ORIGINAL ARTICLE

Body temperature and its effect on leukocyte mobilization,cytokines and markers of neutrophil activation duringand after exercise

Jonathan Peake Æ Jeremiah J. Peiffer Æ Chris R. Abbiss Æ Kazunori Nosaka ÆMitsuharu Okutsu Æ Paul B. Laursen Æ Katsuhiko Suzuki

Accepted: 11 October 2007

� Springer-Verlag 2007

Abstract We investigated the influence of rectal temperature

on the immune system during and after exercise. Ten well-

trained male cyclists completed exercise trials (90 min cycling

at 60% _VO2 max þ 16:1 - km time trial) on three separate

occasions: once in 18�C and twice in 32�C. Twenty minutes

after the trials in 32�C, the cyclists sat for *20 min in cold

water (14�C) on one occasion, whereas on another occasion

they sat at room temperature. Rectal temperature increased

significantly during cycling in both conditions, and was

significantly higher after cycling in 32�C than in 18�C

(P \ 0.05). Leukocyte counts increased significantly dur-

ing cycling but did not differ between the conditions. The

concentrations of serum interleukin (IL)-6, IL-8 and IL-10,

plasma catecholamines, granulocyte-colony stimulating

factor, myeloperoxidase and calprotectin increased signif-

icantly following cycling in both conditions. The

concentrations of serum IL-8 (25%), IL-10 (120%), IL-1

receptor antagonist (70%), tumour necrosis factor-a (17%),

plasma myeloperoxidase (26%) and norepinephrine

(130%) were significantly higher after cycling in 32�C than

in 18�C. During recovery from exercise in 32�C, rectal

temperature was significantly lower in response to sitting in

cold water than at room temperature. However, immune

changes during 90 min of recovery did not differ signifi-

cantly between sitting in cold water and at room

temperature. The greater rise in rectal temperature during

exercise in 32�C increased the concentrations of serum IL-

8, IL-10, IL-1ra, TNF-a and plasma myeloperoxidase,

whereas the greater decline in rectal temperature during

cold water immersion after exercise did not affect immune

responses.

Keywords Exercise � Hyperthermia � Cytokines �Stress hormones � Cold water immersion

Introduction

Exercise of fixed duration in hot (‡28�C) versus temperate/

cold (£18�C) conditions stimulates greater systemic

mobilization of neutrophils, lymphocytes and natural killer

cells, and systemic release of cytokines (e.g., IL-6, IL-1ra,

IL-12 and TNF-a) (Brenner et al. 1996; Cross et al. 1996;

McFarlin and Mitchell 2003; Mitchell et al. 2002; Niess

et al. 2003; Rhind et al. 1999, 2004; Severs et al. 1996;

Starkie et al. 2005). These temperature-related differences

may disappear during exercise to fatigue. The effects of

heat stress on neutrophil, lymphocyte and natural killer cell

activity are equivocal (Brenner et al. 1999; Laing et al.

2005; McFarlin and Mitchell 2003; Mitchell et al. 2002;

Niess et al. 2003). These immune responses are mediated

by increases in rectal temperatures and the release of cat-

echolamines, cortisol and growth hormone during exercise.

Circulating leukocyte, neutrophil, monocyte and CD16+

cell counts remain elevated for a longer period following

J. Peake (&)

School of Human Movement Studies, University of Queensland,

Brisbane, QLD 4072, Australia

e-mail: jpeake@hms.uq.edu.au

J. Peake � K. Suzuki

Faculty of Human Sciences, Waseda University,

Tokorozawa, Japan

J. J. Peiffer � C. R. Abbiss � K. Nosaka � P. B. Laursen

School of Exercise, Biomedical and Health Sciences,

Edith Cowan University, Joondalup, WA, Australia

M. Okutsu � K. Suzuki

Consolidated Research Institute for Advanced Science

and Medical Care, Waseda University, Tokyo, Japan

123

Eur J Appl Physiol

DOI 10.1007/s00421-007-0598-1

exercise in hot versus temperate conditions (Mitchell et al.

2002; Niess et al. 2003; Severs et al. 1996). Heat stress also

induces a greater rise in core temperature, plasma epi-

nephrine concentration, and leukocyte, granulocyte and

monocyte cell counts during subsequent bouts of exercise

on the same day (Brenner et al. 1996, 1997; Severs et al.

1996). These effects could be related to sustained elevation

of rectal temperature and catecholamine concentrations

after exercise in the heat (Brenner et al. 1996, 1997; Niess

et al. 2003). Modulating rectal temperature after exercise

provides insight into the influence of rectal temperature on

immune responses during recovery. One study has inves-

tigated the influence of cold exposure on immune changes

following moderate exercise (1 h cycling at 55% _VO2peak)

in hot conditions (Brenner et al. 1999). However, cold

exposure following more strenuous and prolonged exercise

in hot conditions may have different effects, because this

type of exercise induces a greater rise in rectal temperature

and greater immune disturbances (Niess et al. 2003).

