Noelia Guayerbas

Changes with ageing in several leukocyte functions of male and female rats

The impairment of the immune system with aging, or ‘immunosenescence’, appears to contribute to the increased morbidity and mortality of aged subjects. T cell functions and Natural Killer activity seem to be the immune responses most affected by ageing. Since the immune system works more efficiently in females than in males, we have studied the changes of several immune functions with age in rats of both sexes. In addition, we have investigated if ovariectomy, a model of menopause in rats, produces a loss of this gender-related advantage. In the present work, the changes with age (2, 6, 12, 14, 18, 22 and 24 months old) in lymphocyte chemotaxis, T lymphoproliferative response to the mitogen ConA, IL-2 release and Natural Killer activity of cells from axillary nodes and spleen of male and female rats as well as of females ovariectomized at 12 months of age have been studied. An age-related decrease was found in all investigated functions, with a slightly different evolution depending on the immune organ and gender considered. In general, the data obtained show that a certain degree of immunosenescence takes place with age in rats, with males being less immunocompetent than intact age-matched females, but showing an immune response similar to that of ovariectomized animals.

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.

An impairment of phagocytic function is linked to a shorter life span in two strains of prematurely aging mice.

In previous cross-sectional studies on Swiss mouse populations we have shown that at the same chronological age, animals that take longer to explore a simple T-maze (slow mice) are hyper-emotional and show an impairment of the immune system than those which quickly explore the maze (fast mice). Therefore, we have proposed that the slow mice are a model of prematurely aging mice (PAM). In the present work we have carried out a longitudinal study of age-dependent changes in key functions of phagocytic cells (peritoneal macrophages) such as phagocytosis and superoxide anion production in both male and female Swiss (outbred strain) and BALB/c (inbred strain) PAM and non-prematurely aging mice (NPAM). Gender differences were found showing the females better phagocytic and digestion capacities with concomitant longer life span. The PAM showed an impaired phagocytosis capacity and intracellular superoxide anion production as well as an increase of its extracellular production as compared to NPAM, which could be related to the shortened life span of those animals in both sexes and strains.

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.

Relation of behaviour and macrophage function to life span in a murine model of premature immunosenescence

According to our previous work, mice of the same strain and age show striking inter-individual differences in behaviour when exposed to a T-maze test. Further, the animals exploring the maze slowly (slow mice) or staying at the starting point (freezing behaviour), which show high levels of emotionality/anxiety in other standard behavioural tests, have a less competent immune system (earlier immunosenescence) than those which explore it quickly (fast mice). The present longitudinal study on OF-1 Swiss female mice confirms and extends the above findings. Thus, the animals showing a lower performance in the T-test (slow mice) which is accompanied by a poor neuromuscular coordination in a tightrope test, have a shorter life span than the good performers (fast mice). Moreover, the slow mice have a less competent immune system as regards the following functions of peritoneal macrophages: adherence to substrate, chemotaxis, ingestion of particles and superoxide anion production. This suggests that, at the same chronological age and as regards their immune competence, the slow mice are biologically older than the fast mice. This agrees with current ideas on the close functional relationship between the nervous and the immune system in the physiological adaptation to stress, and supports the concept that an optimum level of performance of these two systems is needed to attain a long life span.

Vitamin E ingestion improves several immune functions in elderly men and women

The effects of diet supplementation with the antioxidant vitamin E (200 mg daily) on several blood neutrophil, lymphocyte and natural killer cell functions have been investigated in healthy elderly men and women before supplementation, after 3 months of supplementation and 6 months after the end of supplementation (post-supplementation). In parallel, samples of healthy adult men and women were used as age controls. In elderly men and women, an impairment of immune functions was observed in comparison with the respective adult controls and the intake of vitamin E resulted in a significant enhancement of immune parameters in both elderly men and women, bringing their values close to those in the adults. These effects were not found in post-supplementation samples in several but not in all functions. The present findings suggest that supplementation with vitamin E can produce an improvement of immune functions and therefore of health in aged people.

Effect of acupuncture treatment on the immune function impairment found in anxious women.

It is presently accepted that emotional disturbances lead to immune system impairment, and that therefore their treatment could restore the immune response. Thus, the aim of the present work was to study the effect of an acupuncture treatment, designed specifically to relieve the emotional symptoms stemming from anxiety, on several functions (adherence, chemotaxis, phagocytosis, basal and stimulated superoxide anion levels, lymphocyte proliferation in response to phytohemagglutinin A (PHA) and natural killer (NK) activity) of leukocytes (neutrophils and lymphocytes) from anxious women. The acupuncture protocol consisted of manual needle stimulation of 19 acupoints, with each session lasting 30 min. It was performed on 34 female 30-60 year old patients, suffering from anxiety, as determined by the Beck Anxiety Inventory (BAI). Before and 72 hours after receiving the first acupuncture session, peripheral blood samples were drawn. In 12 patients, samples were also collected immediately after the first single acupuncture session and one month after the end of the whole acupuncture treatment, which consisted of 10 sessions during a year, until the complete remission of anxiety. Twenty healthy non-anxious women in the same age range were used as controls. The results showed that the most favorable effects of acupuncture on the immune functions appear 72 hours after the single session and persist one month after the end of the complete treatment. Impaired immune functions in anxious women (chemotaxis, phagocytosis, lymphoproliferation and NK activity) were significantly improved by acupuncture, and augmented immune parameters (superoxide anion levels and lymphoproliferation of the patient subgroup whose values had been too high) were significantly diminished. Acupuncture brought the above mentioned parameters to values closer to those of healthy controls, exerting a modulatory effect on the immune system.

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.

Changes in the antioxidant content of mononuclear leukocytes from mice with endotoxin-induced oxidative stress

Oxidative stress, associated with a high production of reactive oxygen species (ROS) by immune cells, is involved in the endotoxic shock caused by endotoxin. This oxidative stress is linked to the inability of the immune cells to maintain adequate levels of antioxidants with free radical-scavenging action. Glutathione (GSH) and ascorbic acid (AA) are intracellular and extracellular antioxidants (ROS scavengers) that improve the leukocyte functions. Therefore, in the present work we have determined the reduced GSH and AA content in axillary nodes, spleen, thymus and peritoneal mononuclear leukocytes from BALB/c mice subjected to lethal endotoxic shock produced by intraperitoneal injection of E. coli lipopolysaccharide (LPS, 100 mg/kg), at several times (0, 2, 4, 12 and 24 h) after LPS injection. Endotoxic shock decreased the levels of AA in the leukocytes from the three organs as well as the levels of GSH in axillary nodes and spleen cells while it increased the GSH levels in thymus and peritoneum. These results are in agreement with the oxidative stress and the altered function previously observed in those leukocytes, and they suggest that antioxidant administration may be useful for the treatment of endotoxic shock and other oxidative stress situations with altered immunological responses.