A diet supplemented with thiolic anti-oxidants improves leucocyte function in two strains of prematurely ageing mice.

1. According to previous studies, Swiss mice of the same age showed striking interindividual differences in behaviour when exposed to a T-maze test, with a slow performance being linked to an impaired immune function, hyperemotional response to stress and a shorter life span compared with mice that quickly explore the maze. These facts led us to propose the slow mice as a model of prematurely ageing mice (PAM). 2. In the present study, we investigated whether this prematurely ageing model could be found in other strains of mice, such as BALB/c mice, by analysing several lymphocytes functions, such as adherence, chemotaxis, proliferative response to the mitogen concanavalin A (Con A), interleukin (IL)-2 release and natural killer (NK) activity. In addition, we tested the probable beneficial effects on these functions of dietary supplementation with thioproline (TP) plus N-acetylcysteine (NAC; 0.1% w/w of each anti-oxidant) in female Swiss and BALB/c mice. 3. Our model of premature ageing, previously reported in Swiss mice, has also been reproduced in the inbred BALB/c mouse strain, in which PAM showing an immunosenescence in several lymphocyte functions, such as lower chemotaxis, proliferative response to Con A, IL-2 release and NK activity, as well as higher adherence, were observed. A short-term (5 week) ingestion of TP + NAC by female Swiss and BALB/c mice improved leucocyte function, increasing chemotaxis, the proliferative response to Con A, IL-2 release and NK activity and decreasing the adherence of lymphocytes. These effects are greatest in cells from PAM of both strains. 4. In conclusion, our model of premature ageing has been reproduced in an inbred strain. In addition, the ingestion of a diet supplemented with two thiolic anti-oxidants, such as NAC and TP, has been shown to be beneficial to the immune response in PAM.

Effect of aging on the modulation of macrophage functions by neuropeptides.

The existence of a functional connection between the nervous and the immune system is supported by increasing recent evidence. In previous work we have shown that peptides from the nervous system, such as gastrin-releasing peptide (GRP), neuropeptide Y (NPY) and sulfated cholecystokinin octapeptide (CCK-8s), have modulatory effects on the immune functions in adult animals. Since the immunodepression found in aging organisms may be related to changes in the neuroimmune network, the aim of the present work was to study the changes with aging in the effect of CCK-8s, GRP and NPY on peritoneal macrophage functions (adherence to tissues, mobility, ingestion of foreign particles and superoxide anion production) from BALB/c mice of three different ages: adult (24+/-2 weeks old), mature (50+/-2 weeks old) and old (72+/-2 weeks old). The results show that the increase in adherence capacity produced by neuropeptides in cells from adult and mature animals disappears in old mice. The stimulatory effect of GRP and NPY on mobility, ingestion and superoxide production in macrophages from adult mice disappears (GRP) or changes to inhibition (NPY) in cells from old animals. The decrease of these functions caused by CCK-8s in adult or mature animals continues in old mice. These data suggest that the modulation by neuropeptides of the macrophage function changes with the age of animals.

Changes in several functions of murine peritoneal macrophages by N-acetylcysteine and thioproline ingestion. Comparative effect between two strains of mice.

The administration of the thiol compounds, N-acetylcysteine (NAC) and in particular thioproline (thiazolidine-4-carboxylic acid) at 0.1% w/w concentration in the diet, improves lymphocyte functions in old female Swiss mice, as has been shown in our previous studies. In the present work, adult mice from two different strains, namely BALB/c (an inbred strain) and OF1-Swiss (noninbred strain), were fed a diet supplemented with the above dose of each thiol compound jointly for five weeks. At 28 weeks of age, peritoneal cell suspensions were obtained and different steps of the phagocytic process, the most representative activity of macrophages, as well as interleukin-1beta (IL-1beta) production, were studied. Thus, adherence to substrate, mobility directed to a chemoattractant gradient (chemotaxis), ingestion of inert particles and superoxide anion production were analysed. The results show that diet supplementation with NAC plus thioproline increased all macrophage functions studied with the exception of superoxide anion production, which was decreased. These effects were more evident in macrophages from Swiss mice, whereas in BALB/c mice the stimulation of phagocytosis and IL-1beta production was lower and no differences were seen after treatment in adherence and superoxide anion production. These data suggest that immune function can be improved in adult mice by administration of the above thiol compounds, especially in the noninbred strain of OF1-Swiss mice.

Effects in vitro of several antioxidants on the natural killer function of aging mice.

