INFLAMMATORY RESPONSE AND NEUTROPHIL

FUNCTIONS IN PLAYERS AFTER A FUTSAL MATCH

NIVALDO R. DE MOURA,1 MARIA F. CURY-BOAVENTURA,1 VINICIUS C. SANTOS,1
ADRIANA C. LEVADA-PIRES,2 JOSE RICARDO BORTOLON,1 JARLEI FIAMONCINI,2
TANIA C. PITHON-CURI,1 RUI CURI,2 AND ELAINE HATANAKA1
1

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
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2012 National Strength and Conditioning Association

(4.8-fold) released in nonstimulated cells. However, in the

stimulated condition, the exercise decreased neutrophil function, diminishing the release of ROS by phorbol myristate
acetatestimulated neutrophils (1.5-fold), and the phagocytic
capacity (1.6-fold). We concluded that playing futsal induces
inflammation, primes and activates neutrophils, and reduces the
efficiency of neutrophil phagocytosis immediately after a match.

KEY WORDS indoor soccer, leukocytes, inflammation, cytokines

INTRODUCTION

utsal is a dynamic sport characterized by aerobic

and anaerobic metabolic demands. A competitive
futsal season includes weekly microcycles of
training, tapering, competition, and recovery. During a futsal match, players may suffer lesions resulting from
muscle fatigue and contact and collision among players.
Additionally, the demands involved in playing 23 matches per
week elevate the stress imposed on players, thereby increasing
the risk of injury and diminishing their performance because of
fatigue, muscle damage, and inflammation (7).
Strenuous physical exercise can result in muscle injury,
promoting release of the skeletal muscle enzymes creatine
kinase (CK), and lactate dehydrogenase (LDH). Both CK and
LDH are found in cellular cytosol, so the appearance of these
enzymes in serum indicates cellular lesion (5). Inflammatory
response to cellular lesion with soreness is initiated by fluid,
plasma protein, and leukocyte infiltration into the injured
muscle. Cytokines (e.g., tumor necrosis factor-alpha [TNF-a],
interleukin [IL]-1b, IL-6) and chemokines (IL-8) are mediators
that participate in the onset of the inflammatory response
(6,17,19). However, when inflammation and strenuous exercise
are prolonged, the initial inflammatory response results in the
production of antiinflammatory cytokines such as IL-1ra, IL-4,
and IL-10, which may lead to immunosuppression and
heightened susceptibility to invasive microorganisms, negatively affecting the athletes performance. Exercise-induced
muscle damage is also associated with phagocyte (neutrophils
and macrophages) infiltration into muscle, production of
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Neutrophil Functions in Futsal Players

reactive oxygen species (ROS), and systemic elevation in the
production of cytokines and other inflammatory mediators
such as leukotrienes and prostaglandins (17).
Futsal athletes seem to be prone to illness, particularly during
playing seasons. The aim of this study was to investigate how
playing a futsal match affects the functional characteristics of the
innate immune response, particularly the production of
inflammatory mediators, leukocyte functions and the possible
relationship with host defense capacity. Specific knowledge
about subclinical systemic inflammation and neutrophil
function in athletes after a match may help coaches, doctors,
and nutritionists devise strategies for controlling the inflammatory process, avoiding infection, and tissue injury. Regardless of
the origin, however, excessive or deficient neutrophil function in
players can be controlled. Thus, if one considers that
incremental levels of proinflammatory cytokine and acutephase proteins are an important characteristic of acute-phase
response, it is reasonable to suppose that the administration of
antioxidants and antiinflammatories, for example, n-3 fatty
acids, may be a useful adjunct to athlete health management. A
knowledge of the immune system alterations that occur during
training and matches may enable the maintenance and
improvement of the performance of futsal athletes. Although
futsal has gained worldwide status as a sport, few studies have
focused on the health of futsal players and on strategies to
prevent lesions. Chronic or acute inflammation modifies the
metabolism of fatty acids, carbohydrates and iron, calcium
concentrations, and the number and functions of circulating
leukocytes (19). Therefore, studies exploring different aspects
of lesion and inflammation are important to improve the
performance of athletes.
This work involved an investigation of the effects of a futsal
match on the players serum CK and LDH activities. After
examining possible muscle lesions by measuring muscle
enzyme activity, we determined the systemic inflammatory
status of the athletes based on their serum levels of C-reactive
protein (CRP), proinflammatory (IL-1b, TNF-a, IL-8, and
IL-6), and antiinflammatory (IL-10 and IL-1ra) cytokines.
Changes in the production of cytokines may lead to
inappropriate activation of leukocytes and even increased
susceptibility to invasive microorganisms, impairing the
athletes health. Therefore, the study was completed by
measuring neutrophil functions (phagocytic capacity, release
of ROS and cytokines) and the proportion of cells with signs
of necrosis and apoptosis.

