EFFECT OF TRANSPORT STRESS ON PERIPHERAL BLOOD LYMPHOCYTE SUBSETS AND Th CYTOKINES IN PIGS

In order to investigate transport stress on porcine peripheral blood lymphocyte subsets and Th cytokines, blood samples were collected from pigs before and after transport. Creatine kinase (CK), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), glucose, and cortisol in the serum were measured. The number of leukocytes and lymphocytes, percentages of lymphocyte subsets, as well as Th cell cytokines level and their mRNA expression were detected, respectively. After transport, the level of CK, glucose and interleukin (IL)-4 increased significantly (p<0.01), LDH, Th memory cells, natural killer and interferon (IFN)increased significantly (p<0.05), cortisol, number of leukocytes and lymphocytes decreased (p<0.01), percentages of T cells, naïve Th cells and cytotoxic T lymphocytes decreased significantly (p<0.05). The mRNA expressions IL-2 and IFNwere down regulated, p<0.01 and p<0.05, respectively. While IL-4, IL6, and IL-10 were up regulated, but only IL-10 displayed a highly significant difference (p<0.01). These data suggested that transport could cause immune suppression in pigs, which influences cellular immunity more than humoral immunity, and humoral immunity may play an important role in transport stress. Proper measures should be taken to reduce susceptibility of infection after transport.


INTRODUCTION
Transport stress is a complex stress, in which many stressors are involved, such as upload and offload, crowding, rocking, noise, temperature, food and water deprivation (Earley et al., 2010).Stress response occurs easily in pigs due to transport (Von Borell, 2001), causing pale, soft, and exudative meat (Pérez et al., 2002).Severe stress may even result in death, with mortality rates reported up to 0.1%-1.0%(Allen et al., 1974;Averós, 2008;Warriss et al., 1989).The serum concentrations of creatine kinase (CK) (Hong et al., 2007), lactate dehydrogenase (LDH), glucose, and cortisol can be affected by transport stress (Yoshioka et al., 2004); transport stress can also increase amounts of heat shock protein (HSP)70 and Hsp70 mRNA expression in the heart and kidney tissues of transported pigs (Hong et al., 2007).
Stress inhibits the immune system function of animals, which can increase morbidity (Salak-Johnson and McGlone, 2007).Immune suppression caused by transport stress has also been reported (Frank et al., 1984).Studies have shown that transport stress increases the leukocyte number, but decreases the lymphocyte number in peripheral blood (Dalin et al., 1993), and changes the serum levels of IL-2, IL-6, and IL-10, as well as the expression of their receptors in the thymus (Qiongxia, 2011), which indicates that transport stress may have an effect on the porcine immune system.However, there is limited knowledge about the effects of transport stress on lymphocyte subsets and Th cell cytokines in porcine peripheral blood.
Effects of transport stress on the immune system are an important problem during new stock introduction and transfer of groups to new locations.The age at which introduction or group transferring of pigs occurs in China is almost the same with grown and fattening pigs.Therefore, grown and fattening pigs were employed to study the effects of transport stress on the porcine immune system, by analyzing total lymphocyte number and subsets, Th cell cytokines IFN-g, IL-4, and IL-6, as well as the mRNA expression of Th cell cytokines, aiming at providing more detail of porcine immunity response to transport stress.

Animals and transport
Five castrated male pigs (Sherlock and Landrace cross-breed) with mean body weight of 105.2±2.4 kg from one farmhouse in a Peking suburb were randomly selected for the experiment.All pigs were loaded on the truck under commercial transport conditions.The transport distance was about 130 km lasting about 130 min with a speed of 60-70 km/h, which took place in July in the morning from 9:40 am to 11:50 am.The weather was cloudy and the temperature was between 26 o C-29 o C.
All experimental protocols were approved by the Committee for the Care and Use of Experimental Animals at China Agricultural University.

