Fish & Shellfish Immunology 38 (2014) 196e203
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Endosulfan decreases cytotoxic activity of nonspecific cytotoxic cells and expression of granzyme gene in Oreochromis niloticus Martha Cecilia Téllez-Bañuelos a, *, Pablo Cesar Ortiz-Lazareno b, Luis Felipe Jave-Suárez b, Victor Hugo Siordia-Sánchez a, Alejandro Bravo-Cuellar b, Anne Santerre a, Galina P. Zaitseva a a b
Departamento de Biología Celular y Molecular, Universidad de Guadalajara, Carretera a Nogales Km 15.5, Las Agujas, 45110 Zapopan, Jalisco, Mexico Centro de Investigación Biomédica de Occidente, IMSS, Sierra Mojada 800, Col. Independencia, 44340 Guadalajara, Jalisco, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 October 2013 Received in revised form 23 February 2014 Accepted 7 March 2014 Available online 20 March 2014
The effect of the organochlorinated insecticide endosulfan, on the cytotoxic activity of Nile tilapia nonspecific cytotoxic cells (NCC) was assessed. Juvenile Nile tilapia were exposed to endosulfan (7 ppb) for 96 h and splenic NCC were isolated. Flow cytometric phenotyping of NCC was based on the detection of the NCC specific membrane signaling protein NCCRP-1 by using the monoclonal antibody Mab 5C6; granzyme expression was evaluated by quantitative RT-PCR. The cytotoxic activity of sorted NCC on HL60 tumoral cells was assessed using propidium iodide (PI) staining of DNA in HL-60 nuclei, indicating dead cells. Nile tilapia splenic NCC had the ability to kill HL-60 tumoral cells, however, the exposure to endosulfan significantly reduced, by a 65%, their cytotoxic activity when using the effector:target ratio of 40:1. Additionally, the exposure to endosulfan tended to increase the expression of NCCRP-1, which is involved in NCC antigen recognition and signaling. Moreover, it decreased the expression of the granzyme gene in exposed group as compared with non-exposed group; however significant differences between groups were not detected. In summary, the acute exposure of Nile tilapia to sublethal concentration of endosulfan induces alteration in function of NCC: significant decrease of cytotoxic activity and a tendency to lower granzyme expression, severe enough to compromise the immunity of this species. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Endosulfan Nile tilapia NCC Cytotoxic activity Granzyme
1. Introduction Endosulfan is a potent organochlorinated insecticideeacaricide; the technical grade material consists of two isomers (a and b); endosulfan-sulphate is the main environmental metabolite in water, sediment and tissue [1]. The use of endosulfan is restricted in many countries due to its adverse effects on ecosystems, including aquatic environments. Moreover, this pesticide has been added to the annex A of the Stockholm Convention in 2011 [2]. In teleosts, the nonspecific cytotoxic cells (NCC) were identified in 1983 by Evans et al. (2001) who reported their participation in innate immunity [3]. First described in channel catfish, NCC have been characterized in teleost and are thought to be the evolutionary precursors of the mammalian NK cells [4]. The NCCs are not the only cytotoxic cells found in fish, where lymphocytes,
* Corresponding author. Tel.: þ52 33 3777 1191; fax: þ52 33 3673 8375. E-mail address: [email protected] (M.C. Téllez-Bañuelos). http://dx.doi.org/10.1016/j.fsi.2014.03.012 1050-4648/Ó 2014 Elsevier Ltd. All rights reserved.
monocyte-macrophages and granulocytes also show nonspecific cytotoxic activity [5]. In both cartilaginous and bony fish, macrophages have been shown to display spontaneous cytotoxicity, and antibody-dependent cell-mediated cytotoxic (ADCC) reactions are found in sharks [6]. Nakanishi et al. (2011) also mentioned that three types of cells are involved in cell-mediated cytotoxicity in fish. One is the NCC, originally described in channel catfish and later in other fish species including rainbow trout, carp, damselfish and tilapia. Other type is represented by NK-like cell lines (distinct from NCCs), which were developed from catfish peripheral blood leukocytes. The third NCC type are the neutrophils [7]. NCC have the ability to kill a wide array of targets including virus-infected cells and cancer cells. In vitro, NCC participate in immunosurveillance against Yac-1 cells, HL-60 cells (human promyelocytic cells), and have potential relevance in the control of protozoan parasites (Tetrahymena pyriformis), and bacterial infections [8,9]. NCC are potent immune effector cells and are able to contact target cells, leading to granule exocytosis related with both necrotic and apoptotic death pathways [10,11]. Praveen et al. (2004)
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reported the existence of granule granzyme-like serine proteases in NCC of channel catfish [9]. These proteases might play an important role in the effector functions of NCC and this indicates that cellmediated immunity with granule exocytosis and Fas pathways have been conserved for more than 300 million years. In tilapia, granzymes (in conjunction with pore-forming proteins) constitute the major components of granules of professional killer cells such as cytotoxic T lymphocytes, NK cells and NCC [12]. NCC are small cells (3.5 mm) and presently the best method for their identification is with the monoclonal antibody 5C6 (Mab 5C6), as originally described for catfish. This antibody binds to the NCC receptor protein NCCRP-1, which is involved in NCC antigen recognition and signaling, and recognizes a discrete ligand referred as the NK target antigen (NKTag) [3,9]. A difference between NCC and mammalian NK is that the latter requires in vitro activation by lymphokines to generate cytotoxicity, in contrast, naive NCC are constitutively active and capable of killing freshly isolated targets [8]. Cuesta et al. (2005) demonstrated gilthead seabream NCC heterogeneity: lymphocytes, monocyte/macrophages and acidophilic granulocytes from lymphoid tissues express NCCRP-1, both at gene and protein level [13]. Tilapia is considered the future of aquaculture and has been nicknamed “the aquatic chicken” due to its ability to grow quickly with poor-quality inputs. It is also a good biological model for toxicological and immunotoxicity studies of the effects of pesticides on environmental and animal and human health, due to diverse characteristics, namely high growth rates, efficiency in adapting to diverse diets, great resistance to diseases and handling practices, easy reproduction in captivity at prolific rates and finally, good tolerance to a wide range of environmental conditions [14]. Previous data from our research group indicated that acute (96 h) in vivo exposure to sublethal doses of endosulfan (7 ppb ¼ 1/ 2LC50) induces nonspecific activation of splenic macrophages and exacerbated interleukin-2 synthesis in Nile tilapia [15,16]. Also, experimental data showed that endosulfan per se increased cellular proliferation, but decreased the lymphoproliferative response to mitogenic stimulus with PMA þ ionomycin. Splenocytes exposed to endosulfan for 15e180 min showed significantly higher levels of pERK1/2 than the non-exposed control. Endosulfan mediated a decrease in etoposide-induced apoptosis and provoked cell senescence [17]. In order to further assess the effects of this pesticide on the innate immune response of Nile tilapia, particularly on the immunosurveillance capacity of NCC, flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR) were used to evaluate the signaling of the NCCRP-1, the cytotoxic activity of splenic NCC against HL-60 tumor cells and the expression of the granzyme gene. 2. Material and methods 2.1. Animals Nile tilapia were obtained from the Aquamol aquaculture farm in Jamay, Jalisco, Mexico, and transferred to 40-L glass aquariums (one fish per tank), at 28 2 C with constant aeration for 3 months before initiating experiments. Water quality monitoring included nitrite, ammonia nitrogen and chlorine determinations. Experimental males weighed 200e250 g; their daily food consisted of commercial dry fish pellets (Tilapia Nutripec 2506 AP, Purina, Mexico).
