1 Exercise Physiology and Metabolism Laboratory, Department of Physiology, University of Melbourne, Parkville 3052, Victoria; and 2 Department of Pathology and Immunology, Monash Medical School, Prahran 3181, Victoria, Australia
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ABSTRACT |
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The
present study was undertaken to examine the effect of prolonged running
on monocyte intracellular cytokine production and plasma cytokine
concentration. Blood samples were collected 1 h before,
immediately after, 2 h after, and 24 h after a competitive marathon run. There was no change in the number of cells spontaneously producing tumor necrosis factor (TNF)-; however, there was a decrease in the number of cells producing interleukin (IL)-1
and
IL-6 (P < 0.01) postexercise. In contrast, there was
an increase in the number of monocytes that responded to
lipopolysaccharide stimulation by producing IL-1
, TNF-
, and IL-6
(P < 0.01) immediately and 2 h postexercise;
however, these cells contained less cytokine (P < 0.05). Plasma IL-6, TNF-
, epinephrine, norepinephrine, and cortisol
concentrations were markedly increased (P < 0.01)
postexercise. These data demonstrate that circulating monocytes
are not the source of elevated levels of plasma IL-6 and TNF-
after
prolonged running. In addition, it is likely that stress hormones
result in a decrease in the amount of cytokine produced by
LPS-stimulated cells postexercise.
flow cytometry; epinephrine; cortisol
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INTRODUCTION |
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PLASMA LEVELS OF SEVERAL
CYTOKINES, namely, interleukin (IL)-1 (15), tumor
necrosis factor (TNF)-
(15, 27), and IL-6 (11,
14-16, 22, 24, 28), have been shown to increase during and
after intense prolonged exercise. It has been hypothesized that stress
associated with strenuous exercise alters leukocyte cytokine production
(2). IL-1, TNF, and IL-6 are recognized as key components
of the immune response against infections (2), and
alterations in their production could leave individuals vulnerable to
invading pathogens after exercise. Numerous cells in a variety of
tissues produce proinflammatory cytokines (25); therefore, alterations in plasma concentrations are not necessarily indicative of
changes in production by circulating leukocytes. In support of this,
Ostrowski et al. (16) observed IL-6 mRNA expression in
skeletal muscle subjected to prolonged running. These previous data
provide indirect evidence that increases in plasma cytokines may be due
not only to altered circulating leukocyte production but also to
changes in other tissues, such as contracting muscle. In the present
study, intracellular cytokines in blood leukocytes were analyzed by
flow cytometry to determine the association between exercise and the
number of circulating monocytes producing IL-1
, TNF-
, and IL-6,
as well as the amount of cytokine they produce. This method has the
advantage of rapid analysis of cytokine production by a large number of
individual cells, permitting reliable detection of even small
proportions of cytokine-positive cells (18). Recent work
from our laboratory (23) employed this method to study the
effect of prolonged, submaximal cycling exercise on monocyte cytokine
production. We observed that circulating monocytes are not likely to be
the source of the small increase in plasma IL-6, because no change was
observed in spontaneous monocyte IL-6 production postexercise.
Furthermore, exercise increased the number of monocytes producing
cytokines upon stimulation; however, these cells produced less cytokine
postexercise compared with preexercise. Notably, plasma cortisol levels
were not elevated postexercise. Cortisol infusion has been demonstrated
to decrease IL-1
, TNF-
, and IL-6 production upon stimulation
(7); therefore, elevations in cortisol may affect cytokine
production. As yet, it is not known whether exercise that causes
substantial elevations in plasma cortisol levels has a similar effect
on spontaneous and stimulated monocyte intracellular cytokine production.
Damaged muscle releases cellular fragments into the circulation that, in turn, may activate immune cells (6). It has been reported that muscle and joint trauma results in activation of circulating monocytes, which, in turn, produce large quantities of proinflammatory IL-1, IL-6, and TNF (20). Because cycling was the exercise mode employed in our previous study, it is unlikely that a large amount of muscle damage would have occurred. In addition, the rise in plasma IL-6 was small (<2 pg/ml), whereas studies that have used running (16) report much higher levels (>90 pg/ml). As yet, it is not known whether exercise, which causes substantial elevations in plasma IL-6, has a similar effect on spontaneous and stimulated monocyte intracellular cytokine production.