METHODS
Experimental Approach to the Problem

In this study, we demonstrated that the players participation

in a futsal match induced muscle damage and inflammation,
as indicated by their augmented serum CK and LDH
activities and IL-6 and CRP levels after a match. After
intense prolonged exercise is done, metabolically exhausted
muscle fibers exhibit diminished membrane resistance and

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augmented serum CK and LDH activities in plasma (5). Once

muscle damage occurs, the cells in the inflammatory focus
release a series of mediators that will simulate tissue repair.
As a result, CRP, TNF-a, IL-1b, IL-6, and IL-8 levels also
increase in plasma. In the conditions of this study, no change
was found in the serum levels of TNF-a, IL-1b, IL-8, IL-10,
and IL-1ra measured before and immediately after the
match. The fact that no changes were observed in the plasma
levels of these cytokines is likely because of the low sensitivity
of the methods used for cytokine determination.
Subjects

With the approval of the Ethics Committee of Cruzeiro do Sul

University (protocol number 178/2008), 16 volunteers
participated in the study. All the athletes signed an informed
consent form agreeing to submit to the procedures involved in
the study. The group presented the following characteristics
(mean 6 SD): age 26.4 6 3.2 years, body mass 70.2 6 6.9 kg,
height 172.8 6 5.7 cm, body fat 12.0 6 2.3%, V_ O2peak 59.7 6
5.1 mlkg21min21, and sports experience of 4.4 6 0.9 years.
All the participants played during approximately the same
amount of time (5 minutes playing and 5 minutes recovering,
playing a total of 10 minutes in each period) until they
completed 2 equal periods of 20 minutes. The subjects with
a history of infection, viruses, chronic lesions, diabetes,
rheumatoid arthritis, hormonal dysfunction, lupus, or other
inflammatory and hematology diseases (such as hemoglobinopathies) and who were on medication were excluded
from the study.
Sample Collection

Twenty milliliters of venous blood were collected before and

immediately after a competitive futsal match. The blood
samples were drawn from 1 of the 3 main veins of the
antecubital fossa (the cephalic, basilic, and median cubital). In
each case, the vein was chosen based on the identification of
the optimal site by both visual and tactile exploration. The
blood samples were drawn into 2 BD Vacutainer tubes, the
first containing heparin, which was used for plasma collection
and cell separation, whereas the second one was a dry gel tube
for serum collection. All the samples were collected in the
field (athletic clothing) 1 hour before and immediately after
the match. After collecting the samples, the serum was stored
at room temperature (2530 C), whereas the plasma was
stored on ice. The laboratory tests were performed within
1 hour of venipuncture, which was the time required to
transport the samples from the field to the laboratory. In the
laboratory, the blood was centrifuged (400g, 10 minutes), and
serum and plasma were separated from the cell components.
Neutrophils were isolated immediately and cellular function
was tested. The enzymatic activity of CK and LDH was
measured not later than 48 hours after collecting the
plasma. The storage temperature of 200 ml of plasma was
between 2 and 8 C, away from light because of the
samples low stability. The volume of samples used for CK,
LDH, and polymerase chain reaction (PCR) determination

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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.

was 10 ml for each biochemical marker and 100 ml for

cytokine determination. However, whenever necessary, the
samples were diluted to fall within the linearity range of the
methods. All the dilutions were performed with suitable
dilution reagents, according to the instructions of the kit
manufacturer. The concentration of diluted samples was
determined by multiplying by the dilution factor.
Serum was collected and stored at 280 C before cytokine
and PCR determination by enzyme-linked immunosorbent
assay (ELISA) and immunoturbidimetric assay, respectively.
The samples were stored for not .3 months.

