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Developmental & Comparative Immunology
Developmental and Comparative Immunology 31 (2007) 1233–1241 www.elsevier.com/locate/devcompimm
Short communication
Adenosine arrests apoptosis in lymphocytes but not in phagocytes from primary leucocyte cultures of the teleost fish, Sparus aurata L. Irene Salinas, Alejandro Rodrı´ guez, Jose´ Meseguer, Maria Angeles Esteban Fish Innate Immune System Group, Department of Cell Biology, Faculty of Biology, Campus de Espinardo, University of Murcia, 30100 Murcia, Spain Received 18 December 2006; received in revised form 28 March 2007; accepted 30 March 2007 Available online 4 May 2007
Abstract Adenosine (A) and its derivatives have important biological functions, including the inhibition of immune responses and apoptosis induction. The aim of this work was to investigate whether A and N6-cyclohexyladenosine (CHA) regulate apoptosis in leukocyte cultures from the teleost, gilthead seabream (Sparus aurata L.) by flow cytometry using a double fluorescence staining method. Head kidney leukocytes (HKLs) were cultured for 0, 24 or 48 h. The kinetics of FDA+/PI (viable), FDA/PI (apoptotic) and FDA/PI+ (necrotic) leukocyte subpopulations were followed during the culture process. Apoptosis was induced by addition of resveratrol or staurosporine to the culture media and the study was validated by transmission electron microscopy. A and CHA did not induce but decreased apoptosis of seabream HKLs, in particular HK lymphocytes (L), after 24 h in culture. Results raise the question whether apoptosis is differently modulated by purine nucleosides in fish L and phagocytes and also in fish compared to mammalian cells. r 2007 Elsevier Ltd. All rights reserved. Keywords: Adenosine; N6-cyclohexyladenosine; Apoptosis; Leukocytes; Flow cytometry; Teleost; Seabream (Sparus aurata)
1. Introduction Adenosine (A) and other ATP derived molecules have long been recognised as important multipotent cell modulators both intra- and extracellularly. Their biological functions range from protecting against ischemic damage to downregulating immune responses [1,2]. In addition, they have been shown to Abbreviations: CHA, N6-cyclohexyladenosine; A, adenosine; HKLs, head kidney leukocytes; FDA, fluorescein diacetate; PI, propidium iodide Corresponding author. E-mail address: [email protected] (J. Meseguer).
affect proliferation, survival and apoptosis of many different cell types, ranging from epithelial, endothelial and smooth muscle cells, to cells of the immune and neural lineages from higher vertebrates [3]. In a previous study, we demonstrated that in fish, the innate immune functions of head kidney leukocytes (HKLs) were downregulated in vitro by N6-cyclohexyladenosine (CHA) but not by A, suggesting the presence of purinergic receptors in fish immune cells. Thus, it seems that regulation of immune responses by A compounds already occurs in lower vertebrates [4]. However, nothing is known about the capacity of purine nucleosides to affect apoptosis in teleost fish.
0145-305X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2007.03.014
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Apoptosis or programmed cell death is genetically controlled and takes place during normal physiological processes such as embryonic development, tissue remodelling and immune response regulation. During apoptosis, a well-defined sequence of events occurs, making it possible to distinguish this process from necrosis [5–7]. For instance, the DNA fragmentation that results from apoptosis can easily be visualised in a total DNA electrophoresis, whereas necrotic cells show intact DNA, with a very different electrophoretic pattern [8]. Moreover, cells undergoing apoptosis show characteristic ultrastructural features under transmission electron microscopy (TEM). These include cell shrinkage, aberrant nuclear shape, chromatin condensation and the appearance of hyperchromatic nuclear fragments named apoptotic bodies [5–7]. In addition to DNA degradation-based techniques, other routinely used methods to discriminate cells undergoing apoptosis include the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) [9], mitochondrial membrane hyperpolarisation [10], the Annexin V-PI staining method [8], caspase activation [11] and cytochrome C translocation [12]. The aim of the present study was to investigate the possible role of A and CHA in modulating apoptosis in gilthead seabream (Sparus aurata L.) leukocytes cultured in vitro. To this end, a double fluorescein diacetate/propidium iodide (FDA/PI) staining flow cytometry assay was set up to quantify the apoptosis of seabream leukocytes. 2. Materials and methods 2.1. Animals Twenty specimens (100 g mean body weight) of the hermaphroditic protandrous seawater teleost gilthead seabream (Sparus aurata L.) were obtained from CULMAREX S.A. (Murcia, Spain). Animals were kept in 450–500 l running seawater (28% salinity) aquaria at 2072 1C and with a 12 h light: 12 h dark photoperiod. Animals were acclimated for 30 days prior to the experiment. They were fed daily with 1 g of a commercial pellet diet (Trow Espan˜a, Burgos, Spain) per fish and were starved 24 h prior to sampling. All the investigations of the present paper were conducted in accordance with national and international guidelines for the protection of animal welfare. All protocols applied in this research were
reviewed by the Committee for Animal Care and Welfare of the University of Murcia, Spain. 2.2. Setting up a double staining flow cytometry assay to measure apoptosis in HKLs 2.2.1. Cell isolation Fish were anaesthetised with benzocaine (4% in acetone) (Sigma) and bled from the caudal vein. HKLs were isolated from each fish under sterile conditions. Briefly, head kidneys were excised, cut into small fragments and transferred to 8 ml of sRPMI (RPMI-1640 culture medium (Gibco) with 0.35% sodium chloride (to adjust the medium’s osmolarity to gilthead seabream plasma osmolarity, 353.33 mOs), 100 IU/ml penicillin (Flow), 100 mg/ml streptomycin (Flow), 10 IU/ml heparin (Sigma) and 5% foetal bovine serum (Gibco)). Cell suspensions were obtained by forcing fragments of the organ through a 102 mm nylon mesh. Afterwards, the cell suspension was layered over 51% Percoll gradients. The leukocyte interface with HKLs was collected and washed twice in sRPMI without heparin. HKLs were counted in a Neubauer chamber and adjusted to 2 106 cells/ml of sRPMI. 2.2.2. Cell culture The 500 ml of each HKL suspension previously obtained, were dispensed into flat bottomed 48-well plates and cultured for 0, 24 or 48 h (22 1C, 5% CO2, 85% humidity). Additionally, 10 ml of resveratrol (22.8 mg/ml in absolute ethanol Sigma), staurosporine (1 mg/ml in absolute ethanol, Sigma) were added in triplicate as positive controls for apoptosis. Additionally, controls consisting of absolute ethanol or dimethylsulfoxide (DMSO, Scharlau) only were tested. Positive controls for necrosis consisted of fixed cells. For that, aliquots of 500 ml of each cell suspension were centrifuged and the supernatants discarded. The cell pellets were fixed by adding 2 ml of formaldehyde 10% in PBS (30 min, 4 1C). All assays were done in triplicate. 2.2.3. Apoptosis assay A double staining flow cytometric assay combining FDA (Sigma) and PI was used by adapting the method used for the detection of apoptotic leukocytes in mammalian cells [13,14] and fish cells [15] to the seabream leukocytes. To optimise the FDA staining protocol for gilthead seabream leukocytes, 0.05 g FDA were resuspended in 4 ml of DMSO. This solution was
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used to obtain different working solutions (12.5, 25, 50, 75 or 100 mg FDA/ml in sRPMI). The 10 ml of each FDA concentration were added to each sample followed by 50 ml of PI (400 mg/ml, Sigma) 30 min prior to analysing the cells in the Coulters Epicss XLTM flow cytometer (Beckman Coulter). Analyses were performed on 30,000 cells, which were acquired at a rate of 300 cells/s. Side scatter (granularity), forward scatter (size), FL-1 (green fluorescence) and FL-3 (red fluorescence) for each cell population were represented in dot plots or histograms. To analyse the leukocyte cell subpopulations present in HKL cell suspensions, two regions were established according to the lymphocyte and phagocyte regions previously characterised [16]. This could only be undertaken at 0 and 24 h of culture because the defined subpopulations were no longer identifiable at 48 h and a significant new population consisting of debris from dead leucocytes appeared.
