005). The increasing awareness of the extent to which tissue injury is induced by activated PMN continues to generate interest in the development of therapeutics that can limit extracellular generation of ROS without impairing PMN microbicidal activity against phagocytosed bacteria.

Ultraviolet radiation is well established in a variety of model systems to exert both a local and systemic immunosuppressive effect (Norval, 2001). Urocanic acid, which is naturally present in human and animal skin in the stratum corneum of the epidermis, is a major cutaneous absorber of UV radiation (Norval and ElGhorr, 2002). Urocanic acid is formed as a trans isomer from histidine and, upon UV exposure, is photoisomerized to cis-urocanic acid (cis-UCA). cis-Urocanic acid exerts an array of immunomodulatory properties both in vitro and in vivo and is believed to be partially responsible for the immunosuppressive effects of UV radiation. Evidence demonstrating an immunosuppressive role for cis-UCA include: 1) intradermal administration of cis-UCA inhibits the delayed-type hypersensitivity reaction to herpes simplex virus type 1 (Ross et al., 1986) and tumor antigens (Beissert et al., 2001); 2) cis-UCA suppresses the induction and elicitation of contact hypersensitivity (Lauerma et al., 1995; Hart et al., 1997; Reeve et al., 1998), possibly through the modulation of dermal mast cells (Wille et al., 1999) or epidermal Langerhans cells (Kurimoto and Streilein, 1992); 3) cis-UCA inhibits the proliferation and antigen-presenting activity of T cells from the spleen, and this inhibition correlates with IL-10 upregulation (Holan et al., 1998); and 4) allograft rejection and graft-vs.-host reactions are suppressed following cis-UCA administration (Gruner et al., 1992; Guymer and Mandel, 1993; Filipec et al., 1998). In addition to its suppressive effect on cell-mediated immunity associated with adaptive immune responses, cis-UCA also impairs cell-mediated innate immune responses. cis-Urocanic acid is reported to impair natural killer cell activity (Gilmour et al., 1993), monocyte production of TNF-α (Hart et al., 1993), and human PMN respiratory burst activity (Kivisto et al., 1996). Although there is a large body of evidence that cis-UCA suppresses cell-mediated innate and adaptive immune responses, whether this molecule acts directly to inhibit these immune or inflammatory responses or induces the activation of another immunosuppressive molecule remains unclear.

Based on the diverse immunosuppressive effects that cis-UCA has on different cells of the immune system, this molecule may be an attractive therapeutic agent for diminishing excessive inflammation that can be deleterious to the host. Because activated PMN are known to induce tissue injury, at least in part through ROS generation (Weiss, 1989), and cis-UCA has been demonstrated in one report to inhibit the respiratory burst activity of human PMN (Kivisto et al., 1996), the influence of this molecule on bovine PMN generation of ROS was assessed. In addition, because the actual mechanism of cis-UCA-mediated inhibition of respiratory burst activity has not been reported for any cell type, selective assaying of the effect of cis-UCA on distinct ROS was performed. Finally, because intracellular killing of bacteria by PMN is essential for host control of infection, the effect of cis-UCA on PMN phagocytosis and intracellular killing were evaluated.

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Materials and Methods 

Reagents 

cis-Urocanic acid (BioCis Pharma, Ltd., Turku, Finland) was prepared as a 37.5mM stock solution in Hanks’ balanced salt solution (HBSS) or PBS at pH 6.5. Hanks’ balanced salt solution and PBS, prepared at a pH 6.5 without cis-UCA, were used as vehicle controls in the various in vitro assays. Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione; Sigma Chemical Co., St. Louis, MO) was prepared as a 1 M stock solution in dimethyl sulfoxide (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 10mM stock solutions in DMSO. Methyl cypridina luciferin analog [MCLA; 2-methyl-6-(4-methoxyphenyl)-3,7-dih ydroimidazol[1,2-a]pyrazin-3-one hydrochloride] and 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein di-acetate acetyl ester (CM-H2DCFDA; both from Molecular Probes, Inc.) were prepared as 1mM stock solutions in DMSO. Cytochrome c from bovine heart and superoxide dismutase (SOD) from bovine erythrocytes (both from Sigma Chemical Co.) were prepared as 1mM and 1,000 units/mL of stock solutions, respectively, in calcium- and magnesium-free HBSS (CMF-HBSS).

Cows 

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 animals in this study were approved by the Beltsville Agricultural Research Center's Animal Care and Use Committee.

