Physiology & Behavior 101 (2010) 320–326

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Physiology & Behavior

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b

Psychological stress during exercise: Lymphocyte subset redistribution in firefighters

Chun-Jung Huang a,⁎, Heather E. Webb b, Ryan S. Garten c, Gary H. Kamimori d, Edmund O. Acevedo e
a
Florida Atlantic University, Boca Raton, FL, USA
b
Mississippi State University, Starkville, MS, USA
c
The University of North Carolina at Greensboro, Greensboro, NC, USA
d
Water Reed Army Institute of Research, Silver Springs, MD, USA
e
Virginia Commonwealth University, Richmond, VA, USA

a r t i c l e i n f o a b s t r a c t

Article history: The purpose of this study examined the changes in heart rate (HR), catecholamines (NE, EPI) and
Received 5 January 2010 percentages of blood lymphocyte subsets (CD3+ T cells, CD3+ CD4+ helper T cells, CD3+ CD8+ cytotoxic
Received in revised form 27 March 2010 T cells, CD3− CD56+ NK cells, CD4/CD8 ratio, CD19+ B cells, and total lymphocytes [NK cells+ T cells
Accepted 26 May 2010
+ B cells]) in firefighters exposed to a computerized firefighting strategies and tactics decision-making
challenge while participating in moderate intensity exercise. Furthermore, this study also examined the
Keywords:
possible relationships between catecholamines (NE and EPI) and blood lymphocyte subsets following
Exercise
Psychological stress
combined mental and physical challenge. Ten professional male firefighters participated in two counter-
Firefighters balanced exercise conditions on a cycle ergometer: (1) 37 min of cycle ergometry at 60% VO2max (exercise
Lymphocytes alone condition; EAC) and (2) 37 min of cycle ergometry at 60% VO2max along with 20 min of a computerized
Catecholamines firefighting strategies and tactics decision-making challenge (firefighting strategies condition; FSC). FSC
Heart rate elicited significantly greater HR, NE, and EPI when compared to EAC. Both EAC and FSC elicited increases in
Stress hormone CD3− CD56+ NK cells. The percentages of CD3+ T cells, CD3+ CD4+ helper T cells, CD4/CD8 ratio, CD19+
B cells, and total lymphocytes were lower immediately following both conditions. Following dual challenge
NE AUC was negatively correlated with percentage of CD19+ B cells immediately post challenge, and HR was
negatively associated with the percent change in the CD4/CD8 ratio from pre to post challenge. These
elevations in NE and heart rate simultaneously in response to the dual challenge suggest greater sympathetic
activation that in turn would possibly explain the alteration in the distribution of lymphocyte subsets.
Published by Elsevier Inc.

1. Introduction and catecholamines (epinephrine [EPI]) and norepinephrine ([NE])

Firefighters have been shown to have greater mortality rates
associated with cardiovascular disease (CVD) [1,2]. A recent review by
Kales et al. [2] has summarized that fire suppression activities result in
an increased risk of death to firefighters (12 to 136 times as high)
when compared to nonemergency duties. Other studies have also
shown greater mortality rates for CVD and cerebrovascular accidents
in firefighters than others matched for age and sex [1–3]. Further-
more, approximately half of the deaths in firefighters are associated
with CVD [4]. One aspect of fire suppression activities that may
contribute to these high mortality rates is the combined psychological
and physical challenge [5]. Elenkov et al. [6] have proposed that
stress-induced diseases are due to the inability to adapt and maintain
allostasis among the hypothalamopituitary (HPA) and sympathoa-
drenal (SA) axes. These elevated levels of cortisol from the HPA axis
⁎ Corresponding author. 777 Glades Road, FH11-24D, Boca Raton, Florida 33431.
Tel.: + 1 561 297 1271; fax: + 1 561 297 2839.
E-mail address: chuang5@fau.edu (C.-J. Huang).
from the SA axis are thought to alter immune cell responses such as
leukocytosis [7–9]. This important immune system response coordi-
nates a number of the body's adaptations to the stressor.
A number of experimental protocols utilizing laboratory-induced
acute psychological stressors have observed alterations in innate (e.g.
natural killer [NK] cells and cytotoxic T cells) and adaptive immunity
(e.g. helper T cells and B cells). For example, following acute
psychological stress an elevation was observed in NK and cytotoxic
T cells [10,11], whereas helper T cells and B cells decreased [10–13]. In
addition, in response to physical stress the magnitude of these
immune cell responses is dependent upon exercise intensity and
duration. High-intensity exercise has been shown to induce the
production of stress hormones with corresponding alterations in
various immune cells [14]. Normally, following exercise NK cells
exhibit the greatest fluctuation followed by T cells and B cells [15].
However, the appropriate redistribution of immune cells in response
to psychological and/or physical stress is imperative to effective and
efficient immune function [16].
The release of cortisol and catecholamines in response to psycho-
logical and physical stress can mediate changes in the distribution in

