Modulation of neuropeptide Y and norepinephrine on several leucocyte functions in adult, old and very old mice.

The age-related changes in the communication between the nervous and the immune system have been scarcely investigated, especially in very aged subjects. The present work deals with the in vitro effects of norepinephrine and neuropeptide Y, separately and jointly, on functions such as lymphoproliferation, NK activity, and IL-2 and TNF-alpha release of peritoneal leucocytes from adult (24+/-2 weeks), old (72+/-2 weeks) and very old (128+/-2 weeks) mice. The old mice showed a decrease in proliferation, NK activity and IL-2 release, and an increase in TNF-alpha, whereas in the very old mice these functions were more similar to those of the adults. The effects of neurotransmitters on these functions were different depending on the age of the animals.

Thiolic antioxidant supplementation of the diet reverses age-related behavioural dysfunction in prematurely ageing mice.

We have studied in a model of premature ageing in mice based on their impaired behavioural response in a simple T-maze test the effect of the ingestion of thioproline (TP) plus N-acetylcysteine (NAC) (0.1% w/w of each antioxidant) by female and male mice of Swiss and BALB/c strains on performance in two behaviour tests. The antioxidant treatment (4 weeks in two different periods of life, i.e., adult and old age) protected all animals against early-age-associated behavioural impairment, but this improvement was more evident in the prematurely ageing mice (PAM) in comparison to the control group or non-prematurely ageing mice (NPAM). An improvement of the exploratory activity and neuromuscular coordination after the thiolic antioxidant treatment was found in the PAM, bringing the behavioural parameters to the NPAM levels. These effects could be due to the glutathione precursor role of NAC and TP that replenish the intracellular reduced glutathione (GSH) levels despite advancing age. In conclusion, diet supplementation with thiolic compounds appears to be an effective therapy for protection against early behavioural decline in prematurely ageing mice.

Improvement of the macrophage functions in prematurely ageing mice by a diet supplemented with thiolic antioxidants.

In a model of prematurely ageing mice, based on the different behavioral response in a simple T-maze test, we have studied in the present work the effect of a short-term (five-weeks) ingestion of thioproline (TP) plus n-acetylcysteine (NAC) (0.1% w/w of each antioxidant) on several functions of peritoneal macrophages from female Swiss mice. The antioxidant treatment increased the chemotaxis, phagocytosis and IL-1beta release and decreased the superoxide levels and TNFalpha production. These effects were more striking in macrophages from prematurely-ageing mice (PAM), than in those from the control group or non-prematurely-ageing mice (NPAM), bringing the above functions of the PAM to the NPAM levels. Diet supplementation with these thiolic compounds, namely TP and NAC, seems an effective therapy for protection against early immune decline in prematurely ageing mice.

Changes with age in peritoneal macrophage functions. Implication of leukocytes in the oxidative stress of senescence.

The age-related deterioration of the immune cells contributes to the increased morbidity and mortality of the aged populations. Since ageing is the result of oxidative stress and macrophages are an important source of oxidation, which is produced in order to carry out many of their functions, the changes with age of several macrophage functions as well as of oxidative stress parameters (oxidants, inflammatory mediators and antioxidants) of peritoneal leukocytes have been investigated using young (4 months), adult (7 months), mature (12 months) and old (20 months) female ICR (CD-1) mice. With age the macrophage functions suffer deterioration. Thus, chemotaxis, phagocytosis and intracellular superoxide anion production decrease in old animals. Moreover, the levels of antioxidant defenses, such as superoxide dismutase activity, decrease with ageing. However, the adherence capacity and the release of oxidant and inflammatory mediators, such as extracellular superoxide anion, TNF-alpha and PGE2, as well as oxidized glutathione/ reduced glutathione ratio, increase with age. These results show that these cells suffer oxidative stress with ageing, which results in an increase of the oxidative damage to nuclear DNA. These changes in peritoneal immune cells could contribute to the chronic oxidative stress state linked to senescence.

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.

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+/-1 week old) and aged (75+/-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.

Leukocyte function and life span in a murine model of premature immunosenescence.

