Brain, Behavior, and Immunity 19 (2005) 540–546
www.elsevier.com/locate/ybrbi
Psychological stress activates interleukin-1 gene expression
in human mononuclear cells
Lena Brydon a,¤, Susan Edwards a, Haiyan Jia b, Vidya Mohamed-Ali b, Ian Zachary b,
John F. Martin b, Andrew Steptoe a
a
The Psychobiology Group, Department of Epidemiology and Public Health, University College London, London, UK
b
Department of Medicine, The Rayne Institute, University College London, London, UK
Received 14 October 2004; received in revised form 19 November 2004; accepted 22 December 2004
Available online 19 February 2005
Abstract
The pathophysiological mechanisms underlying the association between psychological stress and cardiovascular disease are
unclear. Interleukin-1 (IL-1) and interleukin-6 (IL-6) are inXammatory cytokines playing a pivotal role in atherosclerosis. IL-1
activates IL-6, and both cytokines are produced by peripheral blood mononuclear cells. One mechanism through which stress could
promote atherosclerosis is by regulating mononuclear cell cytokine gene expression. We studied cardiovascular and cytokine
responses in 32 healthy men participating in two 5-min mental tasks and in 10 controls. Blood pressure and heart rate, assessed using
a Portapres-2, increased signiWcantly following tasks in all participants. Plasma IL-6 levels, determined by ELISA, also increased following tasks, with maximum levels detected 2 h post-stress. Quantitative RT-PCR analysis showed that mononuclear cell IL-1 gene
expression rose signiWcantly at 30 min post-stress and remained elevated at 75 and 120 min. Increases in IL-1 gene expression correlated positively with plasma IL-6 responses, cardiovascular responses, subjective stress ratings, and anxiety symptoms. No changes
were detected in controls. Stress-induced activation of mononuclear IL-1 is a novel mechanism potentially linking stress and heart
disease. This mechanism could also play a role in other inXammatory diseases exacerbated by stress.
 2005 Elsevier Inc. All rights reserved.
Keywords: Stress; Interleukins; Genes; InXammation; Atherosclerosis
1. Introduction
Evidence from epidemiological and observational
studies shows that coronary heart disease (CHD) is
more prevalent in humans and animals subject to
chronic psychosocial stress. For example, people working in jobs with high demands coupled with low control,
caregivers, socially isolated individuals and people with
symptoms of anxiety and depression are at a higher risk
of developing CHD (Hemingway and Marmot, 1999).
Similarly, cynomolgus monkeys housed in unstable,
socially stressful conditions display impaired endothe-
¤
Corresponding author. Fax: +44 20 7916 8542.
E-mail address: [email protected] (L. Brydon).
0889-1591/$ - see front matter  2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbi.2004.12.003
lial function and develop more coronary artery lesions
compared to unstressed controls (Rozanski et al., 1999;
Williams et al., 1991). Heightened cardiovascular reactivity to acute stress predicts future hypertension, left
ventricular mass, and progression of carotid atherosclerosis (Treiber et al., 2003). Furthermore, acute mental
stress has been shown to cause transient vascular endothelial dysfunction (Ghiadoni et al., 2000) and increases
in circulating inXammatory cytokine levels in healthy
humans (Steptoe et al., 2001). Elevated plasma levels of
IL-1 and IL-6 have been reported in patients with
acute coronary syndromes (Ikeda et al., 2001; Simon
et al., 2000). IL-1, produced primarily by monocytes
and macrophages, is a central component of the acute
phase inXammatory response (Dinarello, 1996). It acts,
in collaboration with a network of other cytokines
L. Brydon et al. / Brain, Behavior, and Immunity 19 (2005) 540–546
including IL-6, to coordinate atherosclerosis through a
variety of proinXammatory, prothrombotic processes
including migration and/or activation of monocytes,
macrophages, T-lymphocytes, and platelets, increased
expression of vascular adhesion molecules, Wbroblast
proliferation, and synthesis of C-reactive protein and
Wbrinogen (Dinarello, 1996; Young et al., 2002). It also
stimulates IL-6 production from a variety of cells. IL-1
acts mainly at the autocrine/paracrine level and is diYcult to detect in plasma from healthy individuals. IL-1
and IL-1 lack signal peptides, so are not readily
secreted from cells into the systemic circulation. In contrast, IL-6 is an endocrine molecule and can be easily
measured in plasma (Yudkin et al., 2000). Therefore, IL6 is often used as a surrogate marker for biologically
active IL-1 (Dinarello, 1996).