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of this grade is frequently employed in field practices. The pesticide was dissolved in distilled water (1:100) and this stock solution was added directly to the 40-L tanks to a final concentration of 7 ppb (7 mg L1), which is equivalent to 1/2 the lethal concentration 50 (1/ 2LC50) for juvenile Nile tilapia, for a 96 h time period, without water changes. 2.3. Experimental groups The effect of acute exposure to the sublethal doses of endosulfan on splenic NCC of Nile tilapia (n ¼ 5) was evaluated. A non-exposed control group of five fish was maintained under similar conditions as the exposed group, but without pesticide. All evaluations were performed in duplicate. Exposed and non-exposed fish were anesthetized with clove oil at 100 mg L1 (Sigma St. Louis, MO, USA) [18]; fish were sacrificed and subsequently carefully dissected aseptically to remove the spleen. 2.4. Purification of mononuclear cells from the spleen of tilapia Mononuclear cells were isolated from the spleen. The spleens were disaggregated and passed through a 100 mm nylon mesh. Red blood cells were separated from the cell suspension by density gradient centrifugations, using a modification of the method described by Bishop et al. [19] and Harford et al. [20]; five mL of cell suspension was layered over five mL of Histopaque 1077 (Sigma St. Louis, MO, USA) and centrifuged at 1200 g for 20 min at 28 C. The immune cells were collected from the upper histopaque layer and a second purification was undertaken over 45.5% Percoll (Sigma Chemicals, St. Louis, MO, USA) in PBS at 1200 g for 17 min at 28 C. Cells at the interface were collected, washed once with cold PBS, and resuspended in RPMI-1640 (RPMI) containing 4 mM glutamine (Gibco-Invitrogen, Grand Island, NY, USA) and supplemented (RPMI-S) with 10% fetal calf serum (FCS, Sigma, St. Louis, MO, USA). Cell viability was evaluated using the trypan blue (Sigma St. Louis, MO, USA) dye exclusion method at 0.2% (w/v). Cell concentration was further adjusted with RPMI-S at 2 105 cells per 100 mL, as required for each assay. 2.5. NCC Phenotype analysis Phenotyping of NCC from spleens was realized by flow cytometric examination for surface expression of NCCRP-1 using a modification of the methods of Bishop et al. (2000) and Oumouna et al. (2001) [8,10]. Briefly, NCC (2 105 cells) were incubated for 1 h at 4 C with 1.25 mL (0.25 mg), 2.5 mL (0.5 mg) or 5 mL (1 mg) of Mab 5C6 of mouse IgM isotype (specific for the NCCRP-1 conventional antigen receptor on NCC, Affinity BioreagentsÔ, Inc, Golden, CO, USA). Cells were then washed twice in cold PBS and incubated with 2 mL (1 mg) of a goat anti-mouse IgM conjugated with FITC (Sigma BioSciences, St. Louis, MO, USA) for 30 min at 4 C. NCC were identified and quantified in a BD FACSAria I cell sorter (BD Bioscience, San Jose, CA, USA) with laser excitation at 488 nm to assess the forward scatter (FSC; size) and side scatter (SSC; granularity) parameters. An IgM isotype control was used. Experimental data were processed with the FACSDiva Software (BD Bioscience, San Jose, CA, USA). All experiments with endosulfan-exposed and non-exposed control organisms were performed in duplicate. 2.6. Target cells
2.2. Endosulfan Technical grade endosulfan (35% EC) was locally purchased (manufactured by Agricura, SA de CV, DF, Mexico) since endosulfan
HL-60 is a non-adherent cell line established by Gallagher et al. [21] by leukopheresis from a 36-year-old Caucasian female with acute promyelocytic leukemia. This cell line, which is commercially
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available, is characterized by a lack of lymphoid markers, growth as compact colonies in semisolid culture, and non-adherence to substrate. The target cells were obtained from ATCC, and consisted of HL-60 cells (ATCC CCL-240, Rockville, MD, USA) grown as suspension cultures in RPMI-S at 37 C in a humid incubator with 5% CO2 (NuAire HEPAIII, Plymouth, MN, USA). Cell concentration was adjusted with RPMI-S as required for each cytotoxicity assay. 2.7. Cytotoxic activity of NCC For the assays, Fluorescence Activated Cell Sorting (FACS)-based positive selection, using the Mab anti-NCCRP-1 (Mab 5C6), of a preparation of splenic mononuclear cells (previously depleted of granulocytes by double density gradient) was performed to obtain a highly pure preparation of NCCs. For sorting, the gate was set around a positive population to the NCCRP-1 marker, which was then used to determine the NCC population. Ninety-nine percent of the purified cells were positive for NCCRP-1. Several effector to target (E:T) cell ratios were assayed (1:1, 10:1, 20:1 and 40:1) in a final volume of 200 mL, in 96 well microtiter plates (Corning Incorporated, Corning, NY, USA), using a modification of the methods of Oumouna et al. [10]. The effectors (at different numbers) and targets (at a constant number) were pelleted in Ubottom 96-well microtiter plates by centrifugation and then incubated for 4 h at 37 C in a humidified incubator with 5% CO2. (NuAire HEPAIII, Plymouth, MN, USA). Cells were then harvested and washed twice with PBS and centrifuged at 1200 g for 7 min. 200 mL of PBS were added, and the cells were labeled with 10 mL of propidium iodide (PI) (Sigma, St. Louis, MO, USA) and incubated for 10 min protected from light. Cell populations were analyzed by flow cytometry (FACSAria I cell sorter, BD Bioscience, San Jose, CA, USA) with laser excitation at 488 nm. Back-gating was used to confirm the identity of each cell population; the flow cytometer was set to collect 10,000 events of gated HL-60 target cells (based on FS and SS characteristics). Experimental data were processed with the FACSDiva Software (BD Bioscience, San Jose, CA, USA). The results were expressed as percent of specific PI uptake ¼ [(test binding spontaneous binding/total binding spontaneous binding)] 100. The PI uptake was measured for the target cells mixed with NCCs (test binding), for the target cells (HL-60) alone (spontaneous binding), and for the target cells permeabilized with acetone (total binding). 2.8. Expression of granzyme in NCC Quantitative RT-PCR (qRT-PCR) was used to evaluate the expression of the granzyme gene in splenic NCC from exposed and non-exposed control groups. Total RNA was isolated from splenic NCC and purified with the PureLink RNA Mini Kit (Ambion-Invitrogen Corporation, Carlsbad, CA, USA). The cDNA was synthesized from 1 mg of total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Mannheim, Germany). Assays were performed with 2.0 LightCycler technology using the LightCyclerFastStar DNA Master PLUS SYBR Green I kit (Roche Applied Science, Mannheim, Germany) as recommended by the manufacturers. Analysis of gene expression was performed with LightCycler ver. 4.1 software (Roche Applied Science, Mannheim, Germany). Beta-actin was used as an endogenous control. Relative quantification of target genes was determined by utilizing the DDCP method. Analysis was performed by taking the values obtained from three independent RNA extractions in duplicate. Oligonucleotides (Invitrogen Corporation, Carlsbad, CA, USA) were designed using Oligo-Primer Analysis ver. 6.51 software (Molecular Biology Insights, Inc., USA) utilizing sequences obtained from the GenBank Nucleotide database of the NCBI website. For the granzyme gene
(AY918866: GenBank) the primer sequences were: F:50 ATCATGCTCCTCAAACTCTCG-30 ; R:50 -GGGCCACCAGAATCACC-30 , with a total amplicon size of 290 bp. For b-actin (EF206796.1: GenBank): the primer sequences were: F: 50 -GGCATCGCACCTTCT ACAAT-30 ; R:50 -TCTCACGCTCAGTTGTGGTAGT-30 , with a total amplicon size of 366 bp. For normalizing target gene expression one reference gene was used, b-actin, calculating the Crossing Point (CP) for target (granzyme) and reference gene in each sample and subsequently calculating the DCP value of each sample, i.e., the target gene CP minus the reference gene CP. This facilitated the analysis by taking only the intrinsic values of each sample. It is extremely note-worthy that DCP is inversely proportional to the expression of the target gene. The relative expression was calculated by taking expressions of non-exposed samples as 1. In this context, expression from all non-exposed samples has a relative value of 1, and the SD of non-exposed samples was zero. Taking this value as reference, the values of relative expression for the exposed samples were calculated using the DCp Method [22]. 2.9. Statistical analysis Statistical differences between exposed and non-exposed groups were evaluated using the Student T-test; significance level was set at p < 0.05. Data were analyzed with the SigmaPlot 10.0 software. To calculate variations in the granzyme expression, the median Cp value of the control group (untreated samples) was calculated after normalization with the expression of b-actin. This median value was used to calculate the relative variation of the treated samples. The expression of control samples was set as 1 and the variations were calculated related to this value. The statistical difference between both groups was assayed using the Student Ttest. 3. Results 3.1. Nile tilapia splenic NCC phenotype Mononuclear cells were purified from the spleens of tilapia by using two density gradients (histopaque and percoll). Representative dot plot correlation Forward scatter (FSC) vs side scatter (SSC) of Nile tilapia mononuclear spleen cells are seen in Fig. 1A(1); FSC vs SSC plots of the same isolated cells are shown in Fig. 1A(2). Flow cytometric phenotyping was based on the detection of the NCCspecific-membrane-signaling-protein NCCRP-1 by Mab 5C6. NCC were identified and quantified by dot plot backgating FSC vs SSC plots (Fig. 1A) from Mab 5C6 stained cells (Fig. 1). The optimal antibody concentration was 1 mg (Fig. 1B). Monoclonal 5C6 binding determined by flow cytometry gave a minimum value of almost 15% and a maximum value of 22.7% (18.6 4.1) of total mononuclear splenocytes (Fig. 2B). 3.2. Effect of in vivo exposure to endosulfan on Nile tilapia splenic NCC phenotype The expression of the signaling protein NCCRP-1, given as MFI (Mean Fluorescence Intensities) was evaluated in splenic NCC from tilapia. No significant differences were observed between non-exposed (1886.78 530.44) and endosulfan-exposed (2310.67 866.42) groups (Fig. 2A). Also, the percentage of NCCRP-1þ cells of Nile tilapia was not significantly affected (p > 0.05) by the acute (96 h) in vivo exposure to 7 ppb of endosulfan (22.3 8.7) compared with the non-exposed group (18.6 4.1). Data are shown as mean SD and compared by using the Student T test. Representative histogram of NCCRP-1þ cells in exposed and non-exposed groups are presented (Fig. 2C).