Hence, the aim of the present study was to investigate whether prolonged, strenuous running affects the ability of circulating monocytes to produce cytokines upon stimulation and whether spontaneous cytokine production is responsible, in part, for the increased plasma cytokine concentration. It was hypothesized that exercise would decrease cytokine production by stimulated circulating monocytes and that these cells would not be the source of elevations in plasma cytokines.
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METHODS |
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Subjects. Five male entrants (76.9 ± 3.5 kg; 182 ± 4.7 cm) in the Melbourne Marathon volunteered for this study. Each subject was informed of the experimental protocol and possible risks and signed a letter of informed consent before participating. Subjects had been free from infection for 6 wk before the study, were exempt from symptoms of respiratory illnesses, and were not taking any medication. For 2 mo leading up to the marathon, subjects had been running 84 ± 22 km/wk. On the day of the marathon, the temperature was 21.2°C and the humidity was 71%. The marathon commenced at 8:00 AM, and subjects completed the race in 169 ± 20 min (range 151-205 min). Experiments were approved by the Human Research Ethics Committee of The University of Melbourne.
Experimental procedures.
A resting blood sample was obtained from a vein in the antecubital
fossa ~1 h before commencement of the race (preexercise). Blood was
also collected immediately upon completion of the marathon (postexercise) and after 2 h (2 h postexercise) and 24 h
of recovery (24 h postexercise). During the race, intake of drinks was
ad libitum, and during the 2-h rest period, subjects were given water and Gatorade ad libitum. Preexercise, postexercise, and 2-h
postexercise blood samples were analyzed for alterations in leukocyte
counts and spontaneous and lipopolysaccharide (LPS)-stimulated monocyte cytokine production. In addition, plasma IL-6, TNF-, glucose, lactate, cortisol, catecholamines, and creatine kinase were analyzed in
all blood samples.
Leukocyte counts. Blood (3 ml) was placed in EDTA tubes and analyzed for differential white cell counts as routinely performed by the hematology laboratory at Alfred Hospital (Melbourne, Victoria, Australia). This analysis included the determination of total white blood cell numbers and neutrophil, monocyte, and lymphocyte numbers to detect changes in circulating white blood cell populations with exercise.
Intracellular cytokines.
Blood (2 ml) was placed in heparin sodium tubes and kept at room
temperature until the end of the experiment for measurement of
intracellular cytokine production. The tubes were gently inverted and
rolled periodically. Whole blood was incubated for 4 h with (stimulated) or without (spontaneous) 1 µg of LPS at 37°C in a humidified incubator. Brefeldin-A (10 µg/ml) was added to all samples
at the commencement of incubation to inhibit intracellular transport of
proteins, thus retaining cytokines produced within the cell. Aliquots
(100 µl) of stimulated and nonstimulated blood were then incubated
for 30 min with CD33 (PECy5)-conjugated monoclonal antibody
(Immunotech, Marseille, France) for staining of monocytes. Red blood
cells were lysed (0.15 M ammonium chloride, 10 mM potassium bicarbonate, and 1 mM EDTA) for 10 min, and the samples were spun in a
centrifuge (350 g) for 5 min. The supernatant was decanted, and the pellet was resuspended in 500 µl of 4% paraformaldehyde for
20 min. Samples were again spun (350 g) for 5 min, and the supernatant was decanted. The fixed cells were permeabilized with 500 µl of permeabilizing solution (Becton Dickinson, San Jose, CA) for 20 min, washed (1% fetal calf serum, phosphate-buffered saline, and 0.02 M sodium azide), and spun (350 g) for 5 min, and the
supernatant was decanted. The cells were then incubated with monoclonal
antibodies against IL-6 [fluorescein isothiocyanate (FITC);
Pharmingen, San Diego, CA] IL-1 [r-phycoerythrin (PE)], TNF-
(FITC), and/or control
(
2aFITC/
1PE; Becton Dickinson) for 30 min. After the samples had been washed and then spun (350 g)
for 5 min, the pellet was resuspended in 500 µl of wash buffer. All
incubations took place at room temperature in the dark. The percentage
of cytokine-positive monocytes was determined by flow cytometry
(FACScan; Becton Dickinson). Monocytes were separately gated on viable
cells on a side scatter vs. CD33 (FL3) cytogram. Data for 2 × 103 events within this gate were acquired. Analysis of data
was then performed by using the Cell Quest program (Becton Dickinson)
with gates for positive set on isotype controls (Fig.