Peripheral blood mononuclear cells (PBMC) are a
source of both IL-1 and IL-6. A recent study by Bierhaus et al. showed that psychological stress activates the
expression of the transcription factor, nuclear factor-B
(NF-B) in PBMC of healthy subjects (Bierhaus et al.,
2003). Notably, NF-B upregulates the expression of a
number of inXammatory proteins including cytokines
(Barnes and Karin, 1997). We postulated that one mechanism through which stress could promote atherosclerosis is by regulating cytokine gene expression in these
cells. To test this hypothesis we investigated the eVect of
acute psychological stress on cardiovascular responses,
plasma IL-6 levels, and IL-1 and IL-6 gene expression
in PBMC of 32 healthy men and a separate group of 10
age-matched controls who underwent the same protocol
in the absence of stress.
2. Methods
2.1. Participants and conditions
Forty-eight healthy men aged 30–59 years were
recruited from two London-based Departments of the
British civil service. All participants were of white European origin, non-smokers, had no previous history of
coronary heart disease or any other relevant physical or
mental illness, and were not taking any medication. One
participant withdrew after feeling unwell and cytokine
data were incomplete in 15 other cases due to blood
sampling problems. Thirty-two men were therefore
included in the Wnal analyses. A comparative non-stress
control group of 10 healthy men in the same age range
also took part, in order to determine whether cytokine
levels and cardiovascular activity would change over
time in the absence of any stress task. Participants were
instructed not to drink caVeinated beverages on the
morning of the experiment, and not to consume alcohol
or exercise on the evening before or the day of testing.
They were asked not to take aspirin for 10 days prior to
541
testing, and to avoid a high fat or high protein breakfast. All participants were tested individually in the
morning. The study was approved by the UCL/UCLH
Committee on the Ethics of Human Research. All participants gave their informed consent before entering
the study.
2.2. Physical measures
Before testing, measures of weight, height, waist, and
hip circumference were obtained and body fat mass was
estimated using a Bodystat 1500 bioelectrical impedance
body composition analysis device (Bodystat, Douglas,
Isle of Man).
2.3. Psychological measures
Symptoms of anxiety and depression were assessed in
the stress group using the Hospital Anxiety and Depression (HAD) Scale, a standard measure of symptoms of
anxiety and depression developed for use with medical
patients (Zigmond, 1981). Scores could range from 0 to
21, with higher values reXecting greater anxiety and
depression. Scores of 11 and above indicate borderline
clinical anxiety and depression.
2.4. Mental stress testing procedure
A venous cannula was inserted for periodic collection
of blood samples. Participants then rested for 30 min.
Blood pressure and heart rate were recorded for the last
5 min of the rest period using a Portapres-2. Participants
then rated feelings of stress on a 7-point scale from
1 D low to 7 D high and a baseline blood sample was
drawn. Mental stress was induced by two behavioural
tasks administered under time pressure (Steptoe et al.,
2002). Each task lasted for 5 min. The Wrst was a computerised colour-word interference task, involving the
successive presentation of target words, printed in
another colour. The second was a hand-eye coordination
task involving the tracing of a star with a metal stylus
that could only be seen in mirror image. Participants
were asked to give accuracy priority over speed on both
tasks. Blood pressure and heart rate were recorded
throughout tasks. At the end of each task, participants
rated task diYculty, involvement, controllability, and
feelings of stress on 7-point scales from 1 D low to
7 D high. Participants then rested quietly for the remainder of the experiment. Blood pressure and heart rate
were recorded at 25–30, 70–75, and 115–120 min poststress, and blood samples were drawn at 30, 75, and
120 min post-stress. Control participants followed the
exact same procedure as the other participants but simply rested in place of the mental stress tasks. Psychological questionnaires were administered to the stress group
during the last hour of the session.