The first aim of this study was to examine whether

circulating concentrations of leukocytes (neutrophils,

lymphocytes and monocytes), cytokines (IL-6, IL-8, IL-10,

IL-1ra, TNF-a and G-CSF) and markers of neutrophil

activation (myeloperoxidase and calprotectin) respond

similarly to a rise in rectal temperature under standardized

exercise and ambient conditions. Our rationale for inves-

tigating this issue was two-fold. First, due to variation in

the experimental design of other studies (e.g., ambient

conditions, exercise protocols and the immune variables

measured), uncertainty remains as to whether different

components of the immune system respond similarly to a

rise in rectal temperature during exercise. Second, the

effects of heat stress during exercise on changes in the

circulating concentrations of IL-8, G-CSF, calprotectin and

IL-10 are unknown. Limited information exists concerning

the physiological factors regulating systemic alterations in

IL-8, G-CSF and calprotectin following exercise, but rectal

temperature may be involved. IL-8 and G-CSF are key

chemokines that regulate leukocyte trafficking. Calprotec-

tin also regulates leukocyte chemotaxis and function. IL-10

is a type-2 cytokine with important anti-inflammatory

properties. Considering these roles of IL-8, G-CSF, cal-

protectin and IL-10 in immunity, it is important to

understand how these agents respond to heat stress during

exercise.

The second aim of this study was to compare the effects

of sitting in cold water and at room temperature after

exercise in hot conditions on changes in rectal temperature

and circulating concentrations of leukocytes (neutrophils,

lymphocytes and monocytes), cytokines (IL-6, IL-8, IL-10,

IL-1ra, TNF-a and G-CSF) and markers of neutrophil

activation (myeloperoxidase and calprotectin). Our ratio-

nale for examining this issue was that little is known about

the physiological factors affecting immune changes during

recovery from strenuous exercise. One study has reported

that cold exposure following 1 h cycling at 55% _VO2peak in

35�C increases the circulating concentrations of neutro-

phils, natural killer cells, plasma norepinephrine and IL-6

during recovery (Brenner et al. 1999). Cold exposure fol-

lowing more strenuous and prolonged exercise (2 h at 60–

80% _VO2 max) in hot conditions may have different effects

on the immune system, because this type of exercise

induces a greater rise in rectal temperature and greater

immune disturbances (Niess et al. 2003). We hypothesized

that by reducing rectal temperature and cardiac output, cold

water immersion would reduce the circulating concentra-

tions of epinephrine and norepinephrine during recovery

from exercise. In turn, these effects would reduce the de-

margination of neutrophils and monocytes into the

bloodstream, and reduce the synthesis of cytokines during

recovery from exercise.

Methods

Experimental design and approach to the problem

To examine the influence of rectal temperature on immune

changes during exercise, we recruited a group of male

cyclists. The cyclists completed three exercise trials: two

trials in hot conditions (mean ± SD 32.2 ± 0.7�C, 55 ± 2%

relative humidity) and one trial in temperate conditions

(mean ± SD 18.1 ± 0.4�C, 58 ± 8% relative humidity).

Two trials in 32�C were necessary to compare the effect of

cold water immersion versus sitting at room temperature

during recovery (see details below). We expected that

rectal temperature would be higher after cycling in 32�C

than after cycling in 18�C. Blood was sampled before

exercise, after 90 min and immediately after exercise.

Rectal temperature was measured continuously during

exercise. Data collected at these time points for the two

trials in 32�C were not significantly different. Therefore we

pooled the data from these two trials, and compared this

pooled data with the data collected from the trial in 18�C.

To investigate the influence of rectal temperature on

immune changes after exercise, 20 min following the two

trials in 32�C, the cyclists either sat in cold water for up to

20 min, or sat outside the climate chamber at room tem-

perature (*23�C) for the same period of time. In the

20 min-period immediately after exercise measurements of

quadriceps strength and vasoconstriction were performed

(data presented elsewhere). We expected that rectal tem-

perature would be lower after sitting in cold water than

after sitting at room temperature. Blood was sampled 5 min

after cold water immersion (45 min post-exercise), and

45 min after cold water immersion (90 min post-exercise)

Eur J Appl Physiol

123

(see Fig. 1). Data at these time points were compared

between recovery treatments in cold water and room

temperature.

Subjects

Ten endurance-trained male cyclists with a minimum of

2 years competitive cycling experience were recruited.

Their mean (SD) age was 27 (6.7) years, body mass was

77.9 (6.6) kg, height was 1.81 (0.06) m, sum of seven

skinfolds was 66 (12) mm, _VO2 max was 4.8 (0.3) l min–1

and peak power output was 343 (25) W. The cyclists were

riding between 250 and 300 km week–1 at the time of the

study. All subjects completed a medical questionnaire and

gave written informed consent prior to the study. The

experimental procedure was approved by the Central

Human Research Ethics Committee at Edith Cowan

University.