The aim of the present work is to study the change with aging in the effect in vitro of several antioxidants: thiazolidine-4-carboxylic acid or thioproline, N-acetylcysteine (NAC), ascorbic acid (AA), and alpha-tocopherol (vitamin E, VE) on the natural killer (NK) activity in mononuclear cells from axillary nodes, spleen, thymus and peritoneal leukocytes from BALB/c male mice. Young (8+/-2 weeks), adult (24+/-2 weeks). mature (48+/-2 weeks), and old (72+/-2 weeks) animals were studied. A nonradioactive cytotoxic assay with cells from the murine lymphoma YAC-1 as target cells and a relation effector cells/target cells of 10/1 were used. The concentrations of the different antioxidants were: 1 mM for thioproline and N-acetylcysteine and 5 microM for ascorbic acid and alpha-tocopherol, which induced a maximum effect in our previous dose-response experiments. The results show that, in general, the above antioxidants cause an enhancement of the NK activity at all ages studied, this stimulation being higher with thioproline and N-acetylcysteine than with ascorbic acid and alpha-tocopherol. The effects were similar for the three lymphoid organs and the peritoneum. This stimulation of the NK activity by antioxidants is an important favorable response, especially in old mice, in which age results in a decrease in NK function and, therefore, in a higher incidence of neoplasia.

Effects of age, sex and physical exercise on the phagocytic process of murine peritoneal macrophages.

Different stages of the phagocytic process, i.e. chemotaxis, ingestion of latex beads and superoxide anion production, were measured in peritoneal macrophages from young (12 +/- 2 weeks old) and old (60 +/- 2 weeks old) male and female BALB/C mice, which were subjected to an acute bout of exercise (swimming until exhaustion) or to a period of training exercise (90 min of swimming each day during 20 days). Sedentary male and female mice as well as young and old animals were used as sex and age controls. The results show that both acute and training exercise stimulate the phagocytic process of murine peritoneal macrophages. The macrophage functions from sedentary controls were lower in the old than in the young animals. The chemotaxis and ingestion capacities of macrophages were higher in the female young and old sedentary mice than in their male counterparts. After exercise, the stimulation of the phagocytic process was higher in old and female mice than in young and male animals. In addition, serum corticosterone levels were measured in order to investigate the relations between stress and macrophage activity. Old and female mice as well as animals subjected to exercise showed, in general, higher corticosterone levels than young, male and sedentary animals.

Enhancement of leukocyte functions in aged mice supplemented with the antioxidant thioproline.

Previous research has shown that supplementation of the diet with thioproline (thiazolidine-4-carboxylic acid), an intracellular sulfhydryl antioxidant and free radical scavenger, increases mouse life span and stimulates the immune system. In the present study aged Swiss mice (20 month old) fed thioproline (0.07%,w/w) for 5 weeks were used. Twelve month and 20 month old mice fed standard diet were used as controls. The lymphoproliferative response to the mitogen Concanavalin A (Con A) and the mobility of lymphocytes, both spontaneous and directed to a chemoattractant gradient (chemotaxis), as well as antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) activity of leukocytes, were measured in cells from spleen and thymus. All of the above functions showed a significant decrease in aged (20 months) in comparison to adult mice (12 months). In aged animals, the ingestion of thioproline stimulated significantly the functions studied. Moreover, the age-related stress, revealed by the high corticosterone levels, was significantly decreased in animals fed this antioxidant. These data suggest that thioproline enhances immune response in the aged.

Immune function in aged women is improved by ingestion of vitamins C and E.

We have investigated the effects of supplementation of the diet with the antioxidant vitamins C and E on several functions of the immune response of aged women. Ten healthy women and 20 women (72 +/- 6 years old) suffering two diseases often associated with age (10 with major depression disorders, MDD, and 10 with coronary heart disease, CHD) were administered 1 g of vitamin C and 200 mg of vitamin E daily for 16 weeks. Blood samples were collected before and after treatment for measurement of several immunological functions, namely proliferative response of lymphocytes to the mitogen phytohemagglutinin (20 mg/L) and phagocytic functions of polymorphonuclear (PMN) neutrophils, i.e., adherence to vascular endothelium, chemotaxis, phagocytosis of latex beads, and superoxide anion production. In addition, we also determined the levels of serum cortisol and lipid peroxides. Intake of vitamins resulted in a significant increase in the lymphoproliferative capacity and in the phagocytic functions of PMN neutrophils as well as in a significant decrease of serum levels of lipid peroxides and cortisol, both in the healthy aged women and in the aged women with MDD or CHD. These findings suggest an important role of antioxidant supplementation in the improvement of immune function in aged females as well as in the prevention and treatment of specific diseases associated with age that are quite prevalent in the developed countries.

Relation between exploratory activity and immune function in aged mice: a preliminary study.