542
L. Brydon et al. / Brain, Behavior, and Immunity 19 (2005) 540–546
2.5. Measurement of plasma interleukin-6
Blood samples were drawn into EDTA-Vacutainer
tubes using 21-gauge ButterXy needles, and centrifuged
immediately at 2500 rpm, for 10 min at room temperature. Plasma was removed, aliquoted, and frozen at
¡80 °C until analysis. Plasma IL-6 was measured using a
high sensitivity two-site ELISA from R and D Systems
(Oxford, UK). The limit of detection of this assay was
0.09 pg/ml, with intra- and interassay coeYcients of variation (CVs) of 5.3 and 9.2%.
2.6. Isolation of RNA from PBMC and reverse
transcription
tics), and 2 l cDNA template including serial dilution of
standards and samples, in a Wnal volume of 20 l. The
PCR conditions were as follows: 10 min at 95 °C, 45
cycles of 10 s at 95 °C, 5 s at 61 °C, and 10 s at 72 °C.
SpeciWcity of the ampliWcation products was assessed by
melting curve analysis. Data were analysed with Roche
Molecular Biochemicals Light Cycler Software Version
3 using the second derivative maximum method. Standard curves were produced by plotting the logarithm of
the concentration of DNA standard against the cycle
numbers of the logarithmic linear phase. The amount of
target sequence in the samples was extrapolated from
the standard curve.
2.8. Statistical analyses
Whole blood samples, obtained as above, were
diluted with an equal volume of RPMI 1640 (Life Technologies) and separated on Ficoll Paque Plus (Amersham Pharmacia Biotech) by centrifugation at 2000 rpm
for 30 min at room temperature. Mononuclear cells were
isolated, washed twice with RPMI 1640, then total RNA
was extracted using TRIzol reagent (Life Technologies).
cDNA was synthesised by reverse transcription using
Moloney murine leukaemia virus reverse transcriptase
(Life Technologies) at 42 °C for 60 min, followed by
inactivation of the reverse transcriptase at 99 °C for
5 min.
2.7. Analysis of cytokine gene expression in PBMC
Quantitative real-time PCR was used to measure
expression of the human IL-1 gene and the human IL-6
gene in cDNA samples, using a LightCycler Instrument
(Roche Diagnostics). Ribosomal 18S RNA was ampliWed as a reference gene. Analysis of this gene demonstrated that it was not aVected by mental stress (data not
shown). Primers were designed using Primer 3 software
(Rozen and Skaletsky, 2000) (http://www-genome.wi.
mit.edu/cgi-bin/primer/primer3_www.cgi), based on gene
sequences published in the GenBank human DNA database (Accession No. M15330 (human IL-1),
NM_000600 (human IL-6), and X03205 (human 18S
rRNA)). Sequences of the sense and antisense oligonucleotides and ampliWcation product sizes were: 5⬘-ACG
ATGCACCTGTACGATCA-3⬘ and 5⬘-TCTTTCAA
CACGCAGGACAG-3⬘ and 229 bp for IL-1, 5⬘-AAA
GAGGCACTGGCAGAA-3⬘ and 5⬘-AGCTCTGGCT
TGTTCCTCAC -3⬘ and 183 bp for IL-6, and 5⬘-CGGC
TACCACATCCAAGGAA-3⬘ and 5⬘-GCTGGAATT
ACCGCGGCT-3⬘ and 186 bp for 18S rRNA. DNA
standards were prepared by PCR followed by puriWcation and spectrophotometric determination. PCRs consisted of MgCl2 (2 mM Wnal for 18S rRNA and IL-6 and
4 mM Wnal for IL-1), cytokine-speciWc forward and
reverse primers (0.5 M each Wnal), 1£ LightCyclerFastStart DNA Master SYBR Green I (Roche Diagnos-
Analyses of changes in cardiovascular, subjective, and
gene expression responses were performed using
repeated measures analysis of variance with group
(stress/control) as the between-subject factor, and trial as
the within-subject factor. Multiple regression analyses,
product-moment correlations, and t tests were also carried out as appropriate. All analyses were conducted
using SPSS v.10.
