Veterinary Immunology and Immunopathology 156 (2013) 176–181
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Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Research paper
The effects of ryanodine receptor 1 (RYR1) mutation on plasma cytokines and catecholamines during prolonged restraint in pigs Ziemowit M. Ciepielewski a,∗ , Wojciech Stojek a , Wojciech Glac a , a b ´ ´ , Anna Kwaczynska , Marian Kamyczek b Dorota My´slinska a b
Department of Animal and Human Physiology, University of Gdansk, 80-308 Gdansk, Poland Experimental Station Pawłowice, 64-122 Pawłowice, National Institute of Animal Production, 32-083 Balice, Kraków, Poland
a r t i c l e
i n f o
Article history: Received 14 August 2013 Received in revised form 26 September 2013 Accepted 2 October 2013 Keywords: Catecholamines Cytokines Pig Restraint stress RYR1 gene
a b s t r a c t In the current study, plasma cytokines, including interleukin (IL) 1, IL-2, IL-6, IL-10, IL-12, interferon-␥ (IFN-␥) and tumor necrosis factor-␣ (TNF-␣) and catecholamines (adrenaline and noradrenaline) were evaluated during 4 h restraint and recovery phase in 60 male pigs. Blood samples were collected from three groups of pigs representing different RYR1 genotypes, namely NN homozygotes (wild-type), Nn heterozygous and nn homozygous (mutant). The 4 h restraint evoked an increase in plasma cytokine concentrations in each of the RYR1 genotype groups. During the restraint, the greatest concentrations of plasma IL-6, IL-10, IL-12 and TNF-␣ in nn homozygous pigs and IFN-␥ in NN homozygous were observed. Interleukin 1, IL-2, IL-10, and TNF-␣ measures were positively intercorrelated in each of the three RYR1 genotype group. A positive correlation was seen between all measured cytokines (with the exception of IL-6) and plasma catecholamine concentrations in Nn heterozygous and nn homozygous pigs. The results suggest that of the cytokine parameters evaluated, IL-6, IL-10, IL-12 and TNF-␣ of the nn homozygous group seem to show a stronger stress-related response as compared with those of the other two (NN and Nn) groups. © 2013 Elsevier B.V. All rights reserved.
1. Introduction A major genetic factor that influences stress susceptibility in pigs, which is inherited in an autosomal recessive manner has been mapped to the ryanodine receptor 1 gene (RYR1) also known as the halothane gene (Haln ). The HAL-locus with two alleles N and n is located on pig chromosome 6, with animals of genotypes NN (wild type, homozygous dominant) and Nn (heterozygous, gene mutation carriers) are not sensitive to halothane (Mitchell and Heffron, 1982; Fujii et al., 1991). Homozygous recessive (nn) pigs are sensitive to halothane and under stress
∗ Corresponding author. Tel.: +48 58 523 61 21; fax: +48 58 236121. E-mail address: [email protected] (Z.M. Ciepielewski). 0165-2427/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetimm.2013.10.001
condition (e.g., transport, overcrowding or overheating) develop porcine stress syndrome (PSS) which is characterized by skeletal muscle rigidity, increase in metabolic rate, acidosis and a rise in body temperature (malignant hyperthermia, MH) (Nelson, 2002). Porcine stress syndrome is the only confirmed model of human MH. Susceptibility to PSS has been ascribed to a point mutation in the skeletal muscle calcium release channel encoded by the RYR1 gene, which results the replacement of cytosine (C) with thymine (T) at nucleotide 1843 of the coding sequence and is identical with a mutation found in human MH syndrome (MacLennan and Chen, 1993). The mechanism whereby the single mutation in the RYR1 gene results in muscle dysfunction and influences carcass composition and meat quality has been well elucidated (Van den Maagdenberg et al., 2008; Bates et al., 2012).
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However, information regarding alterations of immune and stress hormone responses in PSS pigs is limited (Edfors-Lilja et al., 1987; Tokarski et al., 1992; Geers et al., 1994; Weaver et al., 2000). In our previous paper we reported that stressinduced changes in natural killer (NK) cell activity and production of stress-related hormones (prolactin, growth hormone and cortisol) were influenced by RYR1 genotype (Ciepielewski et al., 2013). Although the role of cytokines in the inflammatory states is well established, data on cytokines-stress hormones interactions are unequivocal (Woiciechowsky et al., 1999; Correa et al., 2007). In particular, no studies have directly addressed the concentration of plasma cytokines in stress-susceptible pigs in both basal and stress condition. In an effort to gain further understanding of the physiological mechanisms of the stress reaction in pigs of different individual susceptibility to stress we have used the 4 h restraint model. The current paper describes the effects of porcine RYR1 gene mutation on plasma concentrations of interleukin (IL) 1, IL-2, IL-6, IL-10, IL-12, interferon-␥ (IFN-␥) and tumor necrosis factor-␣ (TNF-␣) and plasma adrenaline and noradrenaline response during prolonged (4 h) restraint and the poststress period. 2. Materials and methods 2.1. Animal, housing and restraint procedure All experiments were performed under the approval and guidelines of the Committee on Animal Care and Use of the Local Ethical Committee, Gdansk, Poland. The pigs employed in the study were the same animals used in Ciepielewski et al. (2013) experiment, and the housing, RYR1 genotype determination, surgical procedures, catheterization and restraint procedure were previously described. Briefly, all subjects were domestic pigs derived from the Polish line 990 (an outbred pig line developed in 1984 at the Pig Hybridization Centre in Pawlowice; National Institute of Animal Production, Krakow, Poland). The RYR1 genotype determination was performed exactly as described by Otsu et al. (1992). The genotypes were defined as follows: (1) homozygous recessive (nn; dimutant with 2 copies of the mutation), (2) heterozygous carrier (Nn; monomutant with 1 copy of the mutation, gene mutation carriers), or (3) homozygous dominant (NN; wild type). Finally, 60 male pigs (20 of each RYR1genotype) 13–15 wk old and weighing 28–33 kg were selected for this study. Of the twenty pigs of each genotype, ten were used in the restrained groups: NN, Nn and nn and ten pigs were used in control (non-restrained) groups: NNCON, NnCON and nnCON. 2.2. Plasma cytokine assays Concentrations of plasma IL-1, IL-6, IL-10, IL-12, IFN-␥, and TNF-␣ were determined according to the manufacturer’s protocols using the porcine-cytokine specific ELISA kits (Quantikine, R&D Systems, USA). The limits of detection for the assays were 2.7, 0.7, 1.8, 3.5, 4.7, and 2.8 pg/mL, respectively. Intra- and inter-assay coefficients of variation were 7.2% and 8.7%, 5.1% and 7.4%, 4.2% and 7.2%, 6.4% and
