Immunology 629

Exercise Deactivates Leukocytes in Asthma

Authors R. P. Vieira1, R. A. Silva3, M. C. Oliveira-Junior1, F. R. Greiffo1, A. P. Ligeiro-Oliveira1, M. A. Martins2,

C. R. F. Carvalho3
1
Affiliations Nove de Julho University, São Paulo, Brazil
2
Clinical Medicine (LIM 20), University of São Paulo, Brazil

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3
Physical Therapy (LIM 34), University of São Paulo, Brazil

Key words Abstract reduced leukocytes activation, showed through

●
▶ asthma
▼ decreased expression of Th2 cytokines (IL-4,
●
▶ exercise
Leukocytes play a central role in asthma physi- IL-5, IL-13; p < 0.001), chemokines (CCL5, CCL10;
●
▶ immunology
opathology. Aerobic training (AT) reduces leuko- p < 0.001), adhesion molecules (VCAM-1, ICAM-
●
▶ allergy

●
▶ cytokines cytes recruitment to the airways, but the effects
of AT on some aspects of leukocytes activation in
asthma are unknown. Therefore, the effects of 4
weeks of AT on airway inflammation, pulmonary
and systemic Th2 cytokines levels, leukocytes
expression of pro and anti-inflammatory, pro-
fibrotic, oxidants and anti-oxidants mediators in
an experimental model of asthma was investi-
gated. AT reduced the levels of IL-4, IL-5, IL-13 in
bronchoalveolar lavage fluid (BALF) (p < 0.001),
serum levels of IL-5, while increased BALF and
serum levels of IL-10 (p < 0.001). In addition, AT
1; p < 0.05), reactive oxygen and nitrogen spe-
cies (GP91phox and 3-nitrotyrosine; p < 0.001),
inducible nitric oxide synthase (iNOS; p < 0.001),
nuclear factor kB (NF-kB; p < 0.001) while increased
the expression of anti-inflammatory cytokine
(IL-10; p < 0.001). AT also decreased the expres-
sion of growth factors (TGF-beta, IGF-1, VEGF and
EGFr; p < 0.001). We conclude that AT reduces the
activation of peribronchial leukocytes in a mouse
model of allergic asthma, resulting in decreased
airway inflammation and Th2 response.

Introduction AT in animals’ models of asthma, currently

accepted after revision
September 23, 2013
Bibliography
DOI http://dx.doi.org/
10.1055/s-0033-1358477
Published online:
November 20, 2013
Int J Sports Med 2014; 35:
629–635 © Georg Thieme
Verlag KG Stuttgart · New York
ISSN 0172-4622
Correspondence
Dr. Rodolfo Paula Vieira
Nove de Julho University
Rua Vergueiro
235/249
São Paulo 01504-001
Brazil
Tel.: + 55/11/3061 7180
Fax: + 55/11/3061 7180
rodrelena@yahoo.com.br
▼ experimental models of acute and chronic allergic
A growing number of studies point out the ben-
eficial effects of regular practice of aerobic train-
ing (AT) for the management of asthmatic
individuals [2, 5–7, 9, 13, 19, 23–26, 30–32, 36]. In
summary, these studies demonstrate that AT sig-
nificantly improves asthma symptoms, including
dyspnea and exercise-induced bronchoconstric-
tion (EIB), health-related quality of life, and also
reduces corticosteroid needing as well as reduces
the levels of exhaled nitric oxide, suggesting a
possible anti-inflammatory effects of AT for the
airways [7, 9, 23–26, 30, 31]. More recently, a study
from Mendes et al. (2011) demonstrated for the
first time that AT reduces eosinophilic inflamma-
tion in asthmatic patients, confirming the anti-
inflammatory effects of AT [24]. However, the
mechanisms involved in the anti-inflammatory
effects of AT for asthma remains not fully eluci-
dated.
In this way, a growing number of experimental
studies have been performed aiming to investi-
gate the possible cellular and molecular mecha-
nism underlying the anti-inflammatory effect of
airway inflammation [11, 12, 27–29, 34, 35, 37–39].
In general, these studies have demonstrated that
AT reduces eosinophilic and lymphocytic airway
inflammation, Th2 cytokines production, nuclear
factor kB (NF-kB) activation, while increases the
expression of anti-inflammatory cytokines IL-1ra
and IL-10 [11, 12, 27–29, 34, 35, 37–39]. From these
studies, some initial evidences of the cellular and
molecular effects of AT in experimental models
of acute and chronic allergic airway inflamma-
tion were identified. For instance, Pastva et al.,
2004 and 2005 demonstrated that part of the
anti-inflammatory effects of AT could be attrib-
uted to reduced NF-kB activation and glucocorti-
coid receptor expression in peribronchial
leukocytes and also in airway epithelium [28, 29].
Following Pastva’s study, Vieira et al., 2007 dem-
onstrated that AT also induces the production of
anti-inflammatory cytokine IL-10 [37], a finding
that was further confirmed by Silva et al., 2010,
that elegantly added the stimulatory effect of AT
on IL-1ra expression [35].

