M. De la Fuente

Improvement by several antioxidants of macrophage function in vitro.

The toxic effects of oxygen radicals produced by immune cells can be controlled to certain degree by endogenous antioxidants, because of their scavenger action. This control is specially important in a type of immune cell, i.e.: the phagocyte, which needs oxygen free radicals and uses antioxidants in order to support its functions. Previous studies have shown an stimulation of the immune system with an antioxidant enriched diet. In the present work, we have studied the effects in vitro of several antioxidants: alpha-tocopherol or vitamin E (VE), ascorbic acid (AA), glutathione (GSH), N-acetylcysteine (NAC) and thioproline or thiazolidine-4-carboxylic acid (TCA), at different concentrations, on the various steps of the phagocytic process of murine peritoneal macrophages, i.e.: adherence to substrate, migration (random migration and directed migration or chemotaxis), ingestion and superoxide anion production. The results show an antioxidant-induced stimulation of the phagocytic process of macrophages. Thus, the adherence to substrate was raised, after short incubation times, by a-tocopherol and ascorbic acid. Random migration, chemotaxis, ingestion and superoxide anion production were increased by all the antioxidants used.

Sex-dependent alterations in response to maternal deprivation in rats

We review here our latest results regarding short- and long-term effects of a neonatal maternal deprivation (MD) stress [24h at postnatal day (PND) 9] on diverse psychoneuroimmunoendocrine parameters, pointing out the existence of numerous sexual dimorphisms. Behavioral changes observed in MD animals might be at least in part attributable to neurodevelopmental effects of MD-induced elevated corticosterone levels. Our findings of short-term effects of MD on hippocampal and cerebellar neurons and glial cells appear to support this hypothesis. However, it is important to note that these cellular effects were more marked in males than in females. Moreover, in analyzing the effects of this neonatal stress on the endocannabinoid system (hippocampal endocannabinoid levels and CB1 receptors) we have also found that males were more affected by MD. Since all these sexual dimorphisms were found at an early neonatal age (PND 13), they are attributable to organizational effects of gonadal steroids. We discuss the potential implications of the elevated corticosterone and decreased leptin levels shown by MD animals in their diverse functional alterations, including the above mentioned neural effects as well as the intriguing persistent deficit in their immunological system. We also emphasize the necessity of analyzing the important influence of sex as regards the specific consequences of early life stress.

Stimulation of the phagocytic function of neutrophils in sedentary men after acute moderate exercise

SummaryAll the different stages of phagocytic function in blood polymorphonuclear neutrophils of sedentary (untrained) young men before, immediately after, and 15 min after being subjected to submaximal acute moderate exercise (50% maximal oxygen uptake) on a cycle ergometer were studied. No statistically significant differences were found in adherence or microbicide capacity againstCandida albicans immediately after physical exercise. However, spontaneous mobility, chemotaxis and attachment and phagocytosis ofCandida albicans at 15 min of incubation were significantly stimulated. Fifteen minutes after exercise spontaneous mobility, chemotaxis, and attachment ofCandida albicans had returned to basal values, while phagocytosis ofCandida albicans remained higher than basal values. One can, therefore, conclude that acute moderate exercise stimulates the phagocytic capacity of neutrophils in sedentary young men. In addition, no differences were found in the plasma adrenocorticotropic hormone concentration after exercise. However, immediately after exercise there was a decrease in the serum cortisol concentration, which had returned to basal 15 min later.

Ascorbic acid modulates in vitro the function of macrophages from mice with endotoxic shock

The toxic effects of oxygen radicals produced by immune cells can be controlled to certain degree by endogenous antioxidants because of their scavenger action. This control is specially important in a type of immune cell, i.e., the phagocyte, which produces oxygen-free radicals and uses antioxidants in order to support its functions. Antioxidants, such as ascorbic acid (AA), are free radical scavengers and improve the immune response. In the pathogenesis of endotoxic shock, a disease with high mortality caused by gram-negative bacterial endotoxin, the reactive oxygen species (ROS) produced by phagocytes have been implicated. In a previous study, we observed in peritoneal macrophages from BALB/c mice suffering lethal endotoxic shock caused by intraperitoneal (i.p.) injection of Escherichia coli lipopolysaccharide (LPS; 100 mg/kg) a high production of superoxide anion. Therefore, in the present work, we have studied the in vitro effect of AA, at different concentrations (0.001, 0.01, 0.1, 1 and 2.5 mM), on the various steps of the phagocytic process, i.e., adherence to substrate, chemotaxis, ingestion of particles and superoxide anion production of murine peritoneal macrophages obtained from BALB/c mice with that of endotoxic shock, at 2, 4, 12 and 24 h after LPS injection. The increased adherence, ingestion and superoxide anion production by macrophages from animals with endotoxic shock were lower in the presence of AA, reaching similar values to those of the control animals. The most effective AA concentration in cells from mice with endotoxic shock was 0.01 mM. These data suggest that AA can regulate the phagocytic process in endotoxic shock, principally decreasing free radical production and thus it could reduce endotoxic shock severity.