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9.2%, 4.5% and 7.5%, and 6.9% and 9.2%, respectively. Plasma concentrations of IL-2 were analyzed using Porcine Interleukin 2 (IL-2) ELISA Kit (Shanghai Crystal Day Biotech Co., China). The sensitivity of the assay was 2.5 pg/mL. Intraand inter-assay coefficients of variation were 9.5% and 12%, respectively. 2.3. Plasma catecholamine assays Concentrations of plasma adrenaline and noradrenaline were assayed using a high-performance liquid chromatography procedure with electrochemical detection (Waters Associates) after extraction from the plasma samples by absorption onto surface-activated aluminum oxide (Dirks et al., 1988). Intra- and interassay coefficients of variation were 11.5% and 12.5% for adrenaline and 2.1% and 1.9% for noradrenaline. 2.4. Statistical analysis All data analyses were conducted using General Linear Model. Data were subjected to a two-factor repeated measures ANOVA to determine the significance of main factors (restraint and genotype) and main factors interactions. When ANOVA indicated significant main effects, results were further characterized by Tukey’s b test. All data are expressed as mean ± SEM. The significant threshold was set at P < 0.05. The relationship between cytokine measures and between cytokine and catecholamine measures were estimated by Pearson correlation coefficients, for each genotype separately. 3. Results and discussion Two-way ANOVA of plasma IL-1, IL-2, IL-6, IL-10, IL-12, IFN-␥, and TNF-␣ adrenaline and noradrenaline concentrations revealed significant (P < 0.001) effects of restraint (P < 0.001), genotype (P < 0.001), and restraint × genotype interaction (P < 0.001). 3.1. Plasma cytokines Prior to the restraint, and in control groups (nnCON, NnCON and NNCON), plasma concentrations of IL-1, IL-2, IL-6, IL-10, IL-12 and INF-␥ did not differ between RYR1 genotypes (Figs. 1 and 2). Basal and control concentrations of TNF-␣ were greater (P < 0.05) in nn homozygous pigs than in heterozygous (Nn) and NN homozygous pigs (Fig. 2C). Th 4 h restraint evoked an increase in all measured cytokine concentrations and the significant differences in stress-induced effects on cytokine concentrations between the RYR1 genotypes occurred (Figs. 1 and 2). The greatest response in plasma IL-6, IL-10, IL-12 and TNF- ␣ was observed in nn homozygous pigs (P < 0.05, in comparison with Nn heterozygous and NN homozygous pigs). In NN homozygous pigs the greatest response in plasma IFN-␥ concentration was observed (P < 0.05, in comparison with NN and nn homozygous pigs). The plasma concentrations of IL-1, IL-2, IL-10, IL-12 returned to the control values at 480 min in each of the RYR1 genotype group (Figs. 1A, B and D and 2A). The plasma concentrations of
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Fig. 1. Plasma IL-1 (A), IL-2 (B), IL-6 (C) and IL-10 (D) concentrations during the 4-h restraint stress (0–240 min) and post-stress period (300–480 min) in pigs of different RYR1 genotypes (nn, Nn, NN: stressed groups; nnCON, NnCON, NNCON: control groups; n = 10 in each group). Significant differences between stressed and control pigs are indicated by asterisks (Tukey b test, P < 0.05). IL-1, interleukin-1; IL-2, interleukin-2; IL-6, interleukin-6; IL-10, interleukin-10; NN, homozygous wild type; Nn, heterozygous carrier; nn, homozygous mutant.
IL-6 (Nn and nn pigs), IFN-␥ (NN and Nn pigs) and TNF-␣ (nn pigs) were increased at 480 min in comparison with the respective control groups (Figs. 1C and 2B and C). 3.2. Plasma catecholamines Prior to the restraint, and in control groups (nnCON, NnCON and NNCON), plasma concentrations of adrenaline did not differ between RYR1 genotypes (Fig. 3A). Basal and control concentrations of noradrenaline were lower (P < 0.05) in NN homozygous pigs than in heterozygous (Nn) and NN homozygous pigs (Fig. 3B). As shown in Fig. 3, the 4 h restraint increased plasma adrenaline and noradrenaline concentrations in the RYR1 genotypes compared with the control groups. The increase in plasma noradrenaline concentration in NN homozygous pigs was significantly lower (P < 0.05) than in Nn heterozygous and nn homozygous pigs. In NN homozygous an Nn heterozygous pigs the plasma adrenaline concentrations returned to the control values at 120 min and in nn homozygous pigs at 180 min (Fig. 3A). Plasma noradrenaline concentrations returned to the baseline at 180, 240 and 480 min in NN homozygous, Nn heterozygous and nn homozygous pigs respectively.
3.3. Regression analysis As shown in Table 1, IL-1, IL-2, IL-10, IL-12, IFN-␥, and TNF-␣, measures were positively intercorrelated in nn homozygous and Nn heterozygous pigs. In NN homozygous pigs IL-1, IL-2, IL-10 and TNF-␣ were positively intercorrelated. In all genotypes TNF-␣ measures were positively correlated with IL-6 concentrations. In nn homozygous and Nn heterozygous pigs there was a significant positive correlation between all measured cytokines (with the exception of IL-6) and plasma catecholamine concentrations. In NN homozygous pigs there was a positive correlation between plasma IL-1, IL-10 and TNF-␣ and catecholamine measures. In the present study, pigs subjected to the prolonged, 4 h restraint showed significant differences in stress-induced effects on the plasma cytokine response between the RYR1 genotypes. Pigs homozygous for the RYR1 gene mutation (nn), exhibited the greatest plasma IL-6, IL-10, IL-12 and TNF-␣ concentrations during the 4 h restraint as compared to RYR1 the gene mutation carriers (Nn) and wild type (NN) pigs. The present study provides the first description of the possible influence of the RYR1 gene status on cytokine response during the prolonged restraint stress. The findings corroborate and extend our observation that
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Table 1 Pearson correlation coefficients between plasma cytokines (IL-1, IL-2, IL-6, IL-10, IL-12, IFN-␥, and TNF-␣ and plasma catecholamines (NA, A) during the 4 h restraint (0–240 min) in homozygous mutant (nn) pigs, heterozygous (Nn) pigs and homozygous wild type (NN) pigs (df = 160 for each genotype). Genotype
nn
Nn
NN
IL-1 b
IL-2
IL-6
IL-10
IL-12
IFN-␥
TNF-␣
NA
– 0.185 −0.163 0.710b 0.326b 0.058 −0.298a
– 0.492b 0.470b 0.756b 0.690b 0.368b
– 0.821b 0.639b 0.681b 0.580b
– 0.849b 0.809b 0.529b
– 0.631b 0.308b
– 0.763b
– 0.157 −0.021 0.110 0.481b 0.078 −0.144
– 0.511b 0.835b 0.828b 0.824b 0.562b
– 0.456b 0.290a 0.443b 0.306a
– 0.676b 0.800b 0.632b
– 0.681b 0.361b
– 0.747b
– 0.486b 0.146 0.093
– 0.729b 0.593b
– 0.850b
IL-2 IL-6 IL-10 IL-12 IFN-␥ TNF-␣ NA A
0.732 −0.126 0.520b 0.580b 0.686b 0.431b 0.834b 0.873b
– −0.216 0.482b 0.715b 0.686b 0.677b 0.757b 0.687b
IL-2 IL-6 IL-10 IL-12 IFN-␥ TNF-␣ NA A IL-2 IL-6
0.754b 0.108 0.839b 0.409b 0.736b 0.761b 0.809b 0.558b 0.523b 0.148
– 0.138 0.716b 0.383b 0.545b 0.618b 0.588b 0.281a – −0.215
IL-10 IL-12 IFN-␥ TNF-␣ NA A
b
a
0.658 0.127 0.763b 0.694b 0.497b 0.407b
0.256 0.083 −0.020 0.455b 0.127 0.120
– 0.099 −0.082 0.584b 0.410b 0.057 −0.090
– 0.024 0.422b 0.729b 0.833b 0.826b
– 0.079 0.482b 0.144 0.054
Abbreviations: A: adrenaline; IL-1: interleukin-1; IL-2: interleukin-2; IL-6: interleukin-6; IL-10: interleukin-10; IL-12: interleukin-12; IFN-␥: interferon-␥; NE: noradrenaline; TNF-␣: tumor necrosis factor-␣. a P < 0.01. b P < 0.001.