Exercise testing

Exercise testing was performed using a Velotron Cycle

Ergometer (RacerMate; Seattle, WA, USA) and the Velo-

tron Coaching Software (Version 1.5). The cycle ergometer

was adjusted to the dimensions of each cyclist’s own

bicycle, equipped with aerodynamic handlebars and fitted

with the cyclist’s own pedals, thereby allowing each cyclist

to use their own shoe/cleat system.

On their first visit to the exercise laboratory, the cyclists

performed a _VO2 max test. Gas exchange was measured

throughout the entire test using a ParvoMedics, TrueOne

2400 diagnostic system (Sandy, UT, USA). Heart rate was

recorded with the use of the ParvoMedics system and

compatible chest electrode (Polar Electro OyTM; HQ,

Kempele, Finland). From the _VO2 max test, peak power

output was calculated, and the power output corresponding

to 80% of their individual second ventilatory threshold

(Lucia et al. 2000), or 60% _VO2 max; was established.

Following the _VO2 max test the cyclists completed a

familiarization 16.1-km performance time trial.

After this initial testing, the cyclists returned to the

exercise laboratory for three exercise trials (two trials in

32�C, one trial in 18�C), separated by at least 1 week. The

order of these trials was randomized. Exercise (steady-

state + time trial) was performed in a climate chamber

(2.9 m · 6.8 m · 2.7 m). On each occasion, the cyclists

were required to ride on the cycle ergometer for 90 min at

*60% _VO2 max: Gas analysis was performed every 15 min

during exercise, and workload was adjusted accordingly to

maintain this intensity. This steady-state exercise was fol-

lowed by a 16.1-km performance time trial. The mean (SD)

duration of the time trial in 18�C was 25 min 25 s (1 min

35 s). The mean duration of the two time trials in 32�C was

27 min 40 s (1 min 42 s), and was significantly longer

(P \ 0.05) than the time trial in 18�C.

During all exercise trials, a fan was positioned 1.5 m in

front of the cyclists. The speed of the fan was set at 30 km h–

1 to simulate environmental conditions experienced when

cycling outdoors (Saunders et al. 2005). The cyclists wore

the same lycra cycling shirt and shorts for all trials. The

cyclists were given 750-ml bottles containing water from

which they could drink ad libitum. After exercise, fluid

consumption was calculated as the total volume of water

consumed during exercise. All trials were conducted

between 9:00 and 11:00 a.m. Exercise testing was per-

formed between the months of October and December when

daily ambient temperatures ranged between 12.1 ± 3.3�C

and 23.1 ± 3.9�C.

Cold water recovery protocol

The cold water recovery treatment involved sitting in an

inflatable pool (iCool Portacovery, Australia) filled with

cold water (14.3 ± 0.2�C) to the level of the clavicle. Each

cyclist was asked to remain in the water for 20 min, but he

was allowed to exit the pool earlier if he was feeling

uncomfortably cold. The order of sitting in cold water or at

room temperature during recovery was randomized and

counterbalanced.

Rectal temperature

Rectal temperature was recorded using a sterile disposable

rectal thermistor (Monatherm Thermistor, 400 Series;

Mallinckrodt Medical, St Louis, MO, USA) self-inserted

12 cm past the anal sphincter prior to each exercise trial.

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1.61 - mk t imert lai

2( 5− 82 min)

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Fig. 1 Schematic

representation of the

experimental design (see text

for further explanation). Arrowsindicate blood sampling points

Eur J Appl Physiol

123

Rectal temperature was recorded using a data logger (Grant

Instruments, Shepreth Cambridgshire, UK).

Blood sampling and processing

The blood-sampling schedule is described earlier in the

methods, and depicted in Fig. 1. Due to a limited research

budget, we were not able to analyse blood samples during

recovery from exercise in 18�C. Venous blood samples

were collected from a forearm vein into sterile vacutainers

containing either K2–EDTA for blood cell counts and the

separation of plasma, or serum separation tubes (Becton

Dickinson, Franklin Lakes, NJ, USA). Before the K2–

EDTA tubes were centrifuged, 1 ml whole blood was

removed to obtain complete blood cell counts. The K2–

EDTA tubes were then centrifuged at 400·g for 10 min at

4�C. After blood collection, the serum separation tubes

were left for 15 min at room temperature to clot, and were

then centrifuged at 400·g for 10 min at 4�C. The K2–

EDTA plasma was divided into 1-ml aliquots for the

analysis of catecholamines, G-CSF, myeloperoxidase and

calprotectin. The serum was divided into 0.7-ml aliquots

for the analysis of cortisol, interleukin-1 receptor antago-

nist (IL-1ra), IL-6, IL-8, IL-10 and TNF-a. All plasma and

serum samples were stored at –80�C until the day of

analysis.

Blood analysis

Complete blood cell counts were obtained using a Beckman

Coulter-Counter Gen-S (France SA, Villepinte, France).

Plasma epinephrine and norepinephrine concentrations

were measured by enzyme-linked immunosorbent assay

(ELISA) (Labor Diagnostika Nord, Nordhorn, Germany).