Previous studies show that fast exploration of a T-shaped maze by mature mice may predict an above average longevity. Since the nervous and the immune systems work in a coordinated fashion, and it seems that these two homeostatic systems both influence organismic aging and suffer a senescent decline, we have performed a comparative study of the above behavioral parameter and different functions of three representative immune cells: lymphocytes, macrophages and natural killer (NK) cells obtained from old (76 +/- 1 weeks of age) female OF1-Swiss mice. At 70 weeks of age the mice were divided into a 'fast' and a 'slow' group, containing 100 and 0%, respectively, of animals able to explore the 50 cm-long first arm of the maze in 20 s or less. At 76 +/- 1 weeks of age the animals were sacrificed, the peritoneal cell suspensions were obtained and the immune organs (axillary nodes, spleen and thymus) were isolated. The following leukocyte functions were studied in peritoneal macrophages: adherence to substrate, mobility (spontaneous and chemotaxis), ingestion of particles and superoxide anion production whereas mobility, lymphoproliferative response to the mitogen Con A and NK activity were studied in the immune-organ leukocyte suspensions. The results show that the aged fast mice have better immune functions than the aged slow mice.

Changes with age in the modulation of natural killer activity of murine leukocytes by gastrin-releasing peptide, neuropeptide Y and sulfated cholecystokinin octapeptide.

Several investigations have suggested that the interactions between the nervous and immune systems are modified with age. The aim of the present work was to study the effect of three neuropeptides: gastrin-releasing peptide (GRP), neuropeptide Y (NPY) and sulfated cholecystokinin octapeptide (CCK-8 s) on natural killer (NK) activity of spleen, thymus and axillary node leukocytes from BALB/c male, young (8+/-1 weeks), adult (24+/-2 weeks) and old (72+/-2 weeks) mice. We used cells from murine lymphoma YAC-1 as targets for the cytotoxic assay and three physiological concentrations of the neuropeptides (10(-8), 10(-10) and 10(-12) M). In control samples, in the absence of neuropeptide, we observed a decreased NK activity in young and old mice with respect to the adults in the three organs studied. Regarding the effect of the neuropeptides, GRP stimulates the cytotoxic activity of leukocytes from all locations, in adult animals. At the same age, NPY also stimulates the NK activity of leukocytes from axillary nodes and thymus, whereas it decreases the NK activity of spleen leukocytes from young mice. CCK-8 s has an inhibitory effect on the axillary node leukocytes from young mice and spleen leukocytes from old animals. However, CCK-8 s increased the NK activity of thymus leukocytes from young and adult mice. The results indicate that the highest values of NK activity are found in adult mice, and that the stimulating effect of the three neuropeptides studied on NK activity of leukocytes from adult mice are reduced or disappeared, in general, in old as well as in young animals. Furthermore, the changes observed with ageing in the modulation of NK activity by the neuropeptides studied suggest an altered integration of the nervous and immune systems.

Effects of a long-term training program of increasing intensity on the immune function of indoor Olympic cyclists.

We have studied, on blood samples, the level of immunocompetence (concentration of immune cells, phagocytic process of polymorphonuclear neutrophils, proliferative response of lymphocytes to mitogens), the ascorbic acid content of such immunocompetent cells and the "stress hormone" status (cortisol, ACTH and beta-endorphin) of 10 cyclists, members of the Spanish Indoor Olympic Team and participants in the Olympic Games of Barcelona '92. The study was performed twice during their training for such an event: during the third year of the program (February, 1991) and immediately before the Games (June, 1992). As regards the phagocytic process of neutrophils, we studied the different steps of this process: adherence to endothelium, directed mobility or chemotaxis, ingestion of latex beads and superoxide anion production measured by the nitroblue tetrazolium (NBT) reduction test. We observed a statistically significant increase in chemotaxis and NBT reduction activity just before the Games as compared to the third year of the program, whereas variations were not found in the other parameters. The values of the proliferative capacity of lymphocytes were slightly higher in June '92 than in February '91, but no statistically significant differences were found. The ascorbic acid content decreased strikingly (especially in lymphocytes) immediately before the Games. Regarding the stress hormones and neuropeptides (cortisol, ACTH and beta-endorphin), we observed an increase in serum ACTH and beta-endorphin levels in the last determination (June '92) in comparison to the first one (February '91). These results suggest that, at the end of a long-term training program. no immunosuppression occurs, although an important increase in the concentration of stress hormones (ACTH and beta-endorphin) is found. This is probably caused by the psychological stress associated to the participation in such an important event as the Olympic Games.

Anabolic steroids and lymphocyte function in sedentary and exercise-trained rats.

