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Post-Graduate Program in Human Movement Sciences, Institute of Physical Activity and Sport Sciences (ICAFE), Cruzeiro do
Sul University, Sao Paulo, Brazil; and 2Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of
Sao Paulo, Sao Paulo, Brazil
ABSTRACT
de Moura, NR, Cury-Boaventura, MF, Santos, VC, LevadaPires, AC, Bortolon, JR, Fiamoncini, J, Pithon-Curi, TC, Curi, R,
and Hatanaka, E. Inflammatory response and neutrophil
functions in players after a futsal match. J Strength Cond
Res 26(9): 25072514, 2012Futsal players suffer injuries
resulting from muscle fatigue and contact or collision among
players. Muscle lesions can be detected by measuring muscle
lesion markers such as creatine kinase (CK) and lactate
dehydrogenase (LDH) in plasma. After an initial lesion, there is
an increase in the plasma levels of C-reactive protein (CRP) and
proinflammatory cytokines. These mediators may activate
neutrophils and contribute to tissue damage and increase
susceptibility to invasive microorganisms. In this study, we
investigated the effect of a futsal match on muscle lesion
markers, cytokines, and CRP in elite players. The basal and
stimulated neutrophil responsiveness after a match was also
evaluated based on measurements of neutrophil necrosis,
apoptosis, phagocytic capacity, reactive oxygen species (ROS)
production, and cytokines (tumor necrosis factor-alpha [TNF-a],
interleukin [IL]-8, IL-1b, IL-10, and IL-1ra) production. Blood
samples were taken from 16 players (26.4 6 3.2 years, 70.2 6
6.9 kg, 59.7 6 5.1 mlkg21min21, sports experience of 4.4 6
0.9 years) before and immediately after a match. Exercise
increased the serum activities of CK (2.5-fold) and LDH
(1.3-fold). Playing futsal also increased the serum concentrations of IL-6 (1.6-fold) and CRP (1.6-fold). The TNF-a, IL-1b,
IL-8, IL-1ra, and IL-10 serum levels were not modified in the
conditions studied. The futsal match induced neutrophil
apoptosis, as indicated by phosphatidylserine externalization
(6.0-fold). The exercise induced priming of neutrophils
by increasing ROS (1.3-fold), TNF-a (5.8-fold), and IL-1b
Address correspondence to Dr. Elaine Hatanaka, ehata@usp.br.
26(9)/25072514
Journal of Strength and Conditioning Research
2012 National Strength and Conditioning Association
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METHODS
Experimental Approach to the Problem
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Reagents
TABLE 1. Effect of playing futsal on markers of muscle injury in the players determined
before and immediately after a match.*
Serum markers of lesion and inflammation
Serum activities
CK (UL21)
LDH (UL21)
Serum concentrations
CRP (mgml21)
IL-6 (pgml21)
TNF-a (pgml21)
IL-1b (pgml21)
IL-8 (pgml21)
IL-1ra (pgml21)
IL-10 (pgml21)
Before a match
222 6 53
262 6 22
5.8 6 0.7
0.1 6 0.04
56 6 7.7
13 6 3.6
16 6 4
1,499 6 379
95 6 29
After a match
455 6 89
351 6 50
7.8 6
0.5 6
58 6
14 6
20 6
1,366 6
103 6
1.3
0.17k
7.8
4.0
7
404
26
*CK = creatine kinase; LDH = lactate dehydrogenase; CRP = C-reactive protein; IL-6 =
interleukin-6; TNF-a = tumor necrosis factor-alpha.
The values are presented as mean 6 standard error of 16 players.
p , 0.01, p = 0.02, and p = 0.03, respectively, for CK, LDH, CRP, and IL-6 before and
after a match.
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The experiments were performed within 1 hour of venipuncture. Human neutrophil (.98%) preparations were isolated
from peripheral blood of human donors under endotoxin-free
conditions using Histopaque (Sigma Chemical Co.) according to the manufacturers instructions. Briefly, blood was
diluted vol/vol with 10 mM phosphate buffered saline (PBS)
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Figure 4. Tumor necrosis factor-alpha (TNF-a; picograms per milliliter), interleukin (IL)-8 (picograms per milliliter), IL-1b (picograms per milliliter), and IL-1ra
(picograms per milliliter) release by neutrophils before and immediately after a futsal match. The measurements were taken in the basal and LPS-stimulated
conditions. The values are presented as mean 6 standard error of 16 players. *p , 0.05, **p , 0.01, and ***p , 0.001 for comparison of cytokine production by
neutrophils before a match without lipopolysaccharide (LPS) stimulation and #p , 0.05, ##p , 0.001, and ###p , 0.05 for comparison of cytokine production
by neutrophils before a match with PMA stimulation.
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RESULTS
The players participation in a futsal match increased the
serum activities of CK (2.5-fold) and LDH (1.3-fold) (Table 1).
The serum CK activities were correlated with serum activities
of LDH (p = 0.87) (Figure 1). As shown in Table 1, the futsal
match increased the players serum levels of CRP (1.6-fold)
and IL-6 (1.6-fold). Under the conditions of this study, the
players showed no difference in serum levels of TNF-a,
(Becton Dickinson). Ten thousand events were analyzed per
IL-1b, IL-8, IL-10, and IL-1ra before and immediately after
experiment (11).
the futsal match (Table 1).