pigs mutated for RYR1 gene show the increased NK cell cytotoxicity response to the restraint in comparison with NN homozygous and Nn heterozygous pigs (Ciepielewski et al., 2013). An imbalance in the neuroendocrine–immune relationship may contribute to a number of pathologies, most notably those relating to stress (Steptoe et al., 2007). It is recognized that the immune system is regulated by internal regulatory pathways with own signals (Besedovsky and Rey, 2007; Borghetti et al., 2009). The signals include pro-inflammatory and anti-inflammatory cytokines and circulating concentrations of such cytokines as IL-1, IL6 and TNF-␣ are markers of inflammation (Hansel et al., 2010). These molecules have also been studied as the possible link between the stress response and immune system, as these cytokines have been shown to be involved in response to various stress stimuli and influence and/or modulate the catecholaminergic systems and the HPA axis functions (Haddad et al., 2002; Goshen and Yirmiya, 2009; Dimitrijevic et al., 2012). The concomitant restraintinduced release of pro-and anti-inflammatory cytokines observed in the current study shows that the opposite inflammatory mechanisms were activated in the course of the prolonged stress response. Moreover, these changes were time dependent, and the significant positive correlations between the circulating cytokine measures occurred. It could be suggested that stress can alter immune function by affecting the different subpopulations of T lymphocytes (Appels et al., 2000; Calcagni and Elenkov, 2006). Thus, the stress-induced release of pro-inflammatory cytokines from T helper (Th) 1 cells and the anti-inflammatory cytokines
from Th2 cells could produce the opposite inflammatory (pro-and anti-inflammatory) processes (Wirtz et al., 2007). In the present study, a single exposure to the restraint increased the plasma concentrations of IL-2, IL-12 and IFN␥. These cytokines have been described as the potentiators of the NK cell activity (Knoblock and Canning, 1992). The increase in these cytokines may contribute to modulation of stress-induced changes in NK cell activity observed in our previous study (Ciepielewski et al., 2013). Recent findings in human and animal studies suggest that different stressors may alter the patterns of cytokine production and release in vivo and in vitro (de Groot et al., 2001; Bartolomucci et al., 2003; Steptoe et al., 2007; Voorhees et al., 2013). In experiments performed in pigs the stressors such as transportation (Lv et al., 2011), isolation (Tuchscherer et al., 2009), changes in environmental temperature (Carroll et al., 2012) or lipopolysaccharide injection (Collier et al., 2011) strongly affected the plasma immunoregulatory cytokines concentrations. Although divergent effects of acute stress on cytokine release have been reported, most of the studies show that the cytokine responsiveness of an acute stressor is related to the catheholamine system or to the hypothalamo–pituitary–adrenal (HPA) axis activity (Chesnokova and Melmed, 2002; Szelenyi and Vizi, 2007). In the current study, the positive correlations observed between the IL-1, IL-10, TNF-␣ and circulating catecholamines suggest the possible direct regulatory effect of adrenergic system on cytokine release (e.g. through the adrenergic receptors on lymphocytes). Systemic catecholamine effects are similar to those of glucocorticoids
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Fig. 3. Plasma adrenaline (A) and nordrenaline (B) concentrations during the 4-h restraint stress (0–240 min) and post-stress period (300–480 min) in pigs of different RYR1 genotypes (nn, Nn, NN: stressed groups; nnCON, NnCON, NNCON: control groups; n = 10 in each group. Significant differences between stressed and control pigs are indicated by asterisks (Tukey b test, P < 0.05). NN, homozygous wild type; Nn, heterozygous carrier; nn, homozygous mutant.
Fig. 2. Plasma IL-12 (A), IFN-␥ (B) and TNF-␣ (C) concentrations during the 4-h restraint stress (0–240 min) and post-stress period (300–480 min) in pigs of different RYR1 genotypes (nn, Nn, NN: stressed groups; nnCON, NnCON, NNCON: control groups; n = 10 in each group). Significant differences between stressed and control pigs are indicated by asterisks (Tukey b test, P < 0.05). IL-12, interleukin-12; IFN-␥, interferon-␥; TNF- ␣, tumor necrosis factor-␣; NN, homozygous wild type; Nn, heterozygous carrier; nn, homozygous mutant.
in inhibition of IL-2, IL-12, IFN-␥ and TNF-␣ (PaninaBordignon et al., 1997), however in the present work only IL-6 concentration did not correlate with plasma adrenalin and noradrenalin which suggest more complex mechanism of pro-inflammatory cytokine-catecholamine relationship. In conclusion, it could be suggested that 4 h restraint activates both the pro- and anti-inflammatory cytokines
release but the intensity of the release could be influenced by the initial state of the animal resulted from the possessing of the RYR1 gene mutation. The IL-6, IL-10, IL-12 and TNF-␣ release during the 4 h restraint in nn homozygous pigs seem to show a stronger stress-related response (as compared with those of the other two groups), however the mechanism by which the RYR1 gene mutation affects the immune functions remain elusive.
Conflict of interest statement None of the authors of this paper has any financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
Acknowledgements This work was supported by Grant N N303 2923 34 from Ministry of Science and Higher Education of Poland.
