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Parameters to evaluate the immunostimulant effect of Zymosan A in head kidney leucocytes (HKL) of salmonids/Parametros para la evaluacion del efecto de Zimosan A como inmunoestimulante sobre leucocitos de rinon cefalico (HKL) de salmonidos.


Immunostimulatory capacity of [beta]-glucan has been demonstrated in fish, which is a powerful tool applied to its farming, as it strengthens the immunity, resistance to diseases and stress (Bagni et al., 2000). Innate immunity experiences the greatest reinforcement mediated by this type of pathogen-associated molecular patterns (PAMPs), therefore its presence can increase the number and activity of macrophages, phagocytosis, respiratory burst, cytotoxicity and activity of lysozyme, which has been evaluated in different fish species such as, Pagrus auratus, Sparus aurata and Dicentrarchus labrax (Cook et al., 2003; Rodriguez et al, 2003; Vazzana et al, 2003; Kudrenko et al., 2009). Different routes have been used for administration of [beta]-glucan to fish, been the most common intraperitoneal injection (ip). Danio rerio has demonstrated a remarkable decrease in mortality and increased survival when they have been treated via ip and then challenged with Aeromonas hydrophila (Rodriguez et al, 2003). Furthermore, the combined use of PAMPs such as [beta]-glucan/LPS has also shown improvement in resistance to bacterial pathogens in Cyprinus carpio, and also it has been shown that ip and oral applications are more effective than applications by bathing (Selvaraj et al., 2006).

Although the effect of glucans on immunity in fish is known, both cellular and molecular pathways involved have not been characterized. In general terms, [beta]-glucans are part of a heterogeneous group of glucose polymers, major constituents of the walls of fungi and bacteria, and hence derives its ability to activate the immune system of both invertebrates and vertebrates (Kataoka et al., 2002). The molecular mechanisms that are activated by this type of glucans, vary from the complexity of the organism and the structural characteristics of the [beta]-glucans (Tsoni & Brown, 2008). For example, only [beta]-glucans that have ramifications with [beta]-1 [right arrow] 3 and [beta]P-1[right arrow]6 bonds in its structure trigger the immune system. In addition, the extension of the structure of these molecules influences the immune response; in this way, large particles (such as Curdlan and Zymosan) are able to directly activate leukocytes (Brown & Gordon, 2005). Intermediate particles as phosphoglucans, have immunostimulatory activity in vivo, but not in vitro. On the other hand, small particles or with low molar mass, as laminarin, are biologically inactive both in vitro and in vivo (Adams et al., 2008; Tsoni & Brown, 2008). The effect of glucans on immunity has been best established in higher vertebrates, for example Curdlan, a linear [beta]-glucan has shown the ability to activate macrophages in rats by stimulating the nuclear transcription factor kB, leading to an overexpression of iNOS, TNF-[alpha] and MIP-2 (Kataoka et al, 2002; Ferwerda et al, 2008). For its part, Zymosan A, a [beta]-glucan constituent of the wall of yeasts induced a number of cytokines and membrane markers comparable to lipopolysaccharide (Dowling et al., 2008).

In fish farming, [beta]-glucan has been used as an immunostimulant, however, it is required to improve the quality of these applications, which involves looking for glucans with high immunostimulatory capacity. This can be evaluated in tanks with challenged fish, leading to high costs, impeding efficient molecule screening processes. This work presents a proposal for evaluating in vitro glucan molecules with immunostimulatory activity. The model presents two advantages; first, the lower cost of implementation, because of the use of primary cultures of macrophages from head kidney of trout and second, as a new proposal to assess the immunostimulatory capacity only at phenotypic level by conventional enzymatic quantifications and through the detection of cytokines secreted into the culture medium. The analysis of the results suggests inducer concentrations and incubation times for the evaluation in vitro of [beta]-glucan on immunity of salmonids, with reference to standard Zymozan A.



Rainbow trout (O. mykiss) from Rio Blanco fish farm were stabilized in the Laboratorio Experimental de Acuicultura (LEDA) in 1000 L tanks with filtered fresh water recirculation at a temperature between 17 and 20[degrees]C with constant aeration and commercial diet once daily until they reached the weight of 130 g.

