Fish & Shellfish Immunology 35 (2013) 2032e2039
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Measurement of intracellular nitric oxide (NO) production in shrimp haemocytes by flow cytometry Jian-An Xian a, b,1, Hui Guo a,1, Bin Li a, Yu-Tao Miao a, Jian-Min Ye a, Sheng-Peng Zhang a, Xun-Bin Pan a, Chao-Xia Ye a, An-Li Wang a, *, Xuan-Ming Hao b, * a
Key Laboratory of Ecology and Environment Science in Guangdong Higher Education, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, School of Life Science, South China Normal University, Guangzhou 510631, People’s Republic of China School of Physical Education & Sports Science, South China Normal University, Guangzhou 510631, People’s Republic of China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2013 Received in revised form 16 October 2013 Accepted 16 October 2013 Available online 25 October 2013
A flow cytometric method to measure the production of intracellular nitric oxide (NO) was adapted for use with shrimp haemocytes. We applied fluorescent probe 4-amino-5-methylamino-20 ,70 -difluorofluorescein diacetate (DAF-FM DA) for NO detection in haemocytes from the tiger shrimp Penaeus monodon, and used flow cytometry to quantify fluorescence intensity in individual haemocyte. The optimized protocol for intracellular NO analysis consists to incubate haemocytes with DAF-FM DA at 10 mM for 60 min to determine the mean fluorescence intensity. Result showed that NO was also produced in the untreated shrimp haemocytes. NO level in granular cells and semigranular cells were much higher than that in hyaline cells. Defined by different characteristic of NO content, three subsets of haemocytes were observed. Zymosan A at dose of 10 or 100 particles per haemocyte triggered higher DAF-FM fluorescence intensity in granular and semigranular cells, than PMA that had no significant impact on all three cell types. These results indicate that granular and semigranular cells are the primary cells for NO generation. Cytochalasin B significantly inhibited the NO level induced by zymosan A. NGMonomethyl-L-arginine (L-NMMA) and diphenylene iodonium chloride (DPI) significantly suppressed the DAF-FM fluorescence in haemocytes, but apocynin could not modulate it, indicating that the DAF-FM fluorescence was closely related to the activity of NO-synthase pathway. The NO donor sodium nitroprusside (SNP) improved the DAF-FM fluorescence in haemocytes, while the NO scavenger C-PTIO (2-(4carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) significantly decreased the fluorescence, demonstrating that the fluorescence intensity of DAF-FM is mainly dependent on the intracellular NO level. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Nitric oxide Flow cytometry DAF-FM DA Haemocyte Shrimp
1. Introduction Nitric oxide (NO) has been identified as a vital physiological modulator and a signaling molecule in mammals [1]. In marine invertebrates, biological roles of NO were found to be related to feeding, immune defence, environmental stress, learning, metamorphosis, swimming, symbiosis, haemocyte aggregation and regulation of blood pressure [2]. Recent studies found that NO acts as a cytotoxic molecule contributed to microorganisms killing in
* Corresponding authors. E-mail addresses: [email protected] (A.-L. Wang), [email protected] (X.-M. Hao). 1 These authors contributed equally to this work. 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.10.014
crustaceans [3e6], as in mammalians [1]. However, the information about biological role of NO in crustacean is still scarce. The direct detection of NO content in biological samples is a challenge due to its short half-life and low concentration. Several indirect methods have been applied to detect the NO level. The most prevalent method of NO production assay was the Griess colorimetric reaction, a method that quantifies nitrites (NO 2 ) and nitrates (NO 3 ), the stable products resulting from the degradation of NO [3,4]. However, there are at least two major restriction of this method: 1) This indirect method may not reflect the real NO level. Not all the NO 2 /NO3 in aquatic animals come from NO metabolism. Environmental NO 2 tends to accumulate into the blood through active uptake mechanisms associated with the chloride cells of the gills [7,8]. 2) This indirect method could not detect the instantaneous NO content in individual cell, and it is hard to estimate the major cell source of NO.
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Another common method for NO detection was flow cytometric assay using the fluorescent probe 20 ,70 -dichlorofluorescein diacetate (DCFH-DA) [9,10]. This probe could also be oxidized by reactive oxygen species (ROS) including hydrogen peroxide (H2O2), peroxynitrite, hydroxyl (HO) and peroxyl (ROO) [9]. Specific inhibitors should be included in this indirect method. Thus, result may also be affected by some processes, such as the dose of inhibitor. Moreover with, this qualitative measure is hard to quantify the NO content accurately. The currently used methods are neither sensitive nor specific. A direct method which is more simple, sensitive and accurate is needed. Flow cytometry (FCM) has been proposed for direct detection of intracellular NO production in human [11], mammal [12] and mussel [13] cells using NO-specific fluorescent probe 4,5diaminofluorescein diacetate (DAF-2 DA) or more sensitive probe 4-amino-5-methylamino-20 ,70 -difluorofluorescein diacetate (DAFFM DA). This direct detection could overcome the major restriction of the indirect methods. Furthermore, FCM can provide information at the single-cell level, so it can easily be applied to analyse the production of NO in different cell subpopulations. The aim of the present work was to describe the development of a flow cytometric approach to measure the NO production in shrimp haemocytes using the fluorescent probe DAF-FM DA, and to evaluate the effects of various modulators on the NO production by different haemocyte types. 2. Materials and methods 2.1. Animals
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emission ¼ 515) which exhibits about a 160-fold greater fluorescence quantum efficiency [12]. DAF-FM green fluorescence was measured on FL1 detector of flow cytometer. Results were given as mean of fluorescence in arbitrary units (A. U.). 2.4. DAF-FM DA dose and time responses An initial experiment was performed to define the optimum DAF-FM DA dose to be added to haemocyte suspension; this dose should be sufficient to emit a measurable fluorescent signal on the FL1 detector and to be non-toxic for cells. After haemolymph was extracted as described previously, haemolymph samples were pooled to reduce interindividual variation, and then the cell concentration was adjusted to about 1 106 cells ml1 with AS. The diluted pooled haemolymph sample was divided into four sub-samples of 2 ml, then 20 ml of the DAF-FM DA stock solutions (0.1, 0.5, 1 and 5 mM) was added to yield final concentrations of 1, 5, 10 and 50 mM, respectively. The mixture was incubated in the dark at room temperature. DAF-FM fluorescence of haemocyte was evaluated just after DAF-FM DA addition (t ¼ 0) and at t ¼ 15, 30, 45, 60, 75 and 90 min. Cell viability at the end point of the experiment was determined using propidium iodide (PI, Sigma) stain as described previously [14]. This experiment repeated three times. 2.5. Non-induced nitric oxide production in different haemocyte types
The experimental shrimp Penaeus monodon (7.78 0.82 g) were obtained from a commercial shrimp farm in Nansha, Guangzhou, Guangdong Province, China. They were maintained in the laboratory with diluted seawater at 20&, pH 7.9e8.0 and controlled temperature (24 2 C), with continuous water circulation. Prior to experimental use, animals were acclimated to the laboratory conditions for one week, and fed twice daily with commercial shrimp feed. Only apparently healthy shrimp in the intermoult stage were used.