Vieira RP et al. Exercise Deactivates Leukocytes in Asthma. Int J Sports Med 2014; 35: 629–635
630 Immunology
However, the literature demonstrates that the leukocytes are
responsible also for the release of Th2 cytokines, growth factors
and oxidants, which play a central role in the inflammatory and
remodeling process in asthma [3, 17, 18, 20–22, 33]. Therefore,
the present study investigate the effects of AT on chronic allergic
airway inflammation, focusing on the effects of AT on peribron-
chial leukocytes activation (i. e., expression of pro-inflammatory,
anti-inflammatory, pro-fibrotic, oxidants and anti-oxidants and
growth factors by leukocytes) involved in the inflammatory and
remodeling process in asthma.
Materials and Methods
▼ morphometic analysis
This study was approved by the ethical committee of the School
of Medicine of the University of Sao Paulo. The “Guide for care
and use of laboratory animals” was followed (NIH publication
85-23, revised 1996). In addition, we state that the present man-
uscript is in accordance to the IJSM’s ethical standard [10].
Animals and experimental groups
32 BALB/c male mice (20–25 g) were distributed in control (Con-
trol; n = 8), aerobic training (AT; n = 8), ovalbumin sensitized
(OVA; n = 8) and ovalbumin sensitized + aerobic training
(OVA + AT; n = 8) groups.
We state that the immunohistochemical and the cytokines
measurements in bronchoalveolar lavage fluid (BALF) were per-
formed in the samples of previous study [37–39].
Treadmill training and test protocol
Animals were adapted to treadmill training (15 min, 25 % incli-
nation and 0.2 km/h) during 3 days. In the following day, all ani-
mals were submitted to maximal exercise test, as previously
described [37, 38]. The physical test was repeated 30 days after
the beginning of AT. The results from physical test were pre-
sented in the previous study [39]. The treadmill physical train-
ing was performed during 4 weeks, 5×/week, 60 min per session,
at low intensity (corresponding to 50 % of maximal exercise
capacity reached in the maximal exercise test). The exercise has
started one day after the first OVA or saline inhalation exposure
[39].
Chronic model of allergic asthma
4 intra-peritoneal (i.p.) injections of OVA (20ug per mouse)
adsorbed with aluminum hydroxide or saline solution for con-
trol groups (non-sensitized mice) were performed on days 0, 14,
28 and 42. 21 days after the first i.p. injection, mice were chal-
lenged with aerosolized OVA (1 %) or with a saline solution 3
times a week until the 50th day [37–39].
Anesthesia and animals’ euthanasia
72h after the last inhalation day and exercise test, animals were
anesthetized by intramuscular injection of ketamine (50 mg/kg)
and xylazine (40 mg/kg), and tracheostomized to collect bron-
choalveolar lavage fluid (BALF). The blood was collected through
the abdominal vein for the cytokines quantification, followed by
euthanasia through exsanguinations.
Bronchoalveolar Lavage Fluid (BALF) procedures
Lungs were gently washed with 1.5 ml of saline (administered as
three 0.5 ml volumes) via the tracheal cannula. Total cell counts
