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The role of electron transport in the defence response of the South African abalone, Haliotis midae
Fish & Shellfish Immunology 26 (2009) 171–176
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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
The role of electron transport in the defence response of the South African abalone, Haliotis midae Marike Janse van Rensburg, Vernon E. Coyne* Department of Molecular and Cell Biology, University of Cape Town, Private Bag, Rondebosch 7700, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 June 2008 Received in revised form 18 September 2008 Accepted 26 September 2008 Available online 14 October 2008
In order to establish health management systems for farmed abalone, it is necessary to understand how the abalone immune system functions and responds to stimulation. Two electron transport system genes, cytochrome b and cytochrome c oxidase III, were found to be upregulated in a cDNA microarray experiment performed on haemocytes from immune-stimulated abalone (Arendze-Bailey, unpublished). The current study sought to elucidate the role of these genes, and thus the electron transport system, in the abalone immune response by specifically inhibiting cytochrome b with antimycin A and measuring haemocyte immune parameters in vivo. Antimycin A did not decrease haemocyte cell viability, but halved cellular ATP from 4 1012 nM/cell to 2 1012 nM/cell (p < 0.05, unpaired t-test). Inhibition of electron transport resulted in a 0.6 fold increase in cellular superoxide levels (p < 0.05, unpaired t-test), while phagocytosis dropped by nearly 50% (p < 0.05, ANOVA) and the ability of haemocytes to kill bacteria was also reduced. Since cytochrome b and cytochrome c oxidase III expression is upregulated in immune-stimulated abalone, and inhibition of electron transport resulted in a decreased immune response in vivo, we conclude that the abalone immune response is dependent on electron transport and that oxidative phosphorylation plays a role in the immune response following stimulation. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Haliotis midae Abalone Electron transport Immune system
1. Introduction Aquaculture of abalone and other molluscs is an economically important activity in South Africa as in many developing and developed countries. However, infectious diseases are considered a major limitation to production [1,2]. In order to establish health management systems for farmed abalone, a better understanding of the effectors of the immune system and characterization of the defence response to infection and stress is necessary [3]. Internal defence in invertebrate species is based on an innate, nonlymphoid immune system [4] and immune function is largely effected by phagocytic haemocytes, complemented by an array of killing mechanisms [4]. Phagocytosis is accompanied by a respiratory burst which in bivalves is activated by foreign particles, organisms and their by-products. The respiratory burst produces reactive oxygen species which act as killing agents, and thus, are important for the elimination of pathogens [5]. Animal cells generate energy in the mitochondria through oxidative phosphorylation, a process in which electrons are passed along a series of carrier molecules that constitute the electron
* Corresponding author. Tel.: þ27 21 650 5070; fax: þ27 21 689 7573. E-mail address: [email protected] (V.E. Coyne). 1050-4648/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.09.016
transport chain [6]. The electrons are generated from reduced nicotinamide adenine dinucleotide (NADH) and succinates produced by oxidation of nutrients such as glucose, and are ultimately transferred to molecular oxygen [6]. Mitochondria consume 90% of the oxygen used by the cell and although there are a number of cellular sources of reactive oxygen species (ROS), the electron transport chain is a major source of ROS [7] as it generates a continuous flux of oxygen radicals [8]. It is estimated that 1–2% of all electrons passing through the respiratory chain end up as oxygen radicals [7] with unpaired electrons escaping mainly from complex I and III [9]. Mitochondrial respiration is highly sensitive to antimycin A [10] a fungicidal agent that inhibits the growth of molds and yeasts and is a respiratory inhibitor in mammals and higher plants [11]. Antimycin A inhibits succinate oxidase and NADH oxidase, as well as mitochondrial electron transport between cytochrome b and c1 [12] by blocking electron flow in the Q-cycle of complex III [7]. Antimycin A also enhances the transfer of single electrons to molecular oxygen, producing superoxide [13] and consequently, has been used to investigate the generation of mitochondrial ROS and to model or simulate hypoxia [14]. Previous work in our laboratory has shown that feed supplemented with probiotics leads to an increased immune response in Haliotis midae, reflected by elevated haemocyte phagocytic activity
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[15] and the production of cellular ROS (Macey, unpublished). A cDNA microarray investigation of gene expression in haemocytes from immune-stimulated H. midae identified two electron transport chain genes, namely cytochrome b and cytochrome c oxidase III, that were differentially expressed in response to feeding a probiotic-supplemented feed (Arendze-Bailey, unpublished). Semiquantitative reverse transcription (RT) PCR of RNA isolated from haemocytes from immune-stimulated abalone verified that both cytochrome b and cytochrome c oxidase III are upregulated in response to probiotic treatment. The aim of the present study was to investigate the role of cytochrome b and cytochrome c oxidase III in the abalone immune response. Since cytochrome b and cytochrome c oxidase III code for respiratory complex components of the electron transport chain, we investigated the role of these genes in context to the role that the electron transport chain may play during stimulation of the abalone immune response. In order to accomplish this, we used antimycin A to specifically inhibit cytochrome b, and subsequently, we investigated the effect of inhibiting the electron transport chain on specific haemocyte immune parameters. 2. Materials and methods 2.1. Animals Abalone were maintained at the Marine and Coastal Management Research Aquarium in Sea Point, Cape Town, South Africa. The animals were maintained in 98 l polyethylene tanks containing aerated, continuously flowing natural seawater between 15 and 18 C. Between experiments, the abalone were fed kelp twice a week. 2.2. Collection of haemolymph Haemolymph was collected from the pedal sinus using a 1.5 ml syringe and 26 G ½-inch needle, and stored on ice until used.