Plasma myeloperoxidase and calprotectin concentrations

were measured using an ELISA kit from HyCult Biotech-

nology (Uden, The Netherlands). Plasma G-CSF and serum

cortisol and concentrations were measured using an ELISA

kit from IBL (Hamburg, Germany). The serum concentra-

tions of IL-6, IL-1ra and TNF-a were measured using

Quantikine1 High Sensitivity ELISA kits (R&D Systems,

Minneapolis, MN, USA). Serum IL-8 and IL-10 concen-

trations were measured using OptEIA kits (Becton

Dickinson, San Diego, CA, USA). The sensitivity and co-

efficient of variation of these ELISA kits are presented in

Table 1. In some pre-exercise serum samples, IL-10 con-

centration was below the concentration of the lowest

standard. Therefore, this standard was further diluted so that

the standard curve for IL-10 was in the range of all serum

samples. When the cytokine concentration of serum sam-

ples exceeded the range of the standard curve, samples were

diluted and measured again. ELISA measurements were

performed using a microplate reader (VERSAmax,

Molecular Devices, Sunnyvale, CA, USA). The intra-assay

variation for all measurements was\7%. Leukocyte counts

were adjusted for percentage changes in blood volume,

whereas plasma and serum variables were adjusted

according to percentage changes in plasma and blood vol-

ume, as calculated from hemoglobin and hematocrit (Dill

and Costill 1974).

Statistical analysis

All data were checked for normal distribution using the

Kolmolgorov–Smirnov statistic. Data for rectal tempera-

ture, leukocyte and monocyte counts, and serum cortisol,

plasma G-CSF, myeloperoxidase and calprotectin concen-

trations were normally distributed. Data for neutrophil and

lymphocyte counts, serum IL-6, IL-8 and TNF-a concen-

trations and plasma catecholamine concentrations were

normally distributed after log transformation. Data for

serum IL-1ra and IL-10 concentrations were not normally

distributed. Data for the PRE, 90 min and END time-points

in the two trials in 32�C were not significantly different.

Therefore, we pooled the data for these two trials for

analysis.

For the normally distributed data, a 2 · 3 factor

repeated measures ANOVA was used to determine time

effects and time · condition interactions. Post-hoc analysis

involved using Student’s paired t tests with the false dis-

covery rate procedure for multiple comparisons (Curran-

Everett 2000) to compare differences between specific time

points and conditions. The data for serum IL-1ra and IL-10

concentration were analyzed using non-parametric Fried-

man’s ANOVA on ranks to determine time effects.

Table 1 Coefficient of variation and sensitivity of enzyme-linked

immunosorbent assays

Parameter Intra-assay coefficient

of variation (%)

Sensitivity

Epinephrine 4.3 11 pg ml–1

Norepinephrine 4.0 44 pg ml–1

G-CSF 2.0 1.2 pg ml–1

Cortisol 4.4 2.5 ng ml–1

IL-6 6.6 0.039 pg ml–1

IL-1ra 5.4 22 pg ml–1

TNF-a 6.9 0.12 pg ml–1

IL-8 3.3 0.8 pg ml–1

IL-10 4.0 2 pg ml–1

Myeloperoxidase 4.1 0.4 ng ml–1

Calprotectin 5.0 1.6 ng ml–1

Eur J Appl Physiol

123

Wilcoxon signed rank tests were then used to assess dif-

ferences between specific time points and conditions.

Statistical significance was set at P \ 0.05. Statistical

analysis was carried out using SigmaStat 3.1 software

(Systat, Point Richmond, CA, USA).

Results

Physiological parameters

The cyclists maintained 60% _VO2 max during steady state

exercise in all three trials; oxygen consumption did not

differ significantly between cycling in 18 and 32�C. The

mean ± SD percentage of maximum heart rate was sig-

nificantly higher (P \ 0.0001) during the steady-state

cycling in 32�C (84 ± 2%) than in 18�C (77 ± 4%). Heart

rates were similar during the time trials (91 ± 5%). Fluid

consumption was higher during cycling in 32�C (2.0 ±

0.8 l vs. 1.0 ± 0.5 l, P \ 0.05). Changes in plasma volume

(–9.2 ± 4.2% in 18�C and –9.0 ± 6.2% in 32�C) and body

mass (dehydration) (–0.7 ± 0.9% in 18�C and –1.0 ± 1.1%

in 32�C) were similar between the conditions. Rectal

temperature increased significantly during exercise, and

was significantly higher in response to cycling in 32�C

(Fig. 2) (time · condition interaction effect P \ 0.0001).

During recovery from cycling in 32�C, rectal temperature

decreased to a greater extent (compared with post-exercise)

after sitting in cold water (–2.4 ± 0.4�C) than sitting at

room temperature (–1.6 ± 0.6�C) (Fig. 2) (interaction

effect P = 0.01).

Leukocytes

Blood leukocyte counts all increased significantly follow-

ing cycling in both 18 and 32�C (time effect P \ 0.0001)

but did not differ significantly between the conditions

(Table 2). Cold water immersion did not significantly

influence blood leukocyte counts during recovery from

exercise in 32�C (Table 2).