The futsal match induced phosphatidylserine externalizaPhagocytosis
tion of neutrophils (6.0-fold) (Figure 2A). No alterations in
Neutrophils were suspended in PBS containing 100 mM
loss of membrane integrity (Figure 2B) and DNA fragmenCaCl2, 50 mM MgCl2, and 100 mM glucose to 1.0 3 106
tation (Figure 2C) were observed.
cellsper milliliter with 1.0 3 107 particles of opsonized
In the basal condition, that is, without PMA stimulation,
zymosan per milliliter. The reaction mixture was composed
neutrophils collected immediately after the futsal match
of 10:1 bacteria/neutrophils in a final total volume of 0.1 ml.
spontaneously released higher amounts of ROS (by 1.3-fold,
p = 0.02) than neutrophils
collected from the players
before the match (Figure 3).
Also in the basal condition, that
is, without LPS stimulation,
neutrophils obtained postexercise released higher amounts of
TNF-a (5.8-fold), IL-1b (4.8fold) than in the preexercise
condition (Figure 4). No alterations were observed in the
basal release of IL-8 and IL1ra by neutrophils before and
after a futsal match (Figure 4).
In the stimulated condition,
that is, with LPS stimulation,
postexercise neutrophils released higher amounts of IL8, IL-1b, TNF-a, and IL-1ra
than in the preexercise condition. Cytokine production by
LPS-stimulated
neutrophils
Figure 6. Playing futsal increased the serum levels of creatine kinase (CK), lactate dehydrogenase (LDH),
increased
1.6,
2.2,
4.0,
and 1.8C-reactive protein (CRP), and interleukin-6 (IL-6). The exercise induced neutrophil death, increased the basal
fold in IL-8, IL-1b, TNF-a, and
responsiveness of neutrophils, and diminished neutrophil function in the stimulated condition.
IL-1ra, respectively (Figure 4).
Figure 5. Phagocytic capacity of neutrophils determined before and
immediately after a match. The values are presented as mean 6 standard
error of 16 players. ***p , 0.001 for a comparison of values before and
after a match. The values are presented as mean 6 standard error of 16
players.
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DISCUSSION
Exercise-induced lesions cause the levels of proinflammatory
cytokines such as TNF-a, IL-1b, and IL-6 to increase twofold
to threefold (18). This increase in the levels of proinflammatory cytokines is accompanied by an increase in serum levels of
CRP, a classic acute-phase protein. In the inflammatory state,
the plasma concentration of CRP may increase 1,000-fold in
relation to its basal level (9,14). The CRP synthesis is largely
regulated by inflammation-associated cytokines such as
TNF-a, IL-1b, and IL-6 (9,14). The biological function of
CRP is associated with improved phagocytosis of bacterial
pathogens by phagocytes. C-reactive protein interacts with Fc
receptors in phagocytic cells and acts as an opsonin,
contributing to the phagocytosis of microorganisms. Although
playing a futsal match was found to increase the serum levels
of CRP, we did not observe a concerted mode of action driving
a more powerful response of innate host defense. Our results
indicated that the phagocytic capacity of the players
postmatch neutrophils decreased when exposed to zymosan,
which is a classical stimulus to study phagocytosis.
Neutrophils are the first leukocytes to migrate to the site of
injury. Together with macrophages, they are responsible for
local cleaning, phagocytizing pathogens and removing cellular
debris (1012,19). Under inflammatory conditions, ROS may
be important signaling molecules for the release of cytokines
and the activation and deactivation or death of leukocytes
such as neutrophils. The initial release of proinflammatory
mediators and neutrophil activation are important events of
tissue repair; however, the inflammatory response must be
a self-controlled event. The hypoproduction or hyperproduction of neutrophils and cytokines may lead to increased
susceptibility to invasive microorganisms, impairing the
athletes performance. Specific knowledge about subclinical
systemic inflammation and neutrophil function may help
futsal professionals to devise strategies to control the
processes that can result in infection and tissue injury.
However, regardless of the origin, excessive or deficient
neutrophil function in players must be controlled.
The observed increment in acute-phase proteins and
cytokines may prime and activate neutrophils (12). An
important aspect of neutrophil function is their ability to
synthesize proinflammatory and antiinflammatory cytokines
and growth factors that modulate the inflammatory response.
The exercise described herein increased the basal responsiveness of neutrophils, increasing the release of ROS and TNF-a,
IL-1b in the nonstimulated condition (absence of stimuli). The
increased production of cytokines by cultured neutrophils
observed after a futsal match implicates the match as a priming
agent. Classical priming agents such as TNF-a and serum
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PRACTICAL APPLICATIONS
Specific knowledge about subclinical systemic inflammation and neutrophil function may help athletic coaches,
doctors, and nutritionists devise strategies for controlling
processes that may result in infection and tissue injury.
Whatever the origin, excessive or deficient neutrophil
function in players must be controlled. Thus, if one
considers that incremental levels of proinflammatory
cytokine and acute-phase proteins are an important
characteristic of acute-phase response, it is reasonable to
assume that the administration of antioxidant supplements,
for instance n-3 fatty acids and antiinflammatory drugs, may
be a useful adjunct to athlete health management.
ACKNOWLEDGMENTS
The authors are indebted to Sabrina da Silva Moura and Gustavo
Barquilha Joel for their technical assistance. This research was
VOLUME 26 | NUMBER 9 | SEPTEMBER 2012 |
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