were performed using a hematocytometer (Neubauer chamber)
Vieira RP et al. Exercise Deactivates Leukocytes in Asthma. Int J Sports Med 2014; 35: 629–635
and the differential cell counts (300 cells per lamina) were per-
formed using cytospins preparations stained with May-Grun-
wald-Giemsa [27, 34, 37]. We clarify that the results of total and
differential cell count was already presented in the following
previous study [39].
Cytokines measurements
The levels of IL-4, IL-5, IL-10 and IL-13 were quantified in bron-
choalveolar lavage and in serum by ELISA using commercial kits
(BD Elispot kit, CA, USA) according to the manufacturer recom-
mendation.
Lung histology, immunohistochemistry and
Lungs were fixed in formalin and embedded in paraffin.
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5-micrometer thick sections were stained with hematoxylin and
eosin for lung structure and inflammation analysis [37]. Immu-
nohistochemistry was performed with anti-IL-4, anti-IL-5, anti-
IL10, anti-IL-13, anti-CCL5, anti-CCL10, anti-VCAM-1, anti-ICAM-1,
anti-GP91phox, anti-3-nitrotyrosine, anti-NF-kB, anti-iNOS,
anti-TGF-beta, anti-IGF-1, anti-VEGF and anti-EGFr antibodies
(Santa Cruz Biotechnology, Santa Cruz, CA), using a biotin-
streptavidin-peroxidase method. With a 50-line, 100-point grid
connected to the ocular of the microscope, we assessed the peri-
bronchial density of positive leukocytes for the markers
described above, using a point-counting technique [37]. Count-
ing was performed in 5 complete airways for each animal at
1 000 × magnification. Results were expressed as positive cells
per square millimeter [37].
Statistical analysis
Parametric and nonparametric data were expressed as
means ± SD and as medians ± 95 % confidence interval (95 % CI),
respectively. Comparisons among groups were performed by
one-way analysis of variance followed by the Student-Newman-
Keuls post hoc test (parametric data) or by one-way analysis of
variance on ranks followed by Dunn’s post-hoc test (nonpara-
metric data); the significance level was adjusted to 95 % (p < 0.05).
Results
▼
BALF levels of pro-inflammatory Th2 and anti-
inflammatory cytokines profile
The levels of pro-inflammatory Th2 cytokines (IL-4, IL-5, IL-13)
and anti-inflammatory cytokine (IL-10) in BALF are presented
in ●
▶ Fig. 1a–d, respectively. The results demonstrated that AT
significantly reduced the levels of IL-4, IL-5 and IL-13 when com-
pared with OVA group (p < 0.01). The results also demonstrated
that AT significantly increased the levels of IL-10 in both non-
sensitized (AT) and sensitized (OVA + AT) groups (p < 0.05).
Systemic Th2 (IL-5) and anti-inflammatory (IL-10)
response
The levels of pro-inflammatory Th2 cytokine IL-5 and anti-
inflammatory cytokine IL-10 are presented in ● ▶ Fig. 2a, b,
respectively. The results demonstrated that AT significantly
reduced the levels of IL-5 compared with OVA group (● ▶ Fig. 2a;
p < 0.01). The results also demonstrated that AT significantly
increased the levels of IL-10 in both non-sensitized (AT) and sen-
sitized (OVA + AT) groups (●▶ Fig. 2b; p < 0.05).
 Immunology 631