2.6. Quantitation of intracellular ATP Since ATP is the final product of the electron transport chain, a decrease in ATP would indicate that antimycin A inhibits haemocyte respiration. Haemocyte ATP levels were measured using an ATP Determination Kit (Invitrogen Molecular Probes) which is based on a luciferin/luciferase reaction. A Standard Reaction Solution, containing water, 20 reaction buffer, 0.1 M DTT, 10 mM 1 D-luciferin and 2.5 ml of 5 mg ml firefly luciferase was prepared according to the manufacturer’s instructions. Haemocyte monolayers were treated for 2 h with either 100 ml PBS (control samples) or 100 ml antimycin A at final concentrations of 1 mM, 10 mM and 100 mM (test samples). The haemocyte monolayer was harvested by incubation in 100 ml 10 mM EDTA for 10 min. A 10 ml sample of the detached cells was removed for cell number determination (Section 2.3). The remaining sample was centrifuged for 5 min at 3000 g. The supernatant was removed and the pellet resuspended in cell lysis buffer ((w/v) 20 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5% Triton-X). After incubating for 15 min on ice, the samples were centrifuged and 10 ml of the supernatant was added to 100 ml of Standard Reaction Solution and incubated for 20 min at room temperature in the dark to ensure that the luminescent signal was stable. Following this, relative luminescence was measured in a TD 20/20 luminometer (Turner Designs). A standard curve was constructed each time the experiment was performed. Low-concentration ATP standard solutions were prepared by diluting the standard ATP solution (5 mM) provided with the kit in dH2O. The concentration of the standards ranged from 5 pM to 5 mM. Each standard (10 ml) was added to 100 ml of Standard Reaction Solution, incubated for 20 min in the dark and the luminescence measured. The luminescent readings of the samples could be converted to concentration of ATP using the standard curve. Because the number of cells in each sample had been determined, the ATP per sample was expressed as nM ATP/ cell. 2.7. Haemocyte viability
2.3. Total haemocyte count The total number of circulating haemocytes was determined directly after haemolymph collection. Undiluted haemolymph (100 ml) was added to 200 ml Alsevier’s Buffer ((w/v) 2.08% C6H12O6, 0.8% C6H5Na3O7$2H2O, 0.336% EDTA, 2.24% NaCl, 12% HCHO). The cells were counted using a haemocytometer and a light microscope at 100 magnification. 2.4. Attachment of abalone haemocyte monolayers After extraction, 100 ml haemolymph was placed into each well of a 96-well microtitre plate to obtain a cell density of 2 108 cells ml1 per well and allowed to attach for 20–25 min at room temperature in a humidified chamber. After attachment, the serum was removed, the haemocytes washed with PBS (7.3 mM monosodium phosphate, 180 mM disodium phosphate, 0.15 M sodium chloride, pH 7.2) and kept in 100 ml MHBSS ((w/v) 2.08% C6H12O6, 2.24% NaCl, 0.082% KCl, 0.02% KH2PO4, 0.071% CaCl2, 0.262% MgCl2, 0.314% MgSO4, 0.003% EGTA) until the assays were performed. 2.5. Treatment of haemocyte monolayers with antimycin A A stock solution of antimycin A (Sigma) was prepared in 100% ethanol (100 mg ml1, final concentration) and diluted to the desired concentration in PBS. After incubation with antimycin A for the required time, the antimycin A was removed, the haemocyte monolayers washed in PBS, and the chosen assay performed.
Antimycin A inhibition of electron transport may affect haemocyte viability, resulting in a diminished immune response. To ensure that a decreased immune response was caused by inhibition of electron transport and not a consequence of non-viable haemocytes, a MTT assay was performed on haemocytes treated with 1 mM, 10 mM and 100 mM antimycin A for 120 min. Also included in this experiment were samples treated with the equivalent amount of ethanol used to prepare the antimycin A to ensure that the ethanol did not affect haemocyte viability. After incubation with antimycin A, the supernatant was removed and the haemocytes rinsed with PBS. The haemocyte monolayers were incubated with 20 ml MHBSS and 20 ml MTT (5 mg ml1, Sigma) for 3 h. The supernatant was removed and the haemocytes rinsed twice with MHBSS. Solubilization of the purple precipitated formazan was achieved by adding 100 ml acidic isopropanol (0.4 M HCl in isopropanol) followed by overnight incubation on a plate shaker. Quantification of the formazan product was performed spectrophotometrically at 540 nm using a NanodropÒ ND-1000 UV–Vis spectrophotometer, where the absorbance at 540 nm represents the ability of haemocyte mitochondrial enzymes to reduce MTT, and is therefore a measure of haemocyte viability. 2.8. Quantitation of intracellular superoxide This assay was performed to determine whether treatment with antimycin A causes an increase in cellular superoxide production. Haemocyte monolayers were incubated with 100 ml 2 mg ml1
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nitroblue tetrazolium (NBT) and either PBS (control samples) or 100 ml 100 mM antimcyin (test samples). The samples were incubated in the dark for 2 h, whereafter the supernatant was removed and the haemocytes fixed by incubating in 100 ml 100% methanol for 10 min. The supernatant was removed, the cells air dried and the precipitated formazan dissolved by adding 120 ml 2 M KOH and 140 ml DMSO. The absorbance was determined at 620 nm on a NanodropÒ ND-1000 UV–Vis spectrophotometer. Since NBT is reduced by superoxide, the absorbance is directly related to the intracelluar amount of superoxide in the haemocytes.
2.9. Phagocytosis To determine whether inhibition of the electron transport chain would affect phagocytosis of pathogens, phagocytosis assays were performed comparing control haemocytes incubated in PBS to haemocytes incubated with antimycin A. The phagocytosis assay was performed as described by Macey and Coyne [15]. The time directly after haemocyte attachment and monolayer formation on the glass slide was designated T ¼ 0. The assay was performed at this time point, as well as after the haemocytes had been incubated in either PBS or antimycin A for 2 h.
