Comparative study of β-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.)
Highlights
► The paper compares NBT assay and RT-luminol assay as tools to monitor ROS in carp. ► RT-luminol assay allows tracking of kinetics during the respiratory burst response. ► RT-luminol assay detects the production of H2O2 and oxygen related radicals. ► RT-luminol assay can be used as a tool to monitor ROS involved in wound healing.
Introduction
Multicellular organisms mediate their early defense against pathogens based on their innate immune system, which provides them with the ability to recognize the presence of pathogens and react rapidly against them [1], [2]. The common carp, Cyprinus carpio, has been intensive studied for many purposes. Common carp is worldwide the most cultured fish species for food consumption. It represents one of the most important species used in aquaculture and although many studies have focused on physiological aspects such as nutrition, farming conditions and infectious diseases [3], [4], [5], [6], it is important to develop and improve reliable methods to monitor and control the health status of carp. The respiratory burst is regarded as one of the most important early defense mechanisms as it plays a crucial role in pathogen eradication, but has also been shown to be involved in tissue regeneration. Therefore, the respiratory burst is a significant mechanism that can be used to monitor health status in fish [7], [8], [9].
Several studies have dealt with the ability of phagocytes to recognize pathogens through the detection of pathogen-associated molecular patterns (PAMPs), which are highly conserved molecules not generally expressed in higher organisms [10], [11]. Phagocytes have also been related to the recognition of damage-associated molecular patterns (DAMPs), those being self signals of tissue damage and cell death [10], [12]. The recognition of all these molecules occurs using special receptors called pattern-recognition receptors (PRRs), and trigger a series of events including the respiratory burst [10], [13], [14], [15]. The initiation of the respiratory burst is marked by an increase in oxygen cellular uptake, followed by the one electron reduction of molecular oxygen (O2) to superoxide anions (O2−). This reaction is catalyzed by the membrane-associated enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, using NADPH as the electron donor [9], [16], [17], [18], [19]. Further reduction of oxygen produces hydrogen peroxide (H2O2), which occurs either as a spontaneous dismutation, especially at low pH, or as a catalyzed reaction by a family of enzymes called Superoxide dismutase (SOD). Additional reactions of O2− and H2O2 may lead to the formation of hydroxyl radicals (OH−), especially in the presence of iron through the Fenton or Haber–Weiss reactions. The interaction of H2O2 with myeloperoxidase (MPO) can produce hypochlorous acid and other toxic metabolites if H2O2 is not dismutated to water and molecular oxygen by the enzyme catalase that can act as a natural scavenger [16], [20], [21], [22], [23], [24] (See Fig. 1). Although different techniques for the quantitation of the respiratory burst have been developed through the years, comparisons of the accuracy and reliability to evaluate fish health status among those techniques are scarce.
To date, several methodologies for the measurement of respiratory burst have been described. Initially, Babior et al. (1973) assessed extracellular O2− based on its capability to reduce ferricytochrome c, reading absorbance at 550 nm. The main disadvantage of this methodology was its limitation to follow the kinetics of the reaction. This restrain was overcome by Cohen and Chovaniec (1978) by introducing the continuous recording of absorbance in a cell suspension, however both methods require large amounts of cells (≈2.5 × 106 cells/well) and reagents (≈950 μl/well) [19], [25], [26], [27]. In parallel, Root et al. (1975) formulated a new methodology for the calculation of respiratory burst produced by human granulocytes; in this procedure the loss of fluorescence of scopoletin (7-OH-6-methoxycoumarin), a natural compound found in the root of plants in the genus Scopolia), was evaluated after exposure of H2O2 in the presence of horseradish peroxidase (HRP). This technique provided high detection sensitivity (as little as 0.2 nmol H2O2/ml), but real-time measurements remained problematic due to the rapid diminution of scopoletin concentration in the samples. Furthermore, the technique cannot easily be applied to adherent cells, since it required the establishment of the cultures in flying coverslips which are then placed in the spectrofluorometer cuvette in a certain standard position [28], [29]. Pick and Keisari (1980) and Pick and Mizel (1981) established