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References Appels, A., Bär, F.W., Bär, J., Bruggeman, C., de Baets, M., 2000. Inflammation, depressive symptomtology, and coronary artery disease. Psychosom. Med. 62, 601–605. Bartolomucci, A., Palanza, P., Sacerdote, P., Ceresini, G., Chirieleison, A., Panerai, A.E., Parmigiani, S., 2003. Individual housing induces altered immune-endocrine responses to psychological stress in mice. Psychoneuroendocrinology 28, 540–558. Bates, R.O., Doumit, M.E., Raney, N.E., Helman, E.E., Ernst, C.W., 2012. Association of halothane sensitivity with growth and meat quality in pigs. Animal 6, 1537–1542. Besedovsky, H.O., Rey, A.D., 2007. Physiology and psychoneuroimmunology: a personal view. Brain Behav. Immun. 21, 34–44. Borghetti, P., Saleri, R., Mocchegiani, E., Corradi, A., Martelli, P., 2009. Infection immunity and the neuroendocrine response. Vet. Immunol. Immunopathol. 130, 141–162. Calcagni, E., Elenkov, I., 2006. Stress system activity, innate and T helper cytokines and susceptibility to immune-related diseases. Ann. NY Acad. Sci. 1069, 62–76. Carroll, J.A., Burdick, N.C., Chase Jr., C.C., Coleman, S.W., Spiers, D.E., 2012. Influence of environmental temperature on the physiological endocrine, and immune responses in livestock exposed to a provocative immune challenge. Domest. Anim. Endocrinol. 43, 146–153. Chesnokova, V., Melmed, S., 2002. Minireview: neuro-immuno-endocrine modulation of the hypothalamic–pituitary–adrenal (HPA) axis by gp130 signaling molecules. Endocrinology 143, 1571–1574. Ciepielewski, Z.M., Stojek, W., Glac, W., Wrona, D., 2013. Restraint effects on stress-related hormones and blood natural killer cell cytotoxicity in pigs with a mutated ryanodine receptor. Domest. Anim. Endocrinol. 44, 195–203. Collier, C.T., Williams, P.N., Carroll, J.A., Welsh Jr., T.H., Laurenz, J.C., 2011. Effect of maternal restraint stress during gestation on temporal lipopolysaccharide-induced neuroendocrine and immune responses of progeny. Domest. Anim. Endocrinol. 40, 40–50. Correa, S.G., Maccioni, M., Rivero, V.E., Iribarren, P., Sotomayor, C.E., Riera, C.M., 2007. Cytokines and the immune-neuroendocrine network: what did we learn from infection and autoimmunity? Cytokine Growth Factor Rev. 18, 125–134. de Groot, J., Ruis, M.A., Scholten, J.W., Koolhaas, J.M., Boersma, W.J., 2001. Long-term effects of social stress on antiviral immunity in pigs. Physiol. Behav. 73, 145–158. Dimitrijevic, M., Stanojevic, S., Kustrimovic, N., Leposavic, G., 2012. Endpoint effector stress mediators in neuroimmune interactions: their role in immune system homeostasis and autoimmune pathology. Immunol. Res. 52, 64–80. Dirks, B., Vorwalter, C., Grünert, A., 1988. Basal plasma catecholamine level determination using HPLC-ED and different sample cleanup techniques. Chromatographia 25, 223–229. Edfors-Lilja, I., Lundström, K., Nyberg, L., Rundgren, M., 1987. Influence of the Hal locus and standardized stress on antibody response and in vitro reactivity of peripheral blood lymphocytes in pigs. Vet. Immunol. Immunopathol. 14, 157–171. Fujii, J., Otsu, K., Zorzato, F., de Leon, S., Khanna, V.K., Weiler, J.E., O’Brien, P.J., MacLennan, D.H., 1991. Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia. Science 5018, 448–451. Geers, R., Bleus, E., Van Schie, T., Villé, H., Gerard, H., Janssens, S., Nackaerts, G., Decuypere, E., Jourquin, J., 1994. Transport of pigs different with respect to the halothane gene: stress assessment. J. Anim. Sci. 72, 2552–2558.
181
Goshen, I., Yirmiya, R., 2009. Interleuki-1 (IL-1) A central regulator of stress responses. Front. Neuroendocrinol. 30, 30–45. Haddad, J.J., Saadé, N.E., Safieh-Garabedian, B., 2002. Cytokines and neuro-immune-endocrine interactions: a role for the hypothalamic–pituitary–adrenal revolving axis. J. Neuroimmunol. 133, 1–19. Hansel, A., Hong, S., Camara, R.J.A., von Kanel, R., 2010. Inflammation as a psychophysiological biomarker in chronic psychosocial stress. Neurosci. Biobehav. Rev. 35, 115–121. Knoblock, K.F., Canning, P.C., 1992. Modulation in vitro porcine natural killer cell activity by recombinant interleukin-1␣, interleukin-2 and interleukin-4. Immunology 76, 299–304. Lv, Q., Zhang, S., Zhao, R., 2011. Transportation stress alters the expression of immunoregulatory cytokines in the porcine thymus. Vet. J. 187, 229–233. MacLennan, D.H., Chen, S.R., 1993. The role of the calcium release channel of skeletal muscle sarcoplasmic reticulum in malignant hyperthermia. Ann. NY Acad. Sci. 707, 294–304. Mitchell, G., Heffron, J.J.A., 1982. Porcine stress syndromes. Adv. Food Res. 28, 167–230. Nelson, T.E., 2002. Malignant hyperthermia: a pharmacogenetic disease of Ca++ regulating proteins. Curr. Mol. Med. 2, 347–369. Otsu, K., Phillips, M.S., Khanna, V.K., de Leon, S., MacLennan, D.H., 1992. Refinement of diagnostic assays for a probable causal mutation for porcine and human malignant hyperthermia. Genomics 13, 835– 837. Panina-Bordignon, P., Mazzeo, D., Lucia, P.D., D’Ambrosio, D., Lang, R., Fabbri, L., Self, C., Sinigaglia, F., 1997. Beta2-agonists prevent Th1 development by selective inhibition of interleukin 12. J. Clin. Invest. 100, 1513–1519. Steptoe, A., Hamer, M., Chida, Y., 2007. The effects of acute psychological stress on circulating inflammatory factors in humans: a review and meta-analysis. Brain Behav. Immun. 21, 901–912. Szelenyi, J., Vizi, E.S., 2007. The catecholamine-cytokine balance: interaction between brain and the immune system. Ann. NY Acad. Sci. 1113, 311–324. Tokarski, J., Wrona, D., Piskorzynska, M., Borman, A., Witkowski, J., Jurkowski, M., Kamyczek, M., 1992. The influence of immobilization stress on natural killer cytotoxic activity in halothane susceptible and resistant pigs. Vet. Immunol. Immunopathol. 31, 371–376. Tuchscherer, M., Kanitz, E., Puppe, B., Tuchscherer, A., Viergutz, T., 2009. Changes in endocrine and immune responses of neonatal pigs exposed to a psychosocial stressor. Res. Vet. Sci. 87, 380–388. Van den Maagdenberg, K., Stinckens, A., Claeys, E., Buys, N., De Smet, S., 2008. Effect of the insulin-like growth factor-II and RYR1 genotype in pigs on carcass and meat quality traits. Meat Sci. 80, 293–303. Voorhees, J.L., Tarr, A.J., Wohleb, E.S., Godbout, J.P., Mo, X., Sheridan, J.F., Eubank, T.D., Marsh, C.B., 2013. Prolonged restraint stress increases IL-6, reduces IL-10 and causes persistent depressive-like behaviour that is reversed by recombinant IL-10. PLoS ONE 8, e58488. Weaver, S.A., Dixon, W.T., Schaefer, A.L., 2000. The effects of mutated skeletal ryanodine receptors on hypothalamic–pituitary–adrenal axis function in boars. J. Anim. Sci. 78, 1319–1330. Wirtz, P.H., von Känel, R., Emini, L., Suter, T., Fontana, A., Ehlert, U., 2007. Variations in anticipatory cognitive stress appraisal and differential proinflammatory cytokine expression in response to acute stress. Brain Behav. Immun. 21, 851–859. Woiciechowsky, C., Shoning, B., Lanksch, W.R., Volk, H.D., Docke, W.D., 1999. Mechanisms of brain-mediated systemic anti-inflammatory syndrome causing immunodepression. J. Mol. Med. 77, 769–780.
