1 Copenhagen Muscle Research Centre and Department of Infectious Diseases, and 2 Department of Orthopedic Medicine and Rehabilitation, Rigshospitalet, 2200 Copenhagen N, Denmark; and 3 Nestlé Research Center CH100, Lausanne, Switzerland
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ABSTRACT |
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The purpose of this study was to
investigate the possible role of glutamine in exercise-induced
impairment of lymphocyte function. Ten male athletes
participated in a randomized, placebo-controlled, double-blind
crossover study. Each athlete performed bicycle exercise for 2 h
at 75% of maximum O2 consumption on 2 separate days.
Glutamine or placebo supplements were given orally during and up to
2 h postexercise. The trial induced postexercise neutrocytosis
that lasted at least 2 h. The total lymphocyte count increased by
the end of exercise due to increase of both
CD3+TCR+ and
CD3+TCR
+ T cells as well as
CD3
CD16+CD56+ natural
killer (NK) cells. Concentrations of CD8+ and
CD4+ T cells lacking CD28 and CD95 on their surface
increased more than those of cells expressing these receptors. Within
the CD4+ cells, only CD45RA
memory cells, but
not CD45RA+ naive cells, increased in response to exercise.
Most lymphocyte subpopulations decreased 2 h after exercise.
Glutamine supplementation abolished the postexercise decline in plasma
glutamine concentration but had no effect on lymphocyte trafficking, NK
and lymphokine-activated killer cell activities, T cell proliferation,
catecholamines, growth hormone, insulin, or glucose. Neutrocytosis was
less pronounced in the glutamine-supplemented group, but it is unlikely
that this finding is of any clinical significance. This study does not
support the idea that glutamine plays a mechanistic role in
exercise-induced immune changes.
training; physical activity; immunology; natural killer cells
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INTRODUCTION |
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EXERCISE INDUCES NUMEROUS EFFECTS on the immune system. In essence, a bout of exercise induces mobilization of immunocompetent cells to the circulation. After strenuous exercise the lymphocyte count declines, and lymphocyte proliferative responses and cytotoxic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells decline (10).
The mechanisms underlying exercise-associated immune changes are
multifactorial and include neuroendocrinological factors such as
epinephrine, norepinephrine, growth hormone, cortisol, and
-endorphin (24) as well as physiological factors such
as increased body temperature during exercise
(11).
Metabolic changes during exercise may also play a role. Declined glutamine concentration in plasma as a result of muscular activity has been suggested to influence lymphocyte function (17), and a decreased level of plasma glucose has been suggested to increase stress-hormone levels and thereby influence immune function (20). The purpose of the present study was to explore the possibility that glutamine supplementation might abolish exercise-induced immunosuppression.
It has been established that cells of the immune system obtain their energy by metabolism of glucose. However, it is also known that glutamine constitutes an important fuel for lymphocytes, macrophages, and neutrophils (15, 16, 21). Several lines of evidence suggest that glutamine is used at a very high rate by these cells, even when they are quiescent (16). It has been proposed that the glutamine pathway in lymphocytes may be under external regulation, due partly to the supply of glutamine itself (1).