a b
600 300
*
*
BALF IL-4 (pg/ml)

BALF IL-5 (pg/ml)

400 200

200 100

0 0
Control AT OVA OVA+ AT Control AT OVA OVA+AT

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c d
250 1000

* *
200 800

BALF IL-10 (pg/ml)

BALF IL-13 (pg/ml)

*
150 600

100 400
*

50 200

0 0
Control AT OVA OVA+AT Control AT OVA OVA+AT

Fig. 1 Figure shows the levels of pro and anti-inflammatory cytokines in BALF. In a, b and c, *p < 0.01 when compared with all groups. In d, *p < 0.05 when
compared with Control group.

a b
300 300
* *
*
Serum IL-10 (pg/ml)
Serum IL-5 (pg/ml)

200 200

100 100

0 0
Control AT OVA OVA+ AT Control AT OVA OVA+AT

Fig. 2 Figure shows the levels of pro and anti-inflammatory cytokines in serum. In a, *p < 0.01 when compared with all groups. In b, *p < 0.05 when com-
pared with Control and OVA groups.

Peribronchial leukocytes expression of Th2 and Th1 changed when compared all experimental groups (● ▶ Fig. 3b;

cytokines, chemokines and adhesion molecules p > 0.05). The results also demonstrated that AT significantly
The expression of Th2 cytokines, Th1 cytokines, chemokines and reduced the expression of chemokines (CCL11 and CCL5) when
adhesion molecules are presented in ● ▶ Fig. 3a–d, respectively. compared with OVA group (● ▶ Fig. 3c; p < 0.01). In addition, AT

The results demonstrated that AT significantly reduced the also significantly reduced the expression of adhesion mole-
expression of Th2 cytokines (IL-4, IL-5 and IL-13) by leukocytes cules (VCAM-1 and ICAM-1) when compared with OVA group
when compared with OVA group (● ▶ Fig. 3a; p < 0.001). The (●
▶ Fig. 3d; p < 0.01).

expression of Th1 cytokines (IL-2 and IFN-gamma) were not

Vieira RP et al. Exercise Deactivates Leukocytes in Asthma. Int J Sports Med 2014; 35: 629–635
632 Immunology

a b
800 60
*
*
600
Positive Cells/mm2

Positive Cells/mm2
40

400 *

20
200

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0 0
IL-4 IL-5 IL-13 IL-2 IFN-gama
Control AT OVA OVA+ AT Control OVA

AT OVA+ AT

c d 5 000
4500
*
*
3750
4 000
Positive Cells/mm2
*
Positive Cells/mm2

3000 *
3 000

2250

2 000
1500

1 000
750

0 0
CCL11 CCL5 VCAM-1 ICAM-1
Control OVA Control OVA

AT OVA+ AT AT OVA+ AT

Fig. 3 Figure shows the expression of Th2 and Th1 cytokines, chemokines and adhesion molecules by peribronchial leukocytes. In a, *p < 0.001 when
compared with all groups. In b, no statistically differences were found comparing all groups. In c and d, *p < 0.01 when compared with all groups.

Expression of oxygen and nitrogen reactive species, that AT significantly reduced OVA-induced the expression of
anti-inflammatory cytokine and NF-kB by peribronchial all growth factors investigated, as TGF-beta (p < 0.01), IGF-1
leukocytes (p < 0.001), VEGF (p < 0.001) and EGFr (p < 0.01).
The expression of Gp91Phox and 3-nitrotyrosine (● ▶ Fig. 4a),

iNOS (●▶ Fig. 4b), IL-10 (●▶ Fig. 4c) and NF-kB (●▶ Fig. 4d) are

presented in ●▶ Fig. 4. The results demonstrated that AT signifi- Discussion

cantly reduced the expression of Gp91Phox and 3-nitrotyrosine
(●
▶ Fig. 4a; p < 0.001). The results also demonstrated that AT sig-
nificantly reduced the iNOS expression (● ▶ Fig. 4b; p < 0.001). On
the other hand, AT in sensitized mice significantly increased the
expression of anti-inflammatory cytokine IL-10 (● ▶ Fig. 4c;
p < 0.01). In addition, AT also significantly reduced the NF-kB
expression (● ▶ Fig. 4d; p < 0.001).
Peribronchial leukocytes derived growth factors
●
▶ Fig. 5a–d shows the expression of growth factors TGF-beta,
IGF-1, VEGF and EGFr, respectively. The results demonstrated
▼
The present study showed for the first time that AT inhibit the
lung leukocytes activation seen in an experimental model of
allergic asthma, demonstrated through the reduced expression
of cytokines, chemokines, adhesion molecules, reactive oxygen
and nitrogen species, NF-kB and growth factors by peribronchial
leukocytes, while increases the expression of the anti-inflam-
matory cytokine IL-10.
Leukocytes play a central role in the pathophysiology of asthma
[3, 17, 18, 20–22, 33]. Leukocytes, especially Th2 leukocytes are
differentiated leukocytes responsible for release of Th2