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Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Research paper
The effects of ryanodine receptor 1 (RYR1) mutation on plasma cytokines and catecholamines during prolonged restraint in pigs Ziemowit M. Ciepielewski a,∗ , Wojciech Stojek a , Wojciech Glac a , a b ´ ´ , Anna Kwaczynska , Marian Kamyczek b Dorota My´slinska a b
Department of Animal and Human Physiology, University of Gdansk, 80-308 Gdansk, Poland Experimental Station Pawłowice, 64-122 Pawłowice, National Institute of Animal Production, 32-083 Balice, Kraków, Poland
a r t i c l e
i n f o
Article history: Received 14 August 2013 Received in revised form 26 September 2013 Accepted 2 October 2013 Keywords: Catecholamines Cytokines Pig Restraint stress RYR1 gene
a b s t r a c t In the current study, plasma cytokines, including interleukin (IL) 1, IL-2, IL-6, IL-10, IL-12, interferon-␥ (IFN-␥) and tumor necrosis factor-␣ (TNF-␣) and catecholamines (adrenaline and noradrenaline) were evaluated during 4 h restraint and recovery phase in 60 male pigs. Blood samples were collected from three groups of pigs representing different RYR1 genotypes, namely NN homozygotes (wild-type), Nn heterozygous and nn homozygous (mutant). The 4 h restraint evoked an increase in plasma cytokine concentrations in each of the RYR1 genotype groups. During the restraint, the greatest concentrations of plasma IL-6, IL-10, IL-12 and TNF-␣ in nn homozygous pigs and IFN-␥ in NN homozygous were observed. Interleukin 1, IL-2, IL-10, and TNF-␣ measures were positively intercorrelated in each of the three RYR1 genotype group. A positive correlation was seen between all measured cytokines (with the exception of IL-6) and plasma catecholamine concentrations in Nn heterozygous and nn homozygous pigs. The results suggest that of the cytokine parameters evaluated, IL-6, IL-10, IL-12 and TNF-␣ of the nn homozygous group seem to show a stronger stress-related response as compared with those of the other two (NN and Nn) groups. © 2013 Elsevier B.V. All rights reserved.
1. Introduction A major genetic factor that influences stress susceptibility in pigs, which is inherited in an autosomal recessive manner has been mapped to the ryanodine receptor 1 gene (RYR1) also known as the halothane gene (Haln ). The HAL-locus with two alleles N and n is located on pig chromosome 6, with animals of genotypes NN (wild type, homozygous dominant) and Nn (heterozygous, gene mutation carriers) are not sensitive to halothane (Mitchell and Heffron, 1982; Fujii et al., 1991). Homozygous recessive (nn) pigs are sensitive to halothane and under stress
∗ Corresponding author. Tel.: +48 58 523 61 21; fax: +48 58 236121. E-mail address: [email protected] (Z.M. Ciepielewski). 0165-2427/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetimm.2013.10.001
condition (e.g., transport, overcrowding or overheating) develop porcine stress syndrome (PSS) which is characterized by skeletal muscle rigidity, increase in metabolic rate, acidosis and a rise in body temperature (malignant hyperthermia, MH) (Nelson, 2002). Porcine stress syndrome is the only confirmed model of human MH. Susceptibility to PSS has been ascribed to a point mutation in the skeletal muscle calcium release channel encoded by the RYR1 gene, which results the replacement of cytosine (C) with thymine (T) at nucleotide 1843 of the coding sequence and is identical with a mutation found in human MH syndrome (MacLennan and Chen, 1993). The mechanism whereby the single mutation in the RYR1 gene results in muscle dysfunction and influences carcass composition and meat quality has been well elucidated (Van den Maagdenberg et al., 2008; Bates et al., 2012).
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However, information regarding alterations of immune and stress hormone responses in PSS pigs is limited (Edfors-Lilja et al., 1987; Tokarski et al., 1992; Geers et al., 1994; Weaver et al., 2000). In our previous paper we reported that stressinduced changes in natural killer (NK) cell activity and production of stress-related hormones (prolactin, growth hormone and cortisol) were influenced by RYR1 genotype (Ciepielewski et al., 2013). Although the role of cytokines in the inflammatory states is well established, data on cytokines-stress hormones interactions are unequivocal (Woiciechowsky et al., 1999; Correa et al., 2007). In particular, no studies have directly addressed the concentration of plasma cytokines in stress-susceptible pigs in both basal and stress condition. In an effort to gain further understanding of the physiological mechanisms of the stress reaction in pigs of different individual susceptibility to stress we have used the 4 h restraint model. The current paper describes the effects of porcine RYR1 gene mutation on plasma concentrations of interleukin (IL) 1, IL-2, IL-6, IL-10, IL-12, interferon-␥ (IFN-␥) and tumor necrosis factor-␣ (TNF-␣) and plasma adrenaline and noradrenaline response during prolonged (4 h) restraint and the poststress period. 2. Materials and methods 2.1. Animal, housing and restraint procedure All experiments were performed under the approval and guidelines of the Committee on Animal Care and Use of the Local Ethical Committee, Gdansk, Poland. The pigs employed in the study were the same animals used in Ciepielewski et al. (2013) experiment, and the housing, RYR1 genotype determination, surgical procedures, catheterization and restraint procedure were previously described. Briefly, all subjects were domestic pigs derived from the Polish line 990 (an outbred pig line developed in 1984 at the Pig Hybridization Centre in Pawlowice; National Institute of Animal Production, Krakow, Poland). The RYR1 genotype determination was performed exactly as described by Otsu et al. (1992). The genotypes were defined as follows: (1) homozygous recessive (nn; dimutant with 2 copies of the mutation), (2) heterozygous carrier (Nn; monomutant with 1 copy of the mutation, gene mutation carriers), or (3) homozygous dominant (NN; wild type). Finally, 60 male pigs (20 of each RYR1genotype) 13–15 wk old and weighing 28–33 kg were selected for this study. Of the twenty pigs of each genotype, ten were used in the restrained groups: NN, Nn and nn and ten pigs were used in control (non-restrained) groups: NNCON, NnCON and nnCON. 2.2. Plasma cytokine assays Concentrations of plasma IL-1, IL-6, IL-10, IL-12, IFN-␥, and TNF-␣ were determined according to the manufacturer’s protocols using the porcine-cytokine specific ELISA kits (Quantikine, R&D Systems, USA). The limits of detection for the assays were 2.7, 0.7, 1.8, 3.5, 4.7, and 2.8 pg/mL, respectively. Intra- and inter-assay coefficients of variation were 7.2% and 8.7%, 5.1% and 7.4%, 4.2% and 7.2%, 6.4% and