Blood collection and treatment
Before loading and 2 hrs after unloading, 10 mL of blood was collected from the precaval vein.Each blood sample was divided into two tubes equally with/without heparin sodium anticoagulant (Sanli, Hunan, China), and then kept in an icebox before arriving at the laboratory.Serum was collected from blood samples without added anticoagulant after being centrifuged at 4 o C and 3000 x g for 15 min, which was stored at -20 o C for further analysis.Heparinized blood samples were used for complete blood counts, flow cytometry and extraction of total RNA.

Tests of biochemistry and cortisol
Concentrations of serum CK, LDH, alkaline phosphatase (ALP), and glucose were detected by an automated biochemical analyzer (TBA-40FR, Toshiba, Tokyo, Japan), in which performance rate method was used for CK, LDH, and ALP and glucose oxidase method was used for glucose.The level of cortisol in the serum was measured by immunoassay analyzer (MiniVidas, biomérieux, Fance) with radiation immunofluorescence method.

Determination of number of leukocytes and lymphocytes and lymphocyte subsets in peripheral blood
Leukocyte and lymphocyte numbers were counted by an automated blood cell analyzer (Mek-7222K, Nihon Konden, Tokyo, Japan) with electric-resistivity method.
Flow cytometry was employed to investigate the difference of porcine lymphocyte subsets in peripheral blood between, before, and after transport, which was represented as percentage.Triple-color flow cytometry with CD3, CD4, and CD8 was used to analyze the lymphocytes subsets in porcine peripheral blood lymphocytes (PBL) with monoclonal antibodies anti-pig CD3e-SPRD (PPT3, BD Biosciences, New York, NY, USA), anti-pig CD4a-FITC (74-12-4, BD Biosciences, New York, NY, USA), and anti-pig CD8a-PE (76-2-11, BD Biosciences, New York, NY, USA) according to the methods used in our previous study (Jiefeng et al., 2011).Briefly, 200 mL heparinized blood samples were labeled simultaneously with the anti-pig monoclonal antibodies mentioned above for 30 min in dark at room temperature.Appropriate negative controls and labeled single antibodies were prepared at the same time.All samples were added to 1000 mL RBC lysate solution and incubated in dark at room temperature for 15 min, then centrifuged at 4 o C and 1500 x g for 5 min.The supernatant was discarded.This procedure was repeated if necessary.The sample was washed with 1 mL cold PBS and centrifuged at 4°C and 1500 x g for 5 min and resuspended with 0.5 mL cold PBS to ensure that each sample contained at least 1 000 000 cells/mL.For each sample, 10 000 cells were analyzed using a FACS Calibur TM flow cytometer (BD Biosciences, New York, NY, USA).CD3 + CD4 -CD - indicates g d T cell, CD3 + CD4 + CD8 -indicates naïve Th cell, CD3 + CD4 -CD8 + indicates cytotoxic lymphocytes (CTL), CD3 + CD4 + CD8 + indicates Th memory cells, and CD3 -CD4 -CD8 + indicates natural killer (NK) cells.

Determination of serum cytokines by ELISA
Concentrations of serum IFN-g, IL-4, and IL-6 were measured by commercial ELISA kits (R & D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions; the assay ranges were 5~2,000 pg/L, 1.8~80 ng/L, and 20~6,000 ng/L, respectively.Each sample optical density was read at 450 nm using a microliter plate reader (Synergy HT, Bio-Tek, Winooski, VT, USA) within Acta Veterinaria (Beograd), Vol.63, No. 2-3, 177-190, 2013.10 min.A standard curve was constructed by plotting the absorbance obtained for each reference standard against its concentration on a linear graph, with absorbance values on the (x) axis and concentration on the (y) axis.