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4 106 cells/ml of sRPMI. Aliquots (250 ml) of each cell suspension were dispensed into 48-well flat bottom culture plates. Then, 250 ml of A (200 mM in sRPMI, Sigma) or CHA (200 mM in sRPMI, Sigma) or sRPMI (control) were added to each well in triplicate. The concentration of purinergic nucleosides was chosen according to previous data obtained for higher vertebrates [16] and fish [4]. Positive controls consisted of wells with 10 ml staurosporine (1 mg/ml in absolute ethanol). Plates were maintained for 30 min, 24 h or 48 h under the same conditions as before and the apoptosis assays were conducted as explained above. The preliminary study enabled us to identify 50 mg/ml as the optimal FDA concentration for staining seabream HKLs. Therefore, this concentration was used in the rest of the experiments. Analysis by subpopulations (lymphocytes and phagocytes) was conducted for 0 and 24 h only. 2.4. Statistical analysis
2.2.4. Microscopy study Stained samples were directly observed from culture plates under a phase contrast microscope (Nikon). Photographs were taken by light and fluorescence microscopy using an alternating green (excitation 470, 490 nm, barrier 520–560 nm) and red (excitation 510, 560 nm, barrier 690 nm) filter combination. HKLs were also cultured in culture dishes (9 cm diameter) at the same cell density as above. Cells were incubated without (control) or with staurosporine (1 mg/ml) and samples were collected after 0, 24 and 48 h of culture for examination by transmission electron microscopy (TEM). Cell suspensions were washed and the supernatants removed, while pellets were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2, 2 h, 4 1C) before being postfixed in OsO4 and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in an EM10X Zeiss transmission microscope. The sections were examined to identify normal, apoptotic or necrotic cells. Nuclear chromatin condensation was used as a typical criterion for apoptotic cells. Cells that swelled and showed cytoplasmic vacuolisation as well nuclear destruction were characterised as necrotic. 2.3. Effects of purinergic nucleosides on seabream HKL apoptosis Six samples of HKLs previously isolated as described above were counted and adjusted to
Results are expressed as mean7standard error. Data were statistically analysed by one-way analysis of variance (ANOVA) and Tukey’s comparison of means when necessary. Differences were considered statistically significant when po0.05. 3. Results 3.1. Setting up of a double staining flow cytometry assay to measure apoptosis in seabream leukocytes 3.1.1. FDA labelling of seabream leukocytes Increasing concentrations of FDA resulted in greater percentage of labelled cells. The optimal concentration of FDA to stain seabream HKLs was 50 mg/ml, which stained over 95% of the phagocytes and 83% of the lymphocytes. Higher concentrations did not result in greater staining percentages. It was also observed that different cell types acquired different FDA (green) intensities being the phagocytic subpopulation more intensely stained than the lymphocyte subpopulation as shown in Fig. 1. 3.1.2. Kinetics of the viability of seabream primary leukocyte cultures Following double staining with FDA and PI, three subpopulations were identified and quantified in the dotplots and histograms obtained after the flow cytometric study: (i) viable (FDA+/PI),
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Fig. 1. (a–c) Flow cytometry analysis of a readily isolated head kidney leukocyte suspension after staining with FDA (50 mg/ml) and propidium iodide (PI) for 30 min. (a) Dotplot showing two main subpopulations: lymphocytes (L) and phagocytes (Ph) according to their SSC and FSC values. (b) Dotplot showing events staining with FL1 (green fluorescence, FDA) and FL3 (red fluorescence, PI). Note the presence of three populations according to the double staining pattern: the FDA+/PI (viable) population contains two subpopulations: the phagocytes (Ph) that acquire greater green intensity and the lymphocytes (L) with a weaker fluorescence; the FDA/PI (apoptotic) population and the FDA/PI+ (necrotic) population. (c) Histogram showing FL3 (red fluorescence) after labelling of HKLs with the double staining protocol. Note that almost all cells remained unpermeabilised and are therefore PI. (d) Histogram showing the green fluorescence (FL1) after labelling of HKLs with the double staining protocol. Most cells were FDA+ being notable that lymphocytes (L) and phagocytes (Ph) show different labelling intensities with FDA. (e–g) Fluorescence microscopy study after labelling with the FDA/PI double staining technique a seabream head kidney leukocyte (HKL) suspension cultured for 24 h. Cells belonging to all three staining patterns were observed, 400. (e) Phase contrast micrograph showing a seabream HKL suspension. (f) Green fluorescence micrograph corresponding to the same field shown in a). Note that most cells in (e) were viable and therefore stained green (FDA+). (g) Red fluorescence micrograph corresponding to the same field shown in a) showing few permeabilised (PI+) leukocytes.
(ii) apoptotic (FDA/PI) and (iii) necrotic (FDA/ PI+) (Fig. 1). Positive controls for necrosis consisting of permeabilised cells fixed with formaldehyde were 100% PI+ (data not shown). Head-kidney lymphocytes and phagocytes displayed clearly different viabilities in the same culture conditions, being the percentage of FDA/ PI (apoptotic) lymphocytes always higher than that of phagocytes. The percentage of FDA/PI+ (necrotic) head-kidney lymphocytes and phagocytes
remained low at 0 and 24 h and no significant differences were recorded (see Table 1). After 48 h in culture, viability of seabream HK cultures significantly decreased compared to 0 and 24 h, the total percentage of apoptotic cells reaching 40% and necrotic cells almost 20%. Finally, despite the fact that cells still cleaved FDA at 24 or 48 h, it was observed that the intensity or mean green fluorescence value decreased with culture time (data not shown).
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Table 1 Effect of time in culture on viability of in vitro seabream HKLs cultures 0h
Phagocytes Lymphocytes
24 h
48 h
FDAPI
FDAPI+
FDAPI
FDAPI+
FDAPI
FDAPI+
1.671.0a 14.771.4b
0.870.1a 0.970.2a
2.270.5a 23.774.7b
2.070.7 a 3.770.5a
40.072.0c
19.673b
Percentage of FDA/PI (apoptotic) and FDA/PI+ (necrotic) seabream leukocytes from head kidney (HKLs) after 0, 24 or 48 h in culture. Results are expressed as mean7sd. Data represent two independent experiments using n ¼ 3 in each experiment. One-way analysis of variance (ANOVA) and Tukey’s comparison of means were conducted when necessary. Different letters stand for statistically significant differences (po0.05) between different culture times.