Isolation of Bovine Blood PMN 

Blood was obtained from the tail veins of healthy lactating Holstein cows, collected into Vacutainer glass tubes containing acid–citrate–dextrose (Becton Dickinson Corp, Franklin, Lakes, NJ), inverted ×5, and stored on ice. Polymorphonuclear neutrophils were isolated using a Percoll gradient as previously described (Weber et al., 2001). Briefly, 20mL of blood was transferred to 50-mL polypropylene conical tubes and centrifuged (1,000 ×g) for 20min at 4°C. The plasma and buffy coat were aseptically aspirated and discarded. The remaining cells were suspended in 34mL of ice-cold PBS and the suspension was slowly pipetted down the side of a clean 50-mL polypropylene conical tube containing 10mL of 1.084g/mL of Percoll (Sigma Chemical Co.). The tubes were centrifuged (400×g) for 40min at 22°C. The supernatant, mononuclear cell layer, and Percoll were aseptically aspirated and a pellet composed of PMN and erythrocytes was retained. Erythrocytes were lysed by mixing 1vol.of cells with 2vol.of an ice-cold 0.2% NaCl solution and inverting the tube for 1min. Tonicity was restored by the addition of 0.5vol.of a 3.7% NaCl solution. The tubes were centrifuged (500×g) for 2min at 4°C. The cell pellet was washed twice by resuspension in CMF-HBSS and recentrifugation for 1min at 4°C. 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. Purity of PMN was >90% and viability >95%. Cells suspended in either CMF-HBSS or PBS were maintained on ice until used in the various assays described below.

Luminol Chemiluminescence Assay 

Polymorphonuclear neutrophils (2×105) suspended in 10μL of CMF-HBSS were incubated with 160μL of either HBSS or increasing concentrations of cis-UCA for 10min at 37°C. Ten microliters of luminol (10mM) and 20μL of PMA (400nM) were added and chemiluminescence (CL) was measured every 5min with a Veritas microplate luminometer (Turner Biosystems Inc., Sunnyvale, CA). Background values, defined as the mean CL values of unactivated PMN, were subtracted from all readings.

Caspase Activity Assay 

Caspase activity was measured using a fluorimetric homogeneous caspase activity assay kit (Roche Diagnostics Corp., Indianapolis, IN). Polymorphonuclear neutrophils (4×105) suspended in 50μL of RPMI supplemented with 10% heat-inactivated (56°C for 30min) newborn bovine calf serum (Cambrex Bio Science, Inc, Walkersville, MD) were incubated with 27μL of either PBS or cis-UCA (37.5mM) and 10μL of either HBSS or PMA (400nM). All reaction volumes were adjusted to 100μL with PBS. Fluorescence activity was measured after 0, 1, 2, 4, and 8h on a Synergy HT multimodal plate reader (BioTek Instruments, Inc., Winooski, VT) at an excitation wavelength of 485nm and an emission wavelength of 528nm. Plates were maintained at 37°C between readings.

Trypan Blue Exclusion Assay 

Polymorphonuclear neutrophils (4×105) suspended in 50μL of RPMI supplemented with 10% heat-inactivated newborn bovine calf serum were incubated with 27μL of either PBS or cis-UCA (37.5mM) and 10μL of either HBSS or PMA (400nM). All reaction volumes were adjusted to 100μL with PBS. After various incubation times at 37°C, PMN viability was determined by adding 50μL of each sample to 50μL of trypan blue and scoring the first 100 cells encountered in the field of view of a light microscope as either alive or dead based on the uptake of trypan blue.

MCLA CL Assay 

Polymorphonuclear neutrophils (4×105) suspended in 7.5μL of CMF-HBSS were incubated with 120μL of either HBSS or increasing concentrations of cis-UCA for 10min at 37°C. A 7.5-μL quantity of MCLA (2μM) and a 15-μL quantity of PMA (400nM) were added to the cells and CL was measured every 5min with a microplate luminometer. The fold increase in superoxide production was determined by calculating the ratio of the arbitrary CL values of PMA-activated PMN to the CL values of unactivated PMN.

Cytochrome c Reduction Assay 

Polymorphonuclear neutrophils (2×105) suspended in 20μL of CMF-HBSS were incubated with 160μL of either HBSS or increasing concentrations of cis-UCA for 10min at 37°C. A 10-μL quantity of the 1-mM stock solution of cytochrome c and a 10-μL quantity of PMA (1mM) were added to the cells and absorbance was measured on a plate reader (BioTek Instruments, Inc.). Optical density (OD) was measured at 10-min intervals at a wavelength of 550nm. A background correction reading at 670nm was subtracted from the 550-nm absorbance readings. Superoxide production was calculated as previously described (Sartorelli et al., 2000) using the following equation: superoxide (nmol) = [(OD550 – reference OD670)×100]/6.3. Background values, defined as the mean amount of superoxide (nmol) produced by unactivated PMN, were subtracted from all values. To confirm the specificity of the assay to detect superoxide production, parallel reactions were run that included the addition of 2μL of SOD (1,000 units/mL) to wells containing PMA-stimulated PMN.

DHE Flow Cytometric Assay 

Polymorphonuclear neutrophils (1×106) suspended in 500μL of CMF-HBSS were incubated with 100μL of DHE (200μM) and either 160μL of HBSS or cis-UCA (37.5mM). All reaction volumes were adjusted to 1.8mL with HBSS. As a positive control, reactions substituting the 160μL of HBSS with an equivalent volume of DPI (100μM), the latter of which inhibits the oxidation of DHE (Katsuyama et al., 2002), were run in parallel. Parallel reactions were also set up that included the addition of 20μL of SOD (1,000 units/mL) to PMA-stimulated PMN. Following a 15-min incubation at 37°C in a shaking (30rpm) water bath, 200μL of PMA (400nM) was added to the cells and the incubation was allowed to proceed. At 15 and 45min after the addition of PMA, fluorescence was measured with a flow cytometer (Coulter Electronics, Inc.) equipped with an air-cooled argon ion laser. The laser was set at a 488-nm wavelength, 7.0 to 7.5 A of current, and 15 mW of power, and aligned using fluorospheres (Coulter fullbrite grade 2, 9.56μm diameter; Epics Division of Coulter Corp., Hialeah, FL). A 400-μL quantity of 1% methylene blue was added to each sample immediately prior to assaying on the flow cytometer to quench extracellular fluorescence (Jain et al., 1991). A relative fluorescence index 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).