3. Results
3.1. EVect of psychological stress on cardiovascular
measures and subjective stress ratings
Tasks induced substantial increases in blood pressure
and heart rate (p < .001) in all participants, with average
changes of 28.7 mmHg systolic and 16.6 mmHg diastolic
pressure (Fig. 1), and 7.3 bpm in heart rate (Fig. 2A).
Blood pressure and heart rate diminished following tasks,
but remained above baseline levels throughout the poststress period. Blood pressure rose slightly over the session
in controls while heart rate remained low. Tasks also
induced increases in subjective stress, but stress ratings
returned to low levels during recovery (Fig. 2B). There
were no signiWcant diVerences between groups in blood
pressure or heart rate at baseline, but subjective stress ratings were marginally elevated in the stress group.
3.2. EVect of psychological stress on mononuclear cell
cytokine gene expression
To investigate the role of mononuclear cells in an
inXammatory response to stress, we studied IL-1 and
IL-6 gene expression in PBMC by quantitative RT-PCR.
IL-1 gene expression rose signiWcantly at 30 min poststress (Fig. 3) and remained elevated at 75 and 120 min.
No signiWcant changes were detected in controls (Fig. 3).
IL-1 gene expression in stress and control groups did
not diVer at baseline but did at all other time points
L. Brydon et al. / Brain, Behavior, and Immunity 19 (2005) 540–546
543
Fig. 3. Acute psychological stress activates IL-1 gene expression in
PBMC of healthy volunteers. IL-1 gene expression was measured by
quantitative real-time RT-PCR before (base) and at 30, 75, and
120 min post-stress in 32 volunteers (solid line) and 10 no-stress controls (dotted line). IL-1 mRNA levels are normalised to 18S rRNA
levels determined for each sample by the same technique. Data are presented as means § SEM. *SigniWcant diVerences between groups (all
p < .05).
Fig. 1. Acute psychological stress increases blood pressure in healthy
volunteers. Mean systolic (A) and diastolic (B) blood pressure at baseline, during stressful tasks, and at 30, 75, and 120 min post-stress in 32
male volunteers (solid line) and 10 no-stress controls (dotted line).
Data are presented as means § SEM. *SigniWcant diVerences between
groups (all p < .05).
Fig. 2. Heart rate and subjective stress responses to acute psychological stress in healthy volunteers. Mean heart rate (A) and subjective
stress rating (B) at baseline, during stressful tasks, and at 30, 75, and
120 min post-stress in 32 male volunteers (solid line) and 10 no-stress
controls (dotted line). Data are presented as means § SEM. *SigniWcant diVerences between groups (all p < .05).
(t D 2.22–3.51, all p < .05). In contrast, IL-6 gene expression was unaltered by stress. The fold changes in IL-6
gene expression were not signiWcant, and averaged
1.30 § 0.77, 1.16 § 0.74, and 1.15 § 0.54 at 30, 75, and
120 min post-stress (F D 1.50, p D .24).
3.3. Relationship between mononuclear IL-1 gene
expression and cardiovascular responses, subjective stress
ratings, and anxiety symptoms
There was a signiWcant association between stressinduced increases in IL-1 gene expression and cardiovascular measures. The fold increase in IL-1 gene
expression from baseline to 30 min post-stress correlated
positively with heart rate responses to tasks (r D .32,
p D .05). Similarly, the fold change in gene expression
from baseline to 75 min correlated with heart rate at
75 min (r D .44, p D .005) and the fold change at 2 h correlated with heart rate at 2 h (r D .40, p D .015). Systolic
blood pressure responses to tasks correlated positively
with the fold change in gene expression at 2 h (r D .37,
p D .021). All correlations were independent of age, body
mass index (BMI), and body fat.