Isolation of leucocytes from head kidney

The method described by Braun-Nesje et al. (1981) was used. Briefly, head kidney was removed aseptically and macerated through a 100 urn nylon mesh, using L-15 medium. The resulting suspension was placed on a discontinuous Percoll density gradient of 34/51%. The gradient was centrifuged at 800 g for 40 min. The interface cells were recovered and washed twice with 1X Hank's Balanced Solution for 5 min at 400 g. The concentration and cell viability was estimated with trypan blue. The cells were adjusted to 5 x 106 cells [mL.sup.-1] with L-15 medium supplemented with 2% FBS and antibiotics (100 U [mL.sup.-1] of penicillin/100 mg [mL.sup.-1] streptomycin). 100 [micro]L of cell suspension was seeded to a 96-well plate. The cells were stabilized at 17[degrees]C overnight (ON) prior induction.


After overnight (ON) period, unattached cells were washed with L-15 medium. The attached cells were stimulated with commercial Zymosan A (SIGMA) at 0.1/0.5/1.0/2.0 mg [mL.sup.-1] To quantify nitric oxide, cells were incubated from 2 to 96 h. For respiratory burst, they were incubated for 1 h. Meanwhile, to assess cytokine induction, incubation was performed between 2 and 10 h.

Quantification of nitric oxide

We used the method of Greiss reaction (Neumann et al., 1995), which quantifies the nitrite content in cell supernatants, since NO is unstable and is oxidized to nitrite and nitrate. After incubation at 17[degrees]C, 100 [micro]L of each well supernatant were taken and left in a separate 96-well plate; then 100 [micro]L of Greiss reagent (sulfaniamide 1% and 0.1% ethylenediamine naftil acid 2.5% phosphoric acid) was added. Absorbance was measured at 540 nm in a Versamax plate reader. Nitrite concentration in the sample was calculated from a calibration curve of sodium nitrite at known concentrations.

Respiratory burst

The methodology used was reduction of NBT to formazan (Selvaraj et al., 2005), which forms a blue precipitate inside the cells. Induced monolayer was washed 3 times with L-15 medium. 100 [micro]L of NBT (1 mg [mL.sup.-1]) was added and incubated for 1 h. Then, washed 3 times with Hank's 1X and cells were fixed with methanol 100% for 3 min. The cells were washed twice with 70% methanol and allowed to air dry. The formazan produced was solubilized by adding 120 [micro]L of 2M KOH and 140 [micro]L 100% DMSO. Absorbance was read at 630 nm using KOH/DMSO as blank.

Indirect ELISA

The cytokines IL-1[beta], IL-6 and TNF-[alpha] were tested with antibodies produced and obtained in our laboratory. Polyclonal antibodies developed in mouse were obtained against synthetic epitope sequences of the respective cytokines tested. For the indirect ELISA assay, 100 [micro]L of cell culture medium (50 ng [[micro]L.sup.-1] in 0.06 M carbonate buffer) were loaded and incubated at 4[degrees]C ON in 96 well ELISA plates. Then, washed three times with PBST 0.05% and blocked with BSA (1%) for 2.5 h at 37[degrees]C. Afterwards, washed and incubated with first antibody (100 [micro]L, 1.5 mg [mL.sup.-1]) for 2 h at 37[degrees]C. The second antibody anti-mouse IgG HRP conjugate (100 [micro]L, 1:7000) was incubated for 1.5 h at 37[degrees]C. The plate was revealed incubating with TMB for 30 min at room temperature and reaction was stopped with 50 [micro]L of sulfuric acid. Finally, absorbance was read at 450 nm.

For statistical analysis, calculations of means, standard deviations and analysis of variance (ANOVA) of all the assays was carried out using Microsoft Excel 2007. Differences were considered significant if the P-value was < 0.05.


HKL centrifugation on gradient yielded an interface, between 34% and 51% of Percoll, enriched in viable macrophages which were stabilized in culture. After 24 h of culture, HKL showed typical spherical cell morphology in the early hours, and then the majority of the cells, due to its characteristics of macrophages began to adhere to the bottom of the well. On day 5 the cells were largely adhered and stabilized (Fig. 1).