1). Results are expressed as the
percentage and number of cytokine-producing cells in CD33+
populations. The absolute count was determined by multiplying the
percentage of cytokine-positive monocytes by the concentration of
monocytes in peripheral blood. For quantification of the amount of
cytokine within positive cells, the mean fluorescence intensity of
positive events was obtained.
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Plasma IL-6 and TNF-.
Blood (3 ml) was collected into EDTA tubes and spun for 4 min at 6,000 g. The supernatant was removed and stored at
80°C until
analysis. The concentrations of IL-6 and TNF-
were measured by using
commercially available chemiluminescent ELISA kits (R&D Systems,
Minneapolis, MN), which detect both soluble and receptor-bound IL-6 and
TNF-
. All measurements were performed in duplicate.
Hormones and metabolites.
Blood (5 ml) was placed in heparin lithium tubes for analysis of
cortisol, glucose, and lactate, and blood (2 ml) for catecholamine analysis was placed into tubes containing 20 µl/ml EGTA and reduced glutathione. Blood was then spun for 4 min at 6,000 g. The supernatant was removed and stored at 80°C until
analysis. Cortisol concentration was determined by RIA (Diagnostic
Products, Los Angeles, CA). Samples were analyzed for plasma
catecholamines by using a modification of the single isotope
3H radioenzymatic assay (TRK 995; Amersham, Amersham, UK).
Plasma glucose and lactate were analyzed by using enzymatic automated analysis (EML-105; Electrolyte Metabolite Laboratory, Radiometer, Copenhagen, Denmark).
Creatine kinase.
Blood (2 ml) was collected into heparin lithium tubes, spun for 4 min
at 8,000 rpm, and stored at 80°C until analysis. Samples were
analyzed for creatine kinase by using enzymatic automated analysis
(Hitachi System 747; Boehringer Mannheim Diagnostica, Mannheim, Germany).
Statistical analysis. Analysis of the measured variables revealed that the data were not normally distributed. To ensure homogeneity of the data, data were log transformed before statistical analysis. A one-way analysis of variance (ANOVA) with repeated measures on the time factor was used to compare blood metabolites, hormones, white blood cell counts, and cytokine-positive cells. Newman-Keuls post hoc tests were used to locate differences when the ANOVA revealed a significant interaction. Descriptive data are presented as means ± SD, and comparative data are presented as means ± SE. The level of significance to reject the null hypothesis was set at P < 0.05.
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RESULTS |
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Leukocyte numbers.
There was an increase (P < 0.01) in circulating
monocyte and neutrophil numbers postexercise and 2 h
postexercise compared with preexercise, resulting in an increase
(P < 0.01) in total circulating leukocyte numbers
(Table 1). Lymphocyte numbers remained at
preexercise levels after the marathon and were suppressed
(P < 0.01) 2 h postexercise (Table 1). Cell
counts had returned to resting values at 24 h postexercise.
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Spontaneous cytokine production.
There was a decrease (P < 0.01) in the percentage of
monocytes spontaneously producing TNF- postexercise; however, there was no change in the number of cells producing TNF-
as a result of
exercise (Table 2). In addition, the
amount of TNF-
per cell, as indicated by fluorescence intensity, was
decreased (P < 0.01) postexercise and 2 h
postexercise (Table 2). This decrease indicates that cells entering
circulation during exercise are not spontaneously producing TNF-
and
that previously productive cells continue to produce TNF-
, but in
lesser amounts. The percentages of cells spontaneously producing
IL-1
and IL-6 were decreased (P < 0.01) postexercise and 2 h postexercise, and the numbers of cells
spontaneously producing IL-1
and IL-6 were decreased
(P < 0.01) postexercise (Table 2). The decrease in the
number of cells positive for IL-1
was maintained (P < 0.01) 2 h postexercise (Table 2). This decrease indicates that
monocytes entering the circulation during exercise are not producing
IL-6 and IL-1
spontaneously and that cells previously producing
IL-1
and IL-6 cease production after exercise. Exercise had no
effect on the amount of IL-1
(P = 0.35) and IL-6 (P = 0.52) in positive cells (Table 2).