2.10. Bacterial killing To determine whether inhibition of the electron transport chain has a direct effect on the killing mechanism of haemocytes, a bacterial killing assay was performed using a modification of the method described by Volety et al. [32]. E. coli JM109 was cultured in 5 ml LB media at 37 C overnight with shaking. The overnight culture was centrifuged at 3000 g and resuspended in PBS to yield approximately 1 106 cells ml1. Haemocyte monolayers were incubated with 100 ml of the bacterial cell suspension for 30 min to allow phagocytosis to occur. The bacteria were removed and the monolayers rinsed in PBS to remove residual bacteria. The T ¼ 0 h controls were prepared by rinsing the haemocytes with either 100 ml PBS or 100 ml antimycin A, while the T ¼ 2 h control and test samples were incubated for 2 h in either PBS or 100 ml 100 mM antimycin A, respectively. After incubation, the supernatant was removed and the haemocytes lysed in 100 ml distilled water for 15 min. The lysed samples were transferred to 1.5 ml eppendorf tubes, 100 ml LB was added and the samples were incubated for 2 h at 37 C with shaking. Following incubation, 100 ml MTT (5 mg ml1) was added to each sample. After a 2 h incubation in the dark, the bacterial cells were pelleted, the supernatant removed and the precipitated formazan dissolved in 100 ml acidic isopropanol. Quantification of the formazan was performed spectrophotometrically at 540 nm using a NanodropÒ ND-1000 UV–Vis spectrophotometer. For the purposes of the assay, bacterial killing was defined as the inability of bacteria to proliferate following exposure to haemocytes. The extent of MTT reduction to a purple formazan reflects the effect of the haemocytes on the bacteria. Thus, a high degree of bacterial survival would result in increased levels of formazan.
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3. Results 3.1. Effect of antimycin A on electron transport The effect of antimycin A on haemocyte ATP production was determined using a luciferin/luciferase assay. Haemocytes treated with 1 mM antimycin A had ATP levels of 2 1012 nM ATP/cell which was equivalent to the ATP concentration in haemocytes exposed to 10 mM and 100 mM antimycin A (Fig. 1). The ATP concentration in antimycin A treated haemocytes was almost half that present in the control haemocytes (4 1012 nM ATP/cell), indicating that antimycin A caused a significant reduction in cellular ATP in treated haemocytes. 3.2. Effect of antimycin A on haemocyte viability In order to ensure that the reduced ATP levels in antimycin A treated haemocytes was not due to the onset of cell death, the viability of haemocytes exposed to antimycin A was determined using a MTT reduction assay. Thus, ability of haemocytes incubated in PBS to reduce MTT to a purple formazan was compared to that of haemocytes incubated in 1 mM, 10 mM and 100 mM antimycin A. Antimycin A had no significant effect on haemocyte viability in that the amount of formazan formed by antimycin A treated haemocytes was not significantly different to that of the control haemocytes (Fig. 2). The effect of ethanol in which the antimycin A was dissolved was also determined. The viability of haemocytes incubated in 0.1% and 1% ethanol (equivalent to ethanol concentration in 10 mM and 100 mM antimycin A solutions, respectively) was not significantly reduced in relation to the PBS control. 3.3. The effect of antimycin A on haemocyte production of cellular superoxide Antimycin A resulted in a 1.5 fold increase in formazan (0.088 absorbance units as opposed to 0.057 absorbance units in the control haemocytes) (Fig. 3). Thus, intracellular superoxide increased significantly in haemocytes treated with antimycin A. 3.4. Effect of antimycin A on haemocyte phagocytic activity Phagocytosis of FITC-labelled V. anguillarum was un-affected in haemocytes that were briefly exposed to antimycin A (Fig. 4). However, there was a significant reduction in phagocytosis after exposing the haemocytes to antimycin A, as opposed to PBS, for 2 h.
2.11. Statistical analysis Experimental data is reported as the mean of three biological repeats, each having been measured in triplicate. Error bars represent the standard error of the mean. An unpaired t-test was used to compare two means (p < 0.05), while multiple comparisons were conducted using one-way ANOVA analysis followed by the Tukey post hoc test (p < 0.05) using SigmaStat 3.1.
Fig. 1. The effect of increasing concentrations of antimycin A on haemocyte ATP production. Haemocytes were treated with 1 mM, 10 mM and 100 mM antimycin A (AM) or PBS for 2 h. Each bar represents the mean of 3 biological repeats measured in triplicate, while the vertical lines reflect standard error of the mean. (*) Indicates sample differs significantly (p < 0.05, unpaired t-test) to the PBS control.
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Fig. 2. The effect of antimycin A on haemocyte viability. Control haemocyte samples were treated with either PBS, 0.1% or 1% ethanol (Etoh) in PBS. Test samples were incubated in 1 mM, 10 mM or 100 mM antimycin A (AM). The bars represent the mean of 3 biological repeats measured in triplicate, while the vertical lines represent standard error of the mean.
3.5. Effect of antimycin A on haemocyte destruction of pathogenic bacteria The number of bacteria killed by haemocytes incubated for 0 h in antimycin A did not differ significantly from that observed in haemocytes incubated in PBS for the same time period, with approximately 0.12 absorbance units of formazan detected (Fig. 5). The number of viable bacteria remaining following exposure to haemocytes incubated in PBS for 2 h was significantly less (p < 0.05) than the number of surviving bacteria in either of the 0 h controls (PBS or antimycin A). However, bacterial survival following exposure to haemocytes that had been incubated in antimycin A for 2 h did not differ significantly to the controls (haemocytes incubated in antimycin A for 0 h or PBS for 2 h).
4. Discussion Abalone farming, like other commercial aquaculture enterprises, is constantly at risk due to the devastating impact of infectious disease outbreaks [1]. An understanding of the abalone response to infection is necessary in order to mitigate the high mortality rates associated with infectious diseases in farmed abalone [3]. Previously, we showed that both cytochrome b and cytochrome c oxidase III are upregulated in response to
Fig. 3. The effect of antimycin A on haemocyte production of cellular superoxide. Control haemocytes were incubated in PBS and test samples in 100 mM antimycin A (AM) for 2 h. Each bar represents the mean of 3 biological repeats measured in triplicate, while the vertical lines reflect standard error of the mean. (*) Indicates sample differs significantly (p < 0.05, unpaired t-test) to the PBS control.