Skeletal muscle is the major tissue involved in glutamine production and is known to release glutamine into the blood stream at a high rate. It has been suggested that skeletal muscle plays a vital role in maintaining the plasma glutamine concentration, thus affecting the availability of glutamine to the immune cells. Consequently, the activity of the skeletal muscle may directly influence the immune system. It has been hypothesized (the so-called "glutamine hypothesis") that under intense physical exercise, the demands on muscle and other organs for glutamine is such that the lymphoid system may be forced into a glutamine debt, which temporarily affects its function. Thus factors that directly or indirectly influence glutamine blood levels could theoretically influence the function of lymphocytes, neutrophils, and monocytes (15, 16, 21). After intense long-term exercise and other physical stress conditions, the glutamine concentration in plasma declines (3, 4, 7, 12, 14, 22). In conditions where the plasma glutamine concentration is decreased, provision of glutamine could be advantageous for cells of the immune system. Several studies (8, 32, 35) have examined the effect of glutamine, as a part of total parenteral nutrition, on cells of the immune system. In humans, it was shown that glutamine-enriched intravenous feeding to patients with hematological malignancies in remission decreased the amount of positive microbial cultures and diminished the number of clinical infections (32). In septic rats, it was shown that glutamine supplemented total parenteral nutrition, partially prevented the decrease in lymphocyte blastogenesis, and increased the phagocytic index compared with standard parenteral nutrition (35). Fahr et al. (8) showed that oral glutamine supplementation of tumor-bearing rats decreased the tumor growth, which was associated with an increase in LAK cell activity.
In vitro, optimal lymphocyte proliferation is dependent on the presence of glutamine (23, 29, 31). Furthermore, Rohde et al. (31) showed that the presence of glutamine augmented the LAK cell activity in vitro, and in relation to strenuous exercise, the time course of changes in serum glutamine was paralleled by changes in LAK cell activities (29). In two recent placebo-controlled glutamine intervention studies (28, 30), glutamine was given postexercise. In the latter studies, glutamine abolished the exercise-induced decline in glutamine but did not abolish the postexercise decline in immune function. However, the possibility that the level of glutamine had declined in compartments other than blood during exercise cannot be excluded. In the present study, therefore, glutamine supplementation was given during as well as after exercise to investigate the hypothesis that glutamine given throughout exercise would be able to restore postexercise impairment of the immune system.
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MATERIALS AND METHODS |
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Subjects.
The experimental protocol was approved by the ethical committee of
Copenhagen Community, and written informed consent was obtained from
all subjects. Ten healthy, elite athletes of ages 25-48 yr (mean
age 37 yr) with a maximal oxygen consumption
(O2 max) of 47.4-68.4
ml · min
1 · kg
1 (mean
O2 max 59.6 ml · min
1 · kg
1)
participated in the study.
Experimental design. Each subject performed two exercise trials separated by 2 wk. At each experimental day the subjects reported to the laboratory at 8:00 AM, after an overnight fast. The subjects were told to avoid strenuous exercise the day before the trial and were not allowed to perform any exercise 8 h before the trial. At the first appointment, a training and diet history was obtained, and subjects were asked to keep the same scheme of training and dieting before the second trial. Furthermore, any disease presenting within 1 wk before or during the experiment excluded the subject.
Three days before their first trial, the subjects performed a graded exercise test determiningSupplementation. The study used a randomized, double-blind, placebo-controlled crossover design. The subjects consumed either isocaloric L-glutamine or maltodextrin (placebo) beverages. The placebo drink was based on maltodextrin, and the dosage was chosen to be isoenergetic with the glutamine supplementation. The total carbohydrate was only ~10% of the dosage used in classic carbohydrate supplementation studies (19). After 60 min of exercise, the subjects consumed 0.5 liter of an aqueous solution of either 3.5 g of glutamine or 3.5 g of maltodextrin; the subsequent four doses of the beverage were ingested at intervals of 45 min. The intervals were based on pilot studies showing that this supplementation protocol would give stable plasma glutamine concentrations. Subjects were asked to finish each drink within 4 min. The beverages were prepared in the morning by adding 50-60°C hot water to bags containing either glutamine or placebo provided by the Nestlé Research Center (Lausanne, Switzerland). The beverages were identical in appearance and taste. After having consumed the fifth beverage, the subjects were offered a standardized meal consisting of ~200 g of white bread, 65 g of cheese, 150 g of tomato, 150 g of cucumber, 50 g of lettuce, and 1 banana. Subjects were allowed to drink water ad libitum.
Blood samples. Blood samples were obtained from an antecubital vein at rest before the exercise (start), immediately after the exercise (finish), and 2 h postexercise.