Expression of cytokines mRNA
Extraction of total RNA and reverse transcription.Total RNA was isolated from peripheral blood lymphocytes, which was separated from 1000 mL peripheral blood by a lymphocyte separation medium (solarbio, Beijing, China) followed by the use of phenol and guanidine isothiocyanate-based TRIZOL reagents (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.The concentration and purity of isolated RNA were determined by a spectrophotometer (SmartSpec plus, BIO-RAD, Hercules, CA, USA) based on the OD 260 /OD 280 ratio.The mRNA was reversely transcribed by the following two procedures: firstly, 1.0 mg mRNA was isolated from each blood sample, and 1.0 mL Oligo-dT18 (0.5 mg/mL) and RNase-free water was added to a 13 mL reaction system, which was incubated at 70 o C for 5 min.Secondly, 1.25 mL dNTPs (10 mM), 0.5 mL RNase inhibitor (40 U/mL), 1.0 mL M-MLV transcriptase (200 U/mL), 5.0 mL M-MLV RT reaction buffer (5×) (Promega, Madison, WI, USA) and 4.25 mL RNase-free water were added into the first reaction system, then incubated in 42 o C for 60 min.The reverse transcription products (cDNA) were stored at -20 o C for quantitative RT-PCR.
Semi-quantitative real-time PCR.The mRNA expressions of IFN-g, IL-2, IL-4, IL-6, and IL-10 were semi-quantitatively determined by RT-PCR.Semi-quantitative RT-PCR analysis was carried out using the DNA Engine Mx3000P ® fluorescence detection system against a double-stranded DNA-specific fluorescent dye (Stratagene, La Jolla, CA, USA) according to optimized PCR protocols.b-actin was amplified at the same time with the target genes as the control.The cDNA was subjected to RT-PCR using the primer pairs and reaction condition as listed in Table 1.A 20 mL reaction system was used in the RT-PCR, containing 10 mL of SYBR Green qPCR mix, 0.3 mL of reference dye, 1 mL of each primer (both 10 mmol/L), and 1 mL of cDNA template.Expression levels were determined by the threshold cycle (CT) method as described by the manufacturer of the detection system.This method was applied to each gene by calculating the expression 2-DDCT , in which DDCT was expressed as: (CT gene -CT b-actin ) Post-transport -(CT gene -CT b-actin ) Pre-transport .

Statistical analysis
All data are expressed as mean ± SD (standard deviation).SPSS software (version 11.5; SPSS Inc., Chicago, IL, USA) was employed for statistical analysis including independent-sample T-tests, in which p<0.05 was considered significant, p<0.01 was considered highly significant.

Changes of serum levels of creatine kinase, lactate dehydrogenase, alkaline phosphatase, glucose and cortisol
In the current experiment, the serum concentrations of CK and glucose increased significantly (p<0.01),LDH increased significantly (p<0.05),ALP also increased but not significantly (p>0.05) (Fig. 1A and B), and the serum level of cortisol decreased significantly (p<0.01) (Fig. 1C).

Changes of the number of leukocytes and lymphocytes, and percentages of lymphocyte subsets
The number of both leukocytes and lymphocytes in PBL decreased significantly after transport, (p<0.01;Fig. 2).
All of the percentages of lymphocyte subsets changed significantly posttransport when compared with pre-transport.g d T cells, naïve Th cells and CTL cells decreased significantly (p<0.05), and Th memory cells and NK cells increased significantly (p<0.05) (Fig. 3).

Changes of serum T helper cell cytokines
Serum concentrations of IFN-g, IL-4, and IL-6 were detected before and after transport in the current study.All the concentrations of cytokines increased with different levels after transport.IFN-g increased significantly (p<0.05;Fig. 4 A), and IL-4 increased highly significantly (p<0.01;Fig. 4 B), but there was no significant differences with IL-6 between before and after transport (p>0.05;Fig. 4 C

Evaluation of porcine transport stress
Previous studies have shown that serum concentrations of CK, LDH, and glucose increase after transport (Bao, 2008;Deyi et al., 1996).Dynamic study of cortisol done by Dalin et al. (1993) showed that serum concentration of cortisol increased to peak level after 30 min of transport, decreased immediately after offload, and reached the lowest point at 4 h post transport.Investigations done by Bradshaw et al. (1996) also revealed a decrease of cortisol after transport.Similarly, a CK, LDH, ALP, and glucose increase and cortisol decrease after transport were found within the current study (Fig. 1 A, B, and C), indicating that a stress response occured during transport.

