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.

Cannabidiol-induced lymphopenia does not involve NKT and NK cells.

The major non-psychoactive compound of cannabis plant, cannabidiol, has been reported to be a promising therapeutic agent for many inflammatory, autoimmune and neurodegenerative diseases. In spite of growing interest in therapeutic use of cannabidiol very little is known about its influence on the immune system. Present study aimed to evaluate lymphocyte subsets distribution in peripheral blood after repeated, systemic administration of cannabidiol. Adult male Wistar rats received intraperitoneal injections of vehicle or cannabidiol at dose of 2.5 or 5 mg/kg/day, for 14 consecutive days. Blood samples were collected one hour after the last injection. Three-color immunofluorescent antibody staining procedure (CD3-FITC/CD45RA-PC7/CD161A-APC and CD3-FITC/CD4-PC7/CD8-APC) was used for determination of T, B, NK, NKT, T helper, and T cytotoxic lymphocyte subsets. Total leukocyte number and percentage numbers of leukocyte subpopulations were also assessed. Administration of cannabidiol at dose of 5 mg/kg caused a significant decrease in total leukocyte number and a significant fall in total numbers of T, B, and both T helper and T cytotoxic lymphocyte subsets. This immunosuppressive effect did not affect the total numbers of NK and NKT cells that are responsible for the primary, nonspecific antiviral and antitumor immune response. In contrast, administration of cannabidiol at dose of 2.5 mg/kg increased the total and percentage NKT cells numbers, and the percentage number of NK cells. The results suggest that repeated treatment with cannabidiol inhibits specific immunity by reduction of T, B, T cytotoxic, and T helper cell numbers, and may enhance nonspecific antiviral and antitumor immune response related to NK and NKT cells.

Amphetamine enhances natural killer cytotoxic activity via beta-adrenergic mechanism.

Although addiction to amphetamine (AMPH) is a serious social and medical problem, the data concerning AMPH - immune interactions are still not numerous. To analyze the mechanism of AMPH-induced changes in the function of the immune system, rats were pretreated with beta-adrenergic receptor antagonist propranolol (PROP; 5 mg/kg, i.p.) prior to AMPH (1 mg/kg, i.p.) administration. Natural Killer cells cytotoxicity (NKCC) ((51)Cr-release assay), the number of LGLs (NK cells) (Timonen method), leukocytes, lymphocytes and monocytes, and plasma corticosterone level (CORT) (RIA) were evaluated in the peripheral blood and spleen. In the peripheral blood increases in NKCC (+331 Delta %), as well as in LGL (+33 Delta %) and monocyte (+65 Delta %) number observed after AMPH were partially inhibited by PROP (respectively by 30%, 19%, and 30%) in contrast to lymphopenia (-19 Delta %) and granulocytosis (+65 Delta %) which were not affected by beta-blockade. In the spleen AMPH-induced decreases in NKCC (-25 Delta %) and in all the leukocyte populations number (approximately -30 Delta %) were completely blocked by PROP. Plasma CORT level, highly elevated by AMPH (+337 Delta %), was attenuated nearly by 50% under beta-adrenergic blockade. These data indicate that AMPH-induced enhancement of cytotoxic activity of NK cell is related to beta-adrenergic mechanism.

Stress-induced enhancement of activity of lymphocyte lysosomal enzymes in pigs of different stress-susceptibility.

To evaluate a possible mechanism of stress-induced lymphopenic effect we assessed the activity of lymphocyte lysosomal enzymes (LE) under immobilization. The effects of immobilization stress on LE (AP, acid phosphatase, cathepsin D and L, beta-N-acetyl-glucosamidase) activity in lymphocytes, number of lymphocytes and plasma cortisol (COR) level in the peripheral blood were examined in the cross-bred Pietrain pigs showing genotypic (presence or lack of RyR1 gene mutation) and phenotypic (reactivity to halothane) differences. It was found that immobilization stress evoked an increase in LE which was concomitant with lymphopenia and a rise of COR level. The most pronounced enhancement of LE, which may reflect a tendency to lymphocyte cytolysis, was found in the recessive homozygotes RyR1 (nn) phenotypically defined as stress/halothane susceptible as well as in the heterozygotes RyR1 (Nn) included in the group of stress/halothane resistant. Despite this individual variability the stress-induced increase in LE activity was present in all the animals. It seems that a possibility of destruction (lysis) of lymphocyte cells should not be excluded as one of the causes of stress lymphopenia.