Vieira RP et al. Exercise Deactivates Leukocytes in Asthma. Int J Sports Med 2014; 35: 629–635
 Immunology 633

a b
1500 4500
*
3750
*
Positive Cells/mm2

Positive Cells/mm2
1000 * 3000

2250

500 1500

750

0 0
3-Nitrotyrosine Gp91Phox iNOS

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Control AT OVA OVA+ AT Control AT OVA OVA+AT

c 800 d 2000

*
*
600 1500
Positive Cells/mm2

400 Positive Cells/mm2 1000

200 500

0 0
IL-10 NF-kB
Control AT OVA OVA+AT Control AT OVA OVA+AT

Fig. 4 Figure shows the expression of oxygen and nitrogen reactive species, anti-inflammatory cytokine and NF-kB by peribronchial leukocytes. In a, b and d,
*p < 0.001 when compared with all groups. In c, *p < 0.01 when compared with all groups.

cytokines, i. e., IL-4, IL-5 and IL-13, which exert pro-inflamma-
tory and pro-fibrotic effects on asthma [3, 17, 18, 20–22, 33]. In
summary, IL-4, IL-5 and IL-13 are involved in eosinophils, den-
dritic cells and T-lymphocytes differentiation, proliferation and
activation, exerting their effects both in the lungs as systemi-
cally [3, 17, 18, 20–22, 33]. In the present study we found that AT
significantly reduced not only the expression of IL-4, IL-5 and
IL-13 by leukocytes but also the BALF levels of IL-4, IL-5 and
IL-13, strongly suggesting that the reduced expression of IL-4,
IL-5 and IL-13 by lung leukocytes reflect leukocytes deactiva-
tion. Furthermore, the results also demonstrated that AT signifi-
cantly reduced the serum levels of IL-5, showing that the effects
of AT on allergic response is not limited to the lungs, but also
may involve a systemic component. However, the results found
in the present study may not be applied to circulating leuko-
cytes, an issue that should be investigated in further studies.
Beyond Th2 cytokines, chemokines, as CCL5 and CCL11 present
an important role in chronic allergic airway inflammation [4].
These chemokines regulates eosinophils trafficking to the air-
ways and are present at increased levels in the asthmatic air-
ways and also related with late onset asthmatic response [4]. In
the present study, we demonstrate that AT significantly reduced
the expression of CCL5 and CCL11 by leukocytes, reinforcing the
anti-inflammatory milieu induced by exercise. However, many
other mediators are involved in the eosinophilic trafficking to
the airways, as adhesion molecules. Adhesion molecules, i. e.,
VCAM-1 and ICAM-1 are well studied molecules in inflamma-
tory diseases and are found abundantly in the airways of asth-
matic patients and in animal models of asthma [8, 18, 40].
Increased expression of these molecules is thought to exert a
central role in the eosinophils adhesion and transmigration dur-
ing asthmatic inflammation [3]. Again, the present study shown
that AT significantly reduced the expression of ICAM-1 and
VCAM-1 by peribronchial leukocytes, accounting to the anti-
inflammatory effects of AT.
Following unresolved chronic allergic airway inflammation, air-
way remodeling is a key feature of asthma and is thought to be
irreversible and the main component of airway hyperrespon-
siveness and obstruction [21]. Airway remodeling is character-
ized by hypertrophy and hyperplasia of airway epithelial cells
and smooth muscle, mucus hypersecretion and increased depo-
sition of extra-cellular matrix proteins in airway walls [21]. Dif-
ferent proteins families are involved in the remodeling process
in asthma, as growth factors (TGF-beta, IGF-1, VEGF and EGF),
matrix metalloproteases (MMPs) and tissue inhibitor of matrix
metalloproteases (TIMPs) [1, 16]. Of note, growth factors stimu-
late the synthesis of extra-cellular matrix proteins and are
accredited to be the main mediators involved in remodeling
[1, 16]. In the present study we show for the first time that AT
inhibited the lung expression of TGF-beta, IGF-1, VEGF and EGFr
in OVA-sensitized animals. Therefore, these results explicitly
show that AT may inhibit the airway remodeling process.