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Fig. 1. Splenic NCC phenotyping by flow cytometry. A(1) Representative dotplot correlating FSC vs SSC of Nile tilapia mononuclear spleen cells. A(2) Forward scatter vs side scatter plot of the same isolated cells. B. Representative dotplot FITC intensity from Mab 5C6 stained and fluorescence histogram obtained from mononuclear splenocytes surface NCCRP-1 expression.
Fig. 2. Effect of endosulfan in the NCCRP-1 expression. A. MFI (Mean Fluorescence Intensities) of the expression of protein NCCRP-1 and B. Percentage of NCCRP-1þ cells of Nile tilapia was not significantly affected (p > 0.05) by the acute (96 h) in vivo exposure to 7 ppb of endosulfan. Data are shown as mean SD and compared by using the Student T-test. C. Representative histogram of NCCRP-1þ cells in exposed and non-exposed groups.
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3.3. Effect of endosulfan exposure on the cytotoxic activity of splenic NCC from Nile tilapia In a preliminary assay, cell sorting was used to confirm the cytometric assignments of the NCC and HL-60 populations based on their respective SSC and FSC. The effector cells (NCC) and target cells (HL-60) have three to five fold differences in size and granularity as observed in Fig. 3. We have observed in our own studies, that NCC have different dispersion characteristics (FSC and SSC) than the HL-60 targets (Fig. 3C and D). The use of dispersion-based gating strategies to discriminate and identify these two cellular populations allows the determination of which HL-60 cells have failed to exclude propidium iodide without the risk of counting any dead NCC effector cells in this population (Fig. 3F and G). Once the cytometric assignments of NCC and HL-60 were confirmed, the cytotoxic activity of NCC on HL-60 was assessed using PI staining of DNA in HL-60 nuclei, indicating dead cells. Splenic NCC were purified (Fig. 3A and B) from endosulfan-exposed and non-exposed control fish and co-cultures of splenic NCC (E) and HL-60 (T) cells were set up using different E:T ratios (1:1, 10:1, 20:1 and 40:1). Experimental data showed no significant differences in the lytic activity of NCC of exposed and non-exposed control groups when using E:T ratios of 1:1 (5.1 0.6 and 7.1 1.7, respectively), 10:1 (9.2 0.6 and 11.7 1.6) and 20:1 (11.6 1.7 and 15.9 0.3, respectively). The E:T ratio of 40:1 presented a significant decrease of the lytic activity of NCC from exposed tilapia (17 2.5) compared to non-exposed organisms
(50.9 5.9, p < 0.05) as observed in Fig. 4 A. Panel B shows plots of representative experimental results obtained after co-culture of E and T in all ratio (1:1, 10:1, 20:1 and 40:1), spontaneous and total binding. 3.4. Effect of in vivo endosulfan exposure in the expression of the granzyme gene on splenic NCC The expression of the granzyme gene at transcription level was assessed by qRT-PCR. The expression of non-exposed tilapia was taken as a reference to calculate the relative variation in the expression of exposed tilapia. The DDCP method was used to obtain the expression values. A tendency to decrease the expression of granzyme gene was observed in splenic NCC from exposed tilapia (0.675 0.205) compared to non-exposed organisms (1.0 0.0) as observed in Fig. 5. 4. Discussion This current study extended our earlier finding that endosulfan modulates phagocytic activity, humoral and cellular specific immune responses in tilapia, and gave new information on the effects of this pesticide on NCC function. NCC are NK cell equivalents in catfish (Ictalurus punctatus), tilapia, gilthead seabream and other teleosts [6,13]. The effect of endosulfan on antigen specific receptor protein-1 (NCCRP-1) expression of NCC cells was evaluated, and even though the difference was not significant it did show an
Fig. 3. Cytotoxic Assay. Non-specific cytotoxic cells were purified and sorted from tilapia spleen (A-B). NCC (D) were mixed with HL-60 (C) target cells and following 4 h cocultivation, (E) propidium iodide (PI) was added. Binding to the target cell nuclei was determined by flow cytometry. The PI signal from the HL-60 gate (F) and the NCC gate (G) was obtained using FACSAria I.
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Fig. 4. Effect of endosulfan on the cytotoxic activity of splenic NCC. Panel A. Different E:T cell ratios on PI uptake by HL-60 target cells was evaluated by cytometry after a 4 h coculture with tilapia splenic NCC. Data are shown as mean SE and compared by using the Student T-test. *p < 0.05: statistical differences between study groups. Panel B. shows plots of representative experimental kinetics of PI-detected cytotoxicity, tilapia NCC and HL-60 target obtained after coculture at different E:T ratios.
increase in the exposed group compared to the non-exposed control group. NCCRP-1 may play a dual role in the activation of NCC; first, as an antigen recognition molecule necessary to target cell lysis, and second, as an initiator of cytokine release from NCC. Both
of these processes are required for a competent innate immune response [23]. Téllez-Bañuelos et al. (2010), in Oreochromis niloticus reported an increase of serum IL-2L concentration. In turn, Curran et al. (2001) showed that IL-2 in NK cells regulates activating
Fig. 5. Effect of endosulfan on granzyme gene expression in NCC from tilapia spleen. A. Box plot graphic showing DCP values taking b-actin as reference gene. NCCs from nonexposed tilapia were included for comparison. Significant differences between groups were not detected. B. Graphic showing an average of the relative expression levels of granzyme gene from the non-exposed and from the exposed group. The value obtained in the non-exposed control group was set as 1 and normalized with b-actin as reference gene.