The effects of the administration of suprapharmacological doses of anabolic steroids (AASs) on the immune system were examined in sedentary and exercise-trained rats by testing mobility and proliferative response in cultures of thymus and spleen-derived lymphocytes. Male Wistar rats were exercise-trained following two programmes of treadmill running of 3 months duration, differing in intensity, in the absence of treatment or with simultaneous i.m. administration of a suprapharmacological dose (10 mg/kg/week) of nandrolone decanoate (ND) or stanozolol (ST) during the past two months. At this dose ND reduced body weight gain, promoted a redistribution of immune cells from thymus to spleen, impaired lymphocyte mobility and inhibited the mitogen-induced proliferative response (about 90% inhibition for thymus-derived cells). Stanozolol (ST) treatment was without effect on body weight gain, but it also induced a redistribution of lymphocytes and modified the in vitro lymphocyte activity, although less severely than ND. Application of the high-intensity training programme reduced lymphocyte mobility and proliferation in vitro and a simultaneous treatment with anabolic steroids further impaired some of the immune cell responses. Application of the endurance-directed training programme, however, did not reduce mobility or mitogen-induced proliferation of lymphocytes, and normalized the activity of these cells in anabolic steroid-treated rats. So, endurance exercise, contrary to high-intensity training, could counteract the apparent negative effects of high doses of androgens on lymphocyte function.

Changes with aging, sex and physical exercise in murine natural killer activity and antibody-dependent cellular cytotoxicity.

Antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) activity were measured in leukocytes from the axillary nodes, the spleen and the thymus of young (12 +/- 2 weeks) and aged (60 +/- 2 weeks) male and female BALB/c mice, which had performed an acute bout of exercise (moderate swimming until exhaustion) or a training exercise (90 min of moderate swimming each day for 20 days). The results show that NK and ADCC activity in sedentary mice (controls) were similar in young and aged animals. However, both kinds of exercise resulted in higher cytotoxicity values in aged mice than in young mice. Acute exercise did not have any effect on NK activity in young and aged mice, nor on ADCC activity in young mice as compared to controls, while training exercise stimulated both cytotoxicities in the two age groups. No correlations between serum corticosterone levels and NK or ADCC activity were found. Our results suggest that moderate training exercise improves both NK and ADCC activity during aging.

Changes with aging and physical exercise in ascorbic acid content and proliferative response of murine lymphocytes.

Ascorbic acid content and lymphoproliferative response to phytohemagglutinin were measured in lymphocytes from axillary nodes, spleen and thymus of young (15 +/- 2 weeks) and old (60 +/- 5 weeks) BALB/c mice. Ascorbic acid content in lymphocytes from spleen and thymus was found to be significantly higher and the lymphoproliferative response in the three immunocompetent organs significantly lower in old mice as compared to young mice. Moreover, young and old BALB/c mice were required to maintain a swimming activity until exhaustion (exhaustive exercise) or 90 min of swimming each day for a total of 20 days (continuous exercise). In both young and old mice the stress produced by exhaustive exercise and confirmed by the existence in serum of significantly increased levels of corticosterone compared to controls, caused a significant decrease in ascorbic acid content as well as in lymphoproliferative response. Continuous exercise, characterized by the presence in serum of significantly decreased levels of corticosterone compared to controls, produced the most significant decrease in ascorbic acid content from young and old murine lymphocytes. Moreover, this exercise resulted in a significant increase in lymphoproliferative response. Our results suggest that aging results in an increase in the ascorbic acid content of lymphocytes accompanied by a decline in the lymphoproliferative response in old BALB/c mice.

Modulation of phagocytic function in murine peritoneal macrophages by bombesin, gastrin-releasing peptide and neuromedin C.

Bombesin, as well as the two mammalian bombesin-like peptides gastrin-releasing peptide and neuromedin C, have been shown in this study to stimulate in vitro all steps of the phagocytic process in murine peritoneal macrophages: adherence to substrate, chemotaxis, ingestion of cells (Candida albicans) and inert particles (latex beads), and production of superoxide anion as measured by nitroblue tetrazolium reduction. A dose-response relationship was observed, with maximal stimulation of phagocytic process between 10(-12)M and 10(-9)M. Gastrin-releasing peptide (GRP) and neuromedin C caused a higher activation of adherence, chemotaxis and ingestion of C. albicans than bombesin. The three neuropeptides induced in murine macrophages a significant, but transient, increase of inositol 1,4,5-trisphosphate (IP3) levels at 60 seconds. On the contrary, these neuropeptides produced a rapid, transient and significant decrease of cAMP at 30 seconds. These results suggest that there are close relations between IP3 and cAMP messenger systems and the phagocytic process in murine peritoneal macrophages when these cells are incubated in the presence of bombesin, GRP or neuromedin C.