0031-9384/$ – see front matter. Published by Elsevier Inc.

doi:10.1016/j.physbeh.2010.05.018
 C.-J. Huang et al. / Physiology & Behavior 101 (2010) 320–326
immune cells [7,8]. Specifically, Schedlowski et al. [17] found that
injection of both EPI and NE increased NK cell numbers and decreased
total T cells and helper T cells, but no change was observed in cytotoxic
T cells. These findings indicate that both EPI and NE may be important
modulators in cell-mediated immunity in response to either psycho-
logical or physical stress. Furthermore, Isowa et al. [12] have concluded
that under acute psychological stress the influence of cortisol on
immune function is minimal, and mostly regulated by catecholamines.
In addition, in response to exercise an increase in sympathetic tone may
affect the intensity and duration of an immune response to antigen [18].
Thus, catecholamines appear to affect cell-mediated immunity, and
cortisol may not exert a suppressive effect of immune regulation in
either acute psychological or physical stress.
A recent study by Webb et al. [19] examined healthy individuals in
response to a combination of psychological and physical stress and
found a greater increase for NE in this dual stress condition. This
finding suggests that the SA axis is further activated, beyond the
exercise alone condition, during a dual challenge, and this additional
increase in NE may further alter the components of cellular immunity.
This combined mental and physical stress of conducting fire
suppression activities may contribute to the high mortality rates [5].
An examination of catecholamines (NE and EPI) and immune cell
distribution following a combination of physical and psychological
challenge may provide a greater understanding of the link between
physical activity, psychological stress, and immune responses. Fire-
fighting may provide a paradigm to investigate the physiological
and psychological consequences of combined stress. Therefore, the
purpose of this study was to examine the changes in catecholamines
(NE, EPI) and percentages of blood lymphocyte subsets (CD3+ T cells,
CD3+CD4+ helper T cells, CD3+ CD8+ cytotoxic T cells, CD3− CD56+
NK cells, CD4/CD8 ratio, CD19+ B cells, and total lymphocytes [NK
cells+ T cells+ B cells]) in firefighters exposed to a computerized
firefighting strategies and tactics decision-making challenge (FSTD)
while participating in moderate intensity exercise. Furthermore, this
study also examined the possible relationships between catechola-
mines (NE and EPI) and blood lymphocyte subsets following
combined mental and physical challenge. We hypothesized that a
dual challenge compared to exercise alone would induce a greater
perturbation of catecholamines and lymphocyte subsets.
2. Methods
2.1. Participants
Ten professional male firefighters were recruited following
approval from the Fire Chief. Participant characteristics are reported
in Table 1. To control variability of the dependent measures of interest
and because recent studies have shown that women may have
different immune reactivity [20] and attenuated cardiovascular
responses to mental stress [21], only male firefighters were investi-
gated. Additionally, subjects provided an informed consent and
completed a medical history questionnaire prior to data collection.
All experimental procedures were approved by the University of
Mississippi's Institutional Review Board.
Table 1
Participant descriptive characteristics (N = 10).
Variable Mean ± SEM
Age (years) 32.80 ± 1.36
Height (cm) 179.30 ± 2.41
Weight (kg) 91.71 ± 4.06
BMI (kg/m2) 28.59 ± 0.91
Firefighter experiences (years) 11.33 ± 2.60
VO2max (ml/kg/min) 35.63 ± 1.94
321
Participants in this study were (a) free of cardiorespiratory and
metabolic disorders, (b) free of any known blood disorders (e.g.
anemia, hemophilia), (c) without hearing or vision problems (includ-
ing color-blindness), (d) free of a history of psychological disorders
and/or chronic illnesses, (e) native English speakers (f) no use of any
prescription or nonprescription medication or tobacco products
within the previous 8 h, (g) non-smokers and consuming with average
consumption of less than ten alcoholic beverages per week, (h) no
experience of any major life events within 30 days of participation (e.g.
death in family, divorce, wedding), and (i) not engaged in actual fire
suppression tasks within the previous 72 h. Additionally, prior to each
testing session, participants were asked to fast overnight for at least 8 h
and to abstain from alcohol consumption for at least 48 h. Participants
were also instructed to maintain their normal physical activity levels
throughout the duration of their involvement in the data collection
procedures.
In addition, all firefighters involved in this study had participated
in advanced fire training, beyond the basic firefighter I/II instruction
required of professional firefighters in the State of Mississippi. Thus,
all participants were qualified to assume the duties of Incident