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signals, and Huenecke et al. (2010) reported that NCRs and NKG2D receptors were increased after IL-2 stimulation [16,24,25]. We suggest that acute exposure to sublethal concentration of endosulfan provokes the nonspecific, but non-functional activation of splenic NCC, and that this incomplete activation may partly explain the decrease of cytotoxic activity as described above. The cytotoxic activity of NCC was measured in relation to MFI generated by the PI uptake when target cells (HL-60) and NCC were incubated, and expressed in percentage. It was observed that with a 40:1effector:target ratio, the NCC of fish exposed to endosulfan showed a 65% decrease of their cytotoxicity compared to the control group. To the best of our knowledge, this study is the first to examine the effects of endosulfan on NCC cell activity. Based on the fact that HL-60 is positive for FasR and is sensitive to cytotoxic action of NCC in tilapia mediated by secreted FasL [3,19] we consider that one of the mechanisms by which NCCs lysed the HL60 cells could be a FasL/Fas-mediated mechanism. We cannot discard the possibility that endosulfan might be affecting the expression of FasL in NCCs, in a similar mechanism to that mediated by other organochlorine pesticides. For example, Bursi c et al. (2005) reported that endocrine disrupting chemicals (including organochlorine pesticides) are recognized by the estrogen receptor (ER) and could disrupt FasL expression in testis [26]. Our finding that endosulfan (a purported gamma-aminobutyric acid receptor inhibitor) suppressed NCC lytic activity is congruent with data that suggested that other organochlorine pesticides alter the GABAergic system in fish [27] and human NK cells [28]. In recent years, GABA has been shown to act as an immunomodulatory molecule, and abundant functional GABA-A channel proteins have been found in human peripheral blood mononuclear cells [29], in T cells from mammalian species [30], and GABA receptors have been described in fish [31,32]. The GABA signaling system is the major target site for organochlorine pesticides [1]; it is active in immune cells and appears to modulate a wide variety of functional properties including cell proliferation, cytokine secretion and phagocytic activity [33,34]. It has been demonstrated that endosulfan exposure in mammalian immune cells induced changes in GABAergic activity [35]. On the other hand, endosulfan acts as a xenoestrogen, binding to ER. The demonstration of ER in murine NK cells [24] and of all four ER isoform mRNA expression in piscine immune organs (including spleen), points to the possibility that ER signaling also takes place in the immune system of all species, including fish [36]. This direct signaling of estrogen may occur in addition to indirect effects via the neuroendocrine axes. Environmental estrogen-active compounds such as organochlorine pesticides and estrogens have previously been shown to exert immune disrupting effects in mammals and effectively inhibit NK lytic activity [37e40]. Finally, we measured the in vivo effect of endosulfan on the expression of the granzyme gene of Nile tilapia, observing tendency to decrease in the group exposed to the pesticide compared to the non-exposed group. We hypothesize that endosulfan alters intracellular signaling, releasing granzyme due to its xenoestrogen role. This idea is based on the data of Jiang et al. (2006) and KannanThulasiraman et al. (2002), who described, in cell lines, that estrogen induced the synthesis of a potent inhibitor of granzyme B, mediated by NF-kB and AP-1 transcription factors [41,42]. Moreover, Li et al. (2008) reported that selective inhibition of JNK MAP kinase reduced NKG2D-mediated cytotoxicity toward target cells and furthermore, blocked granzyme B [43]. The suppression of NCC cytotoxicity observed in the present work could be attributed to immunological mechanisms involving less effective target cell recognition, as well as diminished cytotoxicity of other immune cells, including T lymphocytes and mononuclear phagocytes. Additionally, the change in lymphocyte populations, detectable by
proliferative tests [17], might account for the down-regulation of NCC activity. 5. Conclusion The acute exposure of tilapia to a sublethal concentration of endosulfan induces alteration in function of NCC: significant decrease of cytotoxic activity and a tendency to lower granzyme expression, severe enough to compromise the immunity of this species. This effect on the immune system could affect the animals ability to mount well-regulated immune responses to microbial, and self and tumor antigens. Finally, the effects of sublethal doses of endosulfan may be translatable to mammalian species, including human, that are more likely to encounter this pesticide in their normal environment. Acknowledgments This study was supported by the P3E funding from the CMBD of the Universidad de Guadalajara, grant number 137505. We gratefully acknowledge Eduardo Juarez-Carrillo MSc for providing the fish bioassay laboratory. We thank Jesse Haramati Ph. D for his reading of the manuscript. References [1] Rand GM, Carriger JF, Gardinali PR, Castro J. Endosulfan and its metabolite, endosulfan sulfate, in freshwater ecosystems of South Florida: a probabilistic aquatic ecological risk assessment. Ecotoxicology 2010;19:879e900. [2] Dan X, Shuai L, Min Z, Yeqing S. Research progress on biological effect of endosulfan. Biotechnol Bull 2013;1:45e51. [3] Evans DL, Leary III JH, Jaso-Friedmann L. Nonspecific cytotoxic cells and innate immunity: regulation by programmed cell death. Dev Comp Immunol 2001;25:791e805. [4] Fischer U, Koppang EO, Nakanishi T. Teleost T and NK cell immunity. Fish Shellfish Immunol 2013;35:197e206. [5] Cuesta A, Esteban MA, Meseguer J. Natural cytotoxic activity of gilthead seabream (Sparus aurata L.) leucocytes: assessment by flow cytometry and microscopy. Vet Immunol Immuno 1999;71:161e71. [6] Ishimoto Y, Savan R, Endo M, Sakai M. Non-specific cytotoxic cell receptor (NCCRP)-1 type gene in tilapia (Oreochromis niloticus): its cloning and analysis. Fish Shellfish Immunol 2004;16:163e72. [7] Nakanishi T, Toda H, Shibasaki Y, Somamoto T. Cytotoxic T cells in teleost fish. Dev Comp Immunol 2011;35:1317e23. [8] Bishop GR, Jaso-Friedmann L, Evans DL. Activation-induced programmed cell death of nonspecific cytotoxic cells and inhibition by apoptosis regulatory factors. Cell Immunol 2000;1:126e37. [9] Jaso-Friedmann L, Ruiz J, Bishop GR, Evans DL. Regulation of innate immunity in tilapia: activation of nonspecific cytotoxic cells by cytokine-like factors. Dev Comp Immunol 2000;24:25e36. [10] Oumouna M, Jaso-Friedmann L, Evans DL. Flow cytometry-based assay for determination of teleost cytotoxic cell lysis of target cells. Cytometry 2001;45: 259e66. [11] Rauta PR, Nayak B, Das S. Immune system and immune responses in fish and their role in comparative immunity study: a model for higher organisms. Immunol Lett 2012;148:23e33. [12] Praveen K, Evans DL, Jaso-Friedmann L. Evidence for the existence of granzyme-like serine proteases in teleost cytotoxic cells. J Mol Evol 2004;58: 449e59. [13] Cuesta A, Esteban MA, Meseguer J. Molecular characterization of the nonspecific cytotoxic cell receptor (NCCRP-1) demonstrates gilthead seabream NCC heterogeneity. Dev Comp Immunol 2005;29:637e50. [14] Kumar N, Prabhu PAJ, Pal AK, Remya S, Aklakur Md, Rana RS, et al. Antioxidative and immuno-hematological status of Tilapia (Oreochromis mossambicus) during acute toxicity test of endosulfan. Pestic Biochem Phys 2011;99:45e52. [15] Tellez-Banuelos MC, Santerre A, Casas-Solis J, Bravo-Cuellar A, Zaitseva G. Oxidative stress in macrophages from spleen of Nile tilapia (Oreochromis niloticus) exposed to sublethal concentration of endosulfan. Fish Shellfish Immunol 2009;27:105e11. [16] Tellez-Banuelos MC, Santerre A, Casas-Solis J, Zaitseva G. Endosulfan increases seric interleukin-2 like (IL-2L) factor and immunoglobulin M (IgM) of Nile tilapia (Oreochromis niloticus) challenged with Aeromonas hydrophila. Fish Shellfish Immunol 2010;28:401e5. [17] Tellez-Banuelos MC, Ortiz-Lazareno PC, Santerre A, Casas-Solis J, BravoCuellar A, Zaitseva G. Effects of low concentration of endosulfan on