Table 2 Apoptotic inducers and the viability of in vitro seabream HKLs cultures Incubation time (h)
Phagocytes Lymphocytes
0 24 0 24
Resveratrol
Staurosporine
FDAPI
FDAPI+
FDAPI
FDAPI+
9.872.7 10.172.4 35.374.3 57.175.9*
3.571.3 6.772.4 2.970.9 20.074.9*
8.873.1 14.673.5 29.875.1 56.974.2*
2.370.6 3.171.6 1.570.3 13.672.7*
Effects of two apoptotic inducers, resveratrol and staurosporine, in the percentage of FDA/PI (apoptotic) and FDA/PI+ (necrotic) seabream leukocytes from head kidney (HKLs) after 0 and 24 h in culture. Results are expressed as mean7sd. Data represent two independent experiments using n ¼ 3 in each experiment. One-way analysis of variance (ANOVA) and Tukey’s comparison of means were conducted when necessary. Asterisk denotes statistically significant differences (po0.05) between treated leukocytes at 24 h compared to treated leukocytes at 0 h. No statistically significant differences were found between control cultures and treated cultures at 0 h.
3.1.3. Effects of apoptotic inducers on the viability of seabream primary leukocyte cultures The effects of adding resveratrol or staurosporine to seabream primary HKL cultures at 0 and 24 h are summarised in Table 2. At 0 h, there were no statistically significant differences between control cultures and cultures where apoptotic inducers had been added. However, head-kidney phagocytes appeared more resistant to both treatments than head-kidney lymphocytes, since the percentage of FDA/PI only increased from 9.8% to 10.1% after 24 h incubation with resveratrol and from 8.8% to 14.6% in the case of staurosporine. On the contrary, the percentage of apoptotic lymphocytes after 24 h in resveratrol or staurosporine almost doubled the percentage recorded immediately after their addition (around 30% at 0 h). 3.1.4. Microscopy study Observation of HKLs primary cultures from the different assays under the light or fluorescence microscope confirmed the presence of the three populations previously observed by flow cytometry:
FDA+/PI (viable), FDA/PI (apoptotic) and FDA/PI+ (necrotic) cells (Fig. 1e–g). TEM examination of leukocyte cultures revealed that control cultures (without staurosporine) consisted of cells exhibiting the normal morphology observed at the beginning of the experiment. Micrographs showing the ultrastructural characteristics of seabream HKL cultures after 0, 24 or 48 h in the presence of staurosporine match dot plots corresponding to the flow cytometric analysis after FDA/PI labelling in Fig. 2. Leukocytes cultured in the presence of staurosporine were characterised by normal nucleus and cytoplasm morphology at the beginning of the experiment and did not differ from control leukocytes at the same time (Figs. 2a and d). However, the number of cells with typical chromatin condensation and aberrantly shaped nuclei (apoptotic) dramatically increased after 24 h of incubation in the presence of staurosporine), which correlates with the flow cytometry analysis of this suspension (Figs. 2b and e). Apoptotic cells were abundant in HKL cultures incubated with staurosporine,
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Fig. 2. Flow cytometry (a–c) and transmission electron microscopy (d–f) study of an HKL suspension cultured in presence of staurosporine (1 mg/ml). Dotplot after labelling HKL suspension with FDA and PI after 0 h (a), 24 h (b) and 48 h (c) in culture. Transmission electron micrograph of the same HKL suspension cultured for 0 h (d), 24 h (e) and 48 h (f). Note that the FDA+/PI population progressively moves towards the left and, concomitantly, the FDA/PI moves upwards entering the FDA/PI+ quadrant. These shifts in the populations observed under flow cytometry were in agreement with the morphological changes seen under TEM with increasing numbers of apoptotic nuclei and membrane disruption at the end of the experiment.
especially after 48 h of incubation when most of the cells had lost their architecture, and their membrane and cytoplasm were profoundly damaged. They were therefore scored as necrotic (Figs. 2c and f). 3.2. Effect of purinergic nucleosides on apoptosis of seabream HKLs
on the phagocytes (Fig. 3c). However, A increased the number of apoptotic phagocytes at this time, but the effect was not significant. After 48 h of culture, the percentage of total apoptotic seabream leukocytes was still lower than the percentage found in control samples, although the decreases were not statistically significant (Fig. 3a). 4. Discussion
No significant differences were observed after 30 min in contact with the purine nucleosides, neither in the number of lymphocytes nor in the phagocytes, although in both cases A resulted in a slight increase of apoptotic cells (Fig. 3a–c). Both A and CHA reduced apoptosis in HKL cultures after 24 h. There were significantly fewer (p ¼ 0.006) apoptotic cells in those samples incubated with A or CHA (100 mM) than in control samples after 24 h (Fig. 3a). When the analysis was conducted in the different subpopulations, it was observed that only apoptosis of lymphocytes was significantly inhibited (Fig. 3b), whereas no significant effect was observed
A and other purine nucleosides are important metabolites that regulate a variety of biological processes in animal cells and tissues. They are present both extracellularly and inside cells, their concentration increasing upon tissue damage or injury [1]. Their effects even reach the domain of the immune system, and A is considered as a downregulator of immune responses in vertebrates [2,18]. This was recently confirmed in the gilthead seabream, whose leukocyte phagocytic and respiratory burst activities were decreased in vitro when CHA, an A derivative, was present [4].
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45 40 % Apoptotic cells
35
Control CHA A
30 25 20 *
15
*
10 5 0 24h
30 25 20
*
15
*
10 5 0 30min
% Apoptotic phagocytes
% Apoptotic lymphocytes
30min
48h
4 3 2 1 0 30min
24h control
CHA
24h
Adenosine
Fig. 3. Effects of CHA and A on apoptosis of seabream HKLs cultured in vitro. (a) Percentage of total apoptotic seabream HKLs cultured for 30 min, 24 h or 48 h. (b) Percentage of apoptotic lymphocytes after 30 min or 24 h in culture. (c) Percentage of apoptotic phagocytes after 30 min or 24 h in culture. Results are expressed as mean7sd. One-way analysis of variance (ANOVA) and Tukey’s comparison of means were conducted when necessary. Asterisk denotes statistically significant differences (po0.05). Data represent two independent experiments using n ¼ 3 in each experiment.