Isolation of blood mononuclear cells. Blood mononuclear cells (BMNC) were extracted from heparinized blood by using density gradient centrifugation (Lymphoprep; Nyegaard, Oslo, Norway) on Leucosep tubes (Greiner, Frickenhausen, Germany). After isolation, cells were washed three times in RPMI 1640 (GIBCO, Grand Island, NY).
Freezing and thawing of BMNC. BMNC were frozen in a medium consisting of 50% RPMI, 30% fetal calf serum (FCS; GIBCO), and 20% DMSO (Bie and Berntsen, Rødovre, Denmark) and were kept in liquid nitrogen until thawed for analysis. BMNC were thawed in a water bath at 37°C and washed twice immediately afterward in RPMI containing 10% FCS. Viable cells were counted, and the cell concentration was adjusted to 5 × 106 cells/ml with RPMI medium containing 10% FCS. BMNC from both trials for each subject were thawed and analyzed at the same time to eliminate interassay variability between samples. Assays of proliferation, NK and LAK cell activity, and labeling of cell surface markers were performed simultaneously.
Proliferation assay. The BMNC proliferation assay was performed in triplicate in microtiter plates with U-shaped wells (NUNC, Roskilde, Denmark). BMNC were resuspended in RPMI containing 10% normal human serum. Cells (3 × 105 cells/ml) were cultured for 72 h at 37°C, 5% CO2 with either isotonic NaCl, phytohemagglutinin (PHA; 20 µg/ml; Difco Laboratories, Detroit, MI), or interleukin (IL)-2 (Boehringer Mannheim, Mannheim, Germany). During the last 24 h of the culture period, the cells were exposed to [3H]thymidine (NEN, Boston, MA). The cell cultures were collected on glass fiber filters with a harvesting machine (Micromate 196; Packard), and incorporation of [3H]thymidine into the DNA of the cells was measured in a beta counter (Harvester, Matrix 96; Packard). For each triplicate, the mean count per minute was recorded.
NK cell activity.
The activity of NK cells was measured using K562 tumor target cells in
a 51Cr release assay. Triplicates of 100 µl of BMNC
(effector cells) and 100 µl of target cells (1 × 105 cells/ml) were incubated in microtiter plates (NUNC)
with U-shaped wells for 4 h at 37°C and 5% CO2.
Effector cells were added in different concentrations giving
effector-to-target cell (E/T) ratios of 50:1, 25:1, 12.5:1, and 6.25:1.
After incubation, the plates were centrifuged at 700 g for 5 min, 100 µl of supernatant were transferred from each well to new
tubes, and radioactivity was determined in a gamma counter. Spontaneous
release of 51Cr from the target cells was determined by
incubating 100 µl of target cells with 100 µl of medium. Maximum
release was determined by incubation of 100 µl of target cells with
100 µl of medium containing 10% Triton X-100 (Sigma Chemical, St.
Louis, MO). The activity of NK cells was determined as the percentage
of lysis, calculated by the formula
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LAK cell activity. BMNC were incubated with interleukin-2 (IL-2, Proleukin; Chiron, Emmeryville, CA) in quadruplicate in flat-bottomed microtiter plates (NUNC) for 48 h at 37°C and 5% CO2. The final concentration of IL-2 in the wells was 6 × 103 U/ml and 1 × 106 BMNC/ml. The activity of LAK cells was measured in a 51Cr release assay using DAUDI target cells. Target cells (100 µl) at a concentration of 2 × 104 cells/ml and 100 µl of effector cells in various concentrations were added to the wells in microtiter plates giving E/T ratios of 50:1, 25:1, 12.5:1, and 6.25:1. The assay was carried out to completion as described for the NK cell activity assay.
Flow cytometry.