Cytokines

The concentrations of serum IL-6 (Table 2), IL-8, IL-10

and plasma G-CSF (Fig. 3) were significantly elevated

immediately after cycling in both 18 and 32�C (time effect

P \ 0.001). Serum IL-8 and IL-10 concentrations were

significantly higher following cycling in 32 versus 18�C

(interaction effect P \ 0.0001). Serum TNF-a concentra-

tion increased only after 90 min cycling in 32�C, and was

significantly higher than after cycling in 18�C (interaction

effect P \ 0.01) (Table 3). Serum IL-1ra concentration

also increased only during cycling in 32�C, and was sig-

nificantly higher after 90 min cycling in 32 versus 18�C

(Wilcoxon sign ranked test P = 0.017) (Table 3). Plasma

G-CSF concentration tended to be higher following cycling

in 32�C than in 18�C (interaction effect P = 0.06). Cold

water immersion did not significantly influence cytokine

concentrations during recovery from exercise in 32�C

(Table 3, Fig. 3).

Neutrophil activation

The plasma concentrations of myeloperoxidase and cal-

protectin increased significantly following cycling in both

conditions (time effect P \ 0.001) (Fig. 4). Myeloperoxi-

dase increased to a significantly greater extent following

cycling in 32 versus 18�C (interaction effect P = 0.003).

Myeloperoxidase and calprotectin remained elevated dur-

ing recovery from cycling in 32�C (time effect P \ 0.001),

but there was no effect of cold water immersion.

Stress hormones

The plasma concentrations of epinephrine and norepi-

nephrine increased significantly following cycling in both

conditions (Table 4) (time effect P \ 0.001). Norepi-

nephrine was significantly higher in response to cycling in

Fig. 2 Rectal temperature before and after exercise. See Fig. 1 for

details. Data at PRE, 90 min and END were combined for the two

trials in the heat. Data are presented as means ± SD. * Significantly

different from pre-exercise for both conditions, P \ 0.05. # Signif-

icantly different between conditions, P \ 0.05. § Change from post-

exercise significantly different between conditions, P \ 0.05

Eur J Appl Physiol

123

32 versus 18�C (interaction effect P = 0.003). There was a

trend towards higher levels of norepinephrine following

cold water immersion compared to sitting at room tem-

perature (interaction effect P = 0.056). Serum cortisol

concentration decreased significantly from the beginning to

the end of exercise (time effect P \ 0.001), and was not

influenced by cold water immersion (Table 4).

Discussion

The aims of this study were to compare immune responses

to cycling in 18 and 32�C, and the effects of cold water

immersion on the recovery of immune markers following

the exercise in 32�C. In support of our hypothesis, the

concentrations of serum IL-1ra, IL-8, IL-10, TNF-a,

plasma G-CSF, myeloperoxidase and norepinephrine were

greater after cycling in 32�C than in 18�C. Contrary to our

hypothesis, cold water immersion during recovery from

cycling in 32�C had no significant effect on blood leuko-

cyte counts, or the concentrations of cytokines and

neutrophil activation markers.

We have presented new data indicating that the plasma

concentration of myeloperoxidase, but not calprotectin was

higher following cycling in 32�C than in 18�C. Mitchell

et al. (2002) reported that superoxide production by neu-

trophils in vitro is higher after cycling in 38�C than in

22�C. In contrast, Niess et al. (2003) found no difference in

plasma myeloperoxidase concentration after exercise in 28

versus 18�C. Laing et al. (2005) also reported no difference

in plasma elastase concentration or the release of elastase

from neutrophils stimulated with lipopolysaccharide fol-

lowing exercise in 30 versus 20�C. These findings indicate

that the effects of exercise and heat stress vary between

different aspects of neutrophil activation.

The elevated plasma concentration of myeloperoxidase

after exercise likely reflects neutrophil degranulation,

because myeloperoxidase is contained in azurophilic

granules within neutrophils. Myeloperoxidase is an

important component of neutrophil microbicidal defense

because it catalyses the conversion of H2O2 to HOCl,

among other reactions. Myeloperoxidase-derived HOCl

plays a key role in defense against Gram-negative bacteria,

and regulates the release of neutrophil elastase. Inactiva-

tion of elastase may protect against tissue degradation

(Hirche et al. 2005). The higher plasma myeloperoxidase

concentration following cycling in 32�C could be due to

the stimulatory effects of IL-8 and G-CSF on neutrophils

(Hoglund et al. 1997; Topham et al. 1998). Prolactin and

growth hormone also activate MAPK signalling pathways

involved in neutrophil degranulation (Argetsinger and

Carter-Su 1996; Dogusan et al. 2001). We did not measure

changes in prolactin and growth hormone. However, these

hormones may have contributed to the greater increase in

plasma myeloperoxidase concentration after exercise in

32�C, because they increase more during exercise in hot

compared with cool/temperate conditions (Laing et al.

2005; Niess et al. 2003).