Commander at a fire scene.
2.2. Laboratory psychological stress protocol
Firefighters participated in a computer-based Firefighting Strate-
gies and Tactics Drill (FSTD) that served as a mental challenge. The
FSTD was created using Fire Studio Version 3.0 (Digital Combustion,
Inc., 2004) and Authorware 5.0 (Macromedia, 1999) software. Twelve
min after the initiation of exercise in the treatment session, the
subject began to respond to the FSTD and continued for a total of
20 min. For the FSTD, firefighters were asked to respond as the
Incident Commander to multiple choice questions based upon a fire
scene presented on a 1.32 m (52″) monitor. These questions were
relevant to the fire scene that was being depicted on an adjacent
monitor equal in size. Thus, firefighters were presented with two
adjacent monitors: one depicting the fire scene and one presenting
relevant multiple choice questions. A numeric keypad placed at
handlebar level on the right side of the subject within finger reach
was utilized so that participants could respond to each question by
pressing the number of the corresponding correct answer on the
keypad.
Throughout the FSTD, participants were provided information
typical to actual fire suppression pertaining to the context of the fire
that was presented on the adjacent monitor. Participants were
required to acknowledge having read these pieces of information by
pressing a specified key on the numeric keypad before continuing on
with the relevant multiple choice questions. If the participant took
more than 30 s to respond, a confederate was present to prompt the
participant to make a choice from the options presented. To verify
the validity of the emergent fire scenes and questions, two
independent experts (Fire Director, Memphis, TN and senior training
officer, MS State Fire Academy) evaluated the scenes and questions
for accuracy of fire behavior and appropriateness of the questions
and sequence. Each participant was erroneously informed that his
fire chief would be provided with the results of his performance on
the FSTD. At the termination of the study, all participants were
informed that the Fire Chief would not in fact be provided with the
performance results on the FSTD, and that this false information was
intended to enhance the challenge of the FSTD. A similar protocol
(FSTD) has been shown to increase HR approximately 20 beats per
min at rest [22].
2.3. Testing procedures
Three testing sessions comprised the data collection. These sessions
consisted of: (1) an initial session to obtain consent to participate,
322 C.-J. Huang et al. / Physiology & Behavior 101 (2010) 320–326
familiarize participants with all instruments and procedures, and
assess VO2max. Participants reached VO2max when either the primary
criterion of a plateau in VO2 with an increase in workload was met or
2 of the 3 secondary criteria were achieved. The secondary criteria
were (a) reaching predicted maximal heart rate, (b) achieving a
respiratory exchange ratio of greater than 1.15, and (c) reporting a
rating of perceived exertion (15-point Borg Scale) of 19 or 20. (2)
a session that included 37 min of cycle ergometry at 60% VO2max;
the exercise alone condition (EAC), and (3) a session that included
37 min of cycle ergometry at 60% VO2max with 20 min of the FSTD
(firefighting strategies condition; FSC) (see experimental protocol in
Fig. 1). A mental challenge alone condition was not included because
of the likelihood that an adaptation to a single exposure of the
FSTD could diminish its physiological effect during the dual challenge
condition and because professional firefighters are most often exposed
to mental and physical challenges simultaneously. This exercise
intensity (60% VO2max) is similar to the intensity experienced
during fire suppression activities [23–25]. At least 48 h were allowed
to elapse between session 1 and session 2, and a minimum of one
week and maximum of 3 weeks transpired between session 2 and
session 3. Session 2 and session 3 were counterbalanced between
participants. During all testing sessions, a ParvoMedics TrueOne 2400
integrated metabolic measurement system (ParvoMedics, Sandy, UT)
was used to assess oxygen consumption, and a Quinton Q-4500 EKG
(Cardiac Science; Bothell, WA) was used to assess heart rate. Exercise
was performed on a CompuTrainer Pro Cycle ergometer system
(RacerMate Inc., Seattle, WA) with workload controlled by the
CompuTrainer Coaching Software (Version 1.1). Utilizing this software,
a program for controlling workload was specifically written for
each participant.
For session 1, participants visited the lab to participate in a graded
exercise test on a cycle ergometer designed to elicit maximal exertion
within 8–12 min. For session 2 and session 3, the participants reported
to the lab at 7:00 AM, and a venous catheter was inserted by 7:30 AM.
NE and EPI were determined from blood samples obtained at