Haemolymph was extracted as described previously, and then was diluted with AS to obtain a final concentration of about 1 106 cells ml1. This experiment was performed on individual samples, and fifteen shrimp were analysed. Two hundred microlitres of haemocyte suspension from each shrimp was incubated with DAF-FM DA at 10 mM (the optimum dose) for 60 min (the optimum time) in the dark. Three different morphologic haemocyte types (hyaline cells, semigranular cells and granular cells) can be defined basing on the relative size (FSC values) and granularity (SSC values) as described previously [14]. The DAF-FM fluorescence intensity of each haemocyte type was analysed.
2.2. Preparation of haemocyte suspension
2.6. Haemocyte responses to modulators
Haemolymph (300 ml) was extracted from each shrimp with a 25 gauge needle and 2.5 ml syringe containing an equal volume of ice-cold anticoagulant solution (AS, glucose 20.5 g L1, sodium citrate 8 g L1, sodium chloride 4.2 g L1, pH 7.5). The diluted haemolymph from each shrimp was transferred into a separate microcentrifuge tube held on ice.
Effects of two activators, four inhibitors, NO donor and scavenger on DAF-FM fluorescence intensity were determined in the tiger shrimp haemocytes. Species, doses and functions of modulators used in this study are presented in Table 1.
2.3. Measurement of intracellular nitric oxide by flow cytometry FCM was performed with a FACSCalibur instrument (Bectone Dickinson Immunocytometry Systems, San Jose, CA) equipped with a single argon ion laser with filtered emission at 488 nm. Photomultiplier bandpass filters for fluorescence were 530 nm (green fluorescence, FL1). Size scatter height (SSC) and FL1 fluorescence data were collected on log scale, and forward scatter height (FSC) data were collected on linear scales. For each subsample, 10, 000 events were counted. 4-amino-5-methylamino-20 ,70 -difluorofluorescein diacetate (DAF-FM DA, Sigma) is a NO-specific fluorescent probe, which is cell-permeant and passively diffuses across cellular membranes. Once inside cells, it is deacetylated by intracellular esterases to become DAF-FM. After specifically reacting with NO, DAF-FM is further converted to DAF-FM triazole (l excitation ¼ 495, l
2.6.1. Haemocyte responses to activators Two possible NO activating agents were tested: zymosan A (Sigma) and phorbol myristate acetate (PMA, Sigma). Zymosan A stock preparation: 200 mg of zymosan A was suspended in 10 ml of AS, heated in a boiling water bath for 30 min, and then washed twice. The stock suspension was resuspended in a range of volumes of AS to make three suspensions that contained increasingly higher concentrations of zymosan particles. The particle count was checked microscopically, and aliquots were frozen at 20 C. After haemolymph was extracted, pooled and diluted as described previously, zymosan A was added to haemocyte suspensions to obtain approximately 1, 10 and 100 particles per haemocyte. PMA was added to obtain 1 and 10 mg ml1. DAF-FM DA was added to haemocyte suspensions to yield final concentration of 10 mM, 30 min after adding zymosan A or PMA. The haemocyte solutions were incubated in the dark for 60 min at room temperature, and then the DAF-FM fluorescence of each haemocyte type was analysed by flow cytometry.
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Table 1 List and concentration of modulators tested on NO production of P. monodon haemocytes. Modulator Chemicals Activator
Final concentration
Activity
Zymosan
1, 10 and 100 Phagocytosis, particles cell1 ROS/RNS production 1 Activation of PMA 1 and 10 mg ml NOX and NOS molecules Inhibitor DPI 50 mM Inhibitor of NOX, NOS L-NMMA 50 mM NOS inhibitor Apocynin 50 mM NOX inhibitor Cytochalasin B 10 mg ml1 Phagocytosis inhibitor Donor SNP 0.2 and 2 mM Scavenger C-PTIO 0.2 and 2 mM
Ref. [9,10] [9,10,17]
[9,10,17] [9,17] [17] [9] [34] [34,35]
PMA: phorbol myristate acetate; DPI: diphenylene iodonium chloride; L-NMMA: NG-Monomethyl-L-arginine; Fccp: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; SNP: sodium nitroprusside; C-PTIO: 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide; ROS: reactive oxygen species; RNS: reactive nitrogen species; NOX: NADPH-oxidase; NOS: NO-synthase.
2.6.2. Haemocyte responses to inhibitors Four possible inhibitors used in this experiment are presented in Table 1. Haemocytes were pre-incubated with inhibitors for 30 min. Then 10 mM DAF-FM DA was added. After the solutions were incubated in the dark for 60 min at room temperature, DAF-FM fluorescence in haemocytes was analysed. 2.6.3. Haemocyte responses to NO donor SNP or scavenger C-PTIO Effects of the specific NO donor sodium nitroprusside (SNP) and scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1oxyl-3-oxide (C-PTIO) on detection of NO was measured. Haemocytes were pre-incubated with SNP (0.2 and 2 mM) or C-PTIO (0.2 and 2 mM) for 30 min. Then the haemocytes were incubated with 10 mM DAF-FM DA in the dark for 60 min at room temperature. DAF-FM fluorescence in haemocytes was analysed. 2.7. Statistical analyses All data are presented as means standard deviation (S. D.). A multiple-comparison Tukey test was used to examine for significant differences among treatments using the SPSS 13.0 program from SPSS Inc (Chicago, IL, USA). A P value 0.05). Zymosan at dose of 10 or 100 particles per haemocyte significantly induced the NO production in semigranular cells and granular cells. The dose of 10 particles per haemocyte seems to be the best compromise between activation capability and lower particle ratio. Effect of PMA on NO production is presented in Fig. 7. No significant effect of PMA was observed on NO level in all three haemocyte subpopulations, compared to the control treatment (P > 0.05). 3.4. Haemocyte responses to inhibitors Effects of NMMA, DPI and apocynin on the DAF-FM fluorescence of different haemocyte subpopulations are presented in Fig. 8. For hyaline cells, DAF-FM fluorescence was not affected by the chosen inhibitors (P > 0.05). Apocynin also had no effect on the DAF-FM fluorescence of semigranular cells and granular cells (P > 0.05). For semigranular cells, DAF-FM fluorescence was significantly lower in the presence of NMMA and DPI, decreasing 42.3% and
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Fig. 2. DAF-FM fluorescence histogram of different untreated haemocyte subpopulations of P. monodon. A: Total haemocytes; B: Hyaline cells; C: Semigranular cells; D: Granular cells.