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9.2%, 4.5% and 7.5%, and 6.9% and 9.2%, respectively. Plasma concentrations of IL-2 were analyzed using Porcine Interleukin 2 (IL-2) ELISA Kit (Shanghai Crystal Day Biotech Co., China). The sensitivity of the assay was 2.5 pg/mL. Intraand inter-assay coefficients of variation were 9.5% and 12%, respectively. 2.3. Plasma catecholamine assays Concentrations of plasma adrenaline and noradrenaline were assayed using a high-performance liquid chromatography procedure with electrochemical detection (Waters Associates) after extraction from the plasma samples by absorption onto surface-activated aluminum oxide (Dirks et al., 1988). Intra- and interassay coefficients of variation were 11.5% and 12.5% for adrenaline and 2.1% and 1.9% for noradrenaline. 2.4. Statistical analysis All data analyses were conducted using General Linear Model. Data were subjected to a two-factor repeated measures ANOVA to determine the significance of main factors (restraint and genotype) and main factors interactions. When ANOVA indicated significant main effects, results were further characterized by Tukey’s b test. All data are expressed as mean ± SEM. The significant threshold was set at P < 0.05. The relationship between cytokine measures and between cytokine and catecholamine measures were estimated by Pearson correlation coefficients, for each genotype separately. 3. Results and discussion Two-way ANOVA of plasma IL-1, IL-2, IL-6, IL-10, IL-12, IFN-␥, and TNF-␣ adrenaline and noradrenaline concentrations revealed significant (P < 0.001) effects of restraint (P < 0.001), genotype (P < 0.001), and restraint × genotype interaction (P < 0.001). 3.1. Plasma cytokines Prior to the restraint, and in control groups (nnCON, NnCON and NNCON), plasma concentrations of IL-1, IL-2, IL-6, IL-10, IL-12 and INF-␥ did not differ between RYR1 genotypes (Figs. 1 and 2). Basal and control concentrations of TNF-␣ were greater (P < 0.05) in nn homozygous pigs than in heterozygous (Nn) and NN homozygous pigs (Fig. 2C). Th 4 h restraint evoked an increase in all measured cytokine concentrations and the significant differences in stress-induced effects on cytokine concentrations between the RYR1 genotypes occurred (Figs. 1 and 2). The greatest response in plasma IL-6, IL-10, IL-12 and TNF- ␣ was observed in nn homozygous pigs (P < 0.05, in comparison with Nn heterozygous and NN homozygous pigs). In NN homozygous pigs the greatest response in plasma IFN-␥ concentration was observed (P < 0.05, in comparison with NN and nn homozygous pigs). The plasma concentrations of IL-1, IL-2, IL-10, IL-12 returned to the control values at 480 min in each of the RYR1 genotype group (Figs. 1A, B and D and 2A). The plasma concentrations of
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Fig. 1. Plasma IL-1 (A), IL-2 (B), IL-6 (C) and IL-10 (D) concentrations during the 4-h restraint stress (0–240 min) and post-stress period (300–480 min) in pigs of different RYR1 genotypes (nn, Nn, NN: stressed groups; nnCON, NnCON, NNCON: control groups; n = 10 in each group). Significant differences between stressed and control pigs are indicated by asterisks (Tukey b test, P < 0.05). IL-1, interleukin-1; IL-2, interleukin-2; IL-6, interleukin-6; IL-10, interleukin-10; NN, homozygous wild type; Nn, heterozygous carrier; nn, homozygous mutant.
IL-6 (Nn and nn pigs), IFN-␥ (NN and Nn pigs) and TNF-␣ (nn pigs) were increased at 480 min in comparison with the respective control groups (Figs. 1C and 2B and C). 3.2. Plasma catecholamines Prior to the restraint, and in control groups (nnCON, NnCON and NNCON), plasma concentrations of adrenaline did not differ between RYR1 genotypes (Fig. 3A). Basal and control concentrations of noradrenaline were lower (P < 0.05) in NN homozygous pigs than in heterozygous (Nn) and NN homozygous pigs (Fig. 3B). As shown in Fig. 3, the 4 h restraint increased plasma adrenaline and noradrenaline concentrations in the RYR1 genotypes compared with the control groups. The increase in plasma noradrenaline concentration in NN homozygous pigs was significantly lower (P < 0.05) than in Nn heterozygous and nn homozygous pigs. In NN homozygous an Nn heterozygous pigs the plasma adrenaline concentrations returned to the control values at 120 min and in nn homozygous pigs at 180 min (Fig. 3A). Plasma noradrenaline concentrations returned to the baseline at 180, 240 and 480 min in NN homozygous, Nn heterozygous and nn homozygous pigs respectively.
3.3. Regression analysis As shown in Table 1, IL-1, IL-2, IL-10, IL-12, IFN-␥, and TNF-␣, measures were positively intercorrelated in nn homozygous and Nn heterozygous pigs. In NN homozygous pigs IL-1, IL-2, IL-10 and TNF-␣ were positively intercorrelated. In all genotypes TNF-␣ measures were positively correlated with IL-6 concentrations. In nn homozygous and Nn heterozygous pigs there was a significant positive correlation between all measured cytokines (with the exception of IL-6) and plasma catecholamine concentrations. In NN homozygous pigs there was a positive correlation between plasma IL-1, IL-10 and TNF-␣ and catecholamine measures. In the present study, pigs subjected to the prolonged, 4 h restraint showed significant differences in stress-induced effects on the plasma cytokine response between the RYR1 genotypes. Pigs homozygous for the RYR1 gene mutation (nn), exhibited the greatest plasma IL-6, IL-10, IL-12 and TNF-␣ concentrations during the 4 h restraint as compared to RYR1 the gene mutation carriers (Nn) and wild type (NN) pigs. The present study provides the first description of the possible influence of the RYR1 gene status on cytokine response during the prolonged restraint stress. The findings corroborate and extend our observation that
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Table 1 Pearson correlation coefficients between plasma cytokines (IL-1, IL-2, IL-6, IL-10, IL-12, IFN-␥, and TNF-␣ and plasma catecholamines (NA, A) during the 4 h restraint (0–240 min) in homozygous mutant (nn) pigs, heterozygous (Nn) pigs and homozygous wild type (NN) pigs (df = 160 for each genotype). Genotype
nn
Nn
NN
IL-1 b
IL-2
IL-6
IL-10
IL-12
IFN-␥
TNF-␣
NA
– 0.185 −0.163 0.710b 0.326b 0.058 −0.298a
– 0.492b 0.470b 0.756b 0.690b 0.368b
– 0.821b 0.639b 0.681b 0.580b
– 0.849b 0.809b 0.529b
– 0.631b 0.308b
– 0.763b
– 0.157 −0.021 0.110 0.481b 0.078 −0.144
– 0.511b 0.835b 0.828b 0.824b 0.562b
– 0.456b 0.290a 0.443b 0.306a
– 0.676b 0.800b 0.632b
– 0.681b 0.361b
– 0.747b
– 0.486b 0.146 0.093
– 0.729b 0.593b
– 0.850b
IL-2 IL-6 IL-10 IL-12 IFN-␥ TNF-␣ NA A
0.732 −0.126 0.520b 0.580b 0.686b 0.431b 0.834b 0.873b
– −0.216 0.482b 0.715b 0.686b 0.677b 0.757b 0.687b
IL-2 IL-6 IL-10 IL-12 IFN-␥ TNF-␣ NA A IL-2 IL-6
0.754b 0.108 0.839b 0.409b 0.736b 0.761b 0.809b 0.558b 0.523b 0.148
– 0.138 0.716b 0.383b 0.545b 0.618b 0.588b 0.281a – −0.215
IL-10 IL-12 IFN-␥ TNF-␣ NA A
b
a
0.658 0.127 0.763b 0.694b 0.497b 0.407b
0.256 0.083 −0.020 0.455b 0.127 0.120
– 0.099 −0.082 0.584b 0.410b 0.057 −0.090
– 0.024 0.422b 0.729b 0.833b 0.826b
– 0.079 0.482b 0.144 0.054
Abbreviations: A: adrenaline; IL-1: interleukin-1; IL-2: interleukin-2; IL-6: interleukin-6; IL-10: interleukin-10; IL-12: interleukin-12; IFN-␥: interferon-␥; NE: noradrenaline; TNF-␣: tumor necrosis factor-␣. a P < 0.01. b P < 0.001.