The following anti-human mouse monoclonal antibodies were used:
fluorescein isothiocyanate (FITC)-conjugated antibodies anti-CD16 (clone NKP15) and anti-TCR/
(clone WT31), purchased from Becton Dickinson (San José, CA); and anti-CD4 (clone MT310), anti-CD14 (clone TÜK4), and IgG2a negative control (clone
DAK-GO5), purchased from DAKO (Glostrup, Denmark). DAKO also supplied
an IgG1 negative control (clone DAK-GO1) dual-color reagent
conjugated with FITC and R-phycoerythrin (R-PE). R-PE-conjugated
single-color antibodies included anti-CD56 (clone MY31),
anti-TCR
/
(clone 11F2), anti-CD28 (clone CD28.1), and anti-CD95
(clone DX2), purchased from Becton Dickinson, and anti-CD45RA (clone
4KB5) and anti-CD19 (clone HD37), purchased from DAKO. Peridinin
chlorophyll protein-conjugated antibodies included an IgG1
negative control (clone X40), anti-CD3 (clone SK7), and anti-CD8 (clone
SK1), all from Beckton Dickinson.
Catecholamines. Concentrations of epinephrine and norepinephrine were measured in EGTA- (1.5 mg/ml blood) and glutathione-treated plasma (reduced, 1.3 mg/ml blood) (Boehringer Mannheim) by high-performance liquid chromatography (HPLC; Hewlett-Packard) with electrochemical detection.
Amino acids.
Blood was drawn into glass tubes containing EDTA and centrifuged at
2,500 g for 15 min at 4°C. Plasma was stored at 80°C (for a maximum of 6 mo) and analyzed by HPLC.
Clinical chemistry analyses. These tests were carried out using standard laboratory procedures at the Department of Clinical Chemistry, University Hospital of Copenhagen, Denmark. Blood was drawn into tubes containing EDTA for estimation of the concentration of erythrocytes, hemoglobin, glucose, lymphocytes, monocytes, neutrophilic granulocytes, and total blood leukocytes. The analyses were carried out with a cell counter (Technicon H.I.; Miles, Tarrytown, NY).
Muscle enzymes were measured in lithium-heparinized plasma using automated enzyme reactions (Hitachi System 717; Boehringer Mannheim Diagnostica; Mannheim, Germany). Corrections were made for changes in the plasma volume according to the method described by Dill and Costill (5a).Statistical analyses.
To test whether the measured parameters were influenced by time and
interaction between time and treatment, we carried out a special case
of the three-way analysis of variance (ANOVA); because the measurements
at different times were on the same subjects and each subject served as
its own control (the same subjects repeated the exercise protocol 2 times), a paired, repeated-measures design was employed. The employed
model is
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RESULTS |
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Exercise characteristics. Oxygen consumption, heart rate, and workload of subjects did not differ between the two exercise trials. Average oxygen consumption, heart rate, and workload were respectively 74, 76, and 69% of maximal values obtained during the preliminary tests (data not shown).
Effect of exercise.
The concentration of plasma glutamine significantly decreased 15%
2 h after exercise in the placebo group, whereas the glutamine level did not change in the glutamine trial group (Fig.
1). The individual levels of
several amino acids besides glutamine decreased in response to exercise
in both trial groups (data not shown).
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Concentrations of leukocyte subpopulations.
The concentrations of total leukocytes, neutrophils, and
monocytes were elevated during and immediately after exercise compared with preexercise values, whereas the lymphocyte count increased during
exercise and declined below preexercise values 2 h postexercise (Table 1). The concentrations of
lymphocyte subpopulations in relation to exercise did not differ
between the supplementation and placebo trials. Thus only the data from
the placebo trial is shown (Table
2). The total CD3+ cell
concentration increased at the end of exercise due to an increase in
both CD3+TCR+ and
CD+TCR
+ cells; the concentrations of
CD3+, CD3+TCR
+, and
CD3+TCR
+ decreased 2 h postexercise
compared with preexercise values. The concentrations of total
CD8+, CD8+CD45RA+ naive,
CD8+CD45RA
memory,
CD8+CD28+,
CD8+ CD28
,
CD8+CD95+, and
CD8+CD95
cell subpopulations increased at the
end of exercise and decreased 2 h postexercise. The concentrations
of all NK cell subpopulations (CD3
CD16
CD56+,
CD3
CD16+CD56+, and
CD3
CD16+CD56
) increased during
exercise and declined below preexercise values 2 h postexercise.