Increases in IL-1 gene expression were also associated with psychological measures. Subjective stress ratings obtained immediately after tasks correlated
positively with the fold change in gene expression at
30 min (r D .34, p D .034). Furthermore, stress-induced
increases in IL-1 gene expression were greater in participants who reported more anxiety symptoms on the Hospital Anxiety and Depression Scale. Scores of anxiety and
depression on this scale averaged 5.06 § 3.2 and 3.22 § 2.3,
respectively. Although all scores were in the normal
range, there was a signiWcant relationship between
increases in IL-1 gene expression and anxiety symptoms
(r D .38, p D .036), after controlling for age and BMI.
3.4. EVect of psychological stress on plasma IL-6 and
association with mononuclear IL-1 gene expression
Plasma IL-6 increased in response to stress (F D 32.3,
p < .001), with maximum levels detected 2 h post-stress
(Fig. 4). No changes were observed in controls (F D 1.71,
544
L. Brydon et al. / Brain, Behavior, and Immunity 19 (2005) 540–546
Fig. 4. Acute psychological stress stimulates increases in plasma IL-6
in healthy volunteers. Plasma IL-6 was measured by a high sensitivity
two-site ELISA before (base) and at 30, 75, and 120 min post-stress in
32 volunteers (solid line) and 10 no-stress controls (dotted line). Data
are presented as means § SEM. *SigniWcant diVerences between
groups (all p < .05). The increase in IL-6 was signiWcant between baseline and 30 min (p < .05), 75 min (p < .001), and 120 min (p < .001) in
the stress group, but not in controls.
p D .22) (Fig. 4). The concentration of plasma IL-6 was
greater in stress than control groups at all time points
(F D 16.8, p < .001). Importantly, there was a positive
association between plasma IL-6 responses and IL-1
gene expression. The fold increase in IL-1 gene expression from baseline to 30 min post-stress correlated positively with the increase in IL-6 from baseline to 75 min
(r D .40, p D .013) and the increase in IL-6 from baseline
to 2 h post-stress (r D .36, p D .026), independently of age
and BMI.
4. Discussion
Our results show that an acute psychological stressor
increases mononuclear cell IL-1 gene expression and
plasma IL-6, in a group of healthy volunteers. The sensitivity of inXammatory cytokines to psychological stress
is supported by other studies in animal models and
humans. For example, restraint stress was previously
shown to alter kinetics of IL-1 gene expression in cutaneous wounds of mice (Mercado et al., 2002) and to
increase IL1 gene expression in rat hypothalamus
(Minami et al., 1991). Similarly, academic examination
stress was associated with increased LPS-stimulated production of IL-6 from cultured human PBMC (Maes
et al., 1998).
Psychological stress activates the sympathetic nervous system, which regulates heart rate and release of
catecholamines, and the hypothalamic–pituitary–adrenal axis, which regulates release of corticosteroids. The
acute stressor used in this study induced signiWcant
increases in blood pressure and heart rate in all volunteers. The extent of cardiovascular reactivity varied
among individuals. Importantly, stress-induced increases
in IL-1 gene expression were positively correlated with
heart rate and systolic blood pressure reactivity to stress.
This, together with the rapidity of changes in IL-1, suggests that this is a sympathetically driven response. However, the long duration of cytokine responses suggests
that alternative pathways may also be important. IL-1
gene expression is upregulated by the transcription factor, NF-B (Barnes and Karin, 1997). A recent study
showed that stress-induced upregulation of NF-B protein in PBMC from healthy volunteers paralleled
increases in catecholamines and cortisol (Bierhaus et al.,
2003). In contrast, other studies have reported that catecholamines and corticosteroids inhibit the production of
inXammatory cytokines from PBMC (Agarwal and
Marshall, 1998; Mohamed-Ali et al., 2001). It is possible
that other stress-responsive neuroendocrine mediators
are involved, such as substance P and angiotensin II,
which stimulate cytokine production from mononuclear
cells (Brasier et al., 2002; Cuesta et al., 2002).
