The effects of ryanodine receptor (RYR1) mutation on natural killer cell cytotoxicity, plasma cytokines and stress hormones during acute intermittent exercise in pigs.

Stress susceptibility has been mapped to a single recessive gene, the ryanodine receptor 1 (RYR1) gene or halothane (Hal) gene. Homozygous (Hal(nn)), mutated pigs are sensitive to halothane and susceptible to Porcine Stress Syndrome (PSS). Previous studies have shown that stress-susceptible RYR1 gene mutated homozygotes in response to restraint stress showed an increase in natural killer cell cytotoxicity (NKCC) accompanied by more pronounced stress-related hormone and anti-inflammatory cytokine changes. In order to determine the relationship of a RYR1 gene mutation with NKCC, plasma cytokines and stress-related hormones following a different stress model - exercise - 36 male pigs (representing different genotypes according to RYR1 gene mutation: NN, homozygous dominant; Nn, heterozygous; nn, homozygous recessive) were submitted to an intermittent treadmill walking. During the entire experiment the greatest level of NKCC and the greatest concentrations of interleukin (IL-) 6, IL-10, IL-12, interferon (IFN-)γ and tumor necrosis factor-α and stress-related hormones (adrenaline, prolactin, beta-endorphin) were observed in nn pigs, and the greatest concentration of IL-1 and growth hormone in NN pigs. Immunostimulatory effects of intermittent exercise on NKCC in nn pigs were concomitant with increases in IL-2, IL-12 and IFN-γ, the potent NKCC activators. Our findings suggest that stress-susceptible pigs RYR1 gene mutated pigs develop a greater level of NKCC and cytokine production in response to exercise stress. These results suggest that the heterogeneity of immunological and neuroendocrine response to exercise stress in pigs could be influenced by RYR1 gene mutation.

Natural killer cell cytotoxicity, cytokine and neuroendocrine responses to opioid receptor blockade during prolonged restraint in pigs.

This study evaluated porcine natural killer cell cytotoxicity (NKCC), plasma cytokines including interleukin (IL) 1ß, IL-6, IL-10, IL-12 and tumor necrosis factor-α and plasma stress-related hormones including prolactin (PRL), growth hormone (GH), ß-endorphin (BEND), ACTH and cortisol (COR) during a 4h restraint and recovery phase after saline or naloxone (1mg/kg BW) administration. The restraint preceded with saline altered NKCC and IL-12 concentration (an early from 15 to 60 min increase followed by a decrease) and increased other measured cytokines and hormones concentrations. Naloxone pretreatment blocked the suppressive effects of the restraint on NKCC and IL-12 and altered IL-10, IL-6, TNF-α, PRL and ACTH concentrations. Furthermore, in naloxone-injected pigs, a positive correlation was found between NKCC and all measured cytokines (with the exception of IL-6) and BEND, ACTH and COR. Results suggest that naloxone-sensitive opioid pathways could influence the mechanisms underlying the immune system (including NKCC) response during stress.

Restraint effects on stress-related hormones and blood natural killer cell cytotoxicity in pigs with a mutated ryanodine receptor.

A mutation in the ryanodine receptor gene (RYR1) of the calcium release channel is responsible for increased stress susceptibility in pigs. In the present study, the relation of a mutation in RYR1 with the neuroendocrine (stress-related hormone) response and the immune defense represented by natural killer cell cytotoxicity (NKCC) during a 4-h restraint and recovery phase in 60 male pigs was investigated. Blood samples were collected from pigs previously divided into RYR1 genotypes (nn, Nn, NN), based on PCR amplification and restriction analyses. The blood samples collected during the restraint and recovery phases of the experiment were used to determine NKCC ((51)Cr-release assay), large granular lymphocyte number (hematologic method), and plasma concentrations of prolactin (PRL), GH, ACTH, and cortisol (COR) (by specific RIA). The greatest degree of NKCC response (P < 0.05) to restraint stress relative to controls was observed for the stress-susceptible homozygote group (nn). Measures of stress-related hormones were positively correlated with NKCC during the entire experimental period (P < 0.001 for all investigated hormones) in the nn group. Immunostimulatory effects in the early (0-60 min) phase of restraint were associated with increased hormone responses, especially PRL and GH. In the late (180-240 min) phase of stress and the recovery phase (480 min), a decrease in immune response was accompanied by an elevated COR response in all RYR1 genotypes. Moreover, divergent responses of both PRL (greatest in nn, P < 0.001) and GH (greatest in NN, P < 0.001) to the 4-h restraint were observed. Our results suggest that stress-susceptible RYR1-mutated homozygotes develop a greater level of immune defense, including cytotoxic activity of NK cells, and accompanied by more pronounced stress-induced changes in neuroendocrine response than stress-resistant heterozygous (Nn) and homozygous (NN) pigs.