Regarding the concentrations of CD4+ cell
subpopulations, only CD4+CD95
and
CD4+ cells lacking CD28 or CD45RA on the surface increased
in response to exercise. However, the latter two subsets did not
decrease postexercise, in contrast to the
CD4+CD45RA+,
CD4+CD28+,
CD4+CD95+, and
CD4+CD95
cell subpopulations and the total
CD4+ cells. The CD19+ B cells increased during
exercise but returned to preexercise values within 2 h of rest.
Lymphocyte function. The NK and LAK cell activity and the proliferative responses did not differ between the two trials. Thus only data from the placebo trial are shown in Table 3. The NK cell activity increased in response to exercise when expressed as lytic units (LU) and at all E/T ratios and declined at E/T ratios of 25:1 and 50:1 after 2 h of rest. The LAK cell activity increased during exercise only when expressed as LU and declined below preexercise values 2 h postexercise at E/T ratios of 1:6.25, 1:12.5, and 1:25 and when expressed as LU. The PHA-stimulated proliferative response was decreased immediately after exercise, whereas the IL-2-stimulated response declined 2 h postexercise compared with preexercise values. The unstimulated control did not change.
Glucose, insulin, epinephrine, norepinephrine, and growth hormone. The glucose and insulin levels decreased in response to exercise and were below preexercise values immediately after and 2 h postexercise (Table 4). The levels of epinephrine, norepinephrine, and growth hormone were increased after exercise. The epinephrine level was still slightly elevated after 2 h of rest, whereas the level of norepinephrine had returned to preexercise values. The growth hormone level was slightly below preexercise levels 2 h postexercise. The results did not differ between the two groups.
Effect of supplementation. The neutrophil count was significantly less enhanced at the end of exercise in the glutamine group compared with the placebo group (Table 1).
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DISCUSSION |
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The main finding of the present study was that glutamine supplementation during and after 2 h of strenuous bicycle exercise abolished the exercise-induced decline in plasma concentration of glutamine without abolishing the exercise-induced changes in cytotoxic activity, lymphocyte proliferative responses, and lymphocyte trafficking. During glutamine supplementation, however, the exercise-induced increase in neutrophil count was slightly lower. The exercise-induced increase in growth hormone and catecholamines is thought to mediate the exercise-induced neutrocytosis (25). In line with this suggestion, there was a slight trend toward a lower level in growth hormone and epinephrine at the end of exercise in the glutamine group compared with the placebo group. However, these differences between treatments were not significant. Nieman et al. (19) found that carbohydrate supplementation affects neutrophil trafficking. In the present study, the glutamine supplementation was compared with carbohydrate-containing placebo. Thus the effect of glutamine supplementation on neutrophils might have been confounded by the carbohydrate in the placebo. The effect on neutrophils could not be ascribed to an effect of increased carbohydrate or energy supply because the placebo and glutamine supplementations were isocarbohydrated and isoenergetic.
The present study describes several new findings regarding exercise
effects on lymphocyte subpopulations. The NK cell subsets were more
sensitive to exercise stress than any other cell subtypes, and among
the heterogeneous population of NK cells, the
CD3CD16+CD56+ cells were
recruited in the highest number. Among total CD3+ T cells,
cells expressing TCR
and TCR
were both recruited to the
circulation in response to exercise. The CD8+ cells
increased more than the CD4+ cells in response to exercise,
and it appeared that within the CD4+ subset, only
CD4+ cells lacking CD45RA, CD28, and CD95 (Fas) were recruited.