37.8% respectively compared to semigranular cells without inhibitors (P < 0.05). For granular cells, DAF-FM fluorescence significantly decreased in the presence of NMMA and DPI, decreasing 40.2% and 58.3% respectively compared to the control treatment (P < 0.05). Effect of cytochalasin B on the NO level is given in Fig. 9. In the presence of cytochalasin B, no significant modification of NO production was observed in total haemocytes (P > 0.05). As shown previously, zymosan induced the NO production (P < 0.05). When the zymosan combined with cytochalasin B, NO production reduced to the level of control (P > 0.05). 3.5. Haemocyte responses to NO donor SNP and scavenger C-PTIO
Fig. 3. NO production in different untreated haemocyte subpopulations of P. monodon (n ¼ 15 shrimps). HC: Hyaline cells; SGC: Semigranular cells; GC: Granular cells. Different letters indicate significant differences between haemocyte subpopulations (P < 0.05).
Effects of SNP and C-PTIO on DAF-FM fluorescence in haemocytes are presented in Fig. 10. The pre-incubation of SNP (0.2 and 2 mM) significantly increased the DAF-FM fluorescence of haemocytes (P < 0.01 and P < 0.001). Compared to the control, the relative DAF-FM fluorescence in haemocytes pre-incubated with 0.2 and 2 mM SNP was 177% and 387%, respectively. DAF-FM fluorescence of haemocytes decreased significantly in the presence of C-PTIO (0.2 and 2 mM, P < 0.01 and P < 0.001). 4. Discussion Flow cytometry, which can provide information at the singlecell level, has been used in studies of aquatic invertebrate [9,10,15e19]. Respiratory burst activity which is an important defence mechanism in invertebrate has been measured by flow
Table 2 Percentage and fluorescence intensity of DAF-FM-defined haemocyte subpopulations of Penaeus monodon (n ¼ 15 shrimps). Different letters in columns indicate significant differences between haemocyte subpopulations (P < 0.05). Percentage (%) low
Fig. 4. FSC-FL1 dot plot of P. monodon haemocytes stained by DAF-FM DA. R4: DAFFMlow cells; R5: DAF-FMmed cells; R6: DAF-FMhigh cells.
DAF-FM cells (R4) DAF-FMmed cells (R5) high DAF-FM cells (R6)
c
7.88 4.30 64.14 6.21a 22.62 4.47b
NO production (A.U.) 15.33 4.32c 84.90 9.35b 557.48 30.29a
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Fig. 5. DAF-FM fluorescence histogram (A, B, C) and FSC-SSC dot plot (D, E, F) of DAF-FM-defined haemocyte subpopulations of P. monodon. A, D: DAF-FMlow cells; B, E: DAF-FMmed cells; C, F: DAF-FMhigh cells; R1: Region of hyaline cells; R2: Region of semigranular cells; R3: Region of granular cells.
cytometry using DCFH-DA [9,18]. This probe could be oxidized by both ROS and RNS. Thus, this method also been used in NO qualitative assay indirectly. In the present study, we intended to optimize and apply the FCM protocol for direct measurement of intracellular NO level in shrimp haemocytes. This flow cytometric method is based on intracellular non-fluorescent DAF-FM conversion to fluorescent DAF-FM triazole by NO [12]. In the present study, according to the dynamic variation of intracellular DAF-FM fluorescence intensity, for shrimp haemocytes, 10 mM seemed to be the optimal concentration of this probe, and 60 min appeared to be the optimal incubation time. Using this probe, distinct DAF-FM fluorescence was presented in the untreated haemocytes. This result revealed that NO exists in shrimp haemocytes without additional activation. Some studies hypothesized that ROS/RNS production of untreated haemocytes may be induced by the contact of haemocyte membrane with nonself materials such as syringe and plastic tubes, or by various environmental factors before haemolymph collecting [9]. However, since NO is an important physiological and immune molecule [1], the NO generated in the untreated haemocytes could be considered as a normal, constitutive physiological production. To further understand the roles of different haemocyte types in NO synthesis, NO contents in the three shrimp haemocyte types (hyaline cells, semigranular cells and granular cells) were analysed respectively. Using FCM, these three types of shrimp haemocyte could be defined basing on FSC values and SSC values [14,20]. Our results showed that granular cells produced much more NO than semigranular cells and hyaline cells without stimulation. The NO level in granular cells approximately six times and twenty-one times higher than those in semigranular cells and hyaline cells respectively. These results suggest that the three haemocyte types possess different capabilities for NO response, and the granular and semigranular cells are the primary cells for non-induced NO generation. According to the size (FSC) and DAF-FM fluorescence, three DAFFM-defined haemocyte subpopulations could be clearly distinguished (Fig. 4). The relative size and granularity of the NO-defined subpopulations were analysed in the FSC-SSC dot plot. Results
showed that DAF-FMlow cells, DAF-FMmed cells and DAF-FMhigh cells were located in the region of hyaline cells, semigranular cells and granular cells respectively (Fig. 5). These results suggest that the three haemocyte types cloud also be distinguished according to their great differences in NO level. Zymosan A, a common inducer of phagocytosis and ROS/RNS production, was used to determine the induced-NO production in different haemocyte subpopulations in the present study. Ten zymosan particles per haemocyte appeared to be a good dose for this method, since this dose of zymosan increased the DAF-FM fluorescence with the lowest particle. Our results showed that NO production in granular cells and semigranular cells could be activated by zymosan A particles, but that in hyaline cells could not. These facts indicate that granular cells and semigranular cells may play main role in killing pathogens by RNS pathway. These two types of haemocyte in crayfish Astacus astacus have been reported to display cytotoxic capacity towards mammalian tumour and nontumour cell line [21]. Our finding suggests that the NO production capacity in granular and semigranular cells may be contributed to their cytotoxic function. Numerous studies have demonstrated that different haemocyte types carry out different functions in immune defence of crustaceans [22]. Because of the different roles of haemocyte subpopulations in immunity, the different haemocyte count (DHC) may be considered of major importance to evaluate the immune level of crustaceans [23]. Generation of NO has been demonstrated in crustacean haemocytes during phagocytosis [3]. Hyaline cells and semigranular cells were known to be the main cell types of phagocytosis [22]. However, we found that granular cells and semigranular cells were the major NO-response cells to zymosan. It means that phagocytosis of zymosan particle may be not the activation pathway for NO production. Numerous previous studies have reported that zymosan would trigger the cellular responses including exocytosis and activation of prophenoloxidase (proPO) system localized inside granular cells and semigranular cells [24,25]. They claimed that the active compound in zymosan was soluble b-glucans which activated the proPO system by signal transduction of the pattern recognition proteins (PRPs) such as b-1,3-glucan-binding protein
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Fig. 7. NO production in different haemocyte subpopulations exposed to PMA (n ¼ 5 shrimps). HC: Hyaline cells; SGC: Semigranular cells; GC: Granular cells. For each haemocyte subpopulation, different letters indicate significant differences between treatments (P < 0.05, ANOVA).