The present study is the first to investigate the influence

of heat stress during exercise on changes in the plasma

Table 2 Leukocyte counts before and after exercise

PRE 90 min END R1 R2

Cold water Room temp. Cold water Room temp.

Total leukocytes (cells · 109 l–1)

18�C 5.5 (1.8) 8.6 (2.1)* 12.2 (2.7)*

32�C 5.4 (1.0) 9.5 (3.0)* 12.9 (3.4)* 10.4 (2.6)* 9.8 (4.3)* 12.4 (3.2)* 11.2 (4.0)*

Neutrophils (cells · 109 l–1)

18�C 2.9 (0.9) 4.9 (1.1)* 6.8 (1.4)*

32�C 2.8 (0.7) 5.3 (1.3)* 7.4 (1.7)* 7.2 (2.1)* 6.8 (1.9)* 9.3 (2.2)* 8.3 (1.9)*

Lymphocytes (cells · 109 l–1)

18�C 1.7 (0.2) 2.6 (0.4)* 4.0 (0.5)*

32�C 1.8 (0.3) 2.9 (0.6)* 3.9 (0.6)* 1.8 (0.4) 1.7 (0.2) 1.6 (0.2)* 1.5 (0.3)*

Monocytes (cells · 109 l–1)

18�C 0.4 (0.2) 0.6 (0.2)* 0.7 (0.2)*

32�C 0.4 (0.1) 0.6 (0.2)* 0.8 (0.2)* 0.6 (0.1)* 0.5 (0.2)* 0.8 (0.2)* 0.7 (0.2)*

Data at PRE, 90 min and END were combined for the two trials in the heat. Data for total leukocytes and monocyte are presented as means (SD).

Data for neutrophils and lymphocytes are presented as geometric means (95% confidence intervals). PRE, pre-exercise; 90 min, immediately

after steady-state exercise; END, immediately after 16-km time trial; R1, 45 min after end of time trial and immediately after cold water

immersion; R2, 90 min after end of time trial and 45 min after cold water immersion

* Significantly different from pre-exercise, P \ 0.05

Eur J Appl Physiol

123

concentration of calprotectin. Calprotectin (otherwise

known as S100A8/A9) is secreted from monocytes and

neutrophils by activation of protein kinase C, in response to

a variety of inflammatory conditions (Foell et al. 2007). It

is involved in regulating leukocyte chemotaxis, adhesion

and arachidonic acid metabolism in vitro (Kerkhoff et al.

1999; Ryckman et al. 2003). Calprotectin is upregulated in

monocytic cells by non-inflammatory stimuli such as nor-

epinephrine (Suryono et al. 2006) and pro-inflammatory

cytokines (e.g., IL-1b, TNF-a and IFN-c) (Hu et al. 1996;

Xu and Geczy 2000). Anti-inflammatory stimuli such as

glucocorticoids (Hsu et al. 2005) and IL-10 (Xu et al. 2001)

also induce calprotectin production by macrophages, but

this actually may serve to limit inflammation by restrict-

ing recruitment of leukocytes at sites of inflammation

(Harrison et al. 1999). In the present study, plasma cal-

protectin concentration was similar following cycling in 18

and 32�C, whereas the concentrations of norepinephrine,

IL-10 and TNF-a were greater after cycling in 32�C. Fur-

ther research is needed to identify the factors that regulate

alterations in calprotectin during exercise.

We have also presented new data indicating that the

systemic concentrations of IL-8, IL-10 and G-CSF were

greater following exercise in 32�C than in 18�C. Cate-

cholamines may mediate the synthesis of IL-8, IL-10 and

G-CSF during exercise by increasing cAMP synthesis (van

der Poll et al. 1996; van der Poll and Lowry 1997). The

trend (P = 0.06) toward higher plasma G-CSF concentra-

tion after exercise in 32�C is supported by data from

studies of hyperthermia in mice (Ellis et al. 2005). The

precise mechanisms regulating the synthesis of G-CSF

during hyperthermia are currently unclear, but other cyto-

kines (e.g., TNF-a, IL-1b, IL-17, IL-18 and GM-CSF) may

be involved (Ellis et al. 2005).

High serum IL-8 concentration (1,000 pg ml–1) during

fever is predictive of subsequent health complications such

as sepsis, respiratory insufficiency and death (Engel et al.

2005). In our study, serum IL-8 concentration was only in

the range of 15–30 pg ml–1 after exercise in 32�C. How-

ever, greater increases in the systemic level of IL-8 after

more prolonged hyperthermia during exhaustive exercise

could contribute to heat stroke (Lim and Mackinnon 2006).

Elevated serum IL-10 concentration after exercise in hot

conditions may inhibit the synthesis of type-1 cytokines

such as IL-2 and interferon-c, resulting in impaired cell-

mediated immunity (Elenkov and Chrousos 2002). An

increase in circulating G-CSF during fever stimulates

neutrophil mobilization (Ellis et al. 2005), yet in the

present study, higher plasma G-CSF concentration did not

appear to influence neutrophil counts after exercise in

32�C.