−50 min, −30 min, and 0 min, 10 min, 20 min, 32 min, 37 min, and
every 15 min for 1 h post-exercise. CD3+ T cells, CD3+ CD4+ helper
T cells, CD3+ CD8+ cytotoxic T cells, CD3− CD56+ NK cells, CD19+
B cells, and total lymphocytes were analyzed from blood samples at
−30 min, 37 min, and 60 min post-exercise (see experimental
protocol in Fig. 1).
2.4. Blood sampling
Blood draws were performed by a physician-approved licensed
allied health care professional using standard aseptic technique. An
intravenous catheter (Jelco, 20 g, 25 mm) was inserted into an
antecubital vein, and a positive pressure adapter (CLC2000, ICU
Medical, San Clemente, CA) was attached. During each blood draw, the
first 1 ml of blood (with saline from the extension set) was collected
into a discard syringe preceding the sample draw. Each blood draw
was divided as follows: 5 ml sample was collected into tubes
Fig. 1. Time progression of the experimental protocol in minutes. The vertical solid lines
extending from the ordinate represent the start of exercise (Ex), and the end of exercise
(Ex'). The vertical dotted lines extending from the ordinate represent the start of the
mental challenge (S), and the end of the mental challenge (S').
containing ethylene glycol-bis-(beta-amino ethyl ether)-N, N, N′,
N′-tetraacetic acid and glutathione for subsequent catecholamine
analyses and 3 ml of blood was collected into tubes containing acid
citrate dextrose (ACD) for analysis of blood lymphocyte subsets (CD3+
T cells, CD3+CD4+ helper T cells, CD3+ CD8+ cytotoxic T cells, CD3−
CD56+ NK cells, CD19+ B cells, and total lymphocytes) determination.
All blood samples, except for ACD treated samples, were centrifuged
for 20 min at 2500 rpm at 4 °C, and plasma stored at −80 °C for future
analysis.
Isolation of catecholamines from human plasma was accomplished
by alumina extraction using a Chromosystems reagent kit (Chromsys-
tems, Munchen, Germany). Once extracted, plasma catecholamine
concentration was quantified by high performance liquid chromatog-
raphy (HPLC). The Waters (Waters Corp., Milford, MA) HPLC system
consisted of a pump (model 510, WISP autoinjector (Model 712) with
cooling module, column, and an electrochemical detector (Model
460). Data were stored and analyzed using the Waters Millennium
software package (V 2.10). The flow rate was 0.8 ml/min, samples in
the autoinjector were maintained at 4 °C, and column oven was
maintained at 40 °C. The column was a 15 cm reversed phase C-18
with 5 m silica particles. The mobile phase consisted of a mixture of
water, methanol and buffer salts manufactured by Chromsystems
(Part C5001, Germany) and optimized for the separation of the plasma
catecholamines. The sensitivity of the assay was 5 pg/ml on a column
with a signal to noise ratio of 4 is to 1, a between days coefficient of
variation of less than 5%, and a within days variation of less than 3%.
The standard curve for the range of 5 to 5000 pg/ml had a correlation
coefficient of 0.998.
The percentages of blood lymphocyte subsets (CD3+ T cells,
CD3+CD4+ helper T cells, CD3+ CD8+ cytotoxic T cells, CD3− CD56+
NK cells, CD19+ B cells, and total lymphocytes) were assessed within
48 h of collection using flow cytometry (EpicsXL and FC500, Beckman
and Coulter, Miami, FL), with a whole blood flow cytometric assay [26].
2.5. Statistical analyses
Data analysis was performed using the SPSS version 16.0. To assess
differences between the FSC and EAC, a 2 × 6 (condition × time) repeated
measures analysis of variance (ANOVA) was used to examine heart
rate, 2 × 11 (condition × time) repeated measures ANOVAs were used
to examine NE and EPI, and 2 × 3 (condition × time) repeated measures
ANOVAs were used to examine the percentages of blood lymphocyte
subsets (CD3+ T cells, CD3+ CD4+ helper T cells, CD3+ CD8+ cytotoxic
T cells, CD3− CD56+ NK cells, CD4/CD8 ratio, CD19+ B cells, and
total lymphocytes). Significant interactions were further analyzed
utilizing paired t-tests.
To assess overall release of NE and EPI, during the FSC and EAC,
integrated trapezoidal area-under-the-curves (AUCs) were calculated
with a previously published formula by Pruessner et al. [27]. Finally,
Pearson product–moment correlations were utilized to examine
relationships among NE AUC, EPI AUC, heart rate, and blood lymphocyte
subsets. The α-level was set at p ≤ 0.05.
3. Results
3.1. Assessments of autonomic activation
Heart rate revealed a significant condition by time interaction
[F (5, 45) = 6.21, p b 0.001] with a greater increase for FSC at 20 min
[t (9)=3.50, p =0.007], and 32 min [t (9) =3.60, p =0.006] (see Fig. 2A).
NE revealed a significant condition by time interaction [F (10, 90) =
6.28, p b 0.001] with a greater increase for FSC at 32 min [t (9) = 3.07,
p = 0.01] and 37 min [t (9) = 3.31, p = 0.009] (see Fig. 2B). Further-
more, NE AUC revealed significantly higher concentrations in the FSC
compared to the EAC [t (9) = 3.61, p = 0.006].
 C.-J. Huang et al. / Physiology & Behavior 101 (2010) 320–326 323