PMA is a common inducer of ROS/RNS production for vertebrate phagocytes. It is known to activate protein kinase C, involved in activation of NADPH-oxidase and NO-synthase molecules, responsible for the production of ROS and RNS, respectively. However, in the present study, PMA did not show any capacity to modify NO level in either P. monodon haemocyte type. Similar results were shown in some previous studies of crustaceans and bivalves [14,31e33]. These facts suggest that these species of invertebrate including P. monodon may have a different activation pathway of NOS without protein kinase C. In order to further confirm the relationship between DAF-FM fluorescence and different oxidative pathways, impact of specific inhibitors for the NO-synthase and NADPH-oxidase were tested. The apocynin, inhibitor of NADPH-oxidase pathway, could not affect the DAF-FM fluorescence, suggesting that NADPH-oxidase dependent ROS did not contribute to the DAF-FM fluorescence. On the contrary, both NMMA and DPI which can inhibit the NOsynthase pathway significantly reduced the fluorescence intensity, demonstrating that the DAF-FM fluorescence intensity was closely related to the activity of NO-synthase. Interestingly, the reduction of DAF-FM fluorescence could only be observed in
Fig. 6. NO production in different haemocyte subpopulations exposed to zymosan A at different haemocyte: zymosan A ratios (n ¼ 5 shrimps). HC: Hyaline cells; SGC: Semigranular cells; GC: Granular cells. For each graph, different letters indicate significant differences between treatments (P < 0.05, ANOVA).
(bGBP) [26,27]. Thus, we speculate that NO production induced by zymosan may be also one of the cellular responses involved in the PRPs activation pathway. On the other hand, we found that phagocytosis inhibitor cytochalasin B prevented the induction of zymosan, while it did not modify the NO production in untreated haemocytes. Cytochalasin B is also found to inhibit the glucose and glucosamine transport [28], and to affect the cell morphology, cell adhesion and mucopolysaccharide synthesis [29]. Perhaps, in this study, the signal transductions of PRPs were disturbed by cytochalasin B while it inhibited the actin polymerization [30] or other cellular events. Indeed, more researches should be done to help us to understand the activation mechanism of NO production, and the real relationship between phagocytosis and NO production.
Fig. 8. NO production in different haemocyte subpopulations exposed to various inhibitors (n ¼ 5 shrimps). HC: Hyaline cells; SGC: Semigranular cells; GC: Granular cells. For each haemocyte subpopulation, different letters indicate significant differences between treatments (P < 0.05, ANOVA).
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Acknowledgements This research was supported by the National Natural Science Foundation of China (31302164), Guangdong Provincial Natural Science Foundation (S2011020003256 and S2012040008093), China Postdoctoral Science Foundation (2012M511829), Scientific and Technological Planning Project of Guangdong Province (2011B020307010 and 2012B020307004).
References
Fig. 9. NO production in total P. monodon haemocytes at 120 min incubation in AS (control) or after addition of cytochalasin B (Cyt B: 10 mg ml1), zymosan A (ZA: 10 particles haemocyte1) or both chemicals (ZA þ Cyt B). Different letters indicate significant differences between treatments (P < 0.05, ANOVA).
Fig. 10. DAF-FM fluorescence of P. monodon haemocytes after 30 min pre-incubation with NO donor SNP or NO scavenger C-PITO (n ¼ 5 haemolymph pools). *P < 0.05, **P < 0.01, ***P < 0.001.
granular and semigranular cells. These results correlated well with the NO level analysis during phagocytosis of zymosan A, indicating that the NO-synthase pathway was more active in granular and semigranular cells. Effects of NO donor SNP and scavenger C-PTIO on DAF-FM fluorescence were determined. Results showed that SNP significantly increased the DAF-FM fluorescence, whereas C-PTIO reduced the fluorescence, indicating that the DAF-FM fluorescence intensity of haemocytes is strongly based on the NO content in it. Both of these two modulators at 0.2 mM were enough for clear changes in DAF-FM fluorescence, and could be used as positive and negative controls in the future study. In conclusion, our present study clearly demonstrated a direct method of intracellular NO assessment in shrimp haemocytes using flow cytometry. The optimized protocol for intracellular NO analysis consists to incubate haemocytes with DAF-FM DA at 10 mM for 60 min to determine the mean fluorescence intensity. The three P. monodon haemocyte types were shown to possess different capacities of NO production. Granular and semigranular cells were more active in terms of constitutive content and capacity to produce extra NO after activation by zymosan. The modifications of fluorescence by inhibitors, donor and scavenger further demonstrate that the DAF-FM fluorescence intensity is based on the activity of NO-synthase pathway and the intracellular NO production.
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J.-A. Xian et al. / Fish & Shellfish Immunology 35 (2013) 2032e2039 [24] Cárdenas W, Jenkins JA, Dankert JR. A flow cytometric approach to the study of crustacean cellular immunity. J Invertebr Pathol 2000;76:112e9. [25] Unestam T, Söderhäll K. Soluble fragments from fungal cell walls elicit defence reactions in crayfish. Nature 1977;267:45e6. [26] Söderhäll K, Unestam T. Activation of serum prophenoloxidase in arthropod immunity: the specificity of cell wall glucan activation and activation by purified fungal glycoproteins of crayfish phenoloxidase. Can J Microbiol 1979;25:406e14. [27] Lee SY, Söderhäll K. Early events in crustacean innate immunity. Fish Shellfish Immunol 2002;12:421e37. [28] Estensen RD, Plagemann PGW. Cytochalasin B Inhibition of glucose and glucosamine transport. P Natl Acad Sci USA 1972;69:1430e4. [29] Sanger JW, Holtzer H. Cytochalasin B: effects on cell morphology, cell adhesion, and mucopolysaccharide synthesis. P Natl Acad Sci USA 1972;69:253e7. [30] MacLean-Fletcher S, Pollard TD. Mechanism of action of cytochalasin B on actin. Cell 1980;20:329e41.