Fetal bovine serum, Hepes,

Histopaque, lipopolysaccharide
(LPSEscherichia coli 026:B6),
penicillin, RPMI 1640 medium
supplemented with L-glutamine,
sodium bicarbonate, propidium
iodide (PI), hydroethidine, lucigenin, phorbol myristate acetate
(PMA), formyl-methionyl-leucylphenylalanine, and streptomycin
were supplied by Sigma Chemical Co. (St. Louis, MO, USA).
Determination of Creatine
Kinase and Lactate
Dehydrogenase Activities

Serum CK and LDH activities

were measured according to the
methods established by Oliver
and Zammit and Newsholme
(16,20), respectively. The kits
were supplied by Bioclin Diagnostics (Sao Paulo, Brazil), and the measurements were
carried out according to the manufacturers instructions. The
control serum was used to check the accuracy and precision
of the doses, with a maximum error of 5%. The sensitivity of
the methods was 5.0 UL21 for both CK and LDH. The
reference values, 200480 UL21 for the LDH method
and 025 UL21 for the CK method, were obtained by
determining LDH in healthy male and female populations
according to the manufacturers kit. The instruments were
calibrated for all the determinations, and a control serum was
used to check the data obtained.
Determination of Plasma Cytokines

Plasma levels of IL-1b, TNF-a, IL-8, IL-6, IL-10, and

IL-1ra were determined by the ELISA, according to the
manufacturers instructions (DuoSet KitQuantikine, R&D
System, Minneapolis, MN, USA). The IL-6 and IL-10 methods
were linear for protein concentrations in the range of
252,000 pgml21. The TNF-a method was considered linear
for protein concentrations in the range of 6.01,000 pgml21 of
IL-b 5.02,500 pgml21. The IL-1ra and IL-8 were linear at
concentrations in the range of 3.0250 pgml21. A standard
curve was built for each set of samples and cytokines assayed,
yielding a correlation coefficient in the range of 0.980.99.
For these determinations, the intraassay coefficient of variance
was 35%, whereas the interassay coefficient of variance was
810%.
Figure 1. Correlation between serum creatine kinase (CK; units per liter)
(A) and lactate dehydrogenase (LDH; units per liter) activities measured
in the players before and immediately after a match. The values are
presented as mean 6 standard error of 16 players. The serum CK and
LDH activities (shown in Table 1) were correlated (p = 0.87).

Determination of Serum C-Reactive Protein Levels

The CRP levels were determined by a highly sensitive

immunoturbidimetric method (Bioclin Diagnostics, Sao Paulo,
Brazil) according to the manufacturers instructions. The
linearity in the determination of CRP was 5.0200 mgml21,
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Neutrophil Functions in Futsal Players

Figure 3. Reactive oxygen species (ROS) release by neutrophils (in relative

fluorescence units [RFU]) determined before and immediately after a match.
The measurements were carried out in basal and phorbol myristate acetate
(PMA)-stimulated conditions. The values are presented as mean 6 standard
error of 16 players *p , 0.05 for comparison of ROS production by
neutrophils before and after a match without PMA stimulation. ***p , 0.001
for comparison of ROS production by neutrophils before a match with and
without PMA, and #p , 0.05 for a comparison of ROS production by
neutrophils before and after a match with PMA stimulation.

