Exercise Induced Alterations in Rat Monocyte Number, Morphology, and Function.

The purpose of this study was to verify the histophysiological alterations in monocytes and macrophages induced by short periods of exercise. Male Wistar rats (age = 2 months, body weight = 200g) were divided into seven groups (N = 6 each): sedentary control (C), groups exercised (swimming) at low intensity for 5 (5L), 10 (10L), and 15 minutes (15L), and groups exercised at moderate intensity for 5 (5M), 10 (10M) or 15 minutes (15M). At moderate intensity the animals carried a load of 5% of body weight on their backs. Blood monocytes were evaluated for quantity and morphology, and peritoneal macrophages were analyzed for quantity and phagocytic activity. Data were analyzed using ANOVA and Tukey's post hoc test (p ≤ 0.05). Low intensity groups and 5M exhibited an increase in monocyte levels when compared with the control. There was an increase in monocyte cellular area for the 5L, 10L, 5M and 10M groups; monocyte nuclear area increased for the 10L, 5M and 10M groups in comparison with the control. There was an increase in peritoneal macrophages for the 15L, 10M, 15M and decrease for the 5M group. Macrophage phagocytic capacity increased for low intensity groups and for 10M group. The exercise performed for short periods modulated macrophage levels and function, and monocyte levels and morphology, in an intensity-dependent manner. The sum of acute responses observed in this study may exert a protective effect against sickness and may be used to improve health and lifespan.

INTRODUCTION

The present day lack of time, excessive work journeys and stress have led individuals to perform short bouts of exercise (5–25 minutes). Exercise intensity, duration and frequency, and individual levels of physical fitness are important factors in determining immune response to physical effort (14). Exercise may stimulate or lower immune function. Macrophages and monocytes are part of the innate immune system and are quickly activated when there is an infection. Macrophages phagocytize microorganisms, kill some and present them as antigens to lymphocytes. Immunosuppression of these functions potentially exposes the organism to antigens, which may result in complex diseases (1). Regular exercise performed at moderate intensity is commonly associated with increased immune resistance against infections (12). On the other hand, strenuous exercise has been associated with transitory immune suppression, potentially increasing susceptibility to infections (14), especially upper respiratory infections. (6, 7).

Glucocorticoids and catecholamines are released during exercise and stimulate leukocyte chemotaxis, adhesion, phagocytosis and antigens presentation (22, 8, 21). Thyroid, prolactin, growth hormone (GH) and endorphins contribute to exercise-induced stress on phagocytosis (17). Stress hormones, especially glucocorticoids, released during strenuous exercise are immunosuppressive for lymphocytes. In this situation, phagocytosis and other innate immune mechanisms are stimulated and could compensate suppressed lymphocyte activity (17). Exhaustive exercise has been shown to enhance or suppress certain functions of macrophages; it appears that the modulatory effect of exercise on the monocytes and macrophages is dependent on the parameter being measured, source of tissue macrophages, and the intensity and duration of exercise (22). However, very little information exists with regard to the effect of brief periods of acute exercise on monocyte and macrophage function. It was expected that even a short duration exercise (five to fifteen minutes) would increase monocyte number; macrophage function and morphology due to the immunomodulatory stress imposed by an acute session.

Understanding of the role exercise plays in the immune system function is important to enable one to know whether or not to recommend exercise for people with depressed or dysfunctional immune systems. An animal model was used, because it is a more homogeneous population and external variables are more easily controlled. The aim of the study was to investigate the effect of acute exercise performed at low and moderate intensities on the histophysiology of rat macrophages and monocytes.

METHOD

Animals

All experiments were conducted in accordance with the policy of the American College of Sports Medicine on Research with Experimental Animals and was approved by the Federal University of São Carlos Experimental Animals Committee. Two-month-old male Wistar rats (Rattus novergicus var. albinus, Rodentia, Mammalia) with a mean weight of 190–210g were used. Animals had free access to water and were fed a commercial chow for rodents (Labina, Purina, Campinas, SP, Brazil). The animals were maintained in collective cages (5 rats per cage) at a constant temperature of 23(± 2)°C, and a cycle of 12 hours light/12 hours dark, with light from 06:00h to 18:00h (the housing was pathogen-free). Before the beginning of the experiments, the animals underwent 48 hours of adaptation to the laboratory conditions.