Furthermore, although all CD8+ cell subpopulations were
mobilized to the blood, especially cells lacking CD28, CD45RA and CD95 (Fas) were mobilized in response to exercise. Thus memory cells (defined as CD45RO+ or CD45RA) and not naive
(CD45RA+) cells increased in response to exercise. Cell
cultures of CD8+ T cells that have reached replicate
senescence after multiple rounds of cell division lack expression of
the CD28 costimulatory molecule (6). Thus the initial
increase in CD4+ and CD8+ cells after exercise
is not likely to represent repopulation by newly generated cells but
may be a redistribution of activated cells, in agreement with kinetics
of CD4+ repopulation after anti-HIV (human immunodeficiency
virus) treatment (13) and chemotherapy (9)
and CD4 and CD8 repopulation after bone marrow transplantation
(2). When a fixed number of BMNC was stimulated with
mitogen (PHA), the proliferative response declined in response to
exercise in agreement with previous findings (18). The
explanation of the decreased PHA response could be that PHA
preferentially stimulated CD4+ cells and that the
percentage of these cells declined during exercise, because the number
of CD8+ and CD16+ cells increased more.
Furthermore, the novel finding that cells that have reached their
end-proliferative stage constitute a larger proportion among BMNC after
exercise than before also explains the declined proliferative response.
The NK and LAK cell activities are mediated by a heterogeneous
population of cells, but multiple ANOVA shows that the
CD16+CD56+ cells, more than other NK cell
subsets, explain the NK activity, whereas it is less clear which cells
contribute to the LAK cell activity (34). The NK and LAK
cell activity expressed as LU increased almost twofold as did the
percentage of CD16+CD56+ NK cells (data not
shown). The present study thus indicates that the NK cells recruited to
the blood in response to exercise have a normal cytotoxic function.
During the past few years, attempts have been made to identify nutritional supplements that could abolish exercise-induced immune changes (26). Until now, only carbohydrate loading has demonstrated significant findings (26). Thus it has been demonstrated that carbohydrate loading diminishes exercise-mediated effects on neutrophil and lymphocyte trafficking as well as levels of stress hormones (19). Supplementations with antioxidant vitamins (27) or fish oil (33) have not demonstrated any effect on circulating proinflammatory cytokine, lymphocyte, or neutrophil numbers.
The previous finding of an effect of glutamine supplementation on upper respiratory tract infections (5) is not mechanistically explained by an effect of glutamine on lymphocyte function. Thus glutamine supplementation abolished exercise-induced decrease in plasma glutamine but not exercise-induced impaired lymphocyte function.
In conclusion, the present study demonstrated that exercise primarily induces mobilization of cells that have reached their replicative senescence. Furthermore, the study showed that glutamine supplementation during and after exercise abolishes the exercise-induced decline in plasma glutamine concentration without influencing the exercise-induced change in lymphocyte subpopulations, cytotoxic activity, or proliferative capacity. The finding that the exercise-induced neutrocytosis was less pronounced during glutamine supplementation is not likely to be of any clinical significance. Thus the present study adds to previous findings by Rohde et al. (28, 30) showing that glutamine supplementation during as well as after exercise has no effect on exercise-induced lymphocyte function and trafficking.
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ACKNOWLEDGEMENTS |
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The excellent technical assistance of Ruth Rousing, Hanne Willumsen, and Birgit Mollerup is acknowledged.
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FOOTNOTES |
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The project was supported by Nestlé Research Center.
Address for reprint requests and other correspondence: B. K. Pedersen, Dept. of Infectious Diseases M7721, Rigshospitalet, Tagensvej 20, 2200 Copenhagen N, Denmark (E-mail: bkp{at}rh.dk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 September 2000; accepted in final form 18 June 2001.
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