CM-H2DCFDA Fluorescence Assay 

Polymorphonuclear neutrophils (4×105) suspended in 7.5μL of CMF-HBSS were incubated with 120μL of either HBSS or increasing concentrations of cis-UCA for 10min at 37°C. As a positive control, reactions substituting the 120μL of HBSS with an equivalent volume of DPI (10μM), the latter of which inhibits NADPH oxidase and corresponding generation of ROS detected with CM-H2DCFDA (Chandel et al., 1998; Andersen et al., 2003; Jackson et al., 2004), were run in parallel. A 7.5-μL quantity of CM-H2DCFDA (200μM) and 15μL of PMA (400nM) were added to the cells and fluorescence was measured every 15min on a fluorescent plate reader (BioTek Instruments, Inc.) at an excitation wavelength of 485nm and an emission wavelength of 528nm as previously described (Wang and Joseph, 1999). Background values, defined as the mean fluorescent values of unactivated PMN, were subtracted from all readings.

PMN Phagocytosis and Killing 

Staphylococcus aureus strain 305 (American Type Culture Collection, Manassas, VA) was inoculated on a blood agar plate (Becton Dickinson Diagnostic Systems, Inc., Sparks, MD) and incubated overnight at 37°C. Ten colonies were transferred from the plate to 10mL of brain–heart infusion broth (Becton Dickinson Diagnostic Systems, Inc.) and incubated overnight at 37°C at 225rpm. The tube was subsequently placed in an ice-water bath and mixed by swirling. A 0.1-mL aliquot from the tube was serially diluted in PBS and 0.1mL quantities of the resulting dilutions were spread on blood agar plates. The plates were incubated overnight at 37°C and the stock culture was maintained at 4°C. After determining the concentration (cfu/mL) of the stock culture based on the colony counts of the spread plates, the stock culture was diluted in PBS to yield a final concentration of 1.55×108 cfu/mL.

To assess PMN phagocytosis and killing, 200μL of Staph. aureus (3.1×107 cfu) was added to tubes with or without 1×106 PMN suspended in 1mL of PBS, 400μL of pooled (n=5 cows), heat-inactivated bovine serum, and 160μL of either cis-UCA (37.5mM), DPI (100μM), or HBSS. The ratio of bacteria to PMN was 31:1. The reaction volume was adjusted to 2mL with PBS and the samples were placed on an orbital shaker for 60min at 39°C. All reactions were set up in duplicate.

To determine the percentage of PMN containing phagocytosed bacteria and the actual number of phagocytosed bacteria per PMN, Wright-stained cytospin centrifuge slides were prepared using a 50-μL aliquot of each reaction (Dulin et al., 1982). The first 100 PMN encountered in the field of view of a light microscope were scored as either positive or negative for intracellular bacteria. For those PMN scored as positive, the number of intracellular bacteria was enumerated.

To evaluate whether cis-UCA affected the bactericidal activity of PMN, 1.9mL of the remaining reaction were sonicated with a Virsonic disrupter (Virtis Co., Gardiner, NY) at a power setting of 35 for 60s (Paape and Guidry, 1977). Rupture of the PMN was verified by microscopic examination. Sonication had no effect on bacterial viability (data not shown). A 0.1-mL aliquot from each sonicated reaction was serially diluted in PBS and 0.1mL quantities of the resulting dilutions were spread on blood agar plates. The plates were incubated for 18h at 37°C and the colonies enumerated. The percentage of bacteria killed was determined by calculating the difference in the number of bacteria incubated in the absence and presence of PMN and dividing this difference by the number of bacteria incubated in the absence of PMN.

Statistical Methods 

For luminol-, MCLA-, cytochrome c-, and CM-H2DCFDA-based assays involving multiple measurements of CL, absorbance, or fluorescence over time, 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). For all in vitro assays, a one-way ANOVA was used to compare the mean responses between experimental groups and activated PMN (i.e., PMN incubated with PMA alone). The Tukey post hoc comparison test was used to determine between which groups significant differences existed. All statistical analyses were performed using GraphPad Prism software. A P-value of <0.05 was considered significant.