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Stimulated cytokine production.
Upon stimulation with LPS, the percentage of IL-1-positive monocytes
was depressed postexercise (P < 0.05), and this
remained low (P < 0.01) at 2 h postexercise
(Table 3). There was no change in the
percentages of TNF-
- or IL-6-positive monocytes postexercise. However, because of the increase in circulating monocyte numbers postexercise and 2 h postexercise (P < 0.01), the
absolute number of cytokine-positive monocytes was increased
(P < 0.01) at these times (Table 3). The amounts of
IL-1
, TNF-
, and IL-6 in positive cells were decreased
(P < 0.05) in stimulated samples postexercise and
2 h postexercise (Table 3).
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Plasma measurements.
Plasma IL-6 (Fig. 2) and TNF- (Fig.
3) concentrations were elevated
(P < 0.01) postexercise and 2 h postexercise, and
the increase in plasma IL-6 was maintained 24 h postexercise.
Exercise had no effect on plasma glucose concentration; however, plasma lactate was increased (P < 0.01) in postexercise
samples (Table 4). Plasma epinephrine,
norepinephrine, and cortisol concentrations were elevated
(P < 0.01) postexercise (Table 4). Cortisol and norepinephrine levels remained elevated 2 h postexercise
(P < 0.01) but had returned to preexercise levels by
24 h postexercise. Creatine kinase activity (Table 4) was elevated
postexercise, 2 h postexercise, and 24 h postexercise
(P < 0.01).
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DISCUSSION |
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Numerous studies have examined the effect of exercise on the
plasma concentration of proinflammatory cytokines; however, it is
acknowledged that few studies have examined the cellular origin of
these cytokines (2). Previous studies have demonstrated that exercise does not increase IL-1, IL-1
, TNF-
, and IL-6 mRNA in blood mononuclear cells, even though plasma concentrations of
IL-1
, TNF-
, and IL-6 increase with exercise (10,
26). Moldoveanu et al. (10) and Ullum et al.
(26) suggested that monocytes were unlikely to be the
source of the rise in plasma IL-6 with exercise. In the present study,
the decrease in percentage and number of cells producing cytokines, as
well as the fluorescence intensity of cytokine-positive cells,
post-exercise provides evidence that circulating monocytes are not
likely to be the source of elevated plasma concentrations of TNF-
or
IL-6.
It has been suggested that the source of this increase in plasma
cytokines is the contracting skeletal muscle. Bruunsgaard et al.
(4) observed higher levels of plasma IL-6 when comparing eccentric with concentric exercise. They also observed an increase in
plasma creatine kinase activity, leading them to conclude that the
cytokine response to exercise is related to muscle damage. Further
research reported plasma IL-6 concentration to be higher after running
(which has a large eccentric component) than after cycling (little
eccentric component) exercise (13). In our previous study,
in which subjects performed 2 h of cycling at 70% peak O2 consumption, plasma IL-6 was <2 pg/ml postexercise
(23), compared with 120 pg/ml in the present study. It is
well recognized that repetitive eccentric contractions cause more
damage to skeletal muscle than concentric contractions (1, 9,
19). Muscle damage invokes an immune response, and it is
possible that this process stimulates local production of inflammatory
cytokines (17). Monocytes are involved in the muscle
tissue inflammatory response to muscle injury (29), and
thus infiltrating monocytes may be the source of elevated plasma IL-6.