A also regulates the cell cycle since it is capable of inhibiting apoptosis in cancer cell lines and in rat primary astrocyte cultures [17,19]. However, nothing is known with respect to the effect of purine nucleosides on apoptosis of fish cells and in particular, on leukocyte primary cultures from teleost fish. The present work employs for the first time the FDA/PI technique to assess apoptosis in seabream cells. Our results show that FDA staining differed between head–kidney phagocytes and lymphocytes, the intensity of green fluorescence being markedly higher in phagocytes. This indicates that phagocytic leukocytes contain more esterases, or more active ones, than lymphocytes, probably due to the presence of numerous cytoplasmic granules which contain esterases, among other lysosomal enzymes, in macrophages and granulocytes [20]. The combi-
nation of FDA and PI made it possible to identify viable, apoptotic and necrotic seabream cells. This technique has been successfully applied to teleostean leucocytes before and validated with the conventional Annexin V apoptosis assay [15]. The use of proapoptotic agents like resveratrol and staurosporine served as a good indicator of cell death in seabream leukocytes. Resveratrol, a stilbene derived from plants, has been shown to inhibit proliferation and trigger apoptosis in a number of human cancer cell lines [21]. Staurosporine is a protein kinase inhibitor commonly used to induce apoptosis [22]. In the present study, both compounds managed to induce apoptosis in seabream lymphocytes at a significantly greater rate than controls after 24 h of culture. The semiquantitative information obtained from the study of seabream leukocyte cultures under
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TEM perfectly correlated with quantitative data from the flow cytometry analysis, as expected. Apoptotic cells showed signs of shrinkage, chromatin condensation and rounded electron dense nuclei, as described by other authors [7]. Studies conducted on carp found quantitative correlation between the FDA/PI technique and Annexin V staining [15]. The kinetic study of apoptosis in seabream leukocyte cultures from head kidney reveals that culture time is a vital factor when immunological or biological studies of fish leukocytes involve cell culture techniques. The number of apoptotic HK phagocytes increased linearly with culture time (18% at 24 h and 28% at 48 h). Thus, it can be concluded that in vitro culture of seabream HK phagocytes results in cell death via apoptosis and the low necrotic numbers (2% at 24 h and 4.3% at 48 h) may represent advanced stages of apoptosis. Additionally, mean green fluorescence in seabream HK leucocyte cultures decreased with time within the population stained FDA+/PI, which is also in agreement with previous data and suggests that the cell metabolic rate decreases with culture time [14]. The double FDA/PI staining flow cytometry assay was set up in order to investigate the possible role of A and CHA in the apoptotic events that occur during leukocyte primary cultures. In contrast with previous data available from established tumour cell lines [19] or in astrocytes isolated from rat brain [17], apoptosis was not induced but arrested in seabream HKL cultures after 24 h. This was true in the case of lymphocytes and not the phagocytes, which display the opposite behaviour but in a non-significant way. The A and CHA dose here assayed is a high biological dose and corresponds to scenarios where a sharp increase of A takes places following tissue damage [2]. Further studies may address the minimum concentration of these molecules required to modulate apoptosis in fish leucocytes. Both purines showed similar effects despite the fact that A was not efficient at downregulating the seabream innate immune parameters, whereas CHA was [4]. The present results warrant further studies into the regulation of seabream immune responses and purine nucleosides, being of special relevance the differential behaviour of lymphocytes and phagocytes here reported. In conclusion, both A and CHA decreased apoptosis of seabream lymphocytes but not phagocytes after 24 h in culture. Our findings reveal major differences between lower and higher vertebrates
concerning the biological role of purine nucleosides during regulation of immune responses and how apoptosis is involved in this regulation. Acknowledgements I. Salinas received Ph.D. funding from Fundacio´n Se´neca, Murcia, Spain. Authors wish to thank all staff from the Tissue Culture Service (SACE) at University of Murcia. References [1] Cronstein BN. Adenosine, an endogenous anti-inflammatory agent. J Appl Physiol 1994;76:5–13. [2] Hasko´ G, Cronstein BN. Adenosine: an endogenous regulator of innate immunity. Trends Immunol 2004;25: 33–9. [3] Jacobson KA, Hoffmann C, Cattabeni F, Abbracchio MP. Adenosine-induced cell death: evidence for receptormediated signalling. Apoptosis 1999;4(3):197–211. [4] Salinas I, Rodriguez A, Esteban MA, Meseguer J. N6Cyclohexyl-adenosine but not adenosine is a modulator of teleost fish innate immune activities. Dev Comp Immunol 2005;30:325–34. [5] Earnshaw WC. Apoptosis: lessons from in vitro systems. Trends Cell Biol 1995;5(6):217–20. [6] Lund PK, Westvik AB, Joo GB, Ovstebo R, Haug KB, Kierulf P. Flow cytometric evaluation of apoptosis, necrosis and recovery when culturing monocytes. J Immunol Methods 2001;252(1):45–55. [7] Guejes L, Zurgil N, Deutsh M, Gilburd B, Shoenfeld Y. The influence of different cultivating conditions on the polymorphonuclear leukocyte apoptotic processes in vitro, I: the morphological characteristics of spontaneous apoptosis. Ultrastruct Pathol 2003;27(1):23–32. [8] Bacso´ Z, Everson RB, Eliason JF. The DNA of annexin V-binding apoptotic cells is highly fragmented. Cancer Res 2000;60(16):4623–8. [9] Louagie H, Cornelissen M, Philippe J, Vral A, Thierens H, De Ridder L. Flow cytometric scoring of apoptosis compared to electron microscopy in gamma irradiated lymphocytes. Cell Biol Int 1998;22(4):277–83. [10] Jo WS, Jeong MH, Jin YH, Jang JY, Nam BH, Son SH, et al. Loss of mitochondrial membrane potential and caspase activation enhance apoptosis in irradiated K562 cells treated with herbimycin. A Int J Radiat Biol 2005;81(7):531–43. [11] Thyrell L, Erickson S, Zhivotovsky B, Pokrovskaja K, Sangfelt O, Castro J, et al. Mechanisms of interferon-alpha induced apoptosis in malignant cells. Oncogene 2002;21(8): 1251–62. [12] Cao Y, Adhikari S, Ang AD, Moore PK, Bhatia M. Mechanism of induction of pancreatic acinar cell apoptosis by hydrogen sulphide. Am J Physiol Cell Physiol 2006;291(3):C503–10. [13] Bartkowiak D, Hogner S, Baust H, Nothdurft W, Rottinger EM. Comparative analysis of apoptosis in HL60 detected by annexin-V and fluorescein-diacetate. Cytometry 1999;37(3): 191–6.
ARTICLE IN PRESS I. Salinas et al. / Developmental and Comparative Immunology 31 (2007) 1233–1241 [14] Sandstro¨m K, Hakansson L, Lukinius A, Venge P. A method to study apoptosis in eosinophils by flow cytometry. J Immunol Methods 2000;240(1–2):55–68. [15] Saha NR, Usami T, Suzuki Y. A double staining flow cytometric assay for the detection of steroid induced apoptotic leucocytes in common carp (Cyprinus carpio). Dev Comp Immunol 2003;27(5):351–63. [16] Esteban MA, Mulero V, Mun˜oz J, Meseguer J. Methodological aspects of assessing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream (Sparus aurata L.) by flow cytometry and electron microscopy. Cell Tissue Res 1998;293(1):133–41. [17] Abbracchio MP, Ceruti S, Barbieri D, Franceschi C, Malorni W, Biondo L, et al. A novel action for adenosine: apoptosis of astroglial cells in rat brain primary cultures. Biochem Biophys Res Commun 1995;213(3):908–15. [18] Straub RH, Pongratz G, Gunzler C, Michna A, Baier S, Kees F, et al. Immunoregulation of IL-6 secretion by
[19]
[20]
[21]
[22]
1241
endogenous and exogenous adenosine and by exogenous purinergic agonists in splenic tissue slices. J Neuroimmunol 2002;125(1–2):73–81. Wang MX, Ren LM. Growth inhibitory effect and apoptosis induced by extracellular ATP and adenosine on human gastric carcinoma cells: involvement of intracellular uptake of adenosine. Acta Pharmacol Sin 2006;27(8):1085–92. Bainton DF, Farquhar MG. Differences in enzyme content of azurophil and specific granules of polymorphonuclear leukocytes: I. Histochemical staining of bone marrow smears. J Cell Biol 1968;39(29):286–98. Aggarwal BB, Bhardwaj A, Aggarwal RS, Seeram NP, Shishodia S, Takada Y. Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res 2004;24(5A):2783–840. Bertrand R, Solary E, O’Connor P, Kohn K, Pommier Y. Induction of a common pathway of apoptosis by staurosporine. Exp. Cell Res 1994;211:314–21.