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[31] Austin KA, Paynter KT. Characterization of chemiluminescence measured in haemocytes of the Eastern oyster, Crassostrea virginica. J Exp Zool 1995;273: 461e73. [32] Torreilles J, Guerin MC, Roch P. Peroxidase-release associated with phagocytosis in Mytilus galloprovincialis haemocytes. Dev Comp Immunol 1997;21: 267e75. [33] Ordas MC, Novoa B, Figueras A. Modulation of the chemiluminescence response of Mediterranean mussel (Mytilus galloprovincialis) haemocytes. Fish Shellfish Immunol 2000;10:611e22. [34] Nagy G, Koncz A, Perl A. T cell activation-induced mitochondrial hyperpolarization is mediated by Ca2þ and redox-dependent production of nitric oxide. J Immunol 2003;171:5188e97. [35] Kim D, Kang YS, Lee Y, Yamaguchi K, Matsuoka K, Lee KW, et al. Detection of nitric oxide (NO) in Marine Phytoplankters. J Biosci Bioeng 2008;105: 414e7.
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Short communication
Measurement of intracellular nitric oxide (NO) production in shrimp haemocytes by flow cytometry Jian-An Xian a, b,1, Hui Guo a,1, Bin Li a, Yu-Tao Miao a, Jian-Min Ye a, Sheng-Peng Zhang a, Xun-Bin Pan a, Chao-Xia Ye a, An-Li Wang a, *, Xuan-Ming Hao b, * a
Key Laboratory of Ecology and Environment Science in Guangdong Higher Education, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, School of Life Science, South China Normal University, Guangzhou 510631, People’s Republic of China School of Physical Education & Sports Science, South China Normal University, Guangzhou 510631, People’s Republic of China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2013 Received in revised form 16 October 2013 Accepted 16 October 2013 Available online 25 October 2013
A flow cytometric method to measure the production of intracellular nitric oxide (NO) was adapted for use with shrimp haemocytes. We applied fluorescent probe 4-amino-5-methylamino-20 ,70 -difluorofluorescein diacetate (DAF-FM DA) for NO detection in haemocytes from the tiger shrimp Penaeus monodon, and used flow cytometry to quantify fluorescence intensity in individual haemocyte. The optimized protocol for intracellular NO analysis consists to incubate haemocytes with DAF-FM DA at 10 mM for 60 min to determine the mean fluorescence intensity. Result showed that NO was also produced in the untreated shrimp haemocytes. NO level in granular cells and semigranular cells were much higher than that in hyaline cells. Defined by different characteristic of NO content, three subsets of haemocytes were observed. Zymosan A at dose of 10 or 100 particles per haemocyte triggered higher DAF-FM fluorescence intensity in granular and semigranular cells, than PMA that had no significant impact on all three cell types. These results indicate that granular and semigranular cells are the primary cells for NO generation. Cytochalasin B significantly inhibited the NO level induced by zymosan A. NGMonomethyl-L-arginine (L-NMMA) and diphenylene iodonium chloride (DPI) significantly suppressed the DAF-FM fluorescence in haemocytes, but apocynin could not modulate it, indicating that the DAF-FM fluorescence was closely related to the activity of NO-synthase pathway. The NO donor sodium nitroprusside (SNP) improved the DAF-FM fluorescence in haemocytes, while the NO scavenger C-PTIO (2-(4carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) significantly decreased the fluorescence, demonstrating that the fluorescence intensity of DAF-FM is mainly dependent on the intracellular NO level. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Nitric oxide Flow cytometry DAF-FM DA Haemocyte Shrimp
1. Introduction Nitric oxide (NO) has been identified as a vital physiological modulator and a signaling molecule in mammals [1]. In marine invertebrates, biological roles of NO were found to be related to feeding, immune defence, environmental stress, learning, metamorphosis, swimming, symbiosis, haemocyte aggregation and regulation of blood pressure [2]. Recent studies found that NO acts as a cytotoxic molecule contributed to microorganisms killing in
* Corresponding authors. E-mail addresses: [email protected] (A.-L. Wang), [email protected] (X.-M. Hao). 1 These authors contributed equally to this work. 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.10.014
crustaceans [3e6], as in mammalians [1]. However, the information about biological role of NO in crustacean is still scarce. The direct detection of NO content in biological samples is a challenge due to its short half-life and low concentration. Several indirect methods have been applied to detect the NO level. The most prevalent method of NO production assay was the Griess colorimetric reaction, a method that quantifies nitrites (NO 2 ) and nitrates (NO 3 ), the stable products resulting from the degradation of NO [3,4]. However, there are at least two major restriction of this method: 1) This indirect method may not reflect the real NO level. Not all the NO 2 /NO3 in aquatic animals come from NO metabolism. Environmental NO 2 tends to accumulate into the blood through active uptake mechanisms associated with the chloride cells of the gills [7,8]. 2) This indirect method could not detect the instantaneous NO content in individual cell, and it is hard to estimate the major cell source of NO.