Our finding that heat stress during exercise increased the

serum concentrations of IL-1ra and TNF-a is consistent

with other reports (Rhind et al. 2004; Starkie et al. 2005).

Catecholamines may stimulate IL-1ra synthesis indirectly

via IL-6 (Sondergaard et al. 2000). The mechanisms

Fig. 3 Plasma granulocyte-colony stimulating factor (G-CSF), serum

interleukin (IL)-8 and serum IL-10 concentrations before and after

exercise. See Fig. 1 for details. Data at PRE, 90 min and END were

combined for the two trials in the heat. Data for G-CSF are presented

as mean ± SD. Data for IL-8 are presented as geometric mean ± 95%

confidence intervals. Data for IL-10 are presented as median ± inter-

quartile ranges. * Significantly different from pre-exercise for both

conditions, P \ 0.05. # Significantly different between conditions,

P \ 0.05

Eur J Appl Physiol

123

contributing to the higher serum TNF-a concentration

during exercise in 32�C are unclear. Catecholamines inhi-

bit TNF-a production in vitro by increasing cAMP

synthesis (van der Poll et al. 1996), and IL-6 inhibits the

synthesis of TNF-a during exercise (Starkie et al. 2003).

Mild endotoxemia during exercise may promote TNF-aproduction (Camus et al. 1998), and this could lead to

exercise-induced heat stroke (Lim and Mackinnon 2006).

Several studies have reported higher plasma IL-6

responses to exercise in ‡35 versus 18�C (Brenner et al.

1999; Rhind et al. 2004; Starkie et al. 2005). In contrast, we

found no significant difference in the systemic IL-6

response to exercise in 32 versus 18�C. This difference

may relate to the epinephrine response to exercise.

Researchers have questioned the role of epinephrine in

stimulating IL-6 release during exercise (Holmes et al.

2004; Steensberg et al. 2001), but epinephrine may play a

role during exercise in hot conditions (i.e., [32�C). Epi-

nephrine stimulates IL-6 synthesis by activating

intracellular cAMP (Chio et al. 2004). The studies above

(Brenner et al. 1999; Rhind et al. 2004; Starkie et al. 2005)

reported that plasma epinephrine concentration is higher

following exercise in ‡35 versus 18�C, whereas we

observed similar plasma epinephrine responses to exercise

in 32 versus 18�C. Other factors such as glycogen depletion

and calcium signalling also contribute to the release of IL-6

from skeletal muscle (Holmes et al. 2004; MacDonald et al.

2003). Because exercise in the heat accelerates depletion of

muscle glycogen (Jentjens et al. 2002), it is somewhat

surprising that we did not observe higher serum IL-6

concentration after exercise in 32�C than in 18�C. Exercise

in [32�C may impair renal blood flow, thereby leading to

reduced clearance and greater accumulation of IL-6 in the

bloodstream.

Table 3 Serum cytokine concentrations before and after exercise

PRE 90 min END R1 R2

Cold water Room temp. Cold water Room temp.

IL-6 (pg ml–1)

18�C 0.5 (0.5) 2.7 (0.9)* 6.1 (2.1)*

32�C 0.4 (0.2) 3.7 (1.4)* 7.1 (2.3)* 4.6 (1.6)* 6.9 (2.9)* 3.0 (1.1)* 3.8 (1.4)*

TNF-a (pg ml–1)

18�C 1.1 (0.1) 1.2 (0.1) 1.2 (0.1)

32�C 1.3 (0.2) 1.5 (0.2)*# 1.4 (0.3) 1.4 (0.3) 1.6 (0.2)* 1.4 (0.3) 1.5 (0.2)

IL-1ra (pg ml–1)

18�C 259 (211) 266 (205) 280 (157)

32�C 246 (161) 347 (304)*# 476 (368)* 721 (843)* 599 (1,527)* 954 (1,883)* 750 (837)*

Data for IL-6 and TNF-a are presented as geometric means (95% confidence intervals). Data for IL-1ra are presented as medians (interquartile

ranges). See Table 1 for details

* Significantly different from pre-exercise, P \ 0.05; # significantly different between conditions, P \ 0.05

Fig. 4 Plasma calprotectin and myeloperoxidase concentrations

before and after exercise. See Fig. 1 for details. Data at PRE,

90 min and END were combined for the two trials in the heat. Data

are presented as mean ± SD. * Significantly different from pre-

exercise for both conditions, P \ 0.05. # Significantly different

between conditions, P \ 0.05

Eur J Appl Physiol

123

The finding that blood leukocyte counts were similar at

the end of cycling in temperate versus hot conditions

contrasts with some (Brenner et al. 1996; Cross et al. 1996;

McFarlin and Mitchell 2003; Mitchell et al. 2002; Rhind

et al. 1999; Severs et al. 1996), but not all studies (Brenner

et al. 1999; Niess et al. 2003; Starkie et al. 2005). The lack

of any significant difference in leukocyte counts may be

attributed to the relatively small difference in rectal tem-

perature at the end of exercise in 32 versus 18�C (Walsh

and Whitham 2006). In turn, this difference in rectal tem-

perature may relate to the capacity for heat dissipation

through convective cooling during exercise. We used a fan

to simulate environmental conditions experienced when

cycling outdoors. Convective airflow generated by the fan

may have limited the rise in core temperature while cycling

in 32�C. Heat dissipation is likely impaired to a greater

extent while cycling in water at 39 or 40�C, and this might

account for the greater leukocytosis reported after exercise

in these conditions (Brenner et al. 1996; Cross et al. 1996;

Rhind et al. 1999; Severs et al. 1996). The cyclists in our

study consumed twice as much fluid on average during

exercise in 32 versus 18�C. The greater fluid consumption

may have offset the effects of heat stress on cardiac output,

and therefore leukocyte mobilisation.