Changes with aging in the modulation by neuropeptide Y of murine peritoneal macrophage functions

Some age-related changes in immune function may be due, at least in part, to a disturbance in the communication between the nervous and immune systems. In the present work, the effects in vitro of neuropeptide Y (NPY) (10(-13) to 10(-7) M) on different peritoneal macrophage functions (adherence to substrate, chemotaxis, phagocytosis, superoxide anion production, and the release of TNFalpha and IL-1beta) have been studied on cells from young (12+/-2 weeks), adult (24+/-2 weeks), mature (50+/-2 weeks) and old (72+/-2 weeks) BALB/c mice. The specificity of these actions was confirmed using two C-terminal fragments of NPY, and the intracellular messengers (protein kinase C and cAMP) involved in the action of the neuropeptide were also analyzed. The results show that the functions studied change with aging and that the effects of NPY on each function, which are carried out through specific receptors, as well as on intracellular pathway, differ depending on age, maintaining the immune functions at physiologically adequate levels in old animals.

Ageing Modulates some Aspects of the Non‐Specific Immune Response of Murine Macrophages and Lymphocytes

The deterioration of the immune system with ageing, which leads to an increased morbidity and mortality from infections, appears to be related to decreases in specific lymphocyte functions. However, the alteration of non‐specific immunity is a more controversial subject. Our purpose was to investigate the age‐related changes of different functions of the non‐specific immune response in peritoneal macrophages (adherence to tissues, mobility directed to a chemical gradient from an infectious focus or chemotaxis, phagocytosis of foreign agents and destruction of these agents by superoxide anion production) and in lymphocytes (adherence and chemotaxis) from peritoneum, axillary lymph nodes, spleen and thymus. We used young (12 weeks), adult (22 weeks), mature (48 weeks) and old (72 weeks) female BALB/c mice. The adherence capacity of macrophages and lymphocytes was greater in adult and old mice than in young animals. The chemotaxis of macrophages showed higher values in cells from young mice than in those from adult mice, increasing again in macrophages from mature and old animals. A similar behaviour was shown by phagocytosis, which reached its highest values in old animals. Anion superoxide production increased with age and again the highest values were obtained in the oldest mice. Conversely, chemotaxis of lymphocytes was higher in the adult and mature animals than in the young and old animals. We conclude that, although there is a decrease in lymphocyte chemotaxis in old animals, the non‐specific immune response of macrophages instead of decreasing, may increase in aged mice with respect to the values seen in adult 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.

The Amount of Thiolic Antioxidant Ingestion Needed to Improve Several Immune Functions is Higher in Aged than in Adult Mice

With aging there is an increase of oxidative stress due to an imbalance between the oxidant production and the antioxidant levels in favor of the former. Since immune cell functions are specially linked to reactive oxygen species (ROS) generation, the oxidant/antioxidant balance is essential for these cells. Although low levels of antioxidants cause a decrease in immune function, very high levels of antioxidant compounds could show prooxidant effects. In the present work, we have studied the effect of diet supplementation, for 4 weeks, with two different doses of two thiolic antioxidants, namely thioproline (TP) and N -acetylcysteine (NAC), at 0.1% (w/w) and 0.3% (w/w, of each antioxidant) on the main immune system cells, i.e.: macrophages, lymphocytes and natural killer (NK) cells of adult (33 u - u 1 week old) and aged (75 u - u 1 week old) female Swiss mice. Two groups of animals, adult and aged mice, fed standard diet were used as controls. The results show that the ingestion of 0.1% doses of thiols improves, in the adult mice, several immune functions such as the chemotaxis capacity of both macrophages and lymphocytes, the phagocytosis of macrophages, the lymphoproliferative response to the mitogen Con A and the NK activity. Moreover, no change was observed in adherence capacity of immune cells, and superoxide production was decreased. By contrast, in aged mice the ingestion of these amounts of antioxidants did not change the immune functions studied with the exception of NK activity, which was stimulated. The ingestion of 0.3% of antioxidants by adult mice only increased some immune functions such as adherence and superoxide production, which are markers of oxidative stress. Other functions such as chemotaxis or lymphoproliferative response decreased. However, the ingestion of these very high amounts of thiols by aged animals increased the phagocytosis, the NK activity and specially the lymphoproliferative response to the mitogen, a function that is very depressed with aging.

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.

Stimulation of the phagocytic function in guinea pig peritoneal macrophages by physical activity stress

SummaryA study was made of all the different stages of the phagocytic function in peritoneal macrophages from male guinea pigs [3 (SD 1) months old] before, immediately after, and 24 h after being subjected to stress from physical activity (swimming until exhaustion). The early (10 min) and late (40 min) adherence to tissue substrates, chemotaxis, attachment and phagocytosis ofCandida albicans, ingestion of inert particles (latex beads), and basal oxidative metabolism [measured by nitroblue tetrazolium (NBT) reduction] were significantly stimulated by the physical activity. After 24 h, late adherence, attachment capacities, and basal oxidative metabolism returned to basal values, whereas early adherence, chemotaxis, phagocytosis of cells and inert particles, and microbicidal capacity (production of superoxide anion measured by NBT reduction in presence of ingested material) remained significantly increased. The stress produced by physical activity, reflected in increased serum corticosterone values, led to a global stimulation of the phagocytic function.