Fig. 4. Effect of antimycin A on haemocyte phagocytic activity. Control haemocytes were incubated in PBS and test samples in 100 mM antimycin A (AM) for 0 and 2 h prior to investigating phagocytosis. Each bar represents the mean of 3 biological repeats measured in triplicate, while the vertical lines reflect standard error of the mean. Different letters indicate significant difference (p < 0.05, ANOVA) between values.
immune-stimulation, implying that the mitochondrial respiratory chain might play a role in the abalone immune response. The aim of this study was to investigate the role of the electron transport system in the abalone immune response, and the reliance of this response on oxidative phosphorylation. This was accomplished by inhibiting electron transport in abalone haemocytes with the use of antimycin A and subsequently investigating the effect on various immune parameters. Inhibition of electron transport through antimycin A treatment has been shown to be harmful to cells by inducing apoptosis [16] and has been shown to lead to the death of numerous cell types including juxtaglomerular cells [6], hepatocytes [14,17] and human lymphoblastoid cells [16]. It was therefore important to ensure that antimycin A treated abalone haemocytes remained viable during the course of the assays employed in this study so that any reduction in immune response could be attributed to inhibition of electron transport. The MTT assay has been widely used to assay the viability of numerous cell types [18,19] including phagocytic cells [20], and to determine the effect of chemical treatment on cell viability [21,22]. Since abalone haemocytes remained viable following a 2 h incubation in 100 mM antimycin A, we applied antimycin A at this concentration and time interval in subsequent assays. Antimycin A has been shown to lead to a marked reduction in cellular ATP levels [6,14,17]. Measuring the effect of antimycin A on cellular ATP would assist in determining whether antimycin A indeed has an inhibitory effect on the electron transport system of H. midae. The level of ATP in haemocytes treated with antimycin A was markedly decreased compared to that of control cells. In fact, the level of ATP in antimycin A treated haemocytes was almost 50% of that in control cells, which is similar to results obtained with other cell types; for example, antimycin A lead to a 15% decrease in hepatocyte ATP [14] and a 40% reduction in ATP in renal epithelial cells [23]. In addition to causing a decrease in cellular ATP, antimycin A is known to significantly increase cellular superoxide production [6]. Similarly, abalone haemocytes treated with antimycin A produced significantly more superoxide in comparison to untreated cells (54% increase). Superoxide detected in the control haemocytes is not unusual. For example, superoxide produced in haemocytes from the white shrimp, Penaeus vannamei, that had not been stimulated by various disease response elicitors was attributed as background superoxide [24]. Production of high levels of superoxide due to antimycin A treatment has been widely demonstrated in various cell types [6,12,25,26] and organisms [27]. Meaney et al.
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[13] showed that mitochondria treated with antimycin A released five times more superoxide into the mitochondrial matrix compared to control samples. Mukhopadhyay et al. [28] showed that a 3–7 fold dose-dependent increase in superoxide production occurred in response to antimycin A in rat and human cells. This effect is explained by the fact that interruption of electron transport results in the formation of reactive oxygen species such as superoxide as a consequence of autoxidation of reduced electron transport components [29]. The effect of antimycin A on phagocytosis was investigated as a direct measure of the importance of the electron transport chain in the abalone immune response. Phagocytosis in haemocytes treated with antimycin A declined from nearly 30% in control cells to 17% in treated cells (Fig. 4). A similar effect has been shown in human monocytes, where treatment with antimycin A led to a 50% decrease in phagocytosis after incubation in 360 mM antimycin A for 45 min [30]. Similarly, antimycin A caused diminished phagocytosis of fluorescent beads in alveolar macrophages [31]. After determining that a functional electron transport system is necessary for phagocytosis of invading pathogens, the effect of an inhibited electron transport system on the bactericidal action of abalone haemocytes was examined (Fig. 5). Bacterial killing by abalone haemocytes was estimated colorimetrically through the reduction of the tetrazolium salt MTT to a purple formazan [32]. The formazan produced during the assay is directly proportional to the number of bacterial colonies detected by agar plate count methods such as those described by Canesi et al. [33,34]. Thus, absorbance can be directly correlated to the number of bacterial colonies that survived exposure to the haemocytes. The bactericidal assay showed that the number of bacteria that survived exposure to haemocytes that had been incubated in antimycin A for 2 h was not significantly less than in the control. If antimycin A had inhibited bacterial killing, one would expect the number of surviving bacteria to be equivalent to that of the 0 h control which represents the starting bacterial titre, and significantly higher than the 2 h PBS control which reflects the number of surviving bacteria following haemocyte killing. What the results do show is that some killing did occur, but not to the same level as that in the 2 h control. A possible explanation for this phenomenon could be, as mentioned earlier, the production of additional superoxide at complex III in response to antimycin A blockage of electron transport. Superoxide is bactericidal [35] and is formed in response to bacterial invasion in numerous marine organisms such as Crassostrea gigas [36], Macrobrachium rosenbergii [37], Lynmaea stagnalis
Fig. 5. The effect of antimycin A on the ability of haemocytes to kill invading bacteria. Control samples were incubated in PBS for 0 and 2 h, respectively. Test samples were incubated in 100 mM antimycin A (AM) for 0 and 2 h. Each bar represents the mean of 3 biological repeats measured in triplicate, while the vertical lines reflect standard error of the mean. Different letters indicate significant difference (p < 0.05, ANOVA) between values.
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[38] and Mercenaria mercenaria [5]. Thus, although some bacterial killing occurred, it was insufficient to conclusively determine whether antimycin A did or did not inhibit abalone haemocyte bacterial killing. Indeed, Watabe and Nakaki [6] mention that superoxide production following treatment with respiratory inhibitors makes it difficult to analyse their effect on cellular functions. The bactericidal assay showed that normal abalone haemocytes recognize and kill E. coli as an invading pathogen, and that antimycin A treatment results in the production of sufficient superoxide to destroy some of the invading bacteria. Since antimycin A has been shown to interfere with the killing of Staphylococcus in alveolar [31] and murine macrophages [39], further investigation is required to unequivocally determine whether inhibition of the electron transport system affects the bactericidal action of abalone haemocytes. This study has shown that the mitochondrial respiratory chain plays a role in the immune response of the abalone due to the increased metabolic needs of the cell during immune-stimulation. Increasing our understanding of how the abalone immune system functions would improve our ability to alleviate and or prevent infectious disease outbreaks in farmed H. midae.
Acknowledgements The study was funded by a National Research Foundation research grant (FA2004040600014) awarded to Vernon Coyne. Marike Janse van Rensburg was supported by a NRF Grant-holder linked postgraduate student bursary.