Evidence exists to suggest that exercise in hot conditions

delays recovery of the immune system (Mitchell et al.

2002; Niess et al. 2003; Severs et al. 1996). This response

may relate to the slower decline in rectal temperature fol-

lowing exercise in the heat. Accordingly, we hypothesized

that reducing rectal temperature after exercise in hot con-

ditions would promote faster recovery of the immune

system, through a decrease in cardiac output and circulat-

ing stress hormones. We observed that rectal temperature

decreased more rapidly after sitting in cold water than at

room temperature. However, cold water immersion did not

influence the pattern of changes in circulating leukocyte

counts, cytokines, myeloperoxidase and calprotectin—at

least in the short-term after exercise. These findings con-

trast with the work of Brenner et al. (1999), who reported

that 2 h exposure to cold air (5�C) after 1 h cycling at 55%_VO2peak in 35�C reduced core temperature, but increased

neutrophil counts, plasma IL-6 and norepinephrine con-

centrations above values observed at the end of exercise.

They also noted that cold exposure without prior exercise

induced a slightly smaller (but significant) rise in neutro-

phil counts, plasma IL-6 and norepinephrine concentrations

(Brenner et al. 1999).

The lack of any significant effect of cold water

immersion in our study could relate to the comparatively

short period of cold exposure. We chose a shorter period of

cold exposure because cold water conducts heat more

effectively than cold air. For ethical reasons, we could not

expect the athletes, who had low body fat, to remain sitting

in the cold water beyond a point that they felt comfortable.

Cold water immersion immediately after exercise may

have had a greater impact on the immune system. In any

case, when compared with the findings reported by Brenner

et al. (1999), our data suggest that alterations in rectal

temperature have less impact on immune responses during

recovery from strenuous exercise compared with moderate

exercise. The extent to which strenuous exercise activates

the immune system may exceed the capacity of cold water

immersion to mitigate immune responses during recovery

from such exercise.

In summary, we have presented new evidence that

heat stress during exercise increased the circulating con-

centrations of IL-1ra, IL-8, IL-10, TNF-a, G-CSF,

myeloperoxidase, whereas heat stress did not influence

calprotectin concentration. Cold water immersion follow-

ing exercise reduced rectal temperature more rapidly than

Table 4 Plasma epinephrine and serum cortisol concentrations before and after exercise

PRE 90 min END REC 1 REC 2

Cold water Room temp. Cold water Room temp.

Epinephrine (pg ml–1)

18�C 30 (14) 100 (59)* 476 (362)*

32�C 29 (16) 161 (105)* 644 (446)* 48 (32)* 51 (15)* 29 (32) 28 (18)

Norepinephrine (pg ml–1)

18�C 427 (108) 1,159 (371)* 2,562 (869)*

32�C 417 (108) 1,736 (506)*# 3,259 (750)* 1,146 (356)* 695 (257)* 1,202 (291)* 701 (238)

Cortisol (ng ml–1)

18�C 99 (12) 84 (19)* 50 (10)*

32�C 102 (12) 73 (16)* 49 (15)* 43 (14)* 42 (11)* 63 (27) 54 (11)

Data at PRE, 90 min and END were combined for the two trials in the heat. Data for epinephrine and norepinephrine are presented as geometric

means (95% confidence intervals). Data for cortisol are presented as means (SD). See Table 1 for details

* Significantly different from pre-exercise, P \ 0.05; # significantly different between trials, P \ 0.05

Eur J Appl Physiol

123

sitting at room temperature, but did not significantly

influence circulating leukocyte counts, cytokine, myelo-

peroxidase and calprotectin concentrations during recovery

from exercise. Future studies could investigate in more

detail (1) the time course of immune responses following

exercise in temperate and hot conditions, and (2) the

influence of cold exposure on immune responses after

moderate exercise versus strenuous exercise.

Acknowledgments This study was supported by a Grant-in-Aid for

SCOE research and Young Scientist (A) from the Ministry of Edu-

cation, Culture, Sports, Science and Technology in Japan (no.

17680047). Additional support was provided by a Computing Health

and Science Faculty Small Grant, and a Visiting Fellow Grant from

Edith Cowan University. At the time that this study was conducted,

Jonathan Peake was a recipient of a postdoctoral fellowship from the

Japanese Society for the Promotion of Science.

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