two detection methods based on the HRP-dependent oxidation of phenol sulfonephthalein (phenol red), and a combination of the phenol red and cytochrome c assay, respectively. These methodologies allowed them to measure respiratory burst in macrophage cultures of guinea pigs. However, the sensitivity of the H2O2 detection was reduced to 1 nmol/ml [19], [29]. The most successful alternative was developed by Baehner and Nathan (1968) who introduced the use of nitroblue tetrazolium (NBT) in the detection of respiratory burst [30]. The NBT assay protocol has been optimized over the years but its principle has remained the same [17], [31], [32], [33], [34], [35], [36], [37]. NBT is a yellow, water soluble substance which is internalized by phagocytes, and then reduced intracellularly to formazan during the respiratory burst. For quantitation, the cell membrane is disrupted, the formazan is dissolved in KOH and the absorbance is read from 509 to 690 nm [30], [31], [33], [34], [37]. NBT has perhaps become the most popular method for monitoring respiratory burst to various stimuli [38]. However, inconveniences associated with the NBT assay such as the impossibility to measure real-time during the respiratory burst process and its laborious protocol which increases the risk of pipetting errors, therefore decreasing accuracy, have remained an issue.
Allen et al. described a different approach for the detection of respiratory burst for human polymorphonuclear leukocytes already in 1972. In this study, the authors describe the occurrence of electronically excited states during the production and transformation of free radicals in the respiratory burst. Furthermore, they observed that after electron relaxation to their initial ground state, energy was release in form of photons. This process is known now as native chemiluminescence and can be amplified for its detection using luminol [39], [40]. Different protocols for the luminol amplification of radical production have been used through the years in different species [41], [42], [43], [44]. This paper, for the first time, compares the popular NBT method with the native chemiluminescence amplification method for use in carp (C. carpio). Using β-glucans to induce a respiratory burst response in head kidney leukocytes, the accuracy, sensitivity and adaptability of both methodologies are examined and compared, and their use to quantitate fish health status is discussed.
Section snippets
Fish
European Common carp (C. carpio carpio) were obtained from the central fish facility ‘De Haar-vissen’ (Wageningen, The Netherlands). R3 × R8 carp are the offspring of a cross between fish of Hungarian origin (R8 strain) and the Polish origin (R3) [45]. The fish used ranged between 50 and 100 g and were kept at 23 °C (±1 °C) with 12:12 h light: dark photoperiod.
Preparation of head kidney cell suspensions
During this study four different head kidney cell suspensions were used, they are referred to throughout the paper as: total head kidney
The RT-luminol assay detects reactive oxygen species additional to those detected by the NBT assay
The NBT assay after stimulation of t-HK cells with MacroGard®, showed an increase of oxygen radical production. Cells treated with catalase did not indicate major changes to the production of oxygen radicals. Conversely, treatment with SOD, showed a markedly reduced magnitude of ROS. As expected, co-treatment with SOD and catalase also decreased ROS production (see Fig. 2A).
The RT-luminol assay showed an increase in the oxygen radicals produced by t-HK cells after stimulation with MacroGard®.
Discussion
The present study compares the use of NBT and RT-luminol assays for the assessment of oxygen radical production in carp after stimulation with β-glucans, a PAMP known to stimulate the respiratory burst in fish and mammalian systems [48], [51]. Both methods were able to detect the production of oxygen radicals after stimulation with MacroGard® and Zymosan, and allowed the detection of dose-dependent changes on the respiratory burst magnitude. On this basis, both methods can be used not only to
Conclusions
Both of the methods compared during this study, showed the capacity to detect and measure the respiratory burst response of carp head kidney cells after stimulation with β-glucans, therefore constitute an indicator of the general fish health status. However, only the RT-luminol assay allowed the tracking of kinetics during the respiratory burst response, offering information about peaks of oxygen radical production. Furthermore, only the RT-luminol assay detected the production of hydrogen
Acknowledgments
This work has received funding from the Seventh Framework Program FP7/2007-2013 under grant agreement n° 214505.10.
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