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Results 

cis-UCA Inhibits Bovine PMN Respiratory Burst Activity 

Luminol-derived CL, which is elicited in response to the generation of an array of intracellular and extracellular ROS, including superoxide anion, hydroxyl radicals, hydrogen peroxide, peroxynitrite, and hypochlorous acid (Saez et al., 2000; Munzel et al., 2002), was used to assess the effect of cis-UCA on PMN-induced respiratory burst (Figure 1). Increases in luminol-dependent CL were evident within 45min of stimulation with PMA and continued to increase until reaching a plateau 60min later (Figure 1A). cis-Urocanic acid dose-dependently inhibited the luminol-dependent CL of PMA-stimulated PMN. Overall peak luminescence for each experimental condition was calculated as the area under the respective curve for the last 30min of CL measurements (Figure 1B). Concentrations as low as 30μM cis-UCA partially blocked PMN generation of ROS by ∼23%. Intermediate concentrations of cis-UCA (0.3 to 1mM) inhibited ∼63% of this response, whereas the highest concentration tested (10mM) completely abrogated luminol-dependent CL.

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  • Figure 1. 

    Effect of cis-urocanic acid (cis-UCA) on bovine neutrophil oxidative burst activity. Blood neutrophils isolated from 5 cows were exposed to increasing concentrations of cis-UCA for 10min. Neutrophils were subsequently stimulated with phorbol 12-myristate,13-acetate (PMA, 40nM) in the presence of luminol, and chemiluminescence values were measured every 5min. Background values, defined as the chemiluminescence of unstimulated neutrophils incubated with luminol, were subtracted from all readings, and the difference is reported as the mean (±SE) chemiluminescence in arbitrary units (A). To analyze the effect of cis-UCA on peak generation of reactive oxygen species, the area under the curve was calculated from plotted data points for each experimental condition for the last 30min of chemiluminescence measurements (B). *,**Decreased (P<0.05 and P<0.01, respectively) relative to neutrophils stimulated with PMA in the absence of cis-UCA.

The Onset of Spontaneous Bovine PMN Apoptosis and Injury Is Not Influenced by cis-UCA 

To determine whether the ability of cis-UCA to inhibit respiratory burst activity in PMN could be attributed to injury or cell death or both that would nonspecifically impair PMN functioning, including respiratory burst activity, the effect of cis-UCA on PMN apoptosis and viability was assayed (Figure 2). Changes in cas-pase activity were used to asses whether cis-UCA influenced the onset of apoptosis in unstimulated and PMA-stimulated PMN apoptosis (Figure 2A). Within 4h of incubation, unstimulated and PMA-stimulated PMN demonstrated increased caspase activity relative to freshly isolated PMN exposed to HBSS or PMA and immediately assayed for caspase activity (time 0). Further increases in caspase activity were evident after 8h of incubation. Regardless of the time point, comparable levels of caspase activity were observed among activated and unactivated PMN regardless of exposure to cis-UCA (10mM).

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  • Figure 2. 

    Effect of cis-urocanic acid (cis-UCA) on bovine neutrophil apoptosis and viability. Blood polymorphonuclear neutrophils (PMN) isolated from 5 cows were stimulated with phorbol 12-myristate,13-acetate (+PMA, 40nM) in the presence or absence of 10mM cis-UCA. Parallel studies were also performed with unstimulated (−PMA) neutrophils. Neutrophils were either immediately (time 0) assayed for caspase activity (A) and trypan blue exclusion (B) or incubated at 37°C for increasing time intervals and subsequently assayed. Caspase activity is reported in mean (±SE) arbitrary fluorescence units (A) and viability is reported as the mean (±SE) percentage of neutrophils that excluded trypan blue (B). *,**Different (P<0.05 and P<0.01, respectively) relative to time 0 measurements of neutrophils exposed to identical conditions.

To evaluate the ability of cis-UCA to induce cell injury, PMN treated in parallel as above were assessed for their ability to exclude trypan blue (Figure 2B). In contrast to the early onset of apoptosis, loss of membrane integrity as assessed by trypan blue uptake was not observed until 24h after incubation. The percentage of injured cells at this time (∼31 to 43%) was comparable regardless of whether PMN were activated with PMA or exposed to cis-UCA or both.

cis-UCA Inhibits Bovine PMN Generation of Extracellular Superoxide Anion 

The MCLA-derived CL, which is a specific indicator of the generation of extracellular superoxide anion (Skatchkov et al., 1998; Munzel et al., 2002), was used to determine whether cis-UCA could inhibit generation of PMN extracellular superoxide (Figure 3). Increases in MCLA-dependent CL peaked within 5min of stimulation with PMA and rapidly decreased thereafter (Figure 3A). cis-Urocanic acid dose-dependently inhibited the MCLA-dependent CL of PMA-stimulated PMN. Overall peak luminescence for each experimental condition, calculated as the area under each respective curve during the first 10min of activation, demonstrated that concentrations of cis-UCA as low as 10μM could inhibit extracellular superoxide generation (Figure 3B). The highest concentration of cis-UCA (1mM) assayed inhibited ∼48% of the PMA-induced MCLA CL.

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  • Figure 3. 

    Effect of cis-urocanic acid (cis-UCA) on bovine neutrophil generation of extracellular superoxide as measured by 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazol[1,2-a]pyrazin-3-one hydrochloride (MCLA)-dependent chemiluminescence. Blood neutrophils isolated from 11 cows were exposed to increasing concentrations of cis-UCA for 10min. Neutrophils were subsequently stimulated with phorbol 12-myristate,13-acetate (PMA, 40nM) in the presence of MCLA, and chemiluminescence values were measured every 5min. The mean (±SE) fold increase in chemiluminescence relative to un-stimulated neutrophils incubated with MCLA is shown (A). To analyze the effect of cis-UCA on peak generation of extracellular superoxide, the area under the curve was calculated from plotted data points for each experimental condition for the first 3 measurements (0 to 10min). **Decreased (P<0.01) relative to neutrophils stimulated with PMA in the absence of cis-UCA.