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The role of electron transport in the defence response of the South African abalone, Haliotis midae Marike Janse van Rensburg, Vernon E. Coyne* Department of Molecular and Cell Biology, University of Cape Town, Private Bag, Rondebosch 7700, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 June 2008 Received in revised form 18 September 2008 Accepted 26 September 2008 Available online 14 October 2008
In order to establish health management systems for farmed abalone, it is necessary to understand how the abalone immune system functions and responds to stimulation. Two electron transport system genes, cytochrome b and cytochrome c oxidase III, were found to be upregulated in a cDNA microarray experiment performed on haemocytes from immune-stimulated abalone (Arendze-Bailey, unpublished). The current study sought to elucidate the role of these genes, and thus the electron transport system, in the abalone immune response by specifically inhibiting cytochrome b with antimycin A and measuring haemocyte immune parameters in vivo. Antimycin A did not decrease haemocyte cell viability, but halved cellular ATP from 4 1012 nM/cell to 2 1012 nM/cell (p < 0.05, unpaired t-test). Inhibition of electron transport resulted in a 0.6 fold increase in cellular superoxide levels (p < 0.05, unpaired t-test), while phagocytosis dropped by nearly 50% (p < 0.05, ANOVA) and the ability of haemocytes to kill bacteria was also reduced. Since cytochrome b and cytochrome c oxidase III expression is upregulated in immune-stimulated abalone, and inhibition of electron transport resulted in a decreased immune response in vivo, we conclude that the abalone immune response is dependent on electron transport and that oxidative phosphorylation plays a role in the immune response following stimulation. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Haliotis midae Abalone Electron transport Immune system
1. Introduction Aquaculture of abalone and other molluscs is an economically important activity in South Africa as in many developing and developed countries. However, infectious diseases are considered a major limitation to production [1,2]. In order to establish health management systems for farmed abalone, a better understanding of the effectors of the immune system and characterization of the defence response to infection and stress is necessary [3]. Internal defence in invertebrate species is based on an innate, nonlymphoid immune system [4] and immune function is largely effected by phagocytic haemocytes, complemented by an array of killing mechanisms [4]. Phagocytosis is accompanied by a respiratory burst which in bivalves is activated by foreign particles, organisms and their by-products. The respiratory burst produces reactive oxygen species which act as killing agents, and thus, are important for the elimination of pathogens [5]. Animal cells generate energy in the mitochondria through oxidative phosphorylation, a process in which electrons are passed along a series of carrier molecules that constitute the electron
* Corresponding author. Tel.: þ27 21 650 5070; fax: þ27 21 689 7573. E-mail address: [email protected] (V.E. Coyne). 1050-4648/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.09.016
transport chain [6]. The electrons are generated from reduced nicotinamide adenine dinucleotide (NADH) and succinates produced by oxidation of nutrients such as glucose, and are ultimately transferred to molecular oxygen [6]. Mitochondria consume 90% of the oxygen used by the cell and although there are a number of cellular sources of reactive oxygen species (ROS), the electron transport chain is a major source of ROS [7] as it generates a continuous flux of oxygen radicals [8]. It is estimated that 1–2% of all electrons passing through the respiratory chain end up as oxygen radicals [7] with unpaired electrons escaping mainly from complex I and III [9]. Mitochondrial respiration is highly sensitive to antimycin A [10] a fungicidal agent that inhibits the growth of molds and yeasts and is a respiratory inhibitor in mammals and higher plants [11]. Antimycin A inhibits succinate oxidase and NADH oxidase, as well as mitochondrial electron transport between cytochrome b and c1 [12] by blocking electron flow in the Q-cycle of complex III [7]. Antimycin A also enhances the transfer of single electrons to molecular oxygen, producing superoxide [13] and consequently, has been used to investigate the generation of mitochondrial ROS and to model or simulate hypoxia [14]. Previous work in our laboratory has shown that feed supplemented with probiotics leads to an increased immune response in Haliotis midae, reflected by elevated haemocyte phagocytic activity
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[15] and the production of cellular ROS (Macey, unpublished). A cDNA microarray investigation of gene expression in haemocytes from immune-stimulated H. midae identified two electron transport chain genes, namely cytochrome b and cytochrome c oxidase III, that were differentially expressed in response to feeding a probiotic-supplemented feed (Arendze-Bailey, unpublished). Semiquantitative reverse transcription (RT) PCR of RNA isolated from haemocytes from immune-stimulated abalone verified that both cytochrome b and cytochrome c oxidase III are upregulated in response to probiotic treatment. The aim of the present study was to investigate the role of cytochrome b and cytochrome c oxidase III in the abalone immune response. Since cytochrome b and cytochrome c oxidase III code for respiratory complex components of the electron transport chain, we investigated the role of these genes in context to the role that the electron transport chain may play during stimulation of the abalone immune response. In order to accomplish this, we used antimycin A to specifically inhibit cytochrome b, and subsequently, we investigated the effect of inhibiting the electron transport chain on specific haemocyte immune parameters. 2. Materials and methods 2.1. Animals Abalone were maintained at the Marine and Coastal Management Research Aquarium in Sea Point, Cape Town, South Africa. The animals were maintained in 98 l polyethylene tanks containing aerated, continuously flowing natural seawater between 15 and 18 C. Between experiments, the abalone were fed kelp twice a week. 2.2. Collection of haemolymph Haemolymph was collected from the pedal sinus using a 1.5 ml syringe and 26 G ½-inch needle, and stored on ice until used.