Figure 2. A) Phosphatidylserine externalization in neutrophils (in

percentage) determined before and immediately after a match. B)
Percentage of neutrophils with no alterations in membrane determined
before and immediately after a match. C) Percentage of neutrophils with
DNA fragmentation determined before and immediately after a match.
The values are presented as mean 6 standard error of 16 players.
***p , 0.001 for comparison of values before and after a match.

with a correlation coefficient of 0.99. The equipment was

calibrated for all the determinations, and the data obtained
were compared with a control serum.
Cell Purification

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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Dulbecco at pH 7.4 and carefully layered on 10 ml of

a commercial gradient of Ficoll-Hypaque (Histopaque,
d = 1.077). The tube was centrifuged at 400g at room
temperature for 20 minutes. The supernatant, rich in
mononuclear cells, was discarded, and 10 ml of 5% dextran
was added to the pellet. The tube was homogenized and kept
on ice for 45 minutes to allow for erythrocyte sedimentation. The resulting supernatant rich in granulocytes was
recovered, washed with PBS Dulbecco, and the pellet
hypotonically treated with 10 ml of distilled water to
promote lysis of contaminated red blood cells. After 1 minute,
isotonicity was restored by adding 5 ml of 2.7% NaCl and 15
ml of PBS Dulbecco. The cells were centrifuged at 400g for 5
minutes at room temperature and suspended in RPMI 1640
medium. The purity of the cell preparation was .98%.
Cell Culture and Cytokine Determination

After purification, neutrophils (2.5 3 106 cells per milliliter)

were suspended in RPMI1640 medium supplemented with
0.3 gL21 glutamine, 2.32 gL21 Hepes, 2 gL21 sodium
bicarbonate, 100 mgml21 streptomycin, 100 IUml21 penicillin, and 10% fetal bovine serum. The cells were then counted
in a Neubauer chamber and immediately cultured at 37 C and
5% CO2, with and without LPS (5 mgml21). After 18 hours,
the supernatants were collected and stored at #280 C
before cytokine determination by ELISA (Quantikine) (13).
Cell Viability Assay (Proportion of Necrotic Cells)

Neutrophil viability was assessed using an FACSCalibur

Cytometer (Becton Dickinson Systems, Franklin Lakes, NJ,
USA). The percentage of viable cells in each sample was

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determined based on PI staining (solution at 0.05% in PBS).
Ten thousand events were analyzed per sample. Fluorescence
of the PI was measured using the FL2 channel (orange-red
fluorescence = 585/42 nm) (15).
Proportion of Cells with DNA Fragmentation

The DNA fragmentation was analyzed by flow cytometry

after DNA staining with PI. The presence of detergent in the
solution permeabilized the cells, which promptly incorporated the dye into DNA. Briefly, after incubation, the cells
were centrifuged at 1,000g for 15 minutes at 4 C. The
resulting pellets were carefully resuspended in 300 ml
hypotonic solution containing 50 mgml21 PI, 0.1% sodium
citrate, and 0.1% Triton X-100. The cells were then incubated
for 30 minutes at 4 C. Ten thousand events were analyzed
per sample. Fluorescence of the PI was measured using the
FL2 channel (orange-red fluorescence = 585/42 nm) (15).
Annexin V Staining of Apoptotic Cells

Neutrophils (2.5 3 106 cells per milliliter) were harvested from

culture plates and centrifuged at 4 C, 200g, for 10 minutes. The

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translocation of phosphatidylserine residues from the inner to

the outer leaflet of plasma membrane, assessed by the reaction
with AnnexinVfluorescein isothiocyanate (FITC; Clontech
Laboratories, Inc., Palo Alto, CA, USA) and 50 ml of PI
(50 mgml21), was used as a measure of apoptosis and scored
in a FACScalibur flow cytometer (Becton Dickinson). Three
cell subpopulations were identified: viable cells (Annexin
V-FITC2/PI2), early apoptotic cells (Annexin V-FITC+/PI2),
and late apoptotic cells (Annexin V-FITC+/PI+) (8).
Flow Cytometric Measurement of Reactive Oxygen
Metabolites Using Hydroethidine