Vieira RP et al. Exercise Deactivates Leukocytes in Asthma. Int J Sports Med 2014; 35: 629–635
634 Immunology

a b
8 000 4000
*
TGF-beta + cells (mm2)

6 000 * 3000

IGF-1 + cells (mm2)

4 000 2000

2 000 1000

0 0
Control AT OVA OVA+AT Control AT OVA OVA+ AT

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
c 3 000 d 5000
*
4000 *

EGFr+ cells (mm2)

VEGF+ cells (mm2)

2 000
3000

2000
1 000

1000

0 0
Control AT OVA OVA+ AT Control AT OVA OVA+ AT

Fig. 5 Figure shows the expression of growth factors by peribronchial leukocytes. In a and d, *p < 0.01 when compared with all groups. In b and c,
*p < 0.001 when compared with all groups.

As part of the anti-inflammatory and anti-fibrotic effects of AT
in chronic allergic airway inflammation, we have investigated
the effects of AT on the expression of reactive oxygen species
(ROS), reactive nitrogen species (RNS), anti-inflammatory cytokine
IL-10 and nuclear transcription factor NF-kB. Regarding the role
of ROS and RNS in the pathogenesis of asthma, the literature
clearly demonstrates that increased ROS and RNS production
modulates Th2 inflammatory and fibrotic response in asthma
[33, 41]. In agreement with the current literature, the present
study demonstrated that OVA sensitized animals presented
increased expression of 3-nitrotyrosine and Gp91phox [33, 41].
On the other hand, AT significantly reduced their expression,
confirming the inhibitory effects of AT on reactive oxygen and
nitrogen species synthesis by leukocytes, which may be involved
in these anti-inflammatory and anti-fibrotic effects of AT in
asthma. In addition, a growing number of studies demonstrates
that IL-10 present anti-inflammatory properties, by inhibiting
the eosinoplilic inflammation and Th2 cytokines release, nota-
bly IL-4, IL-5 and IL-13 [17, 22]. In this way, the present results
demonstrated that AT training significantly increased the
expression of IL-10 by leukocytes as well as increased the levels
IL-10 in the lung and also systemically. However, although we
observe a strong stimulus from AT on IL-10 release by leuko-
cytes, the exact molecular mechanisms of IL-10 mediating the
anti-inflammatory effects of AT in asthma remains to be further
investigated. Finally, we also investigated the effects of AT on
NF-kB expression by peribronchial leukocytes. Several studies
show increased NF-kB expression in airways of asthmatic
patients and in animal models of asthma [8, 14, 15, 35, 38, 39].
These studies show that NF-kB controls not only the expression
of pro-inflammatory cytokines, but also the expression of pro-
fibrotic mediators [8, 14, 15, 35, 38, 39]. Our study has confirmed
that ovalbumin-induced chronic allergic lung inflammation is
followed by increased expression of NF-kB in leukocytes. How-
ever, again, the results of the present study showed that AT sig-
nificantly reduced NF-kB expression, possibly accounting as part
of mechanisms involved in the anti-inflammatory and anti-
fibrotic effects of AT in a mouse model of asthma.
In conclusion, the present study shows that aerobic training
reduces chronic allergic airway inflammation and remodeling in
a mouse model of asthma and these results seem to be partially
mediated by deactivation of peribronchial leukocytes.
Acknowledgements
▼
This study was supported by Fundação de Amparo à Pesquisa do
Estado de São Paulo – FAPESP (Process 05/04413-1 and
07/01026-2) Laboratórios de Investigação Médica da Faculdade
de Medicina da USP – LIM FMUSP, Conselho Nacional de Pesquisa
e Desenvolvimento – CNPq and Coordenação de Pessoal de Nível
Superior – CAPES.

Vieira RP et al. Exercise Deactivates Leukocytes in Asthma. Int J Sports Med 2014; 35: 629–635

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