2.6. Quantitation of intracellular ATP Since ATP is the final product of the electron transport chain, a decrease in ATP would indicate that antimycin A inhibits haemocyte respiration. Haemocyte ATP levels were measured using an ATP Determination Kit (Invitrogen Molecular Probes) which is based on a luciferin/luciferase reaction. A Standard Reaction Solution, containing water, 20 reaction buffer, 0.1 M DTT, 10 mM 1 D-luciferin and 2.5 ml of 5 mg ml firefly luciferase was prepared according to the manufacturer’s instructions. Haemocyte monolayers were treated for 2 h with either 100 ml PBS (control samples) or 100 ml antimycin A at final concentrations of 1 mM, 10 mM and 100 mM (test samples). The haemocyte monolayer was harvested by incubation in 100 ml 10 mM EDTA for 10 min. A 10 ml sample of the detached cells was removed for cell number determination (Section 2.3). The remaining sample was centrifuged for 5 min at 3000 g. The supernatant was removed and the pellet resuspended in cell lysis buffer ((w/v) 20 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5% Triton-X). After incubating for 15 min on ice, the samples were centrifuged and 10 ml of the supernatant was added to 100 ml of Standard Reaction Solution and incubated for 20 min at room temperature in the dark to ensure that the luminescent signal was stable. Following this, relative luminescence was measured in a TD 20/20 luminometer (Turner Designs). A standard curve was constructed each time the experiment was performed. Low-concentration ATP standard solutions were prepared by diluting the standard ATP solution (5 mM) provided with the kit in dH2O. The concentration of the standards ranged from 5 pM to 5 mM. Each standard (10 ml) was added to 100 ml of Standard Reaction Solution, incubated for 20 min in the dark and the luminescence measured. The luminescent readings of the samples could be converted to concentration of ATP using the standard curve. Because the number of cells in each sample had been determined, the ATP per sample was expressed as nM ATP/ cell. 2.7. Haemocyte viability
2.3. Total haemocyte count The total number of circulating haemocytes was determined directly after haemolymph collection. Undiluted haemolymph (100 ml) was added to 200 ml Alsevier’s Buffer ((w/v) 2.08% C6H12O6, 0.8% C6H5Na3O7$2H2O, 0.336% EDTA, 2.24% NaCl, 12% HCHO). The cells were counted using a haemocytometer and a light microscope at 100 magnification. 2.4. Attachment of abalone haemocyte monolayers After extraction, 100 ml haemolymph was placed into each well of a 96-well microtitre plate to obtain a cell density of 2 108 cells ml1 per well and allowed to attach for 20–25 min at room temperature in a humidified chamber. After attachment, the serum was removed, the haemocytes washed with PBS (7.3 mM monosodium phosphate, 180 mM disodium phosphate, 0.15 M sodium chloride, pH 7.2) and kept in 100 ml MHBSS ((w/v) 2.08% C6H12O6, 2.24% NaCl, 0.082% KCl, 0.02% KH2PO4, 0.071% CaCl2, 0.262% MgCl2, 0.314% MgSO4, 0.003% EGTA) until the assays were performed. 2.5. Treatment of haemocyte monolayers with antimycin A A stock solution of antimycin A (Sigma) was prepared in 100% ethanol (100 mg ml1, final concentration) and diluted to the desired concentration in PBS. After incubation with antimycin A for the required time, the antimycin A was removed, the haemocyte monolayers washed in PBS, and the chosen assay performed.
Antimycin A inhibition of electron transport may affect haemocyte viability, resulting in a diminished immune response. To ensure that a decreased immune response was caused by inhibition of electron transport and not a consequence of non-viable haemocytes, a MTT assay was performed on haemocytes treated with 1 mM, 10 mM and 100 mM antimycin A for 120 min. Also included in this experiment were samples treated with the equivalent amount of ethanol used to prepare the antimycin A to ensure that the ethanol did not affect haemocyte viability. After incubation with antimycin A, the supernatant was removed and the haemocytes rinsed with PBS. The haemocyte monolayers were incubated with 20 ml MHBSS and 20 ml MTT (5 mg ml1, Sigma) for 3 h. The supernatant was removed and the haemocytes rinsed twice with MHBSS. Solubilization of the purple precipitated formazan was achieved by adding 100 ml acidic isopropanol (0.4 M HCl in isopropanol) followed by overnight incubation on a plate shaker. Quantification of the formazan product was performed spectrophotometrically at 540 nm using a NanodropÒ ND-1000 UV–Vis spectrophotometer, where the absorbance at 540 nm represents the ability of haemocyte mitochondrial enzymes to reduce MTT, and is therefore a measure of haemocyte viability. 2.8. Quantitation of intracellular superoxide This assay was performed to determine whether treatment with antimycin A causes an increase in cellular superoxide production. Haemocyte monolayers were incubated with 100 ml 2 mg ml1
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nitroblue tetrazolium (NBT) and either PBS (control samples) or 100 ml 100 mM antimcyin (test samples). The samples were incubated in the dark for 2 h, whereafter the supernatant was removed and the haemocytes fixed by incubating in 100 ml 100% methanol for 10 min. The supernatant was removed, the cells air dried and the precipitated formazan dissolved by adding 120 ml 2 M KOH and 140 ml DMSO. The absorbance was determined at 620 nm on a NanodropÒ ND-1000 UV–Vis spectrophotometer. Since NBT is reduced by superoxide, the absorbance is directly related to the intracelluar amount of superoxide in the haemocytes.
2.9. Phagocytosis To determine whether inhibition of the electron transport chain would affect phagocytosis of pathogens, phagocytosis assays were performed comparing control haemocytes incubated in PBS to haemocytes incubated with antimycin A. The phagocytosis assay was performed as described by Macey and Coyne [15]. The time directly after haemocyte attachment and monolayer formation on the glass slide was designated T ¼ 0. The assay was performed at this time point, as well as after the haemocytes had been incubated in either PBS or antimycin A for 2 h.