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Full length article
Endosulfan decreases cytotoxic activity of nonspecific cytotoxic cells and expression of granzyme gene in Oreochromis niloticus Martha Cecilia Téllez-Bañuelos a, *, Pablo Cesar Ortiz-Lazareno b, Luis Felipe Jave-Suárez b, Victor Hugo Siordia-Sánchez a, Alejandro Bravo-Cuellar b, Anne Santerre a, Galina P. Zaitseva a a b
Departamento de Biología Celular y Molecular, Universidad de Guadalajara, Carretera a Nogales Km 15.5, Las Agujas, 45110 Zapopan, Jalisco, Mexico Centro de Investigación Biomédica de Occidente, IMSS, Sierra Mojada 800, Col. Independencia, 44340 Guadalajara, Jalisco, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 October 2013 Received in revised form 23 February 2014 Accepted 7 March 2014 Available online 20 March 2014
The effect of the organochlorinated insecticide endosulfan, on the cytotoxic activity of Nile tilapia nonspecific cytotoxic cells (NCC) was assessed. Juvenile Nile tilapia were exposed to endosulfan (7 ppb) for 96 h and splenic NCC were isolated. Flow cytometric phenotyping of NCC was based on the detection of the NCC specific membrane signaling protein NCCRP-1 by using the monoclonal antibody Mab 5C6; granzyme expression was evaluated by quantitative RT-PCR. The cytotoxic activity of sorted NCC on HL60 tumoral cells was assessed using propidium iodide (PI) staining of DNA in HL-60 nuclei, indicating dead cells. Nile tilapia splenic NCC had the ability to kill HL-60 tumoral cells, however, the exposure to endosulfan significantly reduced, by a 65%, their cytotoxic activity when using the effector:target ratio of 40:1. Additionally, the exposure to endosulfan tended to increase the expression of NCCRP-1, which is involved in NCC antigen recognition and signaling. Moreover, it decreased the expression of the granzyme gene in exposed group as compared with non-exposed group; however significant differences between groups were not detected. In summary, the acute exposure of Nile tilapia to sublethal concentration of endosulfan induces alteration in function of NCC: significant decrease of cytotoxic activity and a tendency to lower granzyme expression, severe enough to compromise the immunity of this species. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Endosulfan Nile tilapia NCC Cytotoxic activity Granzyme
1. Introduction Endosulfan is a potent organochlorinated insecticideeacaricide; the technical grade material consists of two isomers (a and b); endosulfan-sulphate is the main environmental metabolite in water, sediment and tissue [1]. The use of endosulfan is restricted in many countries due to its adverse effects on ecosystems, including aquatic environments. Moreover, this pesticide has been added to the annex A of the Stockholm Convention in 2011 [2]. In teleosts, the nonspecific cytotoxic cells (NCC) were identified in 1983 by Evans et al. (2001) who reported their participation in innate immunity [3]. First described in channel catfish, NCC have been characterized in teleost and are thought to be the evolutionary precursors of the mammalian NK cells [4]. The NCCs are not the only cytotoxic cells found in fish, where lymphocytes,
* Corresponding author. Tel.: þ52 33 3777 1191; fax: þ52 33 3673 8375. E-mail address: [email protected] (M.C. Téllez-Bañuelos). http://dx.doi.org/10.1016/j.fsi.2014.03.012 1050-4648/Ó 2014 Elsevier Ltd. All rights reserved.
monocyte-macrophages and granulocytes also show nonspecific cytotoxic activity [5]. In both cartilaginous and bony fish, macrophages have been shown to display spontaneous cytotoxicity, and antibody-dependent cell-mediated cytotoxic (ADCC) reactions are found in sharks [6]. Nakanishi et al. (2011) also mentioned that three types of cells are involved in cell-mediated cytotoxicity in fish. One is the NCC, originally described in channel catfish and later in other fish species including rainbow trout, carp, damselfish and tilapia. Other type is represented by NK-like cell lines (distinct from NCCs), which were developed from catfish peripheral blood leukocytes. The third NCC type are the neutrophils [7]. NCC have the ability to kill a wide array of targets including virus-infected cells and cancer cells. In vitro, NCC participate in immunosurveillance against Yac-1 cells, HL-60 cells (human promyelocytic cells), and have potential relevance in the control of protozoan parasites (Tetrahymena pyriformis), and bacterial infections [8,9]. NCC are potent immune effector cells and are able to contact target cells, leading to granule exocytosis related with both necrotic and apoptotic death pathways [10,11]. Praveen et al. (2004)
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reported the existence of granule granzyme-like serine proteases in NCC of channel catfish [9]. These proteases might play an important role in the effector functions of NCC and this indicates that cellmediated immunity with granule exocytosis and Fas pathways have been conserved for more than 300 million years. In tilapia, granzymes (in conjunction with pore-forming proteins) constitute the major components of granules of professional killer cells such as cytotoxic T lymphocytes, NK cells and NCC [12]. NCC are small cells (3.5 mm) and presently the best method for their identification is with the monoclonal antibody 5C6 (Mab 5C6), as originally described for catfish. This antibody binds to the NCC receptor protein NCCRP-1, which is involved in NCC antigen recognition and signaling, and recognizes a discrete ligand referred as the NK target antigen (NKTag) [3,9]. A difference between NCC and mammalian NK is that the latter requires in vitro activation by lymphokines to generate cytotoxicity, in contrast, naive NCC are constitutively active and capable of killing freshly isolated targets [8]. Cuesta et al. (2005) demonstrated gilthead seabream NCC heterogeneity: lymphocytes, monocyte/macrophages and acidophilic granulocytes from lymphoid tissues express NCCRP-1, both at gene and protein level [13]. Tilapia is considered the future of aquaculture and has been nicknamed “the aquatic chicken” due to its ability to grow quickly with poor-quality inputs. It is also a good biological model for toxicological and immunotoxicity studies of the effects of pesticides on environmental and animal and human health, due to diverse characteristics, namely high growth rates, efficiency in adapting to diverse diets, great resistance to diseases and handling practices, easy reproduction in captivity at prolific rates and finally, good tolerance to a wide range of environmental conditions [14]. Previous data from our research group indicated that acute (96 h) in vivo exposure to sublethal doses of endosulfan (7 ppb ¼ 1/ 2LC50) induces nonspecific activation of splenic macrophages and exacerbated interleukin-2 synthesis in Nile tilapia [15,16]. Also, experimental data showed that endosulfan per se increased cellular proliferation, but decreased the lymphoproliferative response to mitogenic stimulus with PMA þ ionomycin. Splenocytes exposed to endosulfan for 15e180 min showed significantly higher levels of pERK1/2 than the non-exposed control. Endosulfan mediated a decrease in etoposide-induced apoptosis and provoked cell senescence [17]. In order to further assess the effects of this pesticide on the innate immune response of Nile tilapia, particularly on the immunosurveillance capacity of NCC, flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR) were used to evaluate the signaling of the NCCRP-1, the cytotoxic activity of splenic NCC against HL-60 tumor cells and the expression of the granzyme gene. 2. Material and methods 2.1. Animals Nile tilapia were obtained from the Aquamol aquaculture farm in Jamay, Jalisco, Mexico, and transferred to 40-L glass aquariums (one fish per tank), at 28 2 C with constant aeration for 3 months before initiating experiments. Water quality monitoring included nitrite, ammonia nitrogen and chlorine determinations. Experimental males weighed 200e250 g; their daily food consisted of commercial dry fish pellets (Tilapia Nutripec 2506 AP, Purina, Mexico).