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Another common method for NO detection was flow cytometric assay using the fluorescent probe 20 ,70 -dichlorofluorescein diacetate (DCFH-DA) [9,10]. This probe could also be oxidized by reactive oxygen species (ROS) including hydrogen peroxide (H2O2), peroxynitrite, hydroxyl (HO) and peroxyl (ROO) [9]. Specific inhibitors should be included in this indirect method. Thus, result may also be affected by some processes, such as the dose of inhibitor. Moreover with, this qualitative measure is hard to quantify the NO content accurately. The currently used methods are neither sensitive nor specific. A direct method which is more simple, sensitive and accurate is needed. Flow cytometry (FCM) has been proposed for direct detection of intracellular NO production in human [11], mammal [12] and mussel [13] cells using NO-specific fluorescent probe 4,5diaminofluorescein diacetate (DAF-2 DA) or more sensitive probe 4-amino-5-methylamino-20 ,70 -difluorofluorescein diacetate (DAFFM DA). This direct detection could overcome the major restriction of the indirect methods. Furthermore, FCM can provide information at the single-cell level, so it can easily be applied to analyse the production of NO in different cell subpopulations. The aim of the present work was to describe the development of a flow cytometric approach to measure the NO production in shrimp haemocytes using the fluorescent probe DAF-FM DA, and to evaluate the effects of various modulators on the NO production by different haemocyte types. 2. Materials and methods 2.1. Animals
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emission ¼ 515) which exhibits about a 160-fold greater fluorescence quantum efficiency [12]. DAF-FM green fluorescence was measured on FL1 detector of flow cytometer. Results were given as mean of fluorescence in arbitrary units (A. U.). 2.4. DAF-FM DA dose and time responses An initial experiment was performed to define the optimum DAF-FM DA dose to be added to haemocyte suspension; this dose should be sufficient to emit a measurable fluorescent signal on the FL1 detector and to be non-toxic for cells. After haemolymph was extracted as described previously, haemolymph samples were pooled to reduce interindividual variation, and then the cell concentration was adjusted to about 1 106 cells ml1 with AS. The diluted pooled haemolymph sample was divided into four sub-samples of 2 ml, then 20 ml of the DAF-FM DA stock solutions (0.1, 0.5, 1 and 5 mM) was added to yield final concentrations of 1, 5, 10 and 50 mM, respectively. The mixture was incubated in the dark at room temperature. DAF-FM fluorescence of haemocyte was evaluated just after DAF-FM DA addition (t ¼ 0) and at t ¼ 15, 30, 45, 60, 75 and 90 min. Cell viability at the end point of the experiment was determined using propidium iodide (PI, Sigma) stain as described previously [14]. This experiment repeated three times. 2.5. Non-induced nitric oxide production in different haemocyte types
The experimental shrimp Penaeus monodon (7.78 0.82 g) were obtained from a commercial shrimp farm in Nansha, Guangzhou, Guangdong Province, China. They were maintained in the laboratory with diluted seawater at 20&, pH 7.9e8.0 and controlled temperature (24 2 C), with continuous water circulation. Prior to experimental use, animals were acclimated to the laboratory conditions for one week, and fed twice daily with commercial shrimp feed. Only apparently healthy shrimp in the intermoult stage were used.
Haemolymph was extracted as described previously, and then was diluted with AS to obtain a final concentration of about 1 106 cells ml1. This experiment was performed on individual samples, and fifteen shrimp were analysed. Two hundred microlitres of haemocyte suspension from each shrimp was incubated with DAF-FM DA at 10 mM (the optimum dose) for 60 min (the optimum time) in the dark. Three different morphologic haemocyte types (hyaline cells, semigranular cells and granular cells) can be defined basing on the relative size (FSC values) and granularity (SSC values) as described previously [14]. The DAF-FM fluorescence intensity of each haemocyte type was analysed.
2.2. Preparation of haemocyte suspension
2.6. Haemocyte responses to modulators
Haemolymph (300 ml) was extracted from each shrimp with a 25 gauge needle and 2.5 ml syringe containing an equal volume of ice-cold anticoagulant solution (AS, glucose 20.5 g L1, sodium citrate 8 g L1, sodium chloride 4.2 g L1, pH 7.5). The diluted haemolymph from each shrimp was transferred into a separate microcentrifuge tube held on ice.
Effects of two activators, four inhibitors, NO donor and scavenger on DAF-FM fluorescence intensity were determined in the tiger shrimp haemocytes. Species, doses and functions of modulators used in this study are presented in Table 1.
2.3. Measurement of intracellular nitric oxide by flow cytometry FCM was performed with a FACSCalibur instrument (Bectone Dickinson Immunocytometry Systems, San Jose, CA) equipped with a single argon ion laser with filtered emission at 488 nm. Photomultiplier bandpass filters for fluorescence were 530 nm (green fluorescence, FL1). Size scatter height (SSC) and FL1 fluorescence data were collected on log scale, and forward scatter height (FSC) data were collected on linear scales. For each subsample, 10, 000 events were counted. 4-amino-5-methylamino-20 ,70 -difluorofluorescein diacetate (DAF-FM DA, Sigma) is a NO-specific fluorescent probe, which is cell-permeant and passively diffuses across cellular membranes. Once inside cells, it is deacetylated by intracellular esterases to become DAF-FM. After specifically reacting with NO, DAF-FM is further converted to DAF-FM triazole (l excitation ¼ 495, l
2.6.1. Haemocyte responses to activators Two possible NO activating agents were tested: zymosan A (Sigma) and phorbol myristate acetate (PMA, Sigma). Zymosan A stock preparation: 200 mg of zymosan A was suspended in 10 ml of AS, heated in a boiling water bath for 30 min, and then washed twice. The stock suspension was resuspended in a range of volumes of AS to make three suspensions that contained increasingly higher concentrations of zymosan particles. The particle count was checked microscopically, and aliquots were frozen at 20 C. After haemolymph was extracted, pooled and diluted as described previously, zymosan A was added to haemocyte suspensions to obtain approximately 1, 10 and 100 particles per haemocyte. PMA was added to obtain 1 and 10 mg ml1. DAF-FM DA was added to haemocyte suspensions to yield final concentration of 10 mM, 30 min after adding zymosan A or PMA. The haemocyte solutions were incubated in the dark for 60 min at room temperature, and then the DAF-FM fluorescence of each haemocyte type was analysed by flow cytometry.
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Table 1 List and concentration of modulators tested on NO production of P. monodon haemocytes. Modulator Chemicals Activator
Final concentration
Activity
Zymosan
1, 10 and 100 Phagocytosis, particles cell1 ROS/RNS production 1 Activation of PMA 1 and 10 mg ml NOX and NOS molecules Inhibitor DPI 50 mM Inhibitor of NOX, NOS L-NMMA 50 mM NOS inhibitor Apocynin 50 mM NOX inhibitor Cytochalasin B 10 mg ml1 Phagocytosis inhibitor Donor SNP 0.2 and 2 mM Scavenger C-PTIO 0.2 and 2 mM
Ref. [9,10] [9,10,17]
[9,10,17] [9,17] [17] [9] [34] [34,35]
PMA: phorbol myristate acetate; DPI: diphenylene iodonium chloride; L-NMMA: NG-Monomethyl-L-arginine; Fccp: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; SNP: sodium nitroprusside; C-PTIO: 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide; ROS: reactive oxygen species; RNS: reactive nitrogen species; NOX: NADPH-oxidase; NOS: NO-synthase.