Stress-induced changes in peripheral natural killer cell cytotoxicity in pigs may not depend on plasma cortisol.

The study examined cortisol (COR) involvement in stress-related changes in natural killer cell cytotoxicity (NKCC). The relationship between blood COR level, phasic changes in NKCC, and the number of large granular lymphocytes (LGL) was examined in pigs during the course of 4-h immobilization stress (IMB) and for 6 days after its termination. NKCC was determined using 18-h 51Cr-release assay, LGL number was assessed with a standard hematological method, and plasma COR level was measured by radioimmunoassay. The blood level of COR was increasing during IMB (max 446Delta% at the second hour) and decreased after its termination (max -59Delta% on day 2). Changes in NKCC level and LGL number were biphasic; i.e., an initial increase in both measures (NKCC max 24Delta%, LGL max 18Delta%) in an early phase of stress (0-1h) was followed by their subsequent decrease (NKCC max -35Delta%, LGL max -41Delta%) in the late phase (3-4 h) of stress, which persisted for several days after termination of IMB. Thus, in the early phase of stress, there was a positive correlation between NKCC, LGL number, and COR levels (all elevated); a positive correlation between the measures also occurred after termination of IMB (all decreased). A negative correlation between COR and NKCC, which might be indicative of COR-related immunosuppression, was found only in the late (3-4 h) phase of stress. It is concluded that COR may be only one of multiple factors (possibly antagonistic) determining an actual immune response during stress.

Peripheral blood natural killer cell cytotoxicity after damage to the limbic system in the rat.

The present work was aimed at examining the possible involvement of different parts of the septal area (dorsal, medial, lateral, and septohypothalamic nucleus), the basolateral amygdala, and the bed nucleus of the stria terminalis (BNST) in the regulation of the cytotoxic activity of NK cells (NKCC). The experimental approach included performing electrolytic (or sham) lesions in the tested brain areas and to measuring the peripheral blood NKCC (chromium-51 release assay), the number of leukocytes and lymphocytes, and the plasma corticosterone levels both before and at different time points after the lesion. Lesions were also induced in the three extralimbic structures: the paraventricular hypothalamic nucleus (PVN), the dorsal caudate-putamen, and the cerebellum. To test for a possible effect on NKCC of stress associated with blood collection, anesthesia, cranial surgery, and passing electric current through the brain the proper control experiments were also performed. Lesions of the medial septum and BNST caused gradual depression of NKCC, which peaked on the 10th day after the lesion, followed by a recovery to the baseline on days 21 (medial septum) and 42 (BNST) postinjury. In the respective sham-lesioned groups, mere insertion of electrodes into the medial septum and BNST evoked transient enhancement of NKCC (on the 3rd postlesion day), probably resulting from mechanical stimulation of the nervous tissue. Destruction of the other limbic and extralimbic structures appeared ineffective. After PVN lesions NKCC remained unchanged, despite an approximately 60% decrease in the basal corticosterone level. No adverse effects of the experimental and surgical procedures on NKCC, leukocyte and lymphocyte number, and corticosterone level were found, indicating that electrolytic lesions and other stereotaxic techniques can be safely used to study the brain-immune system interactions. The results obtained raise the question about the interrelationship between the medial septum and the hippocampal formation, BNST, the medial amygdala, and the hypothalamus (both medial and lateral) as a possible circuit involved in the regulation of cellular immune functions.