Aging associates with a decline of physiological functions, including the function of the nervous and the immune system. These aged-related changes occur in various degrees in different members of a mouse outbred population. Accordingly, we have proposed a model of premature immunosenescence in mice, based on the demonstration of premature decline in the behavioral response in a simple T-maze and in several immune functions in Swiss outbred mice. Those mice with a worst (slow) performance in this test (linked to a higher emotional response to stress) show a shorter life span and a decreased immune function when compared to fast mice. In order to provide biomarkers of "biological aging" related to health and survival, the present longitudinal study includes the analysis of several immunological parameters such as, proliferative response to mitogen Con A, NK activity and cytokine (TNFalpha, IL-1beta and IL-2) release by peritoneal leukocytes from female Swiss mice. Slow mice showed a lower proliferative response to Con A, IL-2 and IL-1beta release, an impaired NK activity and an increased TNFalpha production as compared to fast mice. Moreover, the age-associated decline of these functions is more strikingly slow than in fast mice. In summary, we propose the above immunological parameters, that change with aging at a different rate in members of a same population, as useful biomarkers to asses the rate of biological aging in mice.

Behavioral characterization of a mouse model of premature immunosenescence.

In previous studies we have shown that differences in life span among members of Swiss mouse populations appear to be related to their performance in a T-maze, with a slow performance ("slow" mice) being linked to an impaired immune function and a shorter life span when compared to "fast" mice, which led us to propose the slow mice as a model of immunosenescence. In the present study we demonstrate that in a tightrope test of neuromuscular vigor and coordination the slow mice show a worse performance, needing more time to complete the task. Moreover, these animals show a decreased locomotor activity and an increased level of emotionality/anxiety in three standard behavioral tests (the holeboard, the open field and the plus-maze) when compared to fast mice. All these behavioral features were most marked in the slow females. The results also indicate that slow animals show a decreased chemotaxis of macrophages and lymphocytes, as well as a reduced lymphoproliferative response to mitogens. The data supports our claim that slow or hyperemotional mice, in which immune and neurobehavioural functions appear to be impaired, may be a useful model of premature aging.

Changes in the ascorbic acid levels of peritoneal lymphocytes and macrophages of mice with endotoxin-induced oxidative stress.

Ascorbic acid (AA) is an important cytoplasmic antioxidant that mice synthesize in the liver, the intracellular levels of which decrease in an oxidative stress situation such as endotoxic shock. The present work deals with the changes in AA levels, that modulate the immune function, in the two main immune cells, namely macrophages and lymphocytes, from female BALB/c mice suffering endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg). The intake by cells of this antioxidant present in vitro at different concentrations was also studied. The animals show an oxidative stress, standardized in previous studies, that causes mortality at 30 h after LPS injection. The cells were obtained from the peritoneum at 2, 4, 12 and 24 h after LPS or PBS (control) injections and were incubated without or with AA at 0.01, 0.1 and 1 mM for 10, 30, 60, 120 or 180 min. The hepatic AA levels were also studied at 0, 2, 4, 12 and 24 h after LPS injection. The peritoneal cells obtained from animals injected with LPS showed increased AA levels in relation to the control cells at all times after LPS injection, with maximal effect at 12h. The AA levels decreased after this time, in agreement with changes in the AA hepatic levels. The increase was due to the AA of lymphocytes since macrophages showed a decrease in AA at different times after LPS injection. Both cells showed an increase in the intracellular levels of AA when this antioxidant was added in vitro. This takes place mainly at 30-60 min of incubation in cells from controls and at 10 min in cells from treated mice 12-24 h after LPS injection. The incorporation decreased at these times of endotoxic shock, a few hours before death. In all cases AA levels were higher in lymphocytes than in macrophages, and 1 mM was the most effective concentration. These results suggest that the immune cells need appropriate levels of antioxidants, such as AA, under oxidative stress conditions, and that while lymphocytes take and accumulate AA, macrophages use it.

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.

Effect of a diet supplemented with thioproline on murine macrophage function in a model of premature ageing.