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of this grade is frequently employed in field practices. The pesticide was dissolved in distilled water (1:100) and this stock solution was added directly to the 40-L tanks to a final concentration of 7 ppb (7 mg L1), which is equivalent to 1/2 the lethal concentration 50 (1/ 2LC50) for juvenile Nile tilapia, for a 96 h time period, without water changes. 2.3. Experimental groups The effect of acute exposure to the sublethal doses of endosulfan on splenic NCC of Nile tilapia (n ¼ 5) was evaluated. A non-exposed control group of five fish was maintained under similar conditions as the exposed group, but without pesticide. All evaluations were performed in duplicate. Exposed and non-exposed fish were anesthetized with clove oil at 100 mg L1 (Sigma St. Louis, MO, USA) [18]; fish were sacrificed and subsequently carefully dissected aseptically to remove the spleen. 2.4. Purification of mononuclear cells from the spleen of tilapia Mononuclear cells were isolated from the spleen. The spleens were disaggregated and passed through a 100 mm nylon mesh. Red blood cells were separated from the cell suspension by density gradient centrifugations, using a modification of the method described by Bishop et al. [19] and Harford et al. [20]; five mL of cell suspension was layered over five mL of Histopaque 1077 (Sigma St. Louis, MO, USA) and centrifuged at 1200 g for 20 min at 28 C. The immune cells were collected from the upper histopaque layer and a second purification was undertaken over 45.5% Percoll (Sigma Chemicals, St. Louis, MO, USA) in PBS at 1200 g for 17 min at 28 C. Cells at the interface were collected, washed once with cold PBS, and resuspended in RPMI-1640 (RPMI) containing 4 mM glutamine (Gibco-Invitrogen, Grand Island, NY, USA) and supplemented (RPMI-S) with 10% fetal calf serum (FCS, Sigma, St. Louis, MO, USA). Cell viability was evaluated using the trypan blue (Sigma St. Louis, MO, USA) dye exclusion method at 0.2% (w/v). Cell concentration was further adjusted with RPMI-S at 2 105 cells per 100 mL, as required for each assay. 2.5. NCC Phenotype analysis Phenotyping of NCC from spleens was realized by flow cytometric examination for surface expression of NCCRP-1 using a modification of the methods of Bishop et al. (2000) and Oumouna et al. (2001) [8,10]. Briefly, NCC (2 105 cells) were incubated for 1 h at 4 C with 1.25 mL (0.25 mg), 2.5 mL (0.5 mg) or 5 mL (1 mg) of Mab 5C6 of mouse IgM isotype (specific for the NCCRP-1 conventional antigen receptor on NCC, Affinity BioreagentsÔ, Inc, Golden, CO, USA). Cells were then washed twice in cold PBS and incubated with 2 mL (1 mg) of a goat anti-mouse IgM conjugated with FITC (Sigma BioSciences, St. Louis, MO, USA) for 30 min at 4 C. NCC were identified and quantified in a BD FACSAria I cell sorter (BD Bioscience, San Jose, CA, USA) with laser excitation at 488 nm to assess the forward scatter (FSC; size) and side scatter (SSC; granularity) parameters. An IgM isotype control was used. Experimental data were processed with the FACSDiva Software (BD Bioscience, San Jose, CA, USA). All experiments with endosulfan-exposed and non-exposed control organisms were performed in duplicate. 2.6. Target cells
2.2. Endosulfan Technical grade endosulfan (35% EC) was locally purchased (manufactured by Agricura, SA de CV, DF, Mexico) since endosulfan
HL-60 is a non-adherent cell line established by Gallagher et al. [21] by leukopheresis from a 36-year-old Caucasian female with acute promyelocytic leukemia. This cell line, which is commercially
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available, is characterized by a lack of lymphoid markers, growth as compact colonies in semisolid culture, and non-adherence to substrate. The target cells were obtained from ATCC, and consisted of HL-60 cells (ATCC CCL-240, Rockville, MD, USA) grown as suspension cultures in RPMI-S at 37 C in a humid incubator with 5% CO2 (NuAire HEPAIII, Plymouth, MN, USA). Cell concentration was adjusted with RPMI-S as required for each cytotoxicity assay. 2.7. Cytotoxic activity of NCC For the assays, Fluorescence Activated Cell Sorting (FACS)-based positive selection, using the Mab anti-NCCRP-1 (Mab 5C6), of a preparation of splenic mononuclear cells (previously depleted of granulocytes by double density gradient) was performed to obtain a highly pure preparation of NCCs. For sorting, the gate was set around a positive population to the NCCRP-1 marker, which was then used to determine the NCC population. Ninety-nine percent of the purified cells were positive for NCCRP-1. Several effector to target (E:T) cell ratios were assayed (1:1, 10:1, 20:1 and 40:1) in a final volume of 200 mL, in 96 well microtiter plates (Corning Incorporated, Corning, NY, USA), using a modification of the methods of Oumouna et al. [10]. The effectors (at different numbers) and targets (at a constant number) were pelleted in Ubottom 96-well microtiter plates by centrifugation and then incubated for 4 h at 37 C in a humidified incubator with 5% CO2. (NuAire HEPAIII, Plymouth, MN, USA). Cells were then harvested and washed twice with PBS and centrifuged at 1200 g for 7 min. 200 mL of PBS were added, and the cells were labeled with 10 mL of propidium iodide (PI) (Sigma, St. Louis, MO, USA) and incubated for 10 min protected from light. Cell populations were analyzed by flow cytometry (FACSAria I cell sorter, BD Bioscience, San Jose, CA, USA) with laser excitation at 488 nm. Back-gating was used to confirm the identity of each cell population; the flow cytometer was set to collect 10,000 events of gated HL-60 target cells (based on FS and SS characteristics). Experimental data were processed with the FACSDiva Software (BD Bioscience, San Jose, CA, USA). The results were expressed as percent of specific PI uptake ¼ [(test binding spontaneous binding/total binding spontaneous binding)] 100. The PI uptake was measured for the target cells mixed with NCCs (test binding), for the target cells (HL-60) alone (spontaneous binding), and for the target cells permeabilized with acetone (total binding). 2.8. Expression of granzyme in NCC Quantitative RT-PCR (qRT-PCR) was used to evaluate the expression of the granzyme gene in splenic NCC from exposed and non-exposed control groups. Total RNA was isolated from splenic NCC and purified with the PureLink RNA Mini Kit (Ambion-Invitrogen Corporation, Carlsbad, CA, USA). The cDNA was synthesized from 1 mg of total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Mannheim, Germany). Assays were performed with 2.0 LightCycler technology using the LightCyclerFastStar DNA Master PLUS SYBR Green I kit (Roche Applied Science, Mannheim, Germany) as recommended by the manufacturers. Analysis of gene expression was performed with LightCycler ver. 4.1 software (Roche Applied Science, Mannheim, Germany). Beta-actin was used as an endogenous control. Relative quantification of target genes was determined by utilizing the DDCP method. Analysis was performed by taking the values obtained from three independent RNA extractions in duplicate. Oligonucleotides (Invitrogen Corporation, Carlsbad, CA, USA) were designed using Oligo-Primer Analysis ver. 6.51 software (Molecular Biology Insights, Inc., USA) utilizing sequences obtained from the GenBank Nucleotide database of the NCBI website. For the granzyme gene
(AY918866: GenBank) the primer sequences were: F:50 ATCATGCTCCTCAAACTCTCG-30 ; R:50 -GGGCCACCAGAATCACC-30 , with a total amplicon size of 290 bp. For b-actin (EF206796.1: GenBank): the primer sequences were: F: 50 -GGCATCGCACCTTCT ACAAT-30 ; R:50 -TCTCACGCTCAGTTGTGGTAGT-30 , with a total amplicon size of 366 bp. For normalizing target gene expression one reference gene was used, b-actin, calculating the Crossing Point (CP) for target (granzyme) and reference gene in each sample and subsequently calculating the DCP value of each sample, i.e., the target gene CP minus the reference gene CP. This facilitated the analysis by taking only the intrinsic values of each sample. It is extremely note-worthy that DCP is inversely proportional to the expression of the target gene. The relative expression was calculated by taking expressions of non-exposed samples as 1. In this context, expression from all non-exposed samples has a relative value of 1, and the SD of non-exposed samples was zero. Taking this value as reference, the values of relative expression for the exposed samples were calculated using the DCp Method [22]. 2.9. Statistical analysis Statistical differences between exposed and non-exposed groups were evaluated using the Student T-test; significance level was set at p < 0.05. Data were analyzed with the SigmaPlot 10.0 software. To calculate variations in the granzyme expression, the median Cp value of the control group (untreated samples) was calculated after normalization with the expression of b-actin. This median value was used to calculate the relative variation of the treated samples. The expression of control samples was set as 1 and the variations were calculated related to this value. The statistical difference between both groups was assayed using the Student Ttest. 3. Results 3.1. Nile tilapia splenic NCC phenotype Mononuclear cells were purified from the spleens of tilapia by using two density gradients (histopaque and percoll). Representative dot plot correlation Forward scatter (FSC) vs side scatter (SSC) of Nile tilapia mononuclear spleen cells are seen in Fig. 1A(1); FSC vs SSC plots of the same isolated cells are shown in Fig. 1A(2). Flow cytometric phenotyping was based on the detection of the NCCspecific-membrane-signaling-protein NCCRP-1 by Mab 5C6. NCC were identified and quantified by dot plot backgating FSC vs SSC plots (Fig. 1A) from Mab 5C6 stained cells (Fig. 1). The optimal antibody concentration was 1 mg (Fig. 1B). Monoclonal 5C6 binding determined by flow cytometry gave a minimum value of almost 15% and a maximum value of 22.7% (18.6 4.1) of total mononuclear splenocytes (Fig. 2B). 3.2. Effect of in vivo exposure to endosulfan on Nile tilapia splenic NCC phenotype The expression of the signaling protein NCCRP-1, given as MFI (Mean Fluorescence Intensities) was evaluated in splenic NCC from tilapia. No significant differences were observed between non-exposed (1886.78 530.44) and endosulfan-exposed (2310.67 866.42) groups (Fig. 2A). Also, the percentage of NCCRP-1þ cells of Nile tilapia was not significantly affected (p > 0.05) by the acute (96 h) in vivo exposure to 7 ppb of endosulfan (22.3 8.7) compared with the non-exposed group (18.6 4.1). Data are shown as mean SD and compared by using the Student T test. Representative histogram of NCCRP-1þ cells in exposed and non-exposed groups are presented (Fig. 2C).