Inhibition effect of transport stress on lymphocyte subsets
The number of leukocytes and lymphocytes in the peripheral blood is also used as a marker of immune status in transported animals (Obernier and Baldwin, 2006;Stanger et al., 2005) of leukocyte number and decrease of lymphocyte number in the peripheral blood of gilt (Dalin et al., 1993).The function of lymphocytes, and the production of cytokines, can be influenced by a variety of stressors (Meagher et al., 2007).T lymphocytes play a variety of important roles in immune regulation, inflammation, and protective immune response throu responses of lymphocyte subsets (Wilhelm et al., 2009).Our results showed that both leukocyte and total lymphocyte numbers decreased after transport, indicating the porcine immune status was suppressed.In addition, decreased percentages of g d T cells, naïve Th cells and CTL cells, and an increase in Th memory cells and NK cells were found after transport in our study (Fig. 3).g d cells are thought to play an important role in innate and adaptive immune responses (Devilder et al., 2009;Pitard et al., 2008;Tian et al., 2006), including acting as antigen presenting cells by expressing major histocompatibility complex (MHC) II (Takamatsu et al., 2002), releasing cytokines/chemokines, having a cytotoxic function (Carding and Egan, 2002), attacking infected cells, and having immune memory functions (Takamatsu et al., 2006).Early activation of g d T cells can promote the production of IFN-g, which improves the function of macrophages and NK cells, having an early antibiotic function before the response of a b T cells (Hayday, 2000).Our results showed that the percentage of g d T cells decreased after transport (Fig. 2), likely resulting in the reduced function of this lymphocyte subset as mentioned above, therefore providing an opportunity for the invasion of pathogens, which may enhance the risk of infection by microorganisms under transport stress conditions.
Naïve Th cells differentiate into Th 1 or Th 2 cells after stimulation by antigens, providing cellular immune or humoral immune responses (Chaplin, 2010).IL-4 plays an important role in the differentiation of naïve Th cells toward Th 2 (Murphy, 1998); IL-6 can improve the early expression of IL-4 and inhibit the response of naïve Th cells to IFN-g, which results in the differentiation of naïve Th cells into Th 2 (Dienz and Rincon, 2009).The decrease of percentage of Naïve Th cells (Fig. 2) with the increase of serum IL-4 and IL-6 (Fig. 3 A and B) indicates that naïve Th cells may have preferentially differentiated into Th 2 .
CTL responds to stimulation by antigens by cell proliferation, production of IFN-g and tumor necrosis factor (TNF)-a, thus killing target cells (Bassaganya-Riera et al., 2004;Laval et al., 2002;Wolfgang et al., 2003).The expression of CD8 on CTL cells would be decreased in the presence of IL-4 and absence of IFN-g (Durrant and Metzger, 2010).The current study showed that the percentage of CTL decreased after transport (Fig. 2), which altered the immune status of transported animals, therefore increasing susceptibility to attack by pathogens.
The Th memory cell response to re-stimulation by antigen is cell proliferation, production of cytokines (IL-2, IFN-a, and IFN-g), and promotion of B lymphocytes to release immunoglobulins (Charerntantanakul et al., 2006;Hontecillas and Bassaganya-Riera, 2003;Zuckermann and Husmann, 1996).The number of Th memory cells increases with age, likely because of consistent contact with foreign antigens.Moreover, porcine Th memory cells would be induced to proliferate once re-stimulated by antigens (Wasin and James, 2007) The percentage of Th memory cells increased in the current study, likely due to the fact that transported pigs came into contact with antigens.NK cells represent an important cell population of the innate immune system with the ability to spontaneously attack pathogen-infected and malignant body cells, as well as to produce immune-regulatory cytokines (especially IFN-g), participating in the immune response (Wilhelm et al., 2009;Yokoyama, 2008).In vitro studies show that the activity of NK cells can be improved by IL-2 (Yata et al., 2009) and IFN-g (Charley et al., 1985), but can be inhibited by IL-4 (Canning and Knoblock, 1992).Therefore, the increase of serum IFN-g may have resulted in the percentage of NK cells being increased after transport in our experiment.However, the down regulation of IL-2 and IFN-g mRNA, and the up regulation of IL-4 mRNA, suggest that the activity of NK cells might decrease as a consequence, and this may be the reason that transported animals are at high risk and susceptible to pathogens.