Experimental groups

Animals were divided into seven groups (N = 6 each group): a sedentary control group; groups exercised at low intensity for 5 (5L), 10 (10L), or 15 minutes (15L), and groups exercised at moderate intensity for 5 (5M), 10 (10M), or 15 minutes (15M). The exercised groups performed a single acute exercise session.

Physical Exercise

The physical exercise chosen was swimming, accomplished between 14.00h and 17.00h in a tank maintained at 30(±2)°C. The groups exercised at low intensity did not carry additional loads. At moderate intensity the animals carried a load of 5% of body weight on their backs, which corresponds to intensity below the inflection point of the curve of the lactate threshold (9). The animals were sacrificed immediately after the end of the exercise and blood and macrophages were collected. Analyses were carried out after that, on the same day.

Total Circulating Levels of Leukocytes and Monocytes

Blood samples were collected and stored in glass tubes with EDTA and kept on ice for the analysis of blood variables. The total leukocyte count was obtained with Türk’s solution (Merck KGaA, Darmstadt, Germany) and counting was performed in a Neubauer chamber. The circulating levels of monocytes were measured under an optical microscope using a blood smear stained with Giemsa-May-Grünwald (Sigma, St. Louis, MO, USA) (2). The blood counts were blinded, only one of the authors knew what the groups were and this author did not participate in the counting.

Total count and phagocytic capacity of peritoneal macrophages

Peritoneal cavity was washed with 10mL of PBS (pH 7.4 and 4°C) to remove resident macrophages. Cells were counted in a Neubauer Chamber with Trypan Blue colorant (Sigma, St. Louis, MO, USA). A calculation was made to obtain a solution aliquot in 2.0 × 106 cells/mL dilutions. To this aliquot, the following components were added: 50μL of rat serum opsonized Zymosan (Sigma, St. Louis, MO, USA), 100μL of glucose 56mM (Sigma, St. Louis, MO, USA), 200μL of defatted albumin (Sigma, St. Louis, MO, USA), and PBS to complete 1mL. The solution was incubated at 37°C for 40 minutes with low shake, and 100 cells were analyzed under light microscope using Trypan Blue colorant. When three particles of Zymosan were phagocytized by the macrophage, the cell was considered to have increased phagocytic capacity (Figure 1) (19).

Figure 1

Photomicrography of peritoneal macrophage (M), total preparation stained with Trypan Blue. The dense structures observed in the cell cytoplasms are the phagocytized particles of Zymosan (arrow). Around these cells are several Zymosan particles indicated by the thick white arrows.

Morphometry analysis

Initially the blood smears were prepared in duplicate, followed by 2–3 minutes of drying. Thereafter, the staining was done with 3ml of May-Grünwald and Giemsa (Sigma, St. Louis, MO, USA) and the analysis was conducted under a light microscope according to recommendation of Sokol et al. (20). From 20 monocytes taken from each animal, cellular and nuclear areas were obtained by applying an image analyzer system Image Pro Plus Version 4.0 for Windows.

Statistical analysis

Data are presented as mean ± SEM (standard error of the mean). The normality of distribution of all parameters was checked with the Kolmogorov-Smirnov test. Differences were analyzed by using two-way analysis of variance (ANOVA) considering exercise duration and intensity as intervenient factors, followed by Tukey’s post hoc test. A significance level of p ≤ 0.05 was used for all comparisons (duration of exercise with control and between groups and intensity of exercise with control and between groups). The software package used was SPSS for Windows version 10.0.

RESULTS

A significant increase in total leukocytes was observed for the exercised groups when compared with the control group. An increase was observed for the 5M group in comparison the 5L (44%) group. An increase in circulating monocytes was observed for the groups exercised at low intensity and for 5M group, in comparison with the control. Lower monocyte values were evident for the 10M (60%) and 15M (45%) groups when compared with the 10L and 15L groups, respectively (Table 1).

Table 1

Circulating levels of leukocytes, monocytes and peritoneal macrophages, and phagocytic capacity of macrophages for the control and groups exercised for 5, 10 and 15 minutes at low and moderate intensities.

