A. B. Rodriguez

A study of the role of corticosterone as a mediator in exercise‐induced stimulation of murine macrophage phagocytosis.

1. It is generally accepted that physical activity provokes changes in the immune system. Previous studies have demonstrated that the stress of physical activity (swimming until exhaustion) increases the phagocytic activity of peritoneal macrophages. However, the precise mechanisms remain unknown. 2. Two experiments were performed in the present study. (A) Peritoneal macrophages from control mice were incubated with plasma from three different groups of mice: (1) mice subjected to swimming until exhaustion with no previous training, (2) mice subjected to the same activity but with 1 month of training (30 min day‐1), and (3) a control (non‐exercised) group. The differences in the resulting phagocytic (attachment and ingestion) capacity were measured. (B) Changes in the concentration of plasma corticosterone after exercise were also measured, and the effect of incubation with the postexercise plasma corticosterone level on the phagocytic activity of peritoneal macrophages was then studied in vitro. 3. The results were: (A) incubation with plasma from both groups of exercised mice (with and without previous training) led to increased levels of phagocytic capacity (number of C. albicans cells ingested per 100 macrophages). (B) Incubation with a corticosterone concentration of 0.72 mumol l‐1 (similar to that observed in plasma immediately after exercise) raised the phagocytic capacity (144 +/‐ 12 after incubation with 0.72 mumol l‐1 vs. 93 +/‐ 19 after incubation with 0.24 mumol l‐1). This increase was also significantly greater than that observed with 7.2 mumol l‐1 corticosterone. 4. It is concluded that corticosterone may mediate the increased phagocytic function of peritoneal macrophages induced by exercise.

High endogenous melatonin concentrations enhance sperm quality and short‐term in vitro exposure to melatonin improves aspects of sperm motility

Abstract:  Although human seminal fluid contains melatonin and spermatozoa reportedly possess membrane melatonin receptors, there are no experimental studies that have ascertained the relationship between melatonin and male infertility. This study evaluated whether urinary 6‐sulfatoxymelatonin and urinary total antioxidant capacity correlate with different seminal parameters including sperm concentration, motility and morphology. Also, the in vitro effects of melatonin on human sperm motility were investigated. Semen samples from 52 men who were counselled for infertility were obtained. Sperm concentration was determined using the haemocytometer method, motility kinematic parameters were assessed using a computer‐aided semen analysis system, while morphology and vitality were evaluated after Diff‐Quick and Eosin–Nigrosin vital staining, respectively. For the quantification of urinary 6‐sulfatoxymelatonin, a commercial ELISA kit was used, and urinary total antioxidant capacity was evaluated by means of a colorimetric assay kit. For the in vitro effects of melatonin, samples were incubated for 30 min in the presence or absence of 1 mm melatonin. Both urinary 6‐sulfatoxymelatonin and total antioxidant capacity levels positively correlated with sperm concentration, motility and morphology, as well as negatively correlated with the number of round cells. Additionally, 30‐min exposure of sperm to 1 mm melatonin improved the percentage of motile and progressively motile cells and decreased the number of static cells, thereby promoting the proportion of rapid cells. Therefore, melatonin improves semen quality, which is important because melatonin supplementation may be potentially used to obtain a successful assisted reproductive technique outcome.

Correlation between the circadian rhythm of melatonin, phagocytosis, and superoxide anion levels in ring dove heterophils.

Abstract: A functional role for melatonin is its relationship to circadian timing mechanisms. In addition, there has recently been assumed to be a functional connection between the pineal gland and the immune system in mammals and birds, with some findings showing melatonin to be a free radical scavenger and general antioxidant. The present study investigates the possible relationship between the circadian rhythm of melatonin and the ingestion capacity as well as superoxide anion levels of ring dove (Streptopelia risoria) heterophils. In birds, heterophils, with their ability to ingest and kill different antigens, play a central role in the host defence mechanism. All determinations were made during 24 hr periods at 2 hr intervals. Radioimmunoassay showed an increase of melatonin serum levels during the dark period (from 20:00 to 07:00 hr) with a maximum at 04:00 hr, and a significant decline during the hours of light with a minimum at 16:00 hr. Similary, the phagocytic index was enhanced during the night, with the maximum at ∼04:00 hr and the minimum at ∼18:00 hr. The same was the case in relation to phagocytic percentage. However, the superoxide anion levels were lower during darkness (minimum at 04:00 hr) and higher during the light period (maximum at 14:00 hr). In conclusion, our findings show that one pineal‐mediated effect on the immune system may be a direct action of melatonin on phagocytosis and the phagocytic biochemical process, and that this neurohormone might act as an antioxidant.