2.10. Bacterial killing To determine whether inhibition of the electron transport chain has a direct effect on the killing mechanism of haemocytes, a bacterial killing assay was performed using a modification of the method described by Volety et al. [32]. E. coli JM109 was cultured in 5 ml LB media at 37 C overnight with shaking. The overnight culture was centrifuged at 3000 g and resuspended in PBS to yield approximately 1 106 cells ml1. Haemocyte monolayers were incubated with 100 ml of the bacterial cell suspension for 30 min to allow phagocytosis to occur. The bacteria were removed and the monolayers rinsed in PBS to remove residual bacteria. The T ¼ 0 h controls were prepared by rinsing the haemocytes with either 100 ml PBS or 100 ml antimycin A, while the T ¼ 2 h control and test samples were incubated for 2 h in either PBS or 100 ml 100 mM antimycin A, respectively. After incubation, the supernatant was removed and the haemocytes lysed in 100 ml distilled water for 15 min. The lysed samples were transferred to 1.5 ml eppendorf tubes, 100 ml LB was added and the samples were incubated for 2 h at 37 C with shaking. Following incubation, 100 ml MTT (5 mg ml1) was added to each sample. After a 2 h incubation in the dark, the bacterial cells were pelleted, the supernatant removed and the precipitated formazan dissolved in 100 ml acidic isopropanol. Quantification of the formazan was performed spectrophotometrically at 540 nm using a NanodropÒ ND-1000 UV–Vis spectrophotometer. For the purposes of the assay, bacterial killing was defined as the inability of bacteria to proliferate following exposure to haemocytes. The extent of MTT reduction to a purple formazan reflects the effect of the haemocytes on the bacteria. Thus, a high degree of bacterial survival would result in increased levels of formazan.
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3. Results 3.1. Effect of antimycin A on electron transport The effect of antimycin A on haemocyte ATP production was determined using a luciferin/luciferase assay. Haemocytes treated with 1 mM antimycin A had ATP levels of 2 1012 nM ATP/cell which was equivalent to the ATP concentration in haemocytes exposed to 10 mM and 100 mM antimycin A (Fig. 1). The ATP concentration in antimycin A treated haemocytes was almost half that present in the control haemocytes (4 1012 nM ATP/cell), indicating that antimycin A caused a significant reduction in cellular ATP in treated haemocytes. 3.2. Effect of antimycin A on haemocyte viability In order to ensure that the reduced ATP levels in antimycin A treated haemocytes was not due to the onset of cell death, the viability of haemocytes exposed to antimycin A was determined using a MTT reduction assay. Thus, ability of haemocytes incubated in PBS to reduce MTT to a purple formazan was compared to that of haemocytes incubated in 1 mM, 10 mM and 100 mM antimycin A. Antimycin A had no significant effect on haemocyte viability in that the amount of formazan formed by antimycin A treated haemocytes was not significantly different to that of the control haemocytes (Fig. 2). The effect of ethanol in which the antimycin A was dissolved was also determined. The viability of haemocytes incubated in 0.1% and 1% ethanol (equivalent to ethanol concentration in 10 mM and 100 mM antimycin A solutions, respectively) was not significantly reduced in relation to the PBS control. 3.3. The effect of antimycin A on haemocyte production of cellular superoxide Antimycin A resulted in a 1.5 fold increase in formazan (0.088 absorbance units as opposed to 0.057 absorbance units in the control haemocytes) (Fig. 3). Thus, intracellular superoxide increased significantly in haemocytes treated with antimycin A. 3.4. Effect of antimycin A on haemocyte phagocytic activity Phagocytosis of FITC-labelled V. anguillarum was un-affected in haemocytes that were briefly exposed to antimycin A (Fig. 4). However, there was a significant reduction in phagocytosis after exposing the haemocytes to antimycin A, as opposed to PBS, for 2 h.
2.11. Statistical analysis Experimental data is reported as the mean of three biological repeats, each having been measured in triplicate. Error bars represent the standard error of the mean. An unpaired t-test was used to compare two means (p < 0.05), while multiple comparisons were conducted using one-way ANOVA analysis followed by the Tukey post hoc test (p < 0.05) using SigmaStat 3.1.
Fig. 1. The effect of increasing concentrations of antimycin A on haemocyte ATP production. Haemocytes were treated with 1 mM, 10 mM and 100 mM antimycin A (AM) or PBS for 2 h. Each bar represents the mean of 3 biological repeats measured in triplicate, while the vertical lines reflect standard error of the mean. (*) Indicates sample differs significantly (p < 0.05, unpaired t-test) to the PBS control.
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Fig. 2. The effect of antimycin A on haemocyte viability. Control haemocyte samples were treated with either PBS, 0.1% or 1% ethanol (Etoh) in PBS. Test samples were incubated in 1 mM, 10 mM or 100 mM antimycin A (AM). The bars represent the mean of 3 biological repeats measured in triplicate, while the vertical lines represent standard error of the mean.
3.5. Effect of antimycin A on haemocyte destruction of pathogenic bacteria The number of bacteria killed by haemocytes incubated for 0 h in antimycin A did not differ significantly from that observed in haemocytes incubated in PBS for the same time period, with approximately 0.12 absorbance units of formazan detected (Fig. 5). The number of viable bacteria remaining following exposure to haemocytes incubated in PBS for 2 h was significantly less (p < 0.05) than the number of surviving bacteria in either of the 0 h controls (PBS or antimycin A). However, bacterial survival following exposure to haemocytes that had been incubated in antimycin A for 2 h did not differ significantly to the controls (haemocytes incubated in antimycin A for 0 h or PBS for 2 h).
4. Discussion Abalone farming, like other commercial aquaculture enterprises, is constantly at risk due to the devastating impact of infectious disease outbreaks [1]. An understanding of the abalone response to infection is necessary in order to mitigate the high mortality rates associated with infectious diseases in farmed abalone [3]. Previously, we showed that both cytochrome b and cytochrome c oxidase III are upregulated in response to
Fig. 3. The effect of antimycin A on haemocyte production of cellular superoxide. Control haemocytes were incubated in PBS and test samples in 100 mM antimycin A (AM) for 2 h. Each bar represents the mean of 3 biological repeats measured in triplicate, while the vertical lines reflect standard error of the mean. (*) Indicates sample differs significantly (p < 0.05, unpaired t-test) to the PBS control.