Group	Circulating leukocytes	Circulating monocytes	Peritoneal macrophages	Phagocytosis (%)
C	4.12 ± 0.17	1.53 ± 0.3	14.08 ± 0.57	74.8 ± 0.74
5L	8.70 ± 1.07*	5.03 ± 0.71*	14.47 ± 0.4	79.8 ± 0.49*
10L	11.53 ± 1.09*	7.36 ± 0.91*	13.73 ± 0.13	78.8 ± 0.8*
15L	9.50 ± 0.92*	6.35 ± 0.6*	17.77 ± 0.5*	83 ± 0.45*
5M	12.56 ± 0.9*#	5.24 ± 0.3*	10.80 ± 0.54*#	75 ± 0.63#
10M	9.17 ± 0.6*	2.90 ± 0.24#	17.20 ± 0.35*#	79.6 ± 0.68*
15M	11.61 ± 0.61*	3.45 ± 0.69#	20.91 ± 0.56*#	77.4 ± 0.75#

Values are mean ± SEM, cells are expressed × 106. C, control group; 5L, 5 minutes low-intensity exercise, 10L, 10 minutes low-intensity exercise, 15L, 15 minutes low-intensity exercise, 5M, 5 minutes moderate-intensity exercise, 10M, 10 minutes moderate-intensity exercise, 15M, 15 minutes moderate-intensity exercise.

Significant difference when compared with the control group.

Significant difference between exercised groups for the same duration (p ≤ 0.05).

A significant increase in peritoneal macrophages was observed for the 15L, 10M and 15M exercised groups of 26%, 22% and 48%, respectively; the 5M group presented decrease (23%) when compared with the control. In the comparison between groups, a decrease in peritoneal macrophages was observed for the 5M (25%); increase for the 10M (25%) and for the15M (18%) groups when compared with the 5L, 10L and 15L groups, respectively (Table 1).

The groups exercised at low intensity and the 10M group exhibited increased phagocytic capacity in peritoneal macrophages when compared with the control. A significant decrease in phagocytic capacity of peritoneal macrophages was observed for the 5M (6%) and for the 15M (7%) groups in comparison with the 5L and 15L groups (Table 1).

The 5L, 10L, 5M, and 10M groups presented an increase in monocyte cellular area (12%, 9%, 15% and 19%, respectively) when compared with the control. A significant increase in monocyte nuclear area was observed for the 10L (15%), 5M (37%), and 10M (18%) groups in comparison with the control. The 5M group presented a significant increase in nuclear area (23%) in comparison with the 5L group (Figure 2).

Figure 2

Cellular and nuclear morphometry of circulating monocytes. Values are expressed for area in μm2 mean ± SEM. C, control group; 5L, 5 minutes low-intensity exercise, 10L, 10 minutes low-intensity exercise, 15L, 15 minutes low-intensity exercise, 5M, 5 minutes moderate-intensity exercise, 10M, 10 minutes moderate-intensity exercise, 15M, 15 minutes moderate-intensity exercise. *Significant difference when compared with the control group; #Significant difference between exercised groups for the same duration, (p ≤0.05).

DISCUSSION

In parallel with a general leukocytosis, the findings for low intensity exercise were mainly an increase of circulating monocytes and peritoneal macrophage phagocytosis seen for any duration of short-term exercise. On the other hand, for the moderate intensity short-term exercise, the main findings were an increase in the numbers of peritoneal macrophages for ten and fifteen minutes of exercise and an increased phagocytosis for ten minutes of exercise. Concerning circulating monocytes a picture of a transient increase of nuclear and/or cellular morphometry was seen in the five and ten minutes exercises, disappearing in fifteen minutes exercise, irrespective of the intensity.

Leukocytosis is caused by contraction of the spleen and other secondary lymphatic organs, in addition to the modulation of increased plasma catecholamines on immune cell migration and activity. This mechanism induces an alteration in the interaction between cells and the endothelium promoting fast leukocyte demargination during exercise (11, 13). In the present study, a significant increase in total leukocytes was observed for the groups exercised at low and moderate intensities.

Moderate exercise increases the expression of adhesion molecules in monocytes and transendothelial migration induced by oxidized low density lipoprotein (ox-LDL) (21). However, exercise performed at high intensity promotes down-regulation in transendothelial migration (20). This mechanism is partially responsible for the increase observed in peritoneal macrophages for the groups 15L, 10M, and 15M. In this case, monocytes would be stimulated to migrate through the endothelium; the migration of these monocytes could culminate in infiltration into the peritoneum. Hence, the increase in thyroid hormones induced by exercise stimulates macrophage chemotaxis (16), and also low levels of noradrenalin modulate macrophage chemotaxis mediated by the β-adrenergic receptors (8). In addition, β-endorphin improves the chemotaxis of monocytes and macrophages (10).