A previous study has shown that diet supplementation with thioproline (thiazolidine-4-carboxylic acid, TCA), an intracellular sulfhydril antioxidant and free radical scavenger, stimulates lymphocyte functions in old mice. In the present work, the effect of thioproline ingestion on the phagocytic functions of peritoneal macrophages, namely adherence, chemotaxis, phagocytosis and superoxide anion production was studied in adult female OF1-Swiss mice, that were fed thioproline (0.1% w/w) for five weeks, starting this ingestion at the age of 22 +/- 2 weeks. Mice were divided into a fast and a slow group based on their exploratory activity, which was assessed by their performance in a T-shaped maze. Slow mice showed a worse phagocytic activity with respect to fast animals. After thioproline treatment, a stimulation of all the functions studied as well as a neutralization of the superoxide radical were observed. The effect of this antioxidant was stronger in the slow than in the fast group. Thus, a diet supplemented with thioproline may enhance the immune functions, especially when they are depressed.

Modulation of murine macrophage function by N-acetylcysteine in a model of endotoxic shock.

In previous studies we have observed changes in several functions of peritoneal macrophages from BALB/c mice with irreversible endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg), which were associated with a high production of superoxide anion. Since antioxidants, such as N-acetylcysteine (NAC), are free radical scavengers that improve the immune response, in the present work we have studied different functions of peritoneal macrophages from BALB/c mice suffering the endotoxic shock above indicated and administered N-acetylcysteine (150 mg/kg i.p.) at 30 minutes after LPS injection. In the peritoneal macrophages obtained at 2, 4, 12 and 24 h after LPS injection, the following functions were studied: adherence to substrate, mobility, ingestion of particles, and production of superoxide anion and tumour necrosis factor (TNF alpha). The increase in adherence, ingestion and superoxide anion and TNF alpha production shown by macrophages from animals with endotoxic shock was counteracted by NAC injection. Moreover, the survival time of mice with endotoxic shock was increased in the presence of NAC. These data suggest that NAC, administered intraperitoneally, may be useful for the treatment of irreversible endotoxic shock by modulation of the function of macrophages with decreased superoxide anion and TNF alpha production and concomitant increase of survival time.

Age-related changes in the modulatory action of gastrin-releasing peptide, neuropeptide Y and sulfated cholecystokinin octapeptide in the proliferation of murine lymphocytes.

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 in vitro of three neuropeptides: gastrin-releasing peptide (GRP), neuropeptide Y (NPY) and sulfated cholecystokinin octapeptide (CCK-8s) on the spontaneous, as well as on the response to mitogen (concanavalin A), proliferative activity of spleen, thymus and axillary node leukocytes from adult (24 +/- 2 weeks), mature (50 +/- 2 weeks) and old (72 +/- 2 weeks) BALB/c male mice. In control samples, in the absence of neuropeptide, we observed a decreased lymphoproliferation in mature and old mice with respect to the adults in response to mitogen in the three organs studied. As regards, the effect of the neuropeptides, they stimulate the spontaneous proliferation of leukocytes from all locations, in adult animals, an effect that is decreased with ageing (in both mature and old animals). The proliferation in response to mitogen was significantly decreased by the neuropeptides in adults, this effect being progressively reduced with age.

Effects of endotoxic shock in several functions of murine peritoneal macrophages.

Gram negative sepsis and septic shock continue to be a major medical problem, with a complex physiopathology and it is associated with high mortality. Although secretion of cytokines such as tumor necrosis factor-alpha by macrophages is the principal host mediator of septic shock, other characteristic functions of macrophages implicated in their phagocytic capacity have not been studied in the process of endotoxic shock. In the present study we have used an intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg) in order to obtain an endotoxic shock model in adult female BALB/c mice. Peritoneal cell suspensions were obtained at several times (2, 4, 12 or 24 h) after injection and the following functions were studied on the peritoneal macrophages: adherence to substrate, mobility (spontaneous and directed or chemotaxis), ingestion of particles and superoxide anion production. The results showed a stimulation of adherence, ingestion and superoxide production as well as a decrease of chemotaxis in the animals injected with LPS. These effects changed with time after LPS injection. Thus, the increase of adherence and the decrease of mobility were higher during the first hours, whereas the increase in ingestion and superoxide production turned larger with time.