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Fig. 1. Splenic NCC phenotyping by flow cytometry. A(1) Representative dotplot correlating FSC vs SSC of Nile tilapia mononuclear spleen cells. A(2) Forward scatter vs side scatter plot of the same isolated cells. B. Representative dotplot FITC intensity from Mab 5C6 stained and fluorescence histogram obtained from mononuclear splenocytes surface NCCRP-1 expression.
Fig. 2. Effect of endosulfan in the NCCRP-1 expression. A. MFI (Mean Fluorescence Intensities) of the expression of protein NCCRP-1 and B. Percentage of NCCRP-1þ cells of Nile tilapia was not significantly affected (p > 0.05) by the acute (96 h) in vivo exposure to 7 ppb of endosulfan. Data are shown as mean SD and compared by using the Student T-test. C. Representative histogram of NCCRP-1þ cells in exposed and non-exposed groups.
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3.3. Effect of endosulfan exposure on the cytotoxic activity of splenic NCC from Nile tilapia In a preliminary assay, cell sorting was used to confirm the cytometric assignments of the NCC and HL-60 populations based on their respective SSC and FSC. The effector cells (NCC) and target cells (HL-60) have three to five fold differences in size and granularity as observed in Fig. 3. We have observed in our own studies, that NCC have different dispersion characteristics (FSC and SSC) than the HL-60 targets (Fig. 3C and D). The use of dispersion-based gating strategies to discriminate and identify these two cellular populations allows the determination of which HL-60 cells have failed to exclude propidium iodide without the risk of counting any dead NCC effector cells in this population (Fig. 3F and G). Once the cytometric assignments of NCC and HL-60 were confirmed, the cytotoxic activity of NCC on HL-60 was assessed using PI staining of DNA in HL-60 nuclei, indicating dead cells. Splenic NCC were purified (Fig. 3A and B) from endosulfan-exposed and non-exposed control fish and co-cultures of splenic NCC (E) and HL-60 (T) cells were set up using different E:T ratios (1:1, 10:1, 20:1 and 40:1). Experimental data showed no significant differences in the lytic activity of NCC of exposed and non-exposed control groups when using E:T ratios of 1:1 (5.1 0.6 and 7.1 1.7, respectively), 10:1 (9.2 0.6 and 11.7 1.6) and 20:1 (11.6 1.7 and 15.9 0.3, respectively). The E:T ratio of 40:1 presented a significant decrease of the lytic activity of NCC from exposed tilapia (17 2.5) compared to non-exposed organisms
(50.9 5.9, p < 0.05) as observed in Fig. 4 A. Panel B shows plots of representative experimental results obtained after co-culture of E and T in all ratio (1:1, 10:1, 20:1 and 40:1), spontaneous and total binding. 3.4. Effect of in vivo endosulfan exposure in the expression of the granzyme gene on splenic NCC The expression of the granzyme gene at transcription level was assessed by qRT-PCR. The expression of non-exposed tilapia was taken as a reference to calculate the relative variation in the expression of exposed tilapia. The DDCP method was used to obtain the expression values. A tendency to decrease the expression of granzyme gene was observed in splenic NCC from exposed tilapia (0.675 0.205) compared to non-exposed organisms (1.0 0.0) as observed in Fig. 5. 4. Discussion This current study extended our earlier finding that endosulfan modulates phagocytic activity, humoral and cellular specific immune responses in tilapia, and gave new information on the effects of this pesticide on NCC function. NCC are NK cell equivalents in catfish (Ictalurus punctatus), tilapia, gilthead seabream and other teleosts [6,13]. The effect of endosulfan on antigen specific receptor protein-1 (NCCRP-1) expression of NCC cells was evaluated, and even though the difference was not significant it did show an
Fig. 3. Cytotoxic Assay. Non-specific cytotoxic cells were purified and sorted from tilapia spleen (A-B). NCC (D) were mixed with HL-60 (C) target cells and following 4 h cocultivation, (E) propidium iodide (PI) was added. Binding to the target cell nuclei was determined by flow cytometry. The PI signal from the HL-60 gate (F) and the NCC gate (G) was obtained using FACSAria I.
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Fig. 4. Effect of endosulfan on the cytotoxic activity of splenic NCC. Panel A. Different E:T cell ratios on PI uptake by HL-60 target cells was evaluated by cytometry after a 4 h coculture with tilapia splenic NCC. Data are shown as mean SE and compared by using the Student T-test. *p < 0.05: statistical differences between study groups. Panel B. shows plots of representative experimental kinetics of PI-detected cytotoxicity, tilapia NCC and HL-60 target obtained after coculture at different E:T ratios.
increase in the exposed group compared to the non-exposed control group. NCCRP-1 may play a dual role in the activation of NCC; first, as an antigen recognition molecule necessary to target cell lysis, and second, as an initiator of cytokine release from NCC. Both
of these processes are required for a competent innate immune response [23]. Téllez-Bañuelos et al. (2010), in Oreochromis niloticus reported an increase of serum IL-2L concentration. In turn, Curran et al. (2001) showed that IL-2 in NK cells regulates activating
Fig. 5. Effect of endosulfan on granzyme gene expression in NCC from tilapia spleen. A. Box plot graphic showing DCP values taking b-actin as reference gene. NCCs from nonexposed tilapia were included for comparison. Significant differences between groups were not detected. B. Graphic showing an average of the relative expression levels of granzyme gene from the non-exposed and from the exposed group. The value obtained in the non-exposed control group was set as 1 and normalized with b-actin as reference gene.