2.6.2. Haemocyte responses to inhibitors Four possible inhibitors used in this experiment are presented in Table 1. Haemocytes were pre-incubated with inhibitors for 30 min. Then 10 mM DAF-FM DA was added. After the solutions were incubated in the dark for 60 min at room temperature, DAF-FM fluorescence in haemocytes was analysed. 2.6.3. Haemocyte responses to NO donor SNP or scavenger C-PTIO Effects of the specific NO donor sodium nitroprusside (SNP) and scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1oxyl-3-oxide (C-PTIO) on detection of NO was measured. Haemocytes were pre-incubated with SNP (0.2 and 2 mM) or C-PTIO (0.2 and 2 mM) for 30 min. Then the haemocytes were incubated with 10 mM DAF-FM DA in the dark for 60 min at room temperature. DAF-FM fluorescence in haemocytes was analysed. 2.7. Statistical analyses All data are presented as means standard deviation (S. D.). A multiple-comparison Tukey test was used to examine for significant differences among treatments using the SPSS 13.0 program from SPSS Inc (Chicago, IL, USA). A P value 0.05). Zymosan at dose of 10 or 100 particles per haemocyte significantly induced the NO production in semigranular cells and granular cells. The dose of 10 particles per haemocyte seems to be the best compromise between activation capability and lower particle ratio. Effect of PMA on NO production is presented in Fig. 7. No significant effect of PMA was observed on NO level in all three haemocyte subpopulations, compared to the control treatment (P > 0.05). 3.4. Haemocyte responses to inhibitors Effects of NMMA, DPI and apocynin on the DAF-FM fluorescence of different haemocyte subpopulations are presented in Fig. 8. For hyaline cells, DAF-FM fluorescence was not affected by the chosen inhibitors (P > 0.05). Apocynin also had no effect on the DAF-FM fluorescence of semigranular cells and granular cells (P > 0.05). For semigranular cells, DAF-FM fluorescence was significantly lower in the presence of NMMA and DPI, decreasing 42.3% and
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Fig. 2. DAF-FM fluorescence histogram of different untreated haemocyte subpopulations of P. monodon. A: Total haemocytes; B: Hyaline cells; C: Semigranular cells; D: Granular cells.
37.8% respectively compared to semigranular cells without inhibitors (P < 0.05). For granular cells, DAF-FM fluorescence significantly decreased in the presence of NMMA and DPI, decreasing 40.2% and 58.3% respectively compared to the control treatment (P < 0.05). Effect of cytochalasin B on the NO level is given in Fig. 9. In the presence of cytochalasin B, no significant modification of NO production was observed in total haemocytes (P > 0.05). As shown previously, zymosan induced the NO production (P < 0.05). When the zymosan combined with cytochalasin B, NO production reduced to the level of control (P > 0.05). 3.5. Haemocyte responses to NO donor SNP and scavenger C-PTIO
Fig. 3. NO production in different untreated haemocyte subpopulations of P. monodon (n ¼ 15 shrimps). HC: Hyaline cells; SGC: Semigranular cells; GC: Granular cells. Different letters indicate significant differences between haemocyte subpopulations (P < 0.05).
Effects of SNP and C-PTIO on DAF-FM fluorescence in haemocytes are presented in Fig. 10. The pre-incubation of SNP (0.2 and 2 mM) significantly increased the DAF-FM fluorescence of haemocytes (P < 0.01 and P < 0.001). Compared to the control, the relative DAF-FM fluorescence in haemocytes pre-incubated with 0.2 and 2 mM SNP was 177% and 387%, respectively. DAF-FM fluorescence of haemocytes decreased significantly in the presence of C-PTIO (0.2 and 2 mM, P < 0.01 and P < 0.001). 4. Discussion Flow cytometry, which can provide information at the singlecell level, has been used in studies of aquatic invertebrate [9,10,15e19]. Respiratory burst activity which is an important defence mechanism in invertebrate has been measured by flow
Table 2 Percentage and fluorescence intensity of DAF-FM-defined haemocyte subpopulations of Penaeus monodon (n ¼ 15 shrimps). Different letters in columns indicate significant differences between haemocyte subpopulations (P < 0.05). Percentage (%) low
Fig. 4. FSC-FL1 dot plot of P. monodon haemocytes stained by DAF-FM DA. R4: DAFFMlow cells; R5: DAF-FMmed cells; R6: DAF-FMhigh cells.
DAF-FM cells (R4) DAF-FMmed cells (R5) high DAF-FM cells (R6)
c
7.88 4.30 64.14 6.21a 22.62 4.47b
NO production (A.U.) 15.33 4.32c 84.90 9.35b 557.48 30.29a
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Fig. 5. DAF-FM fluorescence histogram (A, B, C) and FSC-SSC dot plot (D, E, F) of DAF-FM-defined haemocyte subpopulations of P. monodon. A, D: DAF-FMlow cells; B, E: DAF-FMmed cells; C, F: DAF-FMhigh cells; R1: Region of hyaline cells; R2: Region of semigranular cells; R3: Region of granular cells.
cytometry using DCFH-DA [9,18]. This probe could be oxidized by both ROS and RNS. Thus, this method also been used in NO qualitative assay indirectly. In the present study, we intended to optimize and apply the FCM protocol for direct measurement of intracellular NO level in shrimp haemocytes. This flow cytometric method is based on intracellular non-fluorescent DAF-FM conversion to fluorescent DAF-FM triazole by NO [12]. In the present study, according to the dynamic variation of intracellular DAF-FM fluorescence intensity, for shrimp haemocytes, 10 mM seemed to be the optimal concentration of this probe, and 60 min appeared to be the optimal incubation time. Using this probe, distinct DAF-FM fluorescence was presented in the untreated haemocytes. This result revealed that NO exists in shrimp haemocytes without additional activation. Some studies hypothesized that ROS/RNS production of untreated haemocytes may be induced by the contact of haemocyte membrane with nonself materials such as syringe and plastic tubes, or by various environmental factors before haemolymph collecting [9]. However, since NO is an important physiological and immune molecule [1], the NO generated in the untreated haemocytes could be considered as a normal, constitutive physiological production. To further understand the roles of different haemocyte types in NO synthesis, NO contents in the three shrimp haemocyte types (hyaline cells, semigranular cells and granular cells) were analysed respectively. Using FCM, these three types of shrimp haemocyte could be defined basing on FSC values and SSC values [14,20]. Our results showed that granular cells produced much more NO than semigranular cells and hyaline cells without stimulation. The NO level in granular cells approximately six times and twenty-one times higher than those in semigranular cells and hyaline cells respectively. These results suggest that the three haemocyte types possess different capabilities for NO response, and the granular and semigranular cells are the primary cells for non-induced NO generation. According to the size (FSC) and DAF-FM fluorescence, three DAFFM-defined haemocyte subpopulations could be clearly distinguished (Fig. 4). The relative size and granularity of the NO-defined subpopulations were analysed in the FSC-SSC dot plot. Results