Developmental & Comparative Immunology
Developmental and Comparative Immunology 31 (2007) 1233–1241 www.elsevier.com/locate/devcompimm
Short communication
Adenosine arrests apoptosis in lymphocytes but not in phagocytes from primary leucocyte cultures of the teleost fish, Sparus aurata L. Irene Salinas, Alejandro Rodrı´ guez, Jose´ Meseguer, Maria Angeles Esteban Fish Innate Immune System Group, Department of Cell Biology, Faculty of Biology, Campus de Espinardo, University of Murcia, 30100 Murcia, Spain Received 18 December 2006; received in revised form 28 March 2007; accepted 30 March 2007 Available online 4 May 2007
Abstract Adenosine (A) and its derivatives have important biological functions, including the inhibition of immune responses and apoptosis induction. The aim of this work was to investigate whether A and N6-cyclohexyladenosine (CHA) regulate apoptosis in leukocyte cultures from the teleost, gilthead seabream (Sparus aurata L.) by flow cytometry using a double fluorescence staining method. Head kidney leukocytes (HKLs) were cultured for 0, 24 or 48 h. The kinetics of FDA+/PI (viable), FDA/PI (apoptotic) and FDA/PI+ (necrotic) leukocyte subpopulations were followed during the culture process. Apoptosis was induced by addition of resveratrol or staurosporine to the culture media and the study was validated by transmission electron microscopy. A and CHA did not induce but decreased apoptosis of seabream HKLs, in particular HK lymphocytes (L), after 24 h in culture. Results raise the question whether apoptosis is differently modulated by purine nucleosides in fish L and phagocytes and also in fish compared to mammalian cells. r 2007 Elsevier Ltd. All rights reserved. Keywords: Adenosine; N6-cyclohexyladenosine; Apoptosis; Leukocytes; Flow cytometry; Teleost; Seabream (Sparus aurata)
1. Introduction Adenosine (A) and other ATP derived molecules have long been recognised as important multipotent cell modulators both intra- and extracellularly. Their biological functions range from protecting against ischemic damage to downregulating immune responses [1,2]. In addition, they have been shown to Abbreviations: CHA, N6-cyclohexyladenosine; A, adenosine; HKLs, head kidney leukocytes; FDA, fluorescein diacetate; PI, propidium iodide Corresponding author. E-mail address: [email protected] (J. Meseguer).
affect proliferation, survival and apoptosis of many different cell types, ranging from epithelial, endothelial and smooth muscle cells, to cells of the immune and neural lineages from higher vertebrates [3]. In a previous study, we demonstrated that in fish, the innate immune functions of head kidney leukocytes (HKLs) were downregulated in vitro by N6-cyclohexyladenosine (CHA) but not by A, suggesting the presence of purinergic receptors in fish immune cells. Thus, it seems that regulation of immune responses by A compounds already occurs in lower vertebrates [4]. However, nothing is known about the capacity of purine nucleosides to affect apoptosis in teleost fish.
0145-305X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2007.03.014
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Apoptosis or programmed cell death is genetically controlled and takes place during normal physiological processes such as embryonic development, tissue remodelling and immune response regulation. During apoptosis, a well-defined sequence of events occurs, making it possible to distinguish this process from necrosis [5–7]. For instance, the DNA fragmentation that results from apoptosis can easily be visualised in a total DNA electrophoresis, whereas necrotic cells show intact DNA, with a very different electrophoretic pattern [8]. Moreover, cells undergoing apoptosis show characteristic ultrastructural features under transmission electron microscopy (TEM). These include cell shrinkage, aberrant nuclear shape, chromatin condensation and the appearance of hyperchromatic nuclear fragments named apoptotic bodies [5–7]. In addition to DNA degradation-based techniques, other routinely used methods to discriminate cells undergoing apoptosis include the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) [9], mitochondrial membrane hyperpolarisation [10], the Annexin V-PI staining method [8], caspase activation [11] and cytochrome C translocation [12]. The aim of the present study was to investigate the possible role of A and CHA in modulating apoptosis in gilthead seabream (Sparus aurata L.) leukocytes cultured in vitro. To this end, a double fluorescein diacetate/propidium iodide (FDA/PI) staining flow cytometry assay was set up to quantify the apoptosis of seabream leukocytes. 2. Materials and methods 2.1. Animals Twenty specimens (100 g mean body weight) of the hermaphroditic protandrous seawater teleost gilthead seabream (Sparus aurata L.) were obtained from CULMAREX S.A. (Murcia, Spain). Animals were kept in 450–500 l running seawater (28% salinity) aquaria at 2072 1C and with a 12 h light: 12 h dark photoperiod. Animals were acclimated for 30 days prior to the experiment. They were fed daily with 1 g of a commercial pellet diet (Trow Espan˜a, Burgos, Spain) per fish and were starved 24 h prior to sampling. All the investigations of the present paper were conducted in accordance with national and international guidelines for the protection of animal welfare. All protocols applied in this research were
reviewed by the Committee for Animal Care and Welfare of the University of Murcia, Spain. 2.2. Setting up a double staining flow cytometry assay to measure apoptosis in HKLs 2.2.1. Cell isolation Fish were anaesthetised with benzocaine (4% in acetone) (Sigma) and bled from the caudal vein. HKLs were isolated from each fish under sterile conditions. Briefly, head kidneys were excised, cut into small fragments and transferred to 8 ml of sRPMI (RPMI-1640 culture medium (Gibco) with 0.35% sodium chloride (to adjust the medium’s osmolarity to gilthead seabream plasma osmolarity, 353.33 mOs), 100 IU/ml penicillin (Flow), 100 mg/ml streptomycin (Flow), 10 IU/ml heparin (Sigma) and 5% foetal bovine serum (Gibco)). Cell suspensions were obtained by forcing fragments of the organ through a 102 mm nylon mesh. Afterwards, the cell suspension was layered over 51% Percoll gradients. The leukocyte interface with HKLs was collected and washed twice in sRPMI without heparin. HKLs were counted in a Neubauer chamber and adjusted to 2 106 cells/ml of sRPMI. 2.2.2. Cell culture The 500 ml of each HKL suspension previously obtained, were dispensed into flat bottomed 48-well plates and cultured for 0, 24 or 48 h (22 1C, 5% CO2, 85% humidity). Additionally, 10 ml of resveratrol (22.8 mg/ml in absolute ethanol Sigma), staurosporine (1 mg/ml in absolute ethanol, Sigma) were added in triplicate as positive controls for apoptosis. Additionally, controls consisting of absolute ethanol or dimethylsulfoxide (DMSO, Scharlau) only were tested. Positive controls for necrosis consisted of fixed cells. For that, aliquots of 500 ml of each cell suspension were centrifuged and the supernatants discarded. The cell pellets were fixed by adding 2 ml of formaldehyde 10% in PBS (30 min, 4 1C). All assays were done in triplicate. 2.2.3. Apoptosis assay A double staining flow cytometric assay combining FDA (Sigma) and PI was used by adapting the method used for the detection of apoptotic leukocytes in mammalian cells [13,14] and fish cells [15] to the seabream leukocytes. To optimise the FDA staining protocol for gilthead seabream leukocytes, 0.05 g FDA were resuspended in 4 ml of DMSO. This solution was
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used to obtain different working solutions (12.5, 25, 50, 75 or 100 mg FDA/ml in sRPMI). The 10 ml of each FDA concentration were added to each sample followed by 50 ml of PI (400 mg/ml, Sigma) 30 min prior to analysing the cells in the Coulters Epicss XLTM flow cytometer (Beckman Coulter). Analyses were performed on 30,000 cells, which were acquired at a rate of 300 cells/s. Side scatter (granularity), forward scatter (size), FL-1 (green fluorescence) and FL-3 (red fluorescence) for each cell population were represented in dot plots or histograms. To analyse the leukocyte cell subpopulations present in HKL cell suspensions, two regions were established according to the lymphocyte and phagocyte regions previously characterised [16]. This could only be undertaken at 0 and 24 h of culture because the defined subpopulations were no longer identifiable at 48 h and a significant new population consisting of debris from dead leucocytes appeared.