To confirm the ability of cis-UCA to inhibit extracellular superoxide, reduction of cytochrome c, which occurs in response to the generation of extracellular superoxide anion (Munzel et al., 2002; Tarpey et al., 2004), was also assayed (Figure 4). When using this assay, increases in superoxide anion production were evident within 10min of PMA stimulation and reached a maximum 10min later (Figure 4A). cis-Urocanic acid inhibited extracellular superoxide anion production of PMA-stimulated PMN as assessed by cytochrome c reduction. Overall superoxide generation for each experimental condition was calculated as the area under each respective curve (Figure 4B). Similar to findings in the MCLA-based assay, the lowest concentration of cis-UCA that could inhibit PMA-induced extracellular superoxide generation was 10μM. Concentrations of cis-UCA ranging from 10μM to 10mM inhibited PMA-stimulated PMN generation of superoxide anion by ∼40 to 50%. To confirm the specificity of this assay to detect extracellular superoxide, PMA-stimulated PMN were also incubated with SOD (10 units/mL) and cytochrome c reduction was assessed. Superoxide dismutase reduced extracellular levels of superoxide generated from PMA-stimulated PMN by ∼90%.

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  • Figure 4. 

    Effect of cis-urocanic acid (cis-UCA) on bovine neutrophil generation of extracellular superoxide as measured by cytochrome c reduction. Blood neutrophils isolated from 5 cows were exposed to increasing concentrations of cis-UCA for 10min. Neutrophils were subsequently stimulated with phorbol 12-myristate,13-acetate (PMA, 50μM) in the presence of cytochrome c, absorbance values were measured, and the amount of superoxide anion production calculated. To demonstrate specificity of the assay, parallel reactions were set up in which PMA-activated neutrophils were incubated with superoxide dismutase (SOD, 10 U/mL). Background values, defined as the mean amount of superoxide (nmol) produced by unactivated neutrophils, were subtracted from all experimental conditions and the difference is reported as the mean (±SE) superoxide production in nmol units (A). To analyze the effect of cis-UCA on the generation of superoxide, the area under the curve was calculated from plotted data points for each experimental condition (B). *,**Decreased (P<0.05 and P<0.01, respectively) relative to neutrophils stimulated with PMA in the absence of cis-UCA.

cis-UCA Does Not Affect Bovine PMN Generation of Intracellular Superoxide, Peroxynitrite, and Hydrogen Peroxide 

Dihydroethidium oxidation, which is specifically elicited in response to the generation of intracellular superoxide anion (Munzel et al., 2002; Walrand et al., 2003; Tarpey et al., 2004), was used to assess the effect of cis-UCA on PMN generation of intracellular superoxide (Figure 5). Increased oxidation of DHE, as indicated by increasing fluorescence, was evident within 15min of stimulation with PMA and remained unchanged 30min later. Incubation with cis-UCA (3mM) or SOD (10 units/mL) had no effect on PMA-induced generation of intracellular superoxide. In contrast, the cell-permeable NADPH oxidase inhibitor, DPI (8μM), completely abrogated the PMA-stimulated PMN generation of intracellular superoxide, as reflected by the absence of DHE oxidation and corresponding generation of fluorescence.

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  • Figure 5. 

    Effect of cis-urocanic acid (cis-UCA) on bovine neutrophil generation of intracellular superoxide. Blood neutrophils isolated from 5 cows were exposed to cis-UCA (3mM), superoxide dismutase (SOD; 10 U/mL), or diphenyleneiodonium chloride (DPI, 8μM) for 15min. Neutrophils were subsequently stimulated with phorbol 12-myristate,13-acetate (PMA, 40nM) in the presence of dihydroethidium and the numbers of cells fluorescing and the intensity of fluorescence were recorded 15 and 45min later on a flow cytometer. The mean (±SE) relative fluorescence index values for each experimental group are shown. *,**Decreased (P<0.05 and P<0.01, respectively) relative to neutrophils stimulated with PMA in the absence of any inhibitor.

Because cis-UCA had no apparent effect on the generation of intracellular superoxide, the oxidation of CM-H2DCFDA, which is elicited in response to the generation of intracellular peroxynitrite anion and hydrogen peroxide (Rothe and Valet, 1990; Crow, 1997), was used to assess whether cis-UCA could affect the generation of other intracellular ROS (Figure 6). Increases in CM-H2DCFDA oxidation were evident within 30min of stimulation with PMA and continued to increase up to 45min later (Figure 6A). cis-Urocanic acid had no apparent effect on PMA-stimulated PMN generation of intracellular peroxynitrite anion and hydrogen peroxide, whereas the NADPH oxidase inhibitor DPI partially blocked this response. Overall oxidation of CM-H2DCFDA for each experimental condition was calculated as the area under each respective curve (Figure 6B). All concentrations of cis-UCA tested (10μM to 1mM) had no significant effect on PMN oxidation of intracellular CM-H2DCFDA, whereas DPI (8μM) inhibited oxidation by ∼66%.