HKL cells stabilized in culture for 5 days, hereinafter macrophages, were induced to generate iNOS in response to 1 mg [mL.sup.-1] of Zymosan A. Initially the production of NO tends to increase slightly at 4 h of induction; however the peak was reached after 72 h of incubation, beginning a descent at 96 h (Fig. 2a). Evaluating the dose-response relationship in the activation of glucan-dependent iNOS, it was found that this is only significant at 72 h using 1 mg [mL.sup.-1] of inducer (Fig. 2b).

Macrophages in culture responded to stimulation of Zymosan A via increasing destructive activity by phagocyte oxidase, which was evidenced indirectly through quantification of reactive oxygen species. There was no dose-response to glucan, using 0.5 mg [mL.sup.-1] of inducer and four times this concentration, a significantly ROS level was quantified compared to control, but showing no significance between them (Fig. 3).



Detection of pro-inflammatory cytokines IL-1[beta], TNF-[alpha] and IL-6 in the culture medium showed a peak of secretion from macrophages after 4 h of induction with Zymosan A, for all cytokines (Fig. 4). Six hours after induction, secretion of IL-1[beta] and TNF-[alpha] tended to stabilize, except when the dose of inducer was higher (1 mg [mL.sup.-1]), secretion levels maintained as detected at 4 h. For IL-6 in all inducer concentrations tested, the stabilization arises from hour 6, where the levels detected are similar to the control situation. All assays showed no dose-response relationship, since the peak of secretion detected at 4 h, showed no significant differences for the same cytokine induced with different concentrations of Zymosan A (Fig. 4).




The study of fish immune responses requires optimal molecular markers to characterize the involvement of cellular and humoral components of both innate and adaptive responses (Randelli et al., 2008; Narvaez et al., 2010). Moreover, the generation of in vitro models has allowed the identification of key pro-inflammatory cytokines expressed in response to challenges with PAMPs and pathogens. Therefore, it has been necessary to establish cell lines (Rojas et al., 2010) or the implementation of laboratory primary cell cultures, in both cases the central idea is to generate in vitro models for the evaluation of immune responses, with the aim of projecting it to the fish and its productive management in aquaculture. In the case of this work, we have tested basic conditions for implementing an in vitro system to assess glucan type immunostimulants, known as potent inducers of the immune response that protects the fish against pathogens (Brown & Gordon, 2005).

From head kidney was obtained a phase of leukocytes (HKL) enriched in macrophages, which were grown successfully. This phase corresponds to a Percoll interface between 34% and 51%, which has been previously characterized in fish and particularly in rainbow trout (Chettri et al., 2010, 2011). This phase is used for in vitro induction, both at 24 h of stabilization, as well as days later, where the population of cells adhering to the plate is in the 80% of the total cells grown at the beginning. HKL culture cells are capable of responding to the challenge using different PAMPs such as LPS, flagellin and Poly I:C, in the case of Zymosan A there has been an up regulation of pro-inflammatory genes like TNF-[alpha], IL1-[beta], IL-6 and the key enzyme in the pathway of prostaglandins, COX-2 (Chettri et al., 2011).

Our results confirm the ability of HKL to respond to the challenge with Zymosan A, in all the tested assays, both at enzyme level as in ELISAs. NO has potent toxic effects and, as such, it is an important component of the available arsenal to animal hosts, their production depending on the activation of iNOS, an enzyme that is normally under the regulation of pro-inflammatory cytokines (Rieger & Barreda, 2011). A classical approach to establish the level of immune response, associated with this path, is the quantification of NO production, which has been previously established in fish macrophages (Tafalla & Novoa, 2000; Jurecka et al., 2009). There are no bibliographic records that indicate the level of NO in response to challenge with Zymosan in HKL of rainbow trout, our results show that at 72 h of incubation, the highest level of quantification is reached, and doses of 1 mg [mL.sup.-1] are the ones which generate significantly higher NO production, showing the highest level of response to Zymosan A. Currently, in our laboratory, we are implementing an ELISA to quantify iNOS directly, test that can be performed in shorter induction time.