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4 106 cells/ml of sRPMI. Aliquots (250 ml) of each cell suspension were dispensed into 48-well flat bottom culture plates. Then, 250 ml of A (200 mM in sRPMI, Sigma) or CHA (200 mM in sRPMI, Sigma) or sRPMI (control) were added to each well in triplicate. The concentration of purinergic nucleosides was chosen according to previous data obtained for higher vertebrates [16] and fish [4]. Positive controls consisted of wells with 10 ml staurosporine (1 mg/ml in absolute ethanol). Plates were maintained for 30 min, 24 h or 48 h under the same conditions as before and the apoptosis assays were conducted as explained above. The preliminary study enabled us to identify 50 mg/ml as the optimal FDA concentration for staining seabream HKLs. Therefore, this concentration was used in the rest of the experiments. Analysis by subpopulations (lymphocytes and phagocytes) was conducted for 0 and 24 h only. 2.4. Statistical analysis
2.2.4. Microscopy study Stained samples were directly observed from culture plates under a phase contrast microscope (Nikon). Photographs were taken by light and fluorescence microscopy using an alternating green (excitation 470, 490 nm, barrier 520–560 nm) and red (excitation 510, 560 nm, barrier 690 nm) filter combination. HKLs were also cultured in culture dishes (9 cm diameter) at the same cell density as above. Cells were incubated without (control) or with staurosporine (1 mg/ml) and samples were collected after 0, 24 and 48 h of culture for examination by transmission electron microscopy (TEM). Cell suspensions were washed and the supernatants removed, while pellets were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2, 2 h, 4 1C) before being postfixed in OsO4 and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in an EM10X Zeiss transmission microscope. The sections were examined to identify normal, apoptotic or necrotic cells. Nuclear chromatin condensation was used as a typical criterion for apoptotic cells. Cells that swelled and showed cytoplasmic vacuolisation as well nuclear destruction were characterised as necrotic. 2.3. Effects of purinergic nucleosides on seabream HKL apoptosis Six samples of HKLs previously isolated as described above were counted and adjusted to
Results are expressed as mean7standard error. Data were statistically analysed by one-way analysis of variance (ANOVA) and Tukey’s comparison of means when necessary. Differences were considered statistically significant when po0.05. 3. Results 3.1. Setting up of a double staining flow cytometry assay to measure apoptosis in seabream leukocytes 3.1.1. FDA labelling of seabream leukocytes Increasing concentrations of FDA resulted in greater percentage of labelled cells. The optimal concentration of FDA to stain seabream HKLs was 50 mg/ml, which stained over 95% of the phagocytes and 83% of the lymphocytes. Higher concentrations did not result in greater staining percentages. It was also observed that different cell types acquired different FDA (green) intensities being the phagocytic subpopulation more intensely stained than the lymphocyte subpopulation as shown in Fig. 1. 3.1.2. Kinetics of the viability of seabream primary leukocyte cultures Following double staining with FDA and PI, three subpopulations were identified and quantified in the dotplots and histograms obtained after the flow cytometric study: (i) viable (FDA+/PI),
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Fig. 1. (a–c) Flow cytometry analysis of a readily isolated head kidney leukocyte suspension after staining with FDA (50 mg/ml) and propidium iodide (PI) for 30 min. (a) Dotplot showing two main subpopulations: lymphocytes (L) and phagocytes (Ph) according to their SSC and FSC values. (b) Dotplot showing events staining with FL1 (green fluorescence, FDA) and FL3 (red fluorescence, PI). Note the presence of three populations according to the double staining pattern: the FDA+/PI (viable) population contains two subpopulations: the phagocytes (Ph) that acquire greater green intensity and the lymphocytes (L) with a weaker fluorescence; the FDA/PI (apoptotic) population and the FDA/PI+ (necrotic) population. (c) Histogram showing FL3 (red fluorescence) after labelling of HKLs with the double staining protocol. Note that almost all cells remained unpermeabilised and are therefore PI. (d) Histogram showing the green fluorescence (FL1) after labelling of HKLs with the double staining protocol. Most cells were FDA+ being notable that lymphocytes (L) and phagocytes (Ph) show different labelling intensities with FDA. (e–g) Fluorescence microscopy study after labelling with the FDA/PI double staining technique a seabream head kidney leukocyte (HKL) suspension cultured for 24 h. Cells belonging to all three staining patterns were observed, 400. (e) Phase contrast micrograph showing a seabream HKL suspension. (f) Green fluorescence micrograph corresponding to the same field shown in a). Note that most cells in (e) were viable and therefore stained green (FDA+). (g) Red fluorescence micrograph corresponding to the same field shown in a) showing few permeabilised (PI+) leukocytes.
(ii) apoptotic (FDA/PI) and (iii) necrotic (FDA/ PI+) (Fig. 1). Positive controls for necrosis consisting of permeabilised cells fixed with formaldehyde were 100% PI+ (data not shown). Head-kidney lymphocytes and phagocytes displayed clearly different viabilities in the same culture conditions, being the percentage of FDA/ PI (apoptotic) lymphocytes always higher than that of phagocytes. The percentage of FDA/PI+ (necrotic) head-kidney lymphocytes and phagocytes
remained low at 0 and 24 h and no significant differences were recorded (see Table 1). After 48 h in culture, viability of seabream HK cultures significantly decreased compared to 0 and 24 h, the total percentage of apoptotic cells reaching 40% and necrotic cells almost 20%. Finally, despite the fact that cells still cleaved FDA at 24 or 48 h, it was observed that the intensity or mean green fluorescence value decreased with culture time (data not shown).
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Table 1 Effect of time in culture on viability of in vitro seabream HKLs cultures 0h
Phagocytes Lymphocytes
24 h
48 h
FDAPI
FDAPI+
FDAPI
FDAPI+
FDAPI
FDAPI+
1.671.0a 14.771.4b
0.870.1a 0.970.2a
2.270.5a 23.774.7b
2.070.7 a 3.770.5a
40.072.0c
19.673b
Percentage of FDA/PI (apoptotic) and FDA/PI+ (necrotic) seabream leukocytes from head kidney (HKLs) after 0, 24 or 48 h in culture. Results are expressed as mean7sd. Data represent two independent experiments using n ¼ 3 in each experiment. One-way analysis of variance (ANOVA) and Tukey’s comparison of means were conducted when necessary. Different letters stand for statistically significant differences (po0.05) between different culture times.