  • View full-size image.
  • Figure 6. 

    Effect of cis-urocanic acid (cis-UCA) on bovine neutrophil generation of intracellular peroxynitrite and hydrogen peroxide. Blood neutrophils isolated from 5 cows were exposed to increasing concentrations of cis-UCA or a fixed concentration of diphenyleneiodonium chloride (8μM) for 10min. Neutrophils were subsequently stimulated with phorbol 12-myristate,13-acetate (PMA, 40nM) in the presence of 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester, and fluorescence values were measured every 15min. Background values, defined as the mean fluorescent values of unactivated neutrophils, were subtracted from all readings and the difference is reported as the mean (±SE) fluorescence in arbitrary units (A). To analyze the effect of cis-UCA on generation of reactive oxygen species, the area under the curve was calculated from plotted data points for each experimental condition (B). **Decreased (P<0.01) relative to neutrophils stimulated with PMA in the absence of inhibitor.

DPI, but Not cis-UCA, Inhibits PMN-Mediated Killing of Staph. aureus 

To determine whether the ability of cis-UCA to selectively block extracellular generation of ROS could interfere with PMN functions required for effective host responses to infection, the influence of cis-UCA on PMN phagocytosis and bacterial killing was assessed (Table 1). Polymorphonuclear neutrophils treated with or without cis-UCA (3mM) or DPI (8μM) were incubated with Staph. aureus for 60min. The percentage of PMN containing phagocytosed bacteria and the number of phagocytosed bacteria within each PMN were subsequently enumerated. Equivalent percentages of PMN that phagocytosed Staph. aureus and equivalent numbers of bacteria phagocytosed were observed regardless of treatment. Relative to untreated (control) PMN, those exposed to cis-UCA demonstrated an equivalent capability to kill Staph. aureus. In contrast, PMN incubated with DPI demonstrated a ∼50% impairment in the ability to kill Staph. aureus relative to control or cis-UCA treated PMN.

Table 1. Effect of cis-urocanic acid (cis-UCA, 3mM) and diphenyleneiodonium chloride (DPI, 8μM) on bovine polymorphonuclear neutrophil (PMN) phagocytosis and killing of Staphylococcus aureus1
IndicatorControlcis-UCADPI
PMN with phagocyted bacteria, %93.0±2.092.4±1.494.6±1.4
Number of phagocyted bacteria:PMN15.3±1.016.0±0.616.0±0.4
Killing of bacteria, %56.3±4.358.2±3.428.2±4.8*

1All data presented as mean±standard error; n=5.

*P<0.01.

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Discussion 

The current study investigated the inhibitory effect of cis-UCA on bovine PMN respiratory burst activity. Consistent with a previous study of human PMN (Kivisto et al., 1996), cis-UCA inhibited luminol-dependent respiratory burst activity in a dose-dependent manner (Figure 1). Whereas the lowest reported concentration of cis-UCA to partially inhibit human PMN respiratory burst activity was 700μM, concentrations as low as 30μM were able to block the same response in bovine PMN to a similar extent (i.e., ∼23% inhibition). At the highest concentration assayed (10mM), cis-UCA completely abrogated bovine PMN respiratory burst activity. The differences in minimum effective inhibitory concentrations between the 2 studies may be due to differential species-dependent sensitivity to cis-UCA. Alternatively, because the 2 studies used different agonists to induce respiratory burst activity, namely, zymosan and PMA, the differences in MIC may be due to the differential activation elicited by these agonists.

Within 6 to 12h of isolation, PMN begin to undergo spontaneous apoptosis (Savill et al., 1989). Correspondingly, within 4h of in vitro incubation following the 2 to 3h PMN isolation procedure, bovine PMN demonstrated an increase in caspase activity that reflected the onset of apoptosis (Figure 2A). Because all the functional assays investigating PMN responses were conducted within 2h of PMN isolation, the onset of spontaneous apoptosis did not present a major concern. However, the possibility that the cis-UCA-mediated decrease in luminol-dependent CL was due to enhancement of PMN apoptotic death by cis-UCA needed to be ruled out. As shown in Figure 2A, the onset and level of spontaneous apoptosis were unaffected in PMN exposed to the highest concentration of cis-UCA (10mM) tested in any assay of ROS production. Disruption of membrane integrity, which was assessed by trypan blue exclusion and is an indicator of cell injury, was not observed until 24h after in vitro incubation of untreated PMN (Figure 2B). Similar to apoptosis, cis-UCA had no effect on the percentage of cells that lost membrane barrier function or on the time at which the onset of this process occurred relative to PMN not exposed to cis-UCA. Together, these data suggest that the cis-UCA-mediated inhibition of respiratory burst activity cannot be ascribed to nonspecific impairment of PMN functioning due to cis-UCA-induced injury or apoptotic cell death or both.