Indeed, IL-6 mRNA has been reported to be expressed in skeletal muscle
after prolonged running (16). Both the identification of
the cytokine-producing cell within the muscle and the question as to
whether the increase in cytokine mRNA results in protein translation
during acute exercise remain to be elucidated. The present study
demonstrates increased levels of plasma IL-6 and TNF- postexercise,
yet the data show a decrease and no change in the number of circulating
monocytes spontaneously producing IL-6 and TNF-
, respectively. In
addition, mean IL-6 fluorescence intensity was not altered in response
to exercise, demonstrating that any cells that were spontaneously producing IL-6 postexercise did not increase their production. A
decrease in mean TNF-
fluorescence intensity postexercise and 2 h postexercise suggests that cells spontaneously producing TNF-
decreased their production at these time points; therefore, it is not
likely that these cells are the source of the increase in plasma
levels. It is possible, however, that these cells were productive in
the early stages of exercise and were in a refractory period at the
time of collection. Despite this possibility, it is not likely that
these previously produced cytokines were still present and
significantly contributing to the elevated cytokine concentration at
the time of collection because cytokines are unstable and are removed
from the circulation. This suggests that IL-6 and TNF-
are coming
from a source other than circulating monocytes. Because prolonged
running was the mode of exercise employed, and high levels of plasma
creatine kinase were observed, local production in damaged skeletal
muscle was a likely source.
Decreased blood flow to the splanchnic bed during exercise may be sufficient to induce an ischemic state resulting in gut wall bacterial translocation (2). Endotoxemia has been observed after marathon running (5); therefore, it is possible that subjects in the present study had elevated levels of endotoxin. Endotoxins are lipopolysaccharides of gram-negative bacteria, and it is possible that this powerful monocyte stimulant contributes to elevations in plasma proinflammatory cytokines. The results from the present study demonstrate a decrease in spontaneous cytokine production by circulating monocytes postexercise; therefore, if endotoxemia does contribute to increased levels of plasma cytokines, it is not due to its stimulatory effect on circulating monocytes.
There are several reports that the risk of upper respiratory tract infections is higher in athletes undertaking heavy training or competing in endurance events compared with that in nonathletes (8, 12). It is possible that exercise modifies leukocyte cytokine production, thereby affecting immune function. In the present study, exercise resulted in an increase in circulating leukocytes; therefore, there were more monocytes in circulation to respond to stimulation. However, it is important to note that monocytes responding to stimulation were producing less cytokine than they were preexercise. Hence, exercise may have resulted in an increase in the number of cytokine-positive cells in response to stimulation, but these cells were producing less cytokine postexercise than preexercise. The overall impact of these observations on immune function and the question as to whether cytokine production is a limiting factor in immune protection postexercise remain unclear.
Neuroendocrine hormones have been shown to regulate the immune
response, and direct neuroimmune communication occurs
(21). Proinflammatory cytokines activate both the
hypothalamic-pituitary-adrenal axis and the sympathoadrenergic system
(24), both of which exert potent anti-inflammatory actions
that limit production of proinflammatory cytokines (2).
Incubation of whole blood with epinephrine (3) or
norepinephrine (26) decreases IL-6 and TNF- production. Furthermore, administration of cortisol at levels comparable to those
in the present study decreased LPS-stimulated IL-1, TNF-
, and IL-6
production (7). In the present study, cells spontaneously producing IL-1
and IL-6 ceased production after exercise, and the
amount of TNF-
produced by each cell was reduced postexercise. In
addition, cells produced less cytokine upon stimulation postexercise. Because there was an increase in the concentration of plasma
epinephrine, norepinephrine, and cortisol postexercise, it is possible
that these hormones had a role to play in decreasing cytokine production.
In conclusion, it is likely that elevations in stress hormones during
exercise cause a decrease in monocyte cytokine production postexercise.
The results of this study indicate that circulating monocytes are not
likely to be the source of elevations in plasma TNF- and IL-6 after
prolonged, strenuous running.
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ACKNOWLEDGEMENTS |
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We thank the subjects for participating in this project. In addition, we thank Shannon Campbell, Jane Dancey, Tanyth DeGooyer, and Stella Sarlos for technical assistance on the day of the marathon.
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FOOTNOTES |
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We also acknowledge the Australian Research Council for financial support and Gatorade for providing product.
Address for reprint requests and other correspondence: M. A. Febbraio, Exercise Physiology and Metabolism Laboratory, Dept. of Physiology, The Univ. of Melbourne, Parkville 3052, Australia (E-mail: m.febbraio{at}physiology.unimelb.edu.au).
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 21 July 2000; accepted in final form 30 October 2000.
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