Table 2 Apoptotic inducers and the viability of in vitro seabream HKLs cultures Incubation time (h)
Phagocytes Lymphocytes
0 24 0 24
Resveratrol
Staurosporine
FDAPI
FDAPI+
FDAPI
FDAPI+
9.872.7 10.172.4 35.374.3 57.175.9*
3.571.3 6.772.4 2.970.9 20.074.9*
8.873.1 14.673.5 29.875.1 56.974.2*
2.370.6 3.171.6 1.570.3 13.672.7*
Effects of two apoptotic inducers, resveratrol and staurosporine, in the percentage of FDA/PI (apoptotic) and FDA/PI+ (necrotic) seabream leukocytes from head kidney (HKLs) after 0 and 24 h in culture. Results are expressed as mean7sd. Data represent two independent experiments using n ¼ 3 in each experiment. One-way analysis of variance (ANOVA) and Tukey’s comparison of means were conducted when necessary. Asterisk denotes statistically significant differences (po0.05) between treated leukocytes at 24 h compared to treated leukocytes at 0 h. No statistically significant differences were found between control cultures and treated cultures at 0 h.
3.1.3. Effects of apoptotic inducers on the viability of seabream primary leukocyte cultures The effects of adding resveratrol or staurosporine to seabream primary HKL cultures at 0 and 24 h are summarised in Table 2. At 0 h, there were no statistically significant differences between control cultures and cultures where apoptotic inducers had been added. However, head-kidney phagocytes appeared more resistant to both treatments than head-kidney lymphocytes, since the percentage of FDA/PI only increased from 9.8% to 10.1% after 24 h incubation with resveratrol and from 8.8% to 14.6% in the case of staurosporine. On the contrary, the percentage of apoptotic lymphocytes after 24 h in resveratrol or staurosporine almost doubled the percentage recorded immediately after their addition (around 30% at 0 h). 3.1.4. Microscopy study Observation of HKLs primary cultures from the different assays under the light or fluorescence microscope confirmed the presence of the three populations previously observed by flow cytometry:
FDA+/PI (viable), FDA/PI (apoptotic) and FDA/PI+ (necrotic) cells (Fig. 1e–g). TEM examination of leukocyte cultures revealed that control cultures (without staurosporine) consisted of cells exhibiting the normal morphology observed at the beginning of the experiment. Micrographs showing the ultrastructural characteristics of seabream HKL cultures after 0, 24 or 48 h in the presence of staurosporine match dot plots corresponding to the flow cytometric analysis after FDA/PI labelling in Fig. 2. Leukocytes cultured in the presence of staurosporine were characterised by normal nucleus and cytoplasm morphology at the beginning of the experiment and did not differ from control leukocytes at the same time (Figs. 2a and d). However, the number of cells with typical chromatin condensation and aberrantly shaped nuclei (apoptotic) dramatically increased after 24 h of incubation in the presence of staurosporine), which correlates with the flow cytometry analysis of this suspension (Figs. 2b and e). Apoptotic cells were abundant in HKL cultures incubated with staurosporine,
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Fig. 2. Flow cytometry (a–c) and transmission electron microscopy (d–f) study of an HKL suspension cultured in presence of staurosporine (1 mg/ml). Dotplot after labelling HKL suspension with FDA and PI after 0 h (a), 24 h (b) and 48 h (c) in culture. Transmission electron micrograph of the same HKL suspension cultured for 0 h (d), 24 h (e) and 48 h (f). Note that the FDA+/PI population progressively moves towards the left and, concomitantly, the FDA/PI moves upwards entering the FDA/PI+ quadrant. These shifts in the populations observed under flow cytometry were in agreement with the morphological changes seen under TEM with increasing numbers of apoptotic nuclei and membrane disruption at the end of the experiment.
especially after 48 h of incubation when most of the cells had lost their architecture, and their membrane and cytoplasm were profoundly damaged. They were therefore scored as necrotic (Figs. 2c and f). 3.2. Effect of purinergic nucleosides on apoptosis of seabream HKLs
on the phagocytes (Fig. 3c). However, A increased the number of apoptotic phagocytes at this time, but the effect was not significant. After 48 h of culture, the percentage of total apoptotic seabream leukocytes was still lower than the percentage found in control samples, although the decreases were not statistically significant (Fig. 3a). 4. Discussion
No significant differences were observed after 30 min in contact with the purine nucleosides, neither in the number of lymphocytes nor in the phagocytes, although in both cases A resulted in a slight increase of apoptotic cells (Fig. 3a–c). Both A and CHA reduced apoptosis in HKL cultures after 24 h. There were significantly fewer (p ¼ 0.006) apoptotic cells in those samples incubated with A or CHA (100 mM) than in control samples after 24 h (Fig. 3a). When the analysis was conducted in the different subpopulations, it was observed that only apoptosis of lymphocytes was significantly inhibited (Fig. 3b), whereas no significant effect was observed
A and other purine nucleosides are important metabolites that regulate a variety of biological processes in animal cells and tissues. They are present both extracellularly and inside cells, their concentration increasing upon tissue damage or injury [1]. Their effects even reach the domain of the immune system, and A is considered as a downregulator of immune responses in vertebrates [2,18]. This was recently confirmed in the gilthead seabream, whose leukocyte phagocytic and respiratory burst activities were decreased in vitro when CHA, an A derivative, was present [4].
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45 40 % Apoptotic cells
35
Control CHA A
30 25 20 *
15
*
10 5 0 24h
30 25 20
*
15
*
10 5 0 30min
% Apoptotic phagocytes
% Apoptotic lymphocytes
30min
48h
4 3 2 1 0 30min
24h control
CHA
24h
Adenosine
Fig. 3. Effects of CHA and A on apoptosis of seabream HKLs cultured in vitro. (a) Percentage of total apoptotic seabream HKLs cultured for 30 min, 24 h or 48 h. (b) Percentage of apoptotic lymphocytes after 30 min or 24 h in culture. (c) Percentage of apoptotic phagocytes after 30 min or 24 h in culture. Results are expressed as mean7sd. One-way analysis of variance (ANOVA) and Tukey’s comparison of means were conducted when necessary. Asterisk denotes statistically significant differences (po0.05). Data represent two independent experiments using n ¼ 3 in each experiment.