Luminol is a cell-permeable compound; thus, CL generated by this molecule reflects the production of both intracellular and extracellular ROS (Briheim et al., 1984; Rest, 1994). Luminol has been used to measure an array of ROS, including superoxide (Faulkner and Fridovich, 1993; Lundqvist and Dahlgren, 1996); hydroxyl radical (Yildiz and Demiryurek, 1998; Nemeth et al., 2002); hydrogen peroxide (Castro et al., 1996; Yildiz and Demiryurek, 1998); peroxynitrite (Radi et al., 1993); and hypochlorous acid (Brestel, 1985; Myhre et al., 2003). However, the findings of many of these studies are contradictory in terms of the specificity of luminol for the various ROS. The contradictions arise, in part, from the difficulty in identifying whether a given ROS directly reacts with luminol or whether it is converted to another ROS, the latter of which is responsible for eliciting luminol-derived CL. Therefore, although luminol-dependent CL is a sensitive indicator of respiratory burst activity, luminol is limited in its ability to discriminate among individual ROS.

Superoxide anion is the most proximally generated ROS by NADPH oxidase and serves as a precursor to the direct or indirect formation of other ROS (Weiss, 1989; Hampton et al., 1998). Because of its critical role in the generation of downstream ROS, the ability of cis-UCA to inhibit generation of superoxide was investigated using 2 distinct assays based on MCLA-dependent CL and cytochrome c reduction. Several studies have established that MCLA-dependent CL is a specific marker of extracellular superoxide production (Nakano et al., 1986; Nishida et al., 1989; Nakano, 1990; Pronai et al., 1992). Measurement of cytochrome c reduction is also a widely used and accepted technique for the detection of extracellular superoxide generation (Munzel et al., 2002). Reduction of cytochrome c is not an absolute specific marker of superoxide production because cellular reductants, such as glutathione, have been reported to reduce cytochrome c (Tarpey et al., 2004). However, the extent of specificity of the assay for superoxide can be readily ascertained by the inclusion of control reactions containing exogenous SOD.

Using 2 distinct assays of extracellular superoxide production, MCLA-dependent CL and cytochrome c reduction, we established that cis-UCA inhibited PMA-induced superoxide generation (Figure 3 and 4). For both assays, the lowest concentration of cis-UCA that inhibited superoxide production was 10μM and the extent of inhibition (∼40 to 50%) was equivalent. The specificity of the cytochrome c assay to detect superoxide was confirmed by the ability of exogenously added SOD to inhibit ∼90% of the reduction of cytochrome c (Figure 4). Thus, 2 independent assays confirmed that cis-UCA inhibits PMN generation of extracellular superoxide.

2-Methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazol [1,2-a]pyrazin-3-one hydrochloride-dependent CL and cytochrome c are limited to the detection of extracellular superoxide, and both of these assays established that cis-UCA inhibited extracellular superoxide generation. To investigate whether cis-UCA inhibited global generation of this ROS, intracellular superoxide generation was specifically measured with the fluorescent dye, DHE. Dihydroethidium is a cell-permeable compound and its oxidation is a specific indicator of super-oxide generation (Rothe and Valet, 1990; Benov et al., 1998; Walrand et al., 2003). At a concentration that maximally inhibited extracellular superoxide production, cis-UCA had no effect on intracellular generation of superoxide in PMA-stimulated PMN (Figure 5). The finding that addition of exogenous SOD, which is cell impermeable, had no effect on DHE-mediated fluorescence emitted from PMA-activated PMN confirmed that the assay detected only intracellular superoxide. The cell-permeable NADPH oxidase inhibitor DPI, which was included as a positive control, completely abrogated the PMA-stimulated generation of intracellular superoxide, consistent with previous reports (Ellis et al., 1988; Hampton and Winterbourn, 1995; Katsuyama et al., 2002). Together, these data suggest that cis-UCA inhibits extracellular, but not intracellular, superoxide production.

To determine whether cis-UCA influenced the generation of other intracellular ROS, the cell-permeable probe CM-H2DCFDA was used to monitor their production in activated PMN. This probe is not oxidized by superoxide (Bass et al., 1983; LeBel et al., 1992) but is reportedly oxidized by other ROS, including peroxynitrite anion and hydrogen peroxide (Rothe and Valet, 1990; Crow, 1997; Kooy et al., 1997). By using CM-H2DCFDA, cis-UCA was determined to have no effect on the production of these ROS (Figure 6). Consistent with its role in inhibiting NADPH oxidase, the cell-permeable compound DPI significantly inhibited CM-H2DCFDA-mediated fluorescence in PMA-activated PMN. Although one cannot rule out that the generation of other intracellular ROS not detected by CM-H2DCFDA is impaired by cis-UCA, these findings suggest that the ability of cis-UCA to inhibit respiratory burst activity is largely mediated by its ability to block extracellular ROS production.