Table 2
Effect of a dual challenge on immune parameters (Mean ± SD).

Variable (%) Condition − 30 min 37 min + 60 min Sig.

CD3+ T cells EAC 76.39 ± 4.73 65.28 ± 8.31 77.25 ± 5.04 time*
FSC 75.12 ± 7.61 67.08 ± 7.40 78.49 ± 5.09 time*
CD3+ CD4+ EAC 45.11 ± 7.42 32.92 ± 6.34 47.80 ± 7.06 time*
helper T cells FSC 44.23 ± 7.05 34.27 ± 6.31 47.84 ± 5.61 time*
CD3+ CD8+ EAC 27.29 ± 5.34 26.84 ± 6.50 26.14 ± 5.29 n.s.
cytotoxic T cells FSC 26.97 ± 5.83 27.25 ± 5.59 26.91 ± 5.27 n.s.
CD3− CD56+ EAC 8.49 ± 4.62 19.02 ± 6.64 6.74 ± 2.12 time*
NK cells FSC 8.70 ± 3.77 17.97 ± 5.88 5.21 ± 2.39 time*
CD4/CD8 ratio EAC 1.75 ± 0.59 1.32 ± 0.49 1.93 ± 0.59 time*
FSC 1.72 ± 0.49 1.32 ± 0.43 1.87 ± 0.52 time*
CD19+ B cells EAC 13.48 ± 5.24 10.18 ± 3.05 14.63 ± 4.82 time*
FSC 13.34 ± 5.30 10.17 ± 3.21 14.10 ± 4.27 time*
Total lymphocytes EAC 98.37 ± 3.94 94.47 ± 4.77 98.64 ± 2.98 time*
FSC 97.15 ± 1.85 95.30 ± 3.30 97.73 ± 1.09 time*

There was no significant condition by time interaction in any immune parameters

between EAC and FSC. In both EAC and FSC, the percentage of CD3− CD56+ NK cells
revealed a significant increase across time, whereas the CD3+ T cells, CD3+ CD4+
helper T cells, CD3+ CD8+ cytotoxic T cells, CD3− CD56+ NK cells, CD4/CD8 ratio,
CD19+ B cells, and total lymphocytes elicited significant decreases. No change was
observed in CD3+ CD8+ cytotoxic T cells. Data are given as mean ± SD. Asterisks
indicate significance across time (*P b 0.001).