Hydroethidine (1 mM) was added to the neutrophil (2.5 3 106

cells per milliliter) incubation medium when required.
Immediately afterward, the cells were treated with PMA
(54 ngml21). The ROS release was monitored for 30 minutes.
The assays were run in PBS buffer supplemented with CaCl2
(1 mM), MgCl2 (1.5 mM), and glucose (10 mM), at 37 C,
in a final volume of 0.3 ml. Fluorescence was measured
using the FL3 channel of a FACSCalibur flow cytometer

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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Neutrophil Functions in Futsal Players

The cells were kept at 37 C under moderate shaking.
Phagocytosis was assessed by a fluorochrome assay using
acridine orange, as previously described (4).
Statistical Analyses

The values are presented as mean 6 standard error of

16 players. The statistical analysis consisted of 1-way analysis of
variance using the post hoc Student-Newman-Keuls multiple
Comparison test (INStat; Graph Pad Software, San Diego, CA,
USA). The significance level was set at p , 0.05. The degree of
linear relationship between CK and LDH variables was
established by Pearsons correlation.

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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In the stimulated condition, that is, neutrophils treated with
PMA, we found that the release of ROS was diminished
(1.5-fold) (Figure 4). These results were accompanied by
decreased (1.6-fold) phagocytic capacity of neutrophils when
exposed to zymosan (Figure 5).

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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amyloid A alone cannot stimulate cell cytokine production,

but they augment the maximal rate of cytokine release
when a second stimulus occurs (12). In the LPS-stimulated
condition, it was found that postexercise neutrophils collected
from futsal players released higher amounts of IL-8, IL-1b,
TNF-a, and IL-1ra than in nonexercised controls.
In inflammatory processes, increased levels of ROS, acutephase proteins, proinflammatory cytokines, and fatty acids
can prime neutrophils and other immune cells (12). Under
these conditions, inflammatory mediators play a role in the
process of resolution or progression of the damaged tissue.
Whatever the origin, the hypoproduction or hyperproduction of cytokines may lead to inappropriate activation and
tissue injury and even to increased susceptibility to invasive
microorganisms, impairing the athletes performance. Our
findings also indicated that playing futsal induces neutrophil
death, possibly by apoptosis, as indicated by phosphatidylserine externalization. Additionally, a single futsal match
diminished neutrophil function in the stimulated condition,
decreasing the release of ROS by PMA-stimulated neutrophils, and lowering the phagocytic capacity. Playing futsal
reduced the efficiency of neutrophils to ward off infection
resulting from exposure to pathogens (Scheme 1).
The current literature on futsal is devoid of information about
the intensity and most beneficial type of training or the amount
of time each player should participate actively in a match to
maximize the benefit while maintaining minimal risk. This
study performed an extensive measurement of systemic
inflammation and neutrophil function in athletes after a futsal
match. In conclusion, playing futsal induces inflammation,
activates neutrophils, and reduces the efficiency of neutrophils
against infection resulting from exposure to pathogens
immediately after a match (Figure 6). These findings may be
important for designing a strategy to protect futsal players
against microorganism infection or to prevent reduced athletic
performance because of subclinical inflammation immediately
after a match.

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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Neutrophil Functions in Futsal Players

supported by the Brazilian research funding agencies Fundacxao
de Amparo a` Pesquisa do Estado de Sao Paulo (FAPESP;
2009/06039-0 and 2008/56105-7) and Conselho Nacional de
Desenvolvimento Cientfico e Tecnologico (CNPq). Conception
and study design were done by N.R. de Moura and E. Hatanaka;
data acquisition, analysis, and interpretation were performed by
N.R. de Moura, E. Hatanaka, M.F. Cury-Boaventura, V.C. Santos,
A.C. Levada-Pires, J.R. Bortolon, J. Fiamoncini, T.C. Pithon-Curi,
and R. Curi; and drafting/revision was done by T.C. Pithon-Curi,
R. Curi, and E. Hatanaka. The authors declare that they have no
conflict of interest.

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