cis-Urocanic acid inhibited extracellular, but not intracellular, superoxide production. Interestingly, cis-UCA was able to completely inhibit luminol-dependent CL, which measures both intracellular and extracellular ROS. Thus, if cis-UCA only blocks the generation of extracellular ROS, one might expect that cis-UCA should only partially inhibit luminol-dependent CL. However, there is considerable controversy regarding the actual identity of the ROS that are directly responsible for evoking luminol-dependent CL. There is evidence that luminol-dependent CL is dependent upon both superoxide generation and myeloperoxidase activity and that reactants formed by the reaction of superoxide with myeloperoxidase-derived radicals are responsible for eliciting CL (Edwards, 1987; Suematsu et al., 1988; Ginsburg et al., 1993). Further, there are reports that luminol-dependent CL is also dependent upon the generation of nitric oxide (Wang et al., 1991; Radi et al., 1993; Catz et al., 1995). Whether cis-UCA inhibits nitric oxide production or myeloperoxidase-derived intracellular ROS not detected by CM-H2DCFDA, or both, remains unknown. Thus, the ability of cis-UCA to completely inhibit luminol-dependent CL may reflect not only the inhibition of extracellular ROS, but also the inhibition of intracellular ROS and NO that contribute to luminol-dependent CL. Further, the ratio of the amount of ROS released from bovine PMN extracellularly vs. that generated intracellularly remains unknown. If the ROS responsible for luminol-dependent CL are predominantly extracellular, then one may expect that cis-UCA may inhibit the vast majority of luminol-dependent CL regardless of the fact that luminol measures both intracellular and extracellular ROS. This latter hypothesis is supported by a report that sodium butyrate inhibits up to 80% of PMA-induced luminol-dependent CL in the absence of detectable decreases in intracellular superoxide (Liu et al., 2001). The definitive finding of the current study, based on 2 distinct assays, is that cis-UCA inhibits extracellular generation of superoxide. Whether the ability of cis-UCA to completely block luminol-dependent CL reflects cis-UCA inhibition of intracellular and extracellular ROS other than superoxide remains unclear.

The finding that cis-UCA exerts an inhibitory effect on extracellular generation of ROS, which are known to induce host tissue injury, suggests a possible therapeutic use for cis-UCA in limiting injury induced by the presence of large numbers of activated PMN. However, enthusiasm for implementation of cis-UCA as a therapeutic to limit host tissue injury would be diminished if cis-UCA impaired the bactericidal activity of PMN. It is known that DPI, which inhibits NADPH oxidase-mediated generation of ROS, impairs PMN bactericidal activity (Ellis et al., 1988; Hampton and Winterbourn, 1995). Therefore, the effect of cis-UCA on phagocytosis and killing of Staph. aureus was investigated. The percentage of PMN that phagocytosed bacteria and the number of bacteria phagocytosed were unaffected by exposure to the highest concentration of cis-UCA demonstrated to inhibit extracellular superoxide production (Table 1). Further, cis-UCA had no effect on the ability of PMN to kill Staph. aureus. Consistent with previous reports (Ellis et al., 1988; Hampton and Winterbourn, 1995), DPI had no effect on PMN phagocytosis of Staph. aureus, but did impair bacterial killing. It is well established that intracellular generation of ROS is critical to PMN bactericidal activity; however, the contribution of extracellular ROS to PMN-mediated bacterial killing remains less clear (Hampton et al., 1998). The impairment of bactericidal activity by DPI is consistent with its ability to inhibit intracellular generation of ROS. Conversely, the lack of impairment of the bactericidal activity of PMN exposed to cis-UCA is consistent with the findings that cis-UCA does not impair intracellular generation of ROS.

Two independent assays have established that cis-UCA inhibits production of extracellular superoxide, whereas intracellular generation of ROS was unaffected. The finding that cis-UCA did not impair bactericidal activity triangulates the finding that much, if not all, of the inhibitory activity of cis-UCA is exerted at the extracellular level. The mechanism of this action remains unknown. One possibility is that cis-UCA scavenges extracellular superoxide similar to SOD. However, unlike SOD, there is evidence that cis-UCA is cell permeable. Thus, if this hypothesis were true, the scavenging ability of cis-UCA would be expected to be preserved intracellularly. The anti-inflammatory properties of cis-UCA extend beyond that of inhibiting respiratory burst activity and include the ability to down-regulate IL-1 and IL-2 production and diminish MHC expression (Rasanen et al., 1989). Thus, rather than simply scavenging ROS, it is more likely that the ability of cis-UCA to impair extracellular ROS generation is mediated through a common mechanism that results in abrogation of an array of inflammatory processes.

In summary, cis-UCA was identified as reducing the extracellular levels of superoxide generated by activated PMN without impairing PMN phagocytotic and bactericidal activity. To our knowledge, this is the first study to investigate the effects of cis-UCA on bovine PMN respiratory burst activity. Further, this is the first study to demonstrate with any cell type that the inhibitory effect of cis-UCA on respiratory burst activity is exerted at the level of extracellular production of ROS and that superoxide is one ROS whose generation is specifically blocked. These findings suggest that cis-UCA may have therapeutic value in disease settings accompanied by massive PMN recruitment, such as mastitis, by limiting the production of extracellular ROS that are potentially damaging to host tissue while preserving PMN activity critical to bacterial clearance.

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Acknowledgment 

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 USDA.

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Supplementary data 

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PII: S0022-0302(06)72464-X

doi:10.3168/jds.S0022-0302(06)72464-X

Journal of Dairy Science
Volume 89, Issue 11 , Pages 4188-4201, November 2006

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