showed that DAF-FMlow cells, DAF-FMmed cells and DAF-FMhigh cells were located in the region of hyaline cells, semigranular cells and granular cells respectively (Fig. 5). These results suggest that the three haemocyte types cloud also be distinguished according to their great differences in NO level. Zymosan A, a common inducer of phagocytosis and ROS/RNS production, was used to determine the induced-NO production in different haemocyte subpopulations in the present study. Ten zymosan particles per haemocyte appeared to be a good dose for this method, since this dose of zymosan increased the DAF-FM fluorescence with the lowest particle. Our results showed that NO production in granular cells and semigranular cells could be activated by zymosan A particles, but that in hyaline cells could not. These facts indicate that granular cells and semigranular cells may play main role in killing pathogens by RNS pathway. These two types of haemocyte in crayfish Astacus astacus have been reported to display cytotoxic capacity towards mammalian tumour and nontumour cell line [21]. Our finding suggests that the NO production capacity in granular and semigranular cells may be contributed to their cytotoxic function. Numerous studies have demonstrated that different haemocyte types carry out different functions in immune defence of crustaceans [22]. Because of the different roles of haemocyte subpopulations in immunity, the different haemocyte count (DHC) may be considered of major importance to evaluate the immune level of crustaceans [23]. Generation of NO has been demonstrated in crustacean haemocytes during phagocytosis [3]. Hyaline cells and semigranular cells were known to be the main cell types of phagocytosis [22]. However, we found that granular cells and semigranular cells were the major NO-response cells to zymosan. It means that phagocytosis of zymosan particle may be not the activation pathway for NO production. Numerous previous studies have reported that zymosan would trigger the cellular responses including exocytosis and activation of prophenoloxidase (proPO) system localized inside granular cells and semigranular cells [24,25]. They claimed that the active compound in zymosan was soluble b-glucans which activated the proPO system by signal transduction of the pattern recognition proteins (PRPs) such as b-1,3-glucan-binding protein
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Fig. 7. NO production in different haemocyte subpopulations exposed to PMA (n ¼ 5 shrimps). HC: Hyaline cells; SGC: Semigranular cells; GC: Granular cells. For each haemocyte subpopulation, different letters indicate significant differences between treatments (P < 0.05, ANOVA).
PMA is a common inducer of ROS/RNS production for vertebrate phagocytes. It is known to activate protein kinase C, involved in activation of NADPH-oxidase and NO-synthase molecules, responsible for the production of ROS and RNS, respectively. However, in the present study, PMA did not show any capacity to modify NO level in either P. monodon haemocyte type. Similar results were shown in some previous studies of crustaceans and bivalves [14,31e33]. These facts suggest that these species of invertebrate including P. monodon may have a different activation pathway of NOS without protein kinase C. In order to further confirm the relationship between DAF-FM fluorescence and different oxidative pathways, impact of specific inhibitors for the NO-synthase and NADPH-oxidase were tested. The apocynin, inhibitor of NADPH-oxidase pathway, could not affect the DAF-FM fluorescence, suggesting that NADPH-oxidase dependent ROS did not contribute to the DAF-FM fluorescence. On the contrary, both NMMA and DPI which can inhibit the NOsynthase pathway significantly reduced the fluorescence intensity, demonstrating that the DAF-FM fluorescence intensity was closely related to the activity of NO-synthase. Interestingly, the reduction of DAF-FM fluorescence could only be observed in
Fig. 6. NO production in different haemocyte subpopulations exposed to zymosan A at different haemocyte: zymosan A ratios (n ¼ 5 shrimps). HC: Hyaline cells; SGC: Semigranular cells; GC: Granular cells. For each graph, different letters indicate significant differences between treatments (P < 0.05, ANOVA).
(bGBP) [26,27]. Thus, we speculate that NO production induced by zymosan may be also one of the cellular responses involved in the PRPs activation pathway. On the other hand, we found that phagocytosis inhibitor cytochalasin B prevented the induction of zymosan, while it did not modify the NO production in untreated haemocytes. Cytochalasin B is also found to inhibit the glucose and glucosamine transport [28], and to affect the cell morphology, cell adhesion and mucopolysaccharide synthesis [29]. Perhaps, in this study, the signal transductions of PRPs were disturbed by cytochalasin B while it inhibited the actin polymerization [30] or other cellular events. Indeed, more researches should be done to help us to understand the activation mechanism of NO production, and the real relationship between phagocytosis and NO production.
Fig. 8. NO production in different haemocyte subpopulations exposed to various inhibitors (n ¼ 5 shrimps). HC: Hyaline cells; SGC: Semigranular cells; GC: Granular cells. For each haemocyte subpopulation, different letters indicate significant differences between treatments (P < 0.05, ANOVA).
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Acknowledgements This research was supported by the National Natural Science Foundation of China (31302164), Guangdong Provincial Natural Science Foundation (S2011020003256 and S2012040008093), China Postdoctoral Science Foundation (2012M511829), Scientific and Technological Planning Project of Guangdong Province (2011B020307010 and 2012B020307004).
References
Fig. 9. NO production in total P. monodon haemocytes at 120 min incubation in AS (control) or after addition of cytochalasin B (Cyt B: 10 mg ml1), zymosan A (ZA: 10 particles haemocyte1) or both chemicals (ZA þ Cyt B). Different letters indicate significant differences between treatments (P < 0.05, ANOVA).
Fig. 10. DAF-FM fluorescence of P. monodon haemocytes after 30 min pre-incubation with NO donor SNP or NO scavenger C-PITO (n ¼ 5 haemolymph pools). *P < 0.05, **P < 0.01, ***P < 0.001.
granular and semigranular cells. These results correlated well with the NO level analysis during phagocytosis of zymosan A, indicating that the NO-synthase pathway was more active in granular and semigranular cells. Effects of NO donor SNP and scavenger C-PTIO on DAF-FM fluorescence were determined. Results showed that SNP significantly increased the DAF-FM fluorescence, whereas C-PTIO reduced the fluorescence, indicating that the DAF-FM fluorescence intensity of haemocytes is strongly based on the NO content in it. Both of these two modulators at 0.2 mM were enough for clear changes in DAF-FM fluorescence, and could be used as positive and negative controls in the future study. In conclusion, our present study clearly demonstrated a direct method of intracellular NO assessment in shrimp haemocytes using flow cytometry. The optimized protocol for intracellular NO analysis consists to incubate haemocytes with DAF-FM DA at 10 mM for 60 min to determine the mean fluorescence intensity. The three P. monodon haemocyte types were shown to possess different capacities of NO production. Granular and semigranular cells were more active in terms of constitutive content and capacity to produce extra NO after activation by zymosan. The modifications of fluorescence by inhibitors, donor and scavenger further demonstrate that the DAF-FM fluorescence intensity is based on the activity of NO-synthase pathway and the intracellular NO production.
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