3.3. Correlations among variables

NE AUC negatively correlated with CD19+ B cells immediately

following FSC (r = −0.671, p = 0.03) (see Fig. 3A), whereas EPI AUC
was not correlated with any variables in neither FSC nor EAC. Finally,
heart rate negatively correlated with the percent change in the CD4/
CD8 ratio immediately following both EAC and FSC (r = −0.689,
p = 0.02; r = −0.664, p = 0.03; respectively) (see Fig. 3B and C).

Fig. 2. Heart rate and catecholamine responses. A significant condition by time
interaction was revealed in heart rate, NE and EPI, with greater increases in FSC at 20 min
(*p = 0.007) and 32 min, (*p = 0.006) for heart rate (panel A), 32 min (*p = 0.01) and
37 min (*p = 0.009) for NE (panel B), and 37 min (*p = 0.05) for EPI (panel C). Points
represent the NE and EPI values during the protocol; vertical lines depict standard errors
of the means (SEM). The vertical solid lines extending from the ordinate represent the
start of exercise (Ex), and the end of exercise (Ex'). The vertical dotted lines extending
from the ordinate represent the start of the mental challenge (S), and the end of the
mental challenge (S').
In addition, a significant condition by time interaction was
revealed for EPI [F (10, 90) = 2.30, p = 0.01] with a greater increase
for FSC at 37 min [t (9) = 1.87, p = 0.05] (see Fig. 2C). Moreover, EPI
AUC did not reveal a significant difference between FSC and EAC.
3.2. Changes in immune parameters
The CD3+ T cells, CD3+ CD4+ helper T cells, CD3+CD8+ cytotoxic
T cells, CD3− CD56+ NK cells, CD4/CD8 ratio, CD19+ B cells, and total
lymphocytes did not reveal a significant condition by time interaction
between EAC and FSC. As shown in Table 2, the CD3− CD56+ NK cells
elicited a significant increase (main effect) from baseline to immediately
post-stress, and this increase returned to baseline at 1 h post-stress
[F (2, 18) = 23.54, p b 0.001]. Furthermore, the percentages of CD3+
T cells [F (2, 18) = 24.49, p b 0.001], CD3 + CD4+ helper T cells [F (2,
18) = 49.02, p b 0.001], CD4/CD8 ratio [F (2, 18) = 28.04, p b 0.001],
CD19+ B cells [F (2, 18) = 27.28, p b 0.001], and total lymphocytes [F (2,
18) = 7.21, p b 0.001] were significantly lower immediately following
both EAC and FSC than pre-stress and recovered at 1 h post-stress (see
Table 2). In addition, there was no significant change in CD3+CD8+
cytotoxic T cells across time.
4. Discussion
This study examined changes in catecholamines (NE and EPI) and
percentages of blood lymphocyte subsets (CD3+ T cells, CD3+ CD4+
helper T cells, CD3+ CD8+ cytotoxic T cells, CD3− CD56+ NK cells,
CD4/CD8 ratio, CD19+ B cells, and total lymphocytes) in firefighters
exposed to a dual challenge. The combination of physical and
psychological challenge activated the sympathoadrenal (SA) axis,
eliciting the release of NE and EPI and elevating heart rate. The dual
stress condition and the exercise alone condition elicited increases in
CD3− CD56+ NK cells. The percentages of CD3+ T cells, CD3+ CD4+
helper T cells, CD4/CD8 ratio, CD19+ B cells, and total lymphocytes
were lower immediately following both conditions. Furthermore,
following dual challenge NE AUC was negatively correlated with
percentage of CD19+ B cells immediately post challenge, and HR was
negatively associated with the percent change in the CD4/CD8 ratio
from pre to post challenge. Interestingly, HR was also related to change
in the CD4/CD8 ratio during the exercise alone condition. However,
the elevations in NE and heart rate simultaneously in response to the
dual challenge suggest greater sympathetic activation that in turn
would possibly explain the alteration in the distribution of lymphocyte
subsets.
Firefighters elicited a greater elevation in heart rate following the
dual challenge. This result supports previous research by Acevedo et al.
[28] and Webb et al. [19] who demonstrated that the addition of a
mental challenge elicited an exacerbation in heart rate response
during exercise, suggesting that a precursor to the heart rate elevation
was an increase in catecholamine levels (NE and EPI). Interestingly,
firefighters elicited greater increases in NE and EPI following the dual
challenge. Ray et al. [29], examining firefighters from India, observed
greater NE and EPI responses in firefighters challenged with a physical
and psychological demand. Furthermore, Webb et al. [19] have
examined healthy individuals in response to a combination of physical
324 C.-J. Huang et al. / Physiology & Behavior 101 (2010) 320–326
Fig. 3. Panel A shows the correlation between NE AUC and percentage of CD19+ B cells