pigs mutated for RYR1 gene show the increased NK cell cytotoxicity response to the restraint in comparison with NN homozygous and Nn heterozygous pigs (Ciepielewski et al., 2013). An imbalance in the neuroendocrine–immune relationship may contribute to a number of pathologies, most notably those relating to stress (Steptoe et al., 2007). It is recognized that the immune system is regulated by internal regulatory pathways with own signals (Besedovsky and Rey, 2007; Borghetti et al., 2009). The signals include pro-inflammatory and anti-inflammatory cytokines and circulating concentrations of such cytokines as IL-1, IL6 and TNF-␣ are markers of inflammation (Hansel et al., 2010). These molecules have also been studied as the possible link between the stress response and immune system, as these cytokines have been shown to be involved in response to various stress stimuli and influence and/or modulate the catecholaminergic systems and the HPA axis functions (Haddad et al., 2002; Goshen and Yirmiya, 2009; Dimitrijevic et al., 2012). The concomitant restraintinduced release of pro-and anti-inflammatory cytokines observed in the current study shows that the opposite inflammatory mechanisms were activated in the course of the prolonged stress response. Moreover, these changes were time dependent, and the significant positive correlations between the circulating cytokine measures occurred. It could be suggested that stress can alter immune function by affecting the different subpopulations of T lymphocytes (Appels et al., 2000; Calcagni and Elenkov, 2006). Thus, the stress-induced release of pro-inflammatory cytokines from T helper (Th) 1 cells and the anti-inflammatory cytokines
from Th2 cells could produce the opposite inflammatory (pro-and anti-inflammatory) processes (Wirtz et al., 2007). In the present study, a single exposure to the restraint increased the plasma concentrations of IL-2, IL-12 and IFN␥. These cytokines have been described as the potentiators of the NK cell activity (Knoblock and Canning, 1992). The increase in these cytokines may contribute to modulation of stress-induced changes in NK cell activity observed in our previous study (Ciepielewski et al., 2013). Recent findings in human and animal studies suggest that different stressors may alter the patterns of cytokine production and release in vivo and in vitro (de Groot et al., 2001; Bartolomucci et al., 2003; Steptoe et al., 2007; Voorhees et al., 2013). In experiments performed in pigs the stressors such as transportation (Lv et al., 2011), isolation (Tuchscherer et al., 2009), changes in environmental temperature (Carroll et al., 2012) or lipopolysaccharide injection (Collier et al., 2011) strongly affected the plasma immunoregulatory cytokines concentrations. Although divergent effects of acute stress on cytokine release have been reported, most of the studies show that the cytokine responsiveness of an acute stressor is related to the catheholamine system or to the hypothalamo–pituitary–adrenal (HPA) axis activity (Chesnokova and Melmed, 2002; Szelenyi and Vizi, 2007). In the current study, the positive correlations observed between the IL-1, IL-10, TNF-␣ and circulating catecholamines suggest the possible direct regulatory effect of adrenergic system on cytokine release (e.g. through the adrenergic receptors on lymphocytes). Systemic catecholamine effects are similar to those of glucocorticoids
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Fig. 3. Plasma adrenaline (A) and nordrenaline (B) concentrations during the 4-h restraint stress (0–240 min) and post-stress period (300–480 min) in pigs of different RYR1 genotypes (nn, Nn, NN: stressed groups; nnCON, NnCON, NNCON: control groups; n = 10 in each group. Significant differences between stressed and control pigs are indicated by asterisks (Tukey b test, P < 0.05). NN, homozygous wild type; Nn, heterozygous carrier; nn, homozygous mutant.
Fig. 2. Plasma IL-12 (A), IFN-␥ (B) and TNF-␣ (C) concentrations during the 4-h restraint stress (0–240 min) and post-stress period (300–480 min) in pigs of different RYR1 genotypes (nn, Nn, NN: stressed groups; nnCON, NnCON, NNCON: control groups; n = 10 in each group). Significant differences between stressed and control pigs are indicated by asterisks (Tukey b test, P < 0.05). IL-12, interleukin-12; IFN-␥, interferon-␥; TNF- ␣, tumor necrosis factor-␣; NN, homozygous wild type; Nn, heterozygous carrier; nn, homozygous mutant.
in inhibition of IL-2, IL-12, IFN-␥ and TNF-␣ (PaninaBordignon et al., 1997), however in the present work only IL-6 concentration did not correlate with plasma adrenalin and noradrenalin which suggest more complex mechanism of pro-inflammatory cytokine-catecholamine relationship. In conclusion, it could be suggested that 4 h restraint activates both the pro- and anti-inflammatory cytokines
release but the intensity of the release could be influenced by the initial state of the animal resulted from the possessing of the RYR1 gene mutation. The IL-6, IL-10, IL-12 and TNF-␣ release during the 4 h restraint in nn homozygous pigs seem to show a stronger stress-related response (as compared with those of the other two groups), however the mechanism by which the RYR1 gene mutation affects the immune functions remain elusive.
Conflict of interest statement None of the authors of this paper has any financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
Acknowledgements This work was supported by Grant N N303 2923 34 from Ministry of Science and Higher Education of Poland.