A also regulates the cell cycle since it is capable of inhibiting apoptosis in cancer cell lines and in rat primary astrocyte cultures [17,19]. However, nothing is known with respect to the effect of purine nucleosides on apoptosis of fish cells and in particular, on leukocyte primary cultures from teleost fish. The present work employs for the first time the FDA/PI technique to assess apoptosis in seabream cells. Our results show that FDA staining differed between head–kidney phagocytes and lymphocytes, the intensity of green fluorescence being markedly higher in phagocytes. This indicates that phagocytic leukocytes contain more esterases, or more active ones, than lymphocytes, probably due to the presence of numerous cytoplasmic granules which contain esterases, among other lysosomal enzymes, in macrophages and granulocytes [20]. The combi-
nation of FDA and PI made it possible to identify viable, apoptotic and necrotic seabream cells. This technique has been successfully applied to teleostean leucocytes before and validated with the conventional Annexin V apoptosis assay [15]. The use of proapoptotic agents like resveratrol and staurosporine served as a good indicator of cell death in seabream leukocytes. Resveratrol, a stilbene derived from plants, has been shown to inhibit proliferation and trigger apoptosis in a number of human cancer cell lines [21]. Staurosporine is a protein kinase inhibitor commonly used to induce apoptosis [22]. In the present study, both compounds managed to induce apoptosis in seabream lymphocytes at a significantly greater rate than controls after 24 h of culture. The semiquantitative information obtained from the study of seabream leukocyte cultures under
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TEM perfectly correlated with quantitative data from the flow cytometry analysis, as expected. Apoptotic cells showed signs of shrinkage, chromatin condensation and rounded electron dense nuclei, as described by other authors [7]. Studies conducted on carp found quantitative correlation between the FDA/PI technique and Annexin V staining [15]. The kinetic study of apoptosis in seabream leukocyte cultures from head kidney reveals that culture time is a vital factor when immunological or biological studies of fish leukocytes involve cell culture techniques. The number of apoptotic HK phagocytes increased linearly with culture time (18% at 24 h and 28% at 48 h). Thus, it can be concluded that in vitro culture of seabream HK phagocytes results in cell death via apoptosis and the low necrotic numbers (2% at 24 h and 4.3% at 48 h) may represent advanced stages of apoptosis. Additionally, mean green fluorescence in seabream HK leucocyte cultures decreased with time within the population stained FDA+/PI, which is also in agreement with previous data and suggests that the cell metabolic rate decreases with culture time [14]. The double FDA/PI staining flow cytometry assay was set up in order to investigate the possible role of A and CHA in the apoptotic events that occur during leukocyte primary cultures. In contrast with previous data available from established tumour cell lines [19] or in astrocytes isolated from rat brain [17], apoptosis was not induced but arrested in seabream HKL cultures after 24 h. This was true in the case of lymphocytes and not the phagocytes, which display the opposite behaviour but in a non-significant way. The A and CHA dose here assayed is a high biological dose and corresponds to scenarios where a sharp increase of A takes places following tissue damage [2]. Further studies may address the minimum concentration of these molecules required to modulate apoptosis in fish leucocytes. Both purines showed similar effects despite the fact that A was not efficient at downregulating the seabream innate immune parameters, whereas CHA was [4]. The present results warrant further studies into the regulation of seabream immune responses and purine nucleosides, being of special relevance the differential behaviour of lymphocytes and phagocytes here reported. In conclusion, both A and CHA decreased apoptosis of seabream lymphocytes but not phagocytes after 24 h in culture. Our findings reveal major differences between lower and higher vertebrates
concerning the biological role of purine nucleosides during regulation of immune responses and how apoptosis is involved in this regulation. Acknowledgements I. Salinas received Ph.D. funding from Fundacio´n Se´neca, Murcia, Spain. Authors wish to thank all staff from the Tissue Culture Service (SACE) at University of Murcia. References [1] Cronstein BN. Adenosine, an endogenous anti-inflammatory agent. J Appl Physiol 1994;76:5–13. [2] Hasko´ G, Cronstein BN. Adenosine: an endogenous regulator of innate immunity. Trends Immunol 2004;25: 33–9. [3] Jacobson KA, Hoffmann C, Cattabeni F, Abbracchio MP. Adenosine-induced cell death: evidence for receptormediated signalling. Apoptosis 1999;4(3):197–211. [4] Salinas I, Rodriguez A, Esteban MA, Meseguer J. N6Cyclohexyl-adenosine but not adenosine is a modulator of teleost fish innate immune activities. Dev Comp Immunol 2005;30:325–34. [5] Earnshaw WC. Apoptosis: lessons from in vitro systems. Trends Cell Biol 1995;5(6):217–20. [6] Lund PK, Westvik AB, Joo GB, Ovstebo R, Haug KB, Kierulf P. Flow cytometric evaluation of apoptosis, necrosis and recovery when culturing monocytes. J Immunol Methods 2001;252(1):45–55. [7] Guejes L, Zurgil N, Deutsh M, Gilburd B, Shoenfeld Y. The influence of different cultivating conditions on the polymorphonuclear leukocyte apoptotic processes in vitro, I: the morphological characteristics of spontaneous apoptosis. Ultrastruct Pathol 2003;27(1):23–32. [8] Bacso´ Z, Everson RB, Eliason JF. The DNA of annexin V-binding apoptotic cells is highly fragmented. Cancer Res 2000;60(16):4623–8. [9] Louagie H, Cornelissen M, Philippe J, Vral A, Thierens H, De Ridder L. Flow cytometric scoring of apoptosis compared to electron microscopy in gamma irradiated lymphocytes. Cell Biol Int 1998;22(4):277–83. [10] Jo WS, Jeong MH, Jin YH, Jang JY, Nam BH, Son SH, et al. Loss of mitochondrial membrane potential and caspase activation enhance apoptosis in irradiated K562 cells treated with herbimycin. A Int J Radiat Biol 2005;81(7):531–43. [11] Thyrell L, Erickson S, Zhivotovsky B, Pokrovskaja K, Sangfelt O, Castro J, et al. Mechanisms of interferon-alpha induced apoptosis in malignant cells. Oncogene 2002;21(8): 1251–62. [12] Cao Y, Adhikari S, Ang AD, Moore PK, Bhatia M. Mechanism of induction of pancreatic acinar cell apoptosis by hydrogen sulphide. Am J Physiol Cell Physiol 2006;291(3):C503–10. [13] Bartkowiak D, Hogner S, Baust H, Nothdurft W, Rottinger EM. Comparative analysis of apoptosis in HL60 detected by annexin-V and fluorescein-diacetate. Cytometry 1999;37(3): 191–6.
ARTICLE IN PRESS I. Salinas et al. / Developmental and Comparative Immunology 31 (2007) 1233–1241 [14] Sandstro¨m K, Hakansson L, Lukinius A, Venge P. A method to study apoptosis in eosinophils by flow cytometry. J Immunol Methods 2000;240(1–2):55–68. [15] Saha NR, Usami T, Suzuki Y. A double staining flow cytometric assay for the detection of steroid induced apoptotic leucocytes in common carp (Cyprinus carpio). Dev Comp Immunol 2003;27(5):351–63. [16] Esteban MA, Mulero V, Mun˜oz J, Meseguer J. Methodological aspects of assessing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream (Sparus aurata L.) by flow cytometry and electron microscopy. Cell Tissue Res 1998;293(1):133–41. [17] Abbracchio MP, Ceruti S, Barbieri D, Franceschi C, Malorni W, Biondo L, et al. A novel action for adenosine: apoptosis of astroglial cells in rat brain primary cultures. Biochem Biophys Res Commun 1995;213(3):908–15. [18] Straub RH, Pongratz G, Gunzler C, Michna A, Baier S, Kees F, et al. Immunoregulation of IL-6 secretion by
[19]
[20]
[21]
[22]
1241
endogenous and exogenous adenosine and by exogenous purinergic agonists in splenic tissue slices. J Neuroimmunol 2002;125(1–2):73–81. Wang MX, Ren LM. Growth inhibitory effect and apoptosis induced by extracellular ATP and adenosine on human gastric carcinoma cells: involvement of intracellular uptake of adenosine. Acta Pharmacol Sin 2006;27(8):1085–92. Bainton DF, Farquhar MG. Differences in enzyme content of azurophil and specific granules of polymorphonuclear leukocytes: I. Histochemical staining of bone marrow smears. J Cell Biol 1968;39(29):286–98. Aggarwal BB, Bhardwaj A, Aggarwal RS, Seeram NP, Shishodia S, Takada Y. Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res 2004;24(5A):2783–840. Bertrand R, Solary E, O’Connor P, Kohn K, Pommier Y. Induction of a common pathway of apoptosis by staurosporine. Exp. Cell Res 1994;211:314–21.