immediately following FSC (r = −0.671; p = 0.03). Panel B shows the correlation
between heart rate and percent change of the CD4/CD8 ratio immediately following
EAC (r = −0.689; p = 0.02). Panel C shows the correlation between heart rate and
percent change of the CD4/CD8 ratio immediately following FSC (r = −0.664 p = 0.03).
and psychological stress and found a greater increase for NE in dual
stress condition. Our study utilized a specific stressor (FSTD) with a
special population (firefighters) and demonstrated significant eleva-
tions in EPI. Additionally, NE AUC revealed significantly higher
concentrations in the FSC compared to the EAC. Thus, these elevations
in NE and EPI suggest that the SA axis are further activated, beyond the
exercise alone condition, during a dual challenge.
Several studies have observed the alterations in innate immunity
following acute stress [10,11]. Our study demonstrated that the
percentage of CD3− CD56+ NK cells exhibited a significant change
across time in both FSC and EAC, with an increase immediately after
stress returning to baseline at 1 h post-stress. This finding is
supported by previous studies assessing NK cells following psycho-
logical stress and physical activity, independently [10,11,30–32].
Although our finding did not show any associations between
catecholamines and CD3− CD56+ NK cells, previous studies have
demonstrated that acute stress-induced elevations in NK cells may be
due to the release of catecholamines via beta-adrenergic activation
[7,8,11,33]. More specially, Schedlowski et al. [17] found that in
human subjects an injection of both EPI and NE increased NK cell
numbers.
In addition, this study did not observe any change in the percentage
of CD3+ CD8+ cytotoxic T cells. Although this finding differs with
earlier studies that have demonstrated elevations in CD8+ cytotoxic
T cells [10,11], Manuck et al. [34] have demonstrated that the
elevations in CD3+ CD8+ cytotoxic T cells are only seen with
heightened catecholamine and cardiovascular responses to acute
stress. These apparently inconsistent findings are likely related to
variations in nature and types of the stressor, and our protocol may
have been ineffective in eliciting the necessary elevation in catecho-
lamines. Further investigation is needed to examine the level of
sympathetic activation necessary to elicit changes in lymphocyte
subset distribution.
Acute stress has also been shown to alter adaptive immunity. In this
study, the percentages of CD3+ T cells, CD3+ CD4+ helper T cells,
CD4/CD8 ratio, CD19+ B cells, and total lymphocytes decreased
significantly immediately post-stress and returned to baseline at 1
h post-stress in both conditions. These results are consistent with
earlier studies demonstrating decreased CD3+ T cells [10,11],
decreased CD3+ CD4+ helper T cells and CD4/CD8 ratio [11],
decreased CD19+ B cells [12], and decreased total lymphocytes [13]
following acute mental stress. Our findings are also supported by
Moyna et al. [35] who demonstrated that the percentage of CD3+
T cells and CD19+ B cells decreased with an elevation in the
percentage of NK cells (CD3− CD16+ CD56+) following incremental
exercise. These results indicate that acute stress enhances cellular
immunity and suppress adaptive immunity and may be further altered
at higher intensities of mental and physical stress. In addition, our
result demonstrated that immediately following the FSC, NE AUC
was negatively correlated with CD19+ B cells. This suggests that an
exacerbation in catecholamines following a dual challenge may
contribute to the alterations of distribution in adaptive immunity,
including CD19+ B cells. Interestingly, this study found that heart rate
was negatively associated with the percentage change of CD4/CD8
ratio immediately post-stress in both conditions. This finding is
relatively consistent with studies in which stress-induced reductions
in plasma volume (hemoconcentration) are related to concomitant
elevations in heart rate, systolic blood pressure, and plasma catecho-
lamines, resulting in decreases in CD4 and CD19 cell concentrations
[36]. Future studies are needed to consider other mechanisms that
are responsible for the immune cell redistribution in response to acute
stress.
Finally, the International Association of Fire Fighters [37] has
recommended that firefighters achieve a maximal oxygen uptake of
at least 42 ml/kg/min. Although this study utilized a within-subject
design and the effect of physical activity level was controlled, the
fitness levels (VO2max) of our firefighters was quite low (35.63 ml/kg/