Fig. 4. Effect of antimycin A on haemocyte phagocytic activity. Control haemocytes were incubated in PBS and test samples in 100 mM antimycin A (AM) for 0 and 2 h prior to investigating phagocytosis. Each bar represents the mean of 3 biological repeats measured in triplicate, while the vertical lines reflect standard error of the mean. Different letters indicate significant difference (p < 0.05, ANOVA) between values.
immune-stimulation, implying that the mitochondrial respiratory chain might play a role in the abalone immune response. The aim of this study was to investigate the role of the electron transport system in the abalone immune response, and the reliance of this response on oxidative phosphorylation. This was accomplished by inhibiting electron transport in abalone haemocytes with the use of antimycin A and subsequently investigating the effect on various immune parameters. Inhibition of electron transport through antimycin A treatment has been shown to be harmful to cells by inducing apoptosis [16] and has been shown to lead to the death of numerous cell types including juxtaglomerular cells [6], hepatocytes [14,17] and human lymphoblastoid cells [16]. It was therefore important to ensure that antimycin A treated abalone haemocytes remained viable during the course of the assays employed in this study so that any reduction in immune response could be attributed to inhibition of electron transport. The MTT assay has been widely used to assay the viability of numerous cell types [18,19] including phagocytic cells [20], and to determine the effect of chemical treatment on cell viability [21,22]. Since abalone haemocytes remained viable following a 2 h incubation in 100 mM antimycin A, we applied antimycin A at this concentration and time interval in subsequent assays. Antimycin A has been shown to lead to a marked reduction in cellular ATP levels [6,14,17]. Measuring the effect of antimycin A on cellular ATP would assist in determining whether antimycin A indeed has an inhibitory effect on the electron transport system of H. midae. The level of ATP in haemocytes treated with antimycin A was markedly decreased compared to that of control cells. In fact, the level of ATP in antimycin A treated haemocytes was almost 50% of that in control cells, which is similar to results obtained with other cell types; for example, antimycin A lead to a 15% decrease in hepatocyte ATP [14] and a 40% reduction in ATP in renal epithelial cells [23]. In addition to causing a decrease in cellular ATP, antimycin A is known to significantly increase cellular superoxide production [6]. Similarly, abalone haemocytes treated with antimycin A produced significantly more superoxide in comparison to untreated cells (54% increase). Superoxide detected in the control haemocytes is not unusual. For example, superoxide produced in haemocytes from the white shrimp, Penaeus vannamei, that had not been stimulated by various disease response elicitors was attributed as background superoxide [24]. Production of high levels of superoxide due to antimycin A treatment has been widely demonstrated in various cell types [6,12,25,26] and organisms [27]. Meaney et al.
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[13] showed that mitochondria treated with antimycin A released five times more superoxide into the mitochondrial matrix compared to control samples. Mukhopadhyay et al. [28] showed that a 3–7 fold dose-dependent increase in superoxide production occurred in response to antimycin A in rat and human cells. This effect is explained by the fact that interruption of electron transport results in the formation of reactive oxygen species such as superoxide as a consequence of autoxidation of reduced electron transport components [29]. The effect of antimycin A on phagocytosis was investigated as a direct measure of the importance of the electron transport chain in the abalone immune response. Phagocytosis in haemocytes treated with antimycin A declined from nearly 30% in control cells to 17% in treated cells (Fig. 4). A similar effect has been shown in human monocytes, where treatment with antimycin A led to a 50% decrease in phagocytosis after incubation in 360 mM antimycin A for 45 min [30]. Similarly, antimycin A caused diminished phagocytosis of fluorescent beads in alveolar macrophages [31]. After determining that a functional electron transport system is necessary for phagocytosis of invading pathogens, the effect of an inhibited electron transport system on the bactericidal action of abalone haemocytes was examined (Fig. 5). Bacterial killing by abalone haemocytes was estimated colorimetrically through the reduction of the tetrazolium salt MTT to a purple formazan [32]. The formazan produced during the assay is directly proportional to the number of bacterial colonies detected by agar plate count methods such as those described by Canesi et al. [33,34]. Thus, absorbance can be directly correlated to the number of bacterial colonies that survived exposure to the haemocytes. The bactericidal assay showed that the number of bacteria that survived exposure to haemocytes that had been incubated in antimycin A for 2 h was not significantly less than in the control. If antimycin A had inhibited bacterial killing, one would expect the number of surviving bacteria to be equivalent to that of the 0 h control which represents the starting bacterial titre, and significantly higher than the 2 h PBS control which reflects the number of surviving bacteria following haemocyte killing. What the results do show is that some killing did occur, but not to the same level as that in the 2 h control. A possible explanation for this phenomenon could be, as mentioned earlier, the production of additional superoxide at complex III in response to antimycin A blockage of electron transport. Superoxide is bactericidal [35] and is formed in response to bacterial invasion in numerous marine organisms such as Crassostrea gigas [36], Macrobrachium rosenbergii [37], Lynmaea stagnalis
Fig. 5. The effect of antimycin A on the ability of haemocytes to kill invading bacteria. Control samples were incubated in PBS for 0 and 2 h, respectively. Test samples were incubated in 100 mM antimycin A (AM) for 0 and 2 h. Each bar represents the mean of 3 biological repeats measured in triplicate, while the vertical lines reflect standard error of the mean. Different letters indicate significant difference (p < 0.05, ANOVA) between values.
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[38] and Mercenaria mercenaria [5]. Thus, although some bacterial killing occurred, it was insufficient to conclusively determine whether antimycin A did or did not inhibit abalone haemocyte bacterial killing. Indeed, Watabe and Nakaki [6] mention that superoxide production following treatment with respiratory inhibitors makes it difficult to analyse their effect on cellular functions. The bactericidal assay showed that normal abalone haemocytes recognize and kill E. coli as an invading pathogen, and that antimycin A treatment results in the production of sufficient superoxide to destroy some of the invading bacteria. Since antimycin A has been shown to interfere with the killing of Staphylococcus in alveolar [31] and murine macrophages [39], further investigation is required to unequivocally determine whether inhibition of the electron transport system affects the bactericidal action of abalone haemocytes. This study has shown that the mitochondrial respiratory chain plays a role in the immune response of the abalone due to the increased metabolic needs of the cell during immune-stimulation. Increasing our understanding of how the abalone immune system functions would improve our ability to alleviate and or prevent infectious disease outbreaks in farmed H. midae.
Acknowledgements The study was funded by a National Research Foundation research grant (FA2004040600014) awarded to Vernon Coyne. Marike Janse van Rensburg was supported by a NRF Grant-holder linked postgraduate student bursary.
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