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References Appels, A., Bär, F.W., Bär, J., Bruggeman, C., de Baets, M., 2000. Inflammation, depressive symptomtology, and coronary artery disease. Psychosom. Med. 62, 601–605. Bartolomucci, A., Palanza, P., Sacerdote, P., Ceresini, G., Chirieleison, A., Panerai, A.E., Parmigiani, S., 2003. Individual housing induces altered immune-endocrine responses to psychological stress in mice. Psychoneuroendocrinology 28, 540–558. Bates, R.O., Doumit, M.E., Raney, N.E., Helman, E.E., Ernst, C.W., 2012. Association of halothane sensitivity with growth and meat quality in pigs. Animal 6, 1537–1542. Besedovsky, H.O., Rey, A.D., 2007. Physiology and psychoneuroimmunology: a personal view. Brain Behav. Immun. 21, 34–44. Borghetti, P., Saleri, R., Mocchegiani, E., Corradi, A., Martelli, P., 2009. Infection immunity and the neuroendocrine response. Vet. Immunol. Immunopathol. 130, 141–162. Calcagni, E., Elenkov, I., 2006. Stress system activity, innate and T helper cytokines and susceptibility to immune-related diseases. Ann. NY Acad. Sci. 1069, 62–76. Carroll, J.A., Burdick, N.C., Chase Jr., C.C., Coleman, S.W., Spiers, D.E., 2012. Influence of environmental temperature on the physiological endocrine, and immune responses in livestock exposed to a provocative immune challenge. Domest. Anim. Endocrinol. 43, 146–153. Chesnokova, V., Melmed, S., 2002. Minireview: neuro-immuno-endocrine modulation of the hypothalamic–pituitary–adrenal (HPA) axis by gp130 signaling molecules. Endocrinology 143, 1571–1574. Ciepielewski, Z.M., Stojek, W., Glac, W., Wrona, D., 2013. Restraint effects on stress-related hormones and blood natural killer cell cytotoxicity in pigs with a mutated ryanodine receptor. Domest. Anim. Endocrinol. 44, 195–203. Collier, C.T., Williams, P.N., Carroll, J.A., Welsh Jr., T.H., Laurenz, J.C., 2011. Effect of maternal restraint stress during gestation on temporal lipopolysaccharide-induced neuroendocrine and immune responses of progeny. Domest. Anim. Endocrinol. 40, 40–50. Correa, S.G., Maccioni, M., Rivero, V.E., Iribarren, P., Sotomayor, C.E., Riera, C.M., 2007. Cytokines and the immune-neuroendocrine network: what did we learn from infection and autoimmunity? Cytokine Growth Factor Rev. 18, 125–134. de Groot, J., Ruis, M.A., Scholten, J.W., Koolhaas, J.M., Boersma, W.J., 2001. Long-term effects of social stress on antiviral immunity in pigs. Physiol. Behav. 73, 145–158. Dimitrijevic, M., Stanojevic, S., Kustrimovic, N., Leposavic, G., 2012. Endpoint effector stress mediators in neuroimmune interactions: their role in immune system homeostasis and autoimmune pathology. Immunol. Res. 52, 64–80. Dirks, B., Vorwalter, C., Grünert, A., 1988. Basal plasma catecholamine level determination using HPLC-ED and different sample cleanup techniques. Chromatographia 25, 223–229. Edfors-Lilja, I., Lundström, K., Nyberg, L., Rundgren, M., 1987. Influence of the Hal locus and standardized stress on antibody response and in vitro reactivity of peripheral blood lymphocytes in pigs. Vet. Immunol. Immunopathol. 14, 157–171. Fujii, J., Otsu, K., Zorzato, F., de Leon, S., Khanna, V.K., Weiler, J.E., O’Brien, P.J., MacLennan, D.H., 1991. Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia. Science 5018, 448–451. Geers, R., Bleus, E., Van Schie, T., Villé, H., Gerard, H., Janssens, S., Nackaerts, G., Decuypere, E., Jourquin, J., 1994. Transport of pigs different with respect to the halothane gene: stress assessment. J. Anim. Sci. 72, 2552–2558.
181
Goshen, I., Yirmiya, R., 2009. Interleuki-1 (IL-1) A central regulator of stress responses. Front. Neuroendocrinol. 30, 30–45. Haddad, J.J., Saadé, N.E., Safieh-Garabedian, B., 2002. Cytokines and neuro-immune-endocrine interactions: a role for the hypothalamic–pituitary–adrenal revolving axis. J. Neuroimmunol. 133, 1–19. Hansel, A., Hong, S., Camara, R.J.A., von Kanel, R., 2010. Inflammation as a psychophysiological biomarker in chronic psychosocial stress. Neurosci. Biobehav. Rev. 35, 115–121. Knoblock, K.F., Canning, P.C., 1992. Modulation in vitro porcine natural killer cell activity by recombinant interleukin-1␣, interleukin-2 and interleukin-4. Immunology 76, 299–304. Lv, Q., Zhang, S., Zhao, R., 2011. Transportation stress alters the expression of immunoregulatory cytokines in the porcine thymus. Vet. J. 187, 229–233. MacLennan, D.H., Chen, S.R., 1993. The role of the calcium release channel of skeletal muscle sarcoplasmic reticulum in malignant hyperthermia. Ann. NY Acad. Sci. 707, 294–304. Mitchell, G., Heffron, J.J.A., 1982. Porcine stress syndromes. Adv. Food Res. 28, 167–230. Nelson, T.E., 2002. Malignant hyperthermia: a pharmacogenetic disease of Ca++ regulating proteins. Curr. Mol. Med. 2, 347–369. Otsu, K., Phillips, M.S., Khanna, V.K., de Leon, S., MacLennan, D.H., 1992. Refinement of diagnostic assays for a probable causal mutation for porcine and human malignant hyperthermia. Genomics 13, 835– 837. Panina-Bordignon, P., Mazzeo, D., Lucia, P.D., D’Ambrosio, D., Lang, R., Fabbri, L., Self, C., Sinigaglia, F., 1997. Beta2-agonists prevent Th1 development by selective inhibition of interleukin 12. J. Clin. Invest. 100, 1513–1519. Steptoe, A., Hamer, M., Chida, Y., 2007. The effects of acute psychological stress on circulating inflammatory factors in humans: a review and meta-analysis. Brain Behav. Immun. 21, 901–912. Szelenyi, J., Vizi, E.S., 2007. The catecholamine-cytokine balance: interaction between brain and the immune system. Ann. NY Acad. Sci. 1113, 311–324. Tokarski, J., Wrona, D., Piskorzynska, M., Borman, A., Witkowski, J., Jurkowski, M., Kamyczek, M., 1992. The influence of immobilization stress on natural killer cytotoxic activity in halothane susceptible and resistant pigs. Vet. Immunol. Immunopathol. 31, 371–376. Tuchscherer, M., Kanitz, E., Puppe, B., Tuchscherer, A., Viergutz, T., 2009. Changes in endocrine and immune responses of neonatal pigs exposed to a psychosocial stressor. Res. Vet. Sci. 87, 380–388. Van den Maagdenberg, K., Stinckens, A., Claeys, E., Buys, N., De Smet, S., 2008. Effect of the insulin-like growth factor-II and RYR1 genotype in pigs on carcass and meat quality traits. Meat Sci. 80, 293–303. Voorhees, J.L., Tarr, A.J., Wohleb, E.S., Godbout, J.P., Mo, X., Sheridan, J.F., Eubank, T.D., Marsh, C.B., 2013. Prolonged restraint stress increases IL-6, reduces IL-10 and causes persistent depressive-like behaviour that is reversed by recombinant IL-10. PLoS ONE 8, e58488. Weaver, S.A., Dixon, W.T., Schaefer, A.L., 2000. The effects of mutated skeletal ryanodine receptors on hypothalamic–pituitary–adrenal axis function in boars. J. Anim. Sci. 78, 1319–1330. Wirtz, P.H., von Känel, R., Emini, L., Suter, T., Fontana, A., Ehlert, U., 2007. Variations in anticipatory cognitive stress appraisal and differential proinflammatory cytokine expression in response to acute stress. Brain Behav. Immun. 21, 851–859. Woiciechowsky, C., Shoning, B., Lanksch, W.R., Volk, H.D., Docke, W.D., 1999. Mechanisms of brain-mediated systemic anti-inflammatory syndrome causing immunodepression. J. Mol. Med. 77, 769–780.