Monocytes located at the marginal pool produce TNF-α and other pro-inflammatory cytokines that coordinate innate and specific immune cell functions, including interactions with endothelial cells, differentiated expression of effective molecules on the cell surface, growth and differentiation (13). Additionally, these cells are quickly and selectively mobilized to the central circulation, as exercise induces an increase in catecholamines (18). In the present study, this increase in circulating monocytes was seen after exercise performed at low intensity and in 5 minutes of exercise performed at moderate intensity. Although catecholamine levels were not measured, the increase in this hormone is a classical alteration observed in the first few minutes of physical exercise. With 10 and 15 minutes of exercise at moderate intensity, circulating monocytes were not changed compared with the sedentary group, but monocyte values were lower as compared with the same duration low intensity groups. No previous studies have evaluated this exercise length and no mechanisms have thus far been proposed in the literature. However, a possible explanation may be a higher migration of circulating monocytes to peritoneal tissue. This may explain the observation in peritoneal monocytes, in which the 10 and 15 minute moderate intensity groups presented higher values in tissue than low intensity groups.

These are possible explanations for the increase in circulating monocytes observed in the present study and for the increase in the cellular and nuclear area of these monocytes verified during the first 5 and 10 minutes of exercise, performed at low and moderate intensities. This response to exercise promotes selective mobilization in monocyte subsets from the marginal pool and may induce morphological alterations. However, with regard to morphological alterations, it is difficult to establish the exact mechanism responsible for the alterations observed and the studies are sparse.

It is important to mention that the present study has limitations, for example, only peritoneal tissue monocytes and macrophages were counted. For future investigations the activated monocytes and peritoneal macrophages should be measured with flow cytometry so that the changes induced by short duration exercise may bring more important clinical perspectives.

In humans, exercise performed at moderate intensity and for a duration of 3 hours will induce increases in IL-1β, IL-6 and TNF-α plasma concentration; however, gene expression and mRNA of these proteins do not change in blood monocytes. It was suggested that activated intratissue cells, such as macrophages, could be responsible for the increase in systemic cytokines (13).

The secretion of these cytokines may contribute to the alteration in monocyte cellular and nuclear area. Exercise increases the plasma concentration of several substances: β-endorphins, glucocorticoids, catecholamines, prolactin, and thyroid hormones. Macrophages and monocytes have surface receptors for these molecules (17). In monocytes, the synthesis and expression of these receptors can be up-regulated, as verified by the increase in adhesion molecules (21) and β-endorphin synthesis (3), mediated by exercise. Consequently, these factors promote alterations in monocyte cellular and nuclear area.

The increase in phagocytosis of tissue macrophages can be correlated with increased corticosterone levels after strenuous exercise (4, 5). Noradrenalin, in synergy with its end metabolite 4-hydroxy-3-metoxyphenyl-glycol (HMPG), modulates the phagocytic process of macrophages mediated by the stimulation of α- and β-adrenoreceptors (8). The increase in prolactin plasma concentrations induced by exercise stimulates chemotaxis, phagocytosis, and microbicidal activity of phagocytes (15). The exercise protocols (intensity and duration) used by previous studies are potentially different from the exercise performed in the present study (short-term), although an increase in peritoneal macrophage phagocytic capacity was observed; the exact mechanism still needs to be addressed.

High concentrations of thyroid hormones and β-endorphins secreted during exercise also modulate chemotaxis and phagocytosis of macrophages (10, 5, 16).

The main findings of this study were the morphological and functional alterations in monocytes, through the increase in cellular and nuclear area and an increase in phagocytic capacity after short periods of exercise. There is a paucity of studies that have analyzed the acute immune responses to 5, 10 and 15 minutes of exercise. The alteration in immune cell morphometry after exercise can bring new investigation perspectives, indicating a research field to be explored in exercise immunology. Exercise performed for a short duration may stimulate monocyte function. However, the consequences of these acute alterations need to be investigated from a chronic perspective. The sum of acute responses observed in this study may exert a protective effect against sickness and may be used to improve health and lifespan (14). Based on the results of the present study, one may conclude that short-term, low and moderate intensity exercise may be interesting for starting a physical activity program for sedentary individuals, since no deleterious alterations were observed in monocyte function.