Melatonin reduces body weight gain and increases nocturnal activity in male Wistar rats

AIM This study evaluated the effect of the administration of melatonin, the chief secretory product of the pineal gland, on the body weight in male Wistar rats. MAIN METHODS The animals were housed for 4months in cages equipped to log horizontal activity within a thermostatically-controlled chamber, under a 12h/12h light/dark photoperiod (lights on at 08:00h). After acclimatization, the animals were divided into two groups: (1) control animals, and (2) melatonin-treated animals. Melatonin was administered in tap water (20μg/ml), and fresh drinking fluid was changed twice weekly. Rats were fed a standard diet ad libitum. KEY FINDINGS Food and water intake, body weight, the amplitude of the activity/rest rhythm (motor activity), and blood melatonin and glucose concentrations were measured. The administration of melatonin did not influence either food or water intake or glucose levels relative to those found in the control animals. However, melatonin administration reduced body weight gain and increased nocturnal locomotor activity. The peak concentration of melatonin was found at night coinciding with the increase in nocturnal activity. SIGNIFICANCE The results show that exogenous melatonin reduces body weight gain without having marked effects on metabolism. This may be due in part to the increased nocturnal activity shown by the animals treated with the indoleamine.

Melatonin controls superoxide anion level: Modulation of superoxide dismutase activity in ring dove heterophils

Rodríguez AB, Nogales G, Ortega E, Barriga C. Melatonin controls superoxide anion level: Modulation of superoxide dismutase activity in ring dove heterophils. J. Pineal Res. 1998; 24:9–14.

The glutathione precursor N-acetylcysteine improves immune function in postmenopausal women

Aging is a chronic oxidation process in which the immune system is involved. Because leukocyte functions is a good health marker and longevity predictor, the effects of daily oral administration of N-acetylcysteine (NAC, 600 mg) on several lymphocyte (adherence, chemotaxis, proliferation, natural killer activity) and neutrophil (adherence, chemotaxis, phagocytosis, superoxide) functions, as well as cytokine levels (interleukin-2, tumor necrosis factor alpha, interleukin-8), were studied in 36 healthy postmenopausal women: 18 aged 50-69 years and 18 aged > 69 years. In addition, plasma and leukocyte oxidative stress markers (glutathione, superoxide, malondialdehyde) were evaluated. These parameters were analyzed within 2 and 4 months of of NAC intake and 3 months after the end of the supplementation. In parallel, samples from 18 healthy adult women aged 30-49 years were used as a control age group. the results showed general impairment of immune function and increased oxidation markers in postmenopausal women as compared with the control group; however, NAC administration significantly improved the parameters studied, bringing their values closer to those of younger women and thus exerting a modulatory, rather than a merely stimulatory, action on the immune system. These effects were also observed 3 months after the end of supplementation. The present finding suggest that a short period of NAC supply (i.e., 2-4 months) at the dose used may lead to prolonged strengthening of immune defence in postmenopausal women, likely by increasing the leukocyte glutathione pool. Thus, NAC could contribute to maintenance of good health and quality of life in postmenopausal women by decreasing the probability of immune system-related diseases, such as infections, in aging.