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signals, and Huenecke et al. (2010) reported that NCRs and NKG2D receptors were increased after IL-2 stimulation [16,24,25]. We suggest that acute exposure to sublethal concentration of endosulfan provokes the nonspecific, but non-functional activation of splenic NCC, and that this incomplete activation may partly explain the decrease of cytotoxic activity as described above. The cytotoxic activity of NCC was measured in relation to MFI generated by the PI uptake when target cells (HL-60) and NCC were incubated, and expressed in percentage. It was observed that with a 40:1effector:target ratio, the NCC of fish exposed to endosulfan showed a 65% decrease of their cytotoxicity compared to the control group. To the best of our knowledge, this study is the first to examine the effects of endosulfan on NCC cell activity. Based on the fact that HL-60 is positive for FasR and is sensitive to cytotoxic action of NCC in tilapia mediated by secreted FasL [3,19] we consider that one of the mechanisms by which NCCs lysed the HL60 cells could be a FasL/Fas-mediated mechanism. We cannot discard the possibility that endosulfan might be affecting the expression of FasL in NCCs, in a similar mechanism to that mediated by other organochlorine pesticides. For example, Bursi c et al. (2005) reported that endocrine disrupting chemicals (including organochlorine pesticides) are recognized by the estrogen receptor (ER) and could disrupt FasL expression in testis [26]. Our finding that endosulfan (a purported gamma-aminobutyric acid receptor inhibitor) suppressed NCC lytic activity is congruent with data that suggested that other organochlorine pesticides alter the GABAergic system in fish [27] and human NK cells [28]. In recent years, GABA has been shown to act as an immunomodulatory molecule, and abundant functional GABA-A channel proteins have been found in human peripheral blood mononuclear cells [29], in T cells from mammalian species [30], and GABA receptors have been described in fish [31,32]. The GABA signaling system is the major target site for organochlorine pesticides [1]; it is active in immune cells and appears to modulate a wide variety of functional properties including cell proliferation, cytokine secretion and phagocytic activity [33,34]. It has been demonstrated that endosulfan exposure in mammalian immune cells induced changes in GABAergic activity [35]. On the other hand, endosulfan acts as a xenoestrogen, binding to ER. The demonstration of ER in murine NK cells [24] and of all four ER isoform mRNA expression in piscine immune organs (including spleen), points to the possibility that ER signaling also takes place in the immune system of all species, including fish [36]. This direct signaling of estrogen may occur in addition to indirect effects via the neuroendocrine axes. Environmental estrogen-active compounds such as organochlorine pesticides and estrogens have previously been shown to exert immune disrupting effects in mammals and effectively inhibit NK lytic activity [37e40]. Finally, we measured the in vivo effect of endosulfan on the expression of the granzyme gene of Nile tilapia, observing tendency to decrease in the group exposed to the pesticide compared to the non-exposed group. We hypothesize that endosulfan alters intracellular signaling, releasing granzyme due to its xenoestrogen role. This idea is based on the data of Jiang et al. (2006) and KannanThulasiraman et al. (2002), who described, in cell lines, that estrogen induced the synthesis of a potent inhibitor of granzyme B, mediated by NF-kB and AP-1 transcription factors [41,42]. Moreover, Li et al. (2008) reported that selective inhibition of JNK MAP kinase reduced NKG2D-mediated cytotoxicity toward target cells and furthermore, blocked granzyme B [43]. The suppression of NCC cytotoxicity observed in the present work could be attributed to immunological mechanisms involving less effective target cell recognition, as well as diminished cytotoxicity of other immune cells, including T lymphocytes and mononuclear phagocytes. Additionally, the change in lymphocyte populations, detectable by
proliferative tests [17], might account for the down-regulation of NCC activity. 5. Conclusion The acute exposure of tilapia to a sublethal concentration of endosulfan induces alteration in function of NCC: significant decrease of cytotoxic activity and a tendency to lower granzyme expression, severe enough to compromise the immunity of this species. This effect on the immune system could affect the animals ability to mount well-regulated immune responses to microbial, and self and tumor antigens. Finally, the effects of sublethal doses of endosulfan may be translatable to mammalian species, including human, that are more likely to encounter this pesticide in their normal environment. Acknowledgments This study was supported by the P3E funding from the CMBD of the Universidad de Guadalajara, grant number 137505. We gratefully acknowledge Eduardo Juarez-Carrillo MSc for providing the fish bioassay laboratory. We thank Jesse Haramati Ph. D for his reading of the manuscript. References [1] Rand GM, Carriger JF, Gardinali PR, Castro J. Endosulfan and its metabolite, endosulfan sulfate, in freshwater ecosystems of South Florida: a probabilistic aquatic ecological risk assessment. Ecotoxicology 2010;19:879e900. [2] Dan X, Shuai L, Min Z, Yeqing S. Research progress on biological effect of endosulfan. Biotechnol Bull 2013;1:45e51. [3] Evans DL, Leary III JH, Jaso-Friedmann L. Nonspecific cytotoxic cells and innate immunity: regulation by programmed cell death. Dev Comp Immunol 2001;25:791e805. [4] Fischer U, Koppang EO, Nakanishi T. Teleost T and NK cell immunity. Fish Shellfish Immunol 2013;35:197e206. [5] Cuesta A, Esteban MA, Meseguer J. Natural cytotoxic activity of gilthead seabream (Sparus aurata L.) leucocytes: assessment by flow cytometry and microscopy. Vet Immunol Immuno 1999;71:161e71. [6] Ishimoto Y, Savan R, Endo M, Sakai M. Non-specific cytotoxic cell receptor (NCCRP)-1 type gene in tilapia (Oreochromis niloticus): its cloning and analysis. Fish Shellfish Immunol 2004;16:163e72. [7] Nakanishi T, Toda H, Shibasaki Y, Somamoto T. Cytotoxic T cells in teleost fish. Dev Comp Immunol 2011;35:1317e23. [8] Bishop GR, Jaso-Friedmann L, Evans DL. Activation-induced programmed cell death of nonspecific cytotoxic cells and inhibition by apoptosis regulatory factors. Cell Immunol 2000;1:126e37. [9] Jaso-Friedmann L, Ruiz J, Bishop GR, Evans DL. Regulation of innate immunity in tilapia: activation of nonspecific cytotoxic cells by cytokine-like factors. Dev Comp Immunol 2000;24:25e36. [10] Oumouna M, Jaso-Friedmann L, Evans DL. Flow cytometry-based assay for determination of teleost cytotoxic cell lysis of target cells. Cytometry 2001;45: 259e66. [11] Rauta PR, Nayak B, Das S. Immune system and immune responses in fish and their role in comparative immunity study: a model for higher organisms. Immunol Lett 2012;148:23e33. [12] Praveen K, Evans DL, Jaso-Friedmann L. Evidence for the existence of granzyme-like serine proteases in teleost cytotoxic cells. J Mol Evol 2004;58: 449e59. [13] Cuesta A, Esteban MA, Meseguer J. Molecular characterization of the nonspecific cytotoxic cell receptor (NCCRP-1) demonstrates gilthead seabream NCC heterogeneity. Dev Comp Immunol 2005;29:637e50. [14] Kumar N, Prabhu PAJ, Pal AK, Remya S, Aklakur Md, Rana RS, et al. Antioxidative and immuno-hematological status of Tilapia (Oreochromis mossambicus) during acute toxicity test of endosulfan. Pestic Biochem Phys 2011;99:45e52. [15] Tellez-Banuelos MC, Santerre A, Casas-Solis J, Bravo-Cuellar A, Zaitseva G. Oxidative stress in macrophages from spleen of Nile tilapia (Oreochromis niloticus) exposed to sublethal concentration of endosulfan. Fish Shellfish Immunol 2009;27:105e11. [16] Tellez-Banuelos MC, Santerre A, Casas-Solis J, Zaitseva G. Endosulfan increases seric interleukin-2 like (IL-2L) factor and immunoglobulin M (IgM) of Nile tilapia (Oreochromis niloticus) challenged with Aeromonas hydrophila. Fish Shellfish Immunol 2010;28:401e5. [17] Tellez-Banuelos MC, Ortiz-Lazareno PC, Santerre A, Casas-Solis J, BravoCuellar A, Zaitseva G. Effects of low concentration of endosulfan on
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