min) compared to the proposed minimum level of 42 ml/kg/min for
safety and effectiveness during fire suppression activities. However,
the VO2max value in our firefighters was also similar to the firefighters'
in the study published by Roberts et al. [38] (35.00 ml/kg/min). In
addition, the norm for male age 30 to 39 years old is 42.60 ml/kg/min
[39]. Epidemiological evidence consistently shows that regular
physical activity reduces the risk of diseases, including cardiovascular
disease [40]. Participation in exercise training may result in beneficial
adaptations of physiological systems that are activated during acute
psychological stress. For example, Hamer et al. [41] demonstrated
that physical fitness (as indexed by lower exercise heart rate) is
correlated with lesser pro-inflammatory immune responses (e.g. IL-6
cytokine) following acute mental stress. This finding suggests that
exercise training program should be implemented to reduce the risks
of CVD in firefighters, and consideration should be given to annual
physical performance evaluations [42]. In addition, although this
study did not examine the loss of work economy, a recent study
by Webb et al. [43] examined VO2, RER, and workload (watts
and distance) in firefighters exposed to a computerized firefighting
strategies and tactics decision-making challenge (FSTD) while
participating at a relative exercise intensity similar to the present
study. These authors found no significant differences between the
 C.-J. Huang et al. / Physiology & Behavior 101 (2010) 320–326
exercise alone condition and the dual stress condition. This data
suggests that dual challenge, compared to exercise alone, does not
elicit a difference in working economy. Thus, the catecholamine and
immune responses observed in the present study likely did not occur
due to a loss in work economy.
In summary, firefighters participating in a firefighting simulation
task (dual challenge) responded with elevations in heart rate, NE, and
EPI. These findings suggest that the combined mental and physical
stress often experienced by firefighters may be a contributing factor to
the elevated risk of cardiovascular disease and increased proportion-
ate morality risks of firefighters [2]. Additionally, these elevations in
NE and heart rate simultaneously may possibly explain the alteration
in the distribution of lymphocyte subsets, resulting in ineffective cell-
mediated immune responses. Firefighters are engaged in fire
suppression activities with standard work to rest ratios; approxi-
mately 15 to 20 min of work, then resting for approximately 10 min
[44]. The repeated work–rest regimen of firefighting may potentially
exacerbate catecholamine and heart rate responses, further contrib-
uting to the perturbation of lymphocyte subsets [29,45]. Finally, this
study has several limitations. First, we did not measure the effects of
hemoconcentration (e.g. blood pressure, hydration status) that may
influence lymphocytes subset redistribution. Second, our study did
not measure cortisol concentrations. A recent study by Kimura et al.
[46] has demonstrated that no significant change was observed in
cortisol concentrations following acute stress, and cortisol was not
correlated with any changes in lymphocyte subsets. This is further
supported by Isowa et al. [12] who have concluded that under acute
stress the influence of cortisol on immune function is minimal, and
mostly regulated by catecholamines. The examination of cortisol in
the lymphocyte subset redistribution following prolonged or repeated
bouts of physical and/or mental stress is warranted. Finally, the
numbers of lymphocytes were not measured in the present study.
Both measurements of numbers and percentages of lymphocytes in
the blood would provide a more clear presentation of lymphocyte
redistribution and immune system activation. Further investigation
addressing these limitations will foster a better understanding of the
immune cell response to a combined psychological and physical
challenge in firefighters. In turn, a greater understanding of these
responses to stress can assist in finding strategies to overcome the
inherent psychobiological challenges associated with mentally and
physically demanding professions.
Acknowledgments
Use of trade names does not constitute endorsement of product.
The opinions or assertions contained herein are the private views
of the authors and are not to be construed as official or reflecting the
opinions of the Department of the Army or the Department of
Defense.
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