Respiratory burst has also been a classical evaluation to establish the level of activation of macrophages in response to a stimulus (Cook et al., 2003). This parameter has been previously established and the test conditions of rainbow trout HKL, determined (Chettri et al., 2010). Our results, in this regard, are a positive control of the Zymosan A induction established in vitro over HKL. The reference concentration of Zymosan A to achieve generation of ROS, in a clearly detectable way, is 1.6 mg [mL.sup.-1]. Our assays proved that over 0.5 mg [mL.sup.-1], HKL are able to increase the production of reactive oxygen species significantly with respect to control, as well as up to 2 mg [mL.sup.-1] tested.

The main objective of this work focused on establishing an analytical model of HKL response to Zymosan A, as a reference to test immunostimulants in salmonids, considering a high-capacity screening system. For this, cytokines secreted into the culture medium were detected in an in vitro assay that has not previously been proposed or established for salmonid HKLs. Normally, fish cytokine assessments are conducted at a transcriptional level, and very few papers consider the use of antibodies as indicators of cytokine production. In trout, there is only one reference of the detection of TNF-[alpha], where it has been shown that LPS stimuli can induce cytokine secretion, without increasing the transcriptional activity of the gene that encodes for it (Roher et al., 2011).

Our results confirm that increased transcriptional activity, previously demonstrated, that occurs in trout HKL in response to Zymosan A (Chettri et al., 2011), also causes increased secretion of pro-inflammatory cytokines TNF-[alpha], IL-1[beta] and IL-6. The result that best represents what has been described at the transcriptional level, is the secretion of IL-6, which at four hours of induction, achieved the highest detection in the culture medium, coinciding exactly with the previously demonstrated at transcriptional level, with the highest level of expression at the same time.

In the case of IL-1P and TNF-[alpha], transcriptional activity described by Chettri et al. (2011), increases to 12 h of incubation which, at protein level, does not correlate with our results, since at 10 h, basal levels of the cytokine in the culture medium are already detected. As to the concentration of inductor, it was proved that low doses of Zymosan A (0.1 mg [mL.sup.-1]) generate secretion of three cytokines, as well as it was shown previously occurring at transcriptional level. It is interesting to note that the samples evaluated by indirect ELISA, considered 50 ng [[micro]L.sup.-1] of total proteins in the culture medium, which is a highly sensitive, replicable and massified method.

In summary we propose a model for evaluating the glucan immunostimulant ability in salmonids, using HKL primary cultures, and evaluating the secretion of pro-inflammatory cytokines in the culture medium. The test dose of inducer can be 0.1 mg mL-1 and the incubation time 4 h. As a positive control of induction, quantification of reactive oxygen species is proposed, through NBT reduction in cells treated with 0.5 mg [mL.sup.-1] of inducer. Finally, as evidence of the destructive capacity of macrophages, we propose the use 1 mg [mL.sup.-1] of inducer for 72 h for the quantification of NO.

The widespread of such screening techniques of potentially useful molecules as immunostimulants, have the potential to increase in a short-term period the supply of aquaculture products with a high capacity to improve the fish farm production.

DOI: 10.3856/vol40-issue3-fulltext-4


This work was supported by Grant D07I1095 of FONDEF (CONICYT-Chile) and Proyecto Investigacion Asociativa (PIA 037.358/2011) Vicerrectoria de Investigacion y Estudios Avanzados, Pontificia Universidad Catolica de Valparaiso. We thank the staff of the Laboratorio de Genetica Aplicada (PUCV) for the fish care during this research.


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Received: 23 February 2011; Accepted: 15 March 2012

Andres Abarca (1), Jorn Bethke (1), Edgar Narvaez (1), Roberto Flores (2), Luis Mercado (1)

(1) Grupo de Marcadores Inmunologicos en Organismos Acuaticos Laboratorio de Genetica e Inmunologia Molecular, Instituto de Biologia Facultad de Ciencias, Pontificia Universidad Catolica de Valparaiso Avenida Universidad 330, Campus Curauma, Valparaiso, Chile

(2) Centro i~mar, Universidad de Los Lagos Camino a Chinquihue Km 6, Puerto Montt, Chile

Corresponding author: Luis Mercado (
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Title Annotation:articulo en ingles; leucocitos de rinon cefalico
Author:Abarca, Andres; Bethke, Jorn; Narvaez, Edgar; Flores, Roberto; Mercado, Luis
Publication:Latin American Journal of Aquatic Research
Date:Nov 1, 2012
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