Different effects of transport stress on T helper cell cytokines
The balance between Th 1 and Th 2 is disrupted by stress, resulting in a new homeostasis (Ilia and Chrousos, 2002).Cytokines play an important role in the differentiation of naïve Th cells into Th 1 or Th 2 (Coffman, 2006).Our studies showed that serum concentrations of IFN-g, IL-4, and IL-6 determined by ELISA increased post-transport (Fig. 4 A, B, and C), suggesting that transport stress induces naïve Th cells to differentiate into Th 1 or Th 2 .The mRNA expression of cytokines was tested by semi-quantitative RT-PCR, showing that the expressions of cellular immune cytokines IFN-g and IL-2 were down regulated, but humoral immune cytokines IL-4, IL-6, and IL-10 were up regulated (Fig. 5).Similar results have been found in a previous study (Qiongxia, 2011) suggesting that cellular immunity was enhanced in the early phase and then subsequently suppressed during transport, but humoral immunity continued to be strengthened.A previous study also showed that transport affects the humoral immune response of suckling calves by elevating the level of IgG 1 (Mackenzie et al., 1997), suggesting humoral immunity may play an important role in transport stress.
IL-6 plays an important role in promoting B lymphocyte differentiation into mature plasma cells, which release immunoglobulins, therefore enhancing humoral immunity (Commins et al., 2010).IL-6 also acts as a marker of bacterial infections in pigs (Caroline et al., 1998).In a previous study, the serum concentration of IL-6 increased after 1 h of transport, and the mRNA expression of IL-6 was up regulated and reached peak level after 4 h of transport (Qiongxia, 2011), which was similar to the findings in our experiment, indicating that bacterial infection might occur in transported animals and that humoral immunity was strengthened.

182Figure 1 .
Figure 1.Changes of serum levels of biochemistry indices and cortisol Mean of biochemical indexes and cortisol concentration in serum between pretransport and post-transport.(A) Serum levels of creatine kinase (CK), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP), all of which increased after transport, p<0.01, p<0.05 and P>0.05, respectively.(B) Serum level of glucose, which increased significantly after transport, p<0.01.(C) Serum level of cortisol, which decreased significantly after transport, p<0.01.Error bars represent the mean as ± SEM. *: p<0.05; **: p<0.01

Figure 2 .Figure 3 .
Figure 2. Number changes of leukocyte and lymphocyte in peripheral blood Mean of leukocyte (LEU) and lymphocyte (LYM) numbers in porcine peripheral blood between pre-transport and post-transport (x10 9 cells/L).Both LEU and LYM in peripheral blood decreased significantly after transport, p<0.01, p<0.01, respectively.Error bars represent the mean as ± SEM. *: p<0.05; **: p<0.01

Figure 4 .
Figure 4. Serum concentrations of T helper cell cytokines in peripheral blood Mean of major Th cell cytokines concentrations in serum.Interferon (IFN)-g, IL-4 and IL-6 increased after transport.There were significant differences of IL-4 and IFN-g between pre-transport and post-transport, p<0.01 and p<0.05, respectively.The difference of IL-6 between pre-transport and post-transport was not significant, p>0.05.Error bars represent the mean as ± SEM. *: p<0.05; **: p<0.01

Table 1 .
Wuren MA et al.: Effect of transport stress on peripheral blood lymphocyte subsets and Th cytokines in pigs Primers employed in the RT-PCR for the study ) used for detection of mRNAs specific for cytokines and housekeeping genes by RT-PCR in pigs.