Physiological concentrations of melatonin and corticosterone affect phagocytosis and oxidative metabolism of ring dove heterophils

A functional connection between the neuroendocrine and the immune systems has been established. Of particular interest is the finding that hormones such as melatonin and corticosterone are able to exert modulating effects on the immune function. Therefore, after determining the circadian rhythms of melatonin and corticosterone, we evaluated the in vitro effect of physiological concentrations of melatonin and corticosterone, separately and together, on the phagocytic function and superoxide anion levels of heterophils in ring dove (Streptopelia risoria). Trials were performed with concentrations corresponding to the nocturnal and diurnal levels reached by each of the hormones (50:300 pg/mL and 100:10 ng/mL for melatonin and corticosterone, diurnal:nocturnal, respectively). The phagocytes were incubated with the hormones both alone and concurrently. At the highest (nocturnal) concentration, melatonin augmented phagocytic function and at the same time inducing a fall in superoxide anion levels. At the highest (diurnal) concentration, corticosterone also enhanced phagocytic function, but without modifying the phagocyte oxidative metabolism. In the presence of both hormones, however, whether with nocturnal or diurnal concentrations, there was a greater increase in phagocytic function and a decrease in superoxide anion levels than was produced by either of the hormones alone. In conclusion, our findings suggest that melatonin and corticosterone may have an additive effect in the modulation of phagocytic function.

Effect of age on adherence and chemotaxis capacities of peritoneal macrophages. Influence of physical activity stress.

The aim of the present research was to study the effects of age on the adherence and chemotaxis capacities of macrophages. Macrophages were obtained from the peritoneum of young and old mice (young, 12 +/- 4 weeks; old, 68 +/- 6 weeks) and young and mature guinea pigs (young, 12 +/- 1 weeks; mature, 108 +/- 2 weeks). Adherence of macrophages was evaluated with a plastic adherence technique, and chemotaxis in a Boyden chamber. The macrophages from old animals showed a higher adherence capacity (studied at 10, 40 and 60 min of incubation), and lower chemotaxis capacity in both mice and guinea pigs. The effect of physical activity stress (swimming until exhaustion) was also studied, both with and without a previous training program, on the adherence and chemotaxis of macrophages from young and old mice. While the physical activity stress (detected by the increase of the serum corticosterone concentration) did not induce changes in adherence or chemotaxis of peritoneal macrophages from young mice, in the old mice, there was a decrease in adherence and an increase in chemotaxis.

Melatonin and aging: in vitro effect of young and mature ring dove physiological concentrations of melatonin on the phagocytic function of heterophils from old ring dove.

We have studied the circadian rhythm of melatonin in the ring dove (Streptopelia risoria) for different age groups: young (1-1.5 years), mature (3-4 years) and old animals (>8 years). Melatonin levels were determined by radioimmunoassay. Results showed a significant decline in plasma melatonin levels in old animals when compared with the concentrations observed in the other two age groups, in which maximum (nocturnal) concentrations were 300 pg/ml and minimum (diurnal) concentrations were 50 pg/ml. We analyzed the in vitro effect of the physiological concentrations found in young and mature animals on the heterophils obtained from old animals, evaluating the capacity for ingestion and destruction of Candida albicans, and the oxidative metabolism associate to phagocytosis by determining the superoxide anion levels. Melatonin induced an increase in both the phagocytosis index and the candidicide capacity. This effect was dose-dependent. In relation with the oxidative metabolism, a decline in superoxide anion levels after incubation with both concentrations of the hormone was observed. Thus our results corroborate in this avian species the decline in plasma melatonin levels with advanced age, as well as the enhancing effect of physiological concentrations of melatonin on the phagocytic function.

Hydrogen peroxide increases the phagocytic function of human neutrophils by calcium mobilisation

We have studied the effect of exogenous administration of hydrogen peroxide (H2O2) on phagocytic activity of human neutrophils. The treatment of cells with increasing concentrations of H2O2 evoke a significant elevation of phagocytic function assayed as phagocytic index, percentage and efficiency; and was similar to that induced by the calcium mobilising agonist formyl-methionyl-leucyl-phenylalanine (fMLP). This stimulatory effect was reduced by pre-treatment of neutrophils with catalase and abolished in neutrophils loaded with the intracellular calcium quelator dimethyl BAPTA. In the absence of extracellular calcium, treatment of cells with H2O2 resulted in a increase in [Ca2+]i, indicating the release of calcium from intracellular stores. H2O2 abolished the typical calcium release stimulated by the physiological agonist fMLP, while depletion of agonist-sensitive calcium pools by fMLP was able to prevent H2O2-induced calcium release. We conclude that H2O2 induces calcium release from agonist-sensitive stores and consequently increase the phagocytosis process.