Baseline stress ratings and plasma IL-6 were marginally higher in the stress group compared to controls.
These diVerences may have been due to an anticipatory
response in the stress group, who knew that they would
be performing challenging tasks. However, we do not
believe that this is a serious confound, since our results
clearly show that there was a signiWcant stress-induced
increase in plasma IL-6 in the stress group only. Plasma
IL-6 responses to stress were positively correlated with
increases in IL-1 gene expression. This association was
delayed inasmuch as the change in IL-1 gene expression at 30 min post-stress correlated with the increase in
IL-6 at 75 min and 2 h post-stress. This suggests that
stress-induced activation of IL-1 stimulates the IL-6
response. Since IL-6 gene expression was unaltered by
stress, it is unlikely that the plasma IL-6 response is due
to increased IL-6 production by PBMC. Stress might
activate translation of pre-formed stocks of IL-6 mRNA
in these cells or release of stored IL-6 protein. However,
the delayed plasma IL-6 response suggests this cytokine
is being synthesised de novo. Instead, we propose that
stress-induced upregulation of PBMC IL-1 leads to
increased intercellular levels of this cytokine which stimulate IL-6 production from a diVerent cell source. Endothelial cells, smooth muscle cells, and adipocytes all
produce IL-6 and interact with PBMC (Flower et al.,
2003; Norioka et al., 1990; Sironi et al., 1989). Furthermore, IL-1 has been shown to stimulate IL-6 production from these cells in vitro (Flower et al., 2003;
Norioka et al., 1990; Sironi et al., 1989).
PBMC are a mixed population of diVerent immune
cell subsets, including monocytes, neutrophils, and lymphocytes. Previous studies have shown that psychological stress induces leukocytosis and a re-distribution of
lymphocyte subsets (Benschop et al., 1996). We therefore
needed to consider whether a change in the relative composition of immune cells in the PBMC fraction would
aVect our measurements. In agreement with previous
Wndings, total blood cell counts measured in samples
L. Brydon et al. / Brain, Behavior, and Immunity 19 (2005) 540–546
taken at baseline, immediately post-stress and at 30 and
75 min post-stress in a subset of 14 of the stress group
participants, showed that stress induced a signiWcant
increase in total leukocytes (data not shown). This was
due to an increase in lymphocyte numbers, and stress
had no eVect on the number of monocytes or neutrophils
in participants’ blood. The quantitative RT-PCR
method used in this study means that diVerences in total
leukocyte numbers would not aVect gene expression
measurements. Since IL-1 is produced primarily by
monocytes, it is likely that increases in IL-1 gene
expression are occurring predominately in these cells
(Toda et al., 2002). However, it is conceivable that lymphocytes could contribute to this eVect, since they also
express IL-1. Importantly, we found no correlation
between IL1 gene expression and the numbers of lymphocytes, monocytes or neutrophils in participants’
blood, suggesting that changes in relative immune cell
numbers in the PBMC fraction do not account for our
Wndings. However, lymphocytes may be further subdivided into natural killer (NK) cells, T cells, and B cells
and psychological stress has been shown to speciWcally
increase the prevalence of NK cells and CD8+ T lymphocytes in the PBMC pool (Benschop et al., 1996). Relevant to this, Richlin et al. recently reported that an
increase in NF-B DNA-binding activity in human
PBMC following acute SNS activation during exercise
stress could be mainly accounted for by an increase in
the relative numbers of NK cells, which constitutively
express higher levels of this transcription factor than
other leukocyte subsets, as opposed to a change in NFB expression within speciWc cells per se (Richlin et al.,
2004). As mentioned previously, NF-B activates the
expression of a number of inXammatory proteins,
including IL-1 and IL-6. Since we did not quantify lymphocyte subsets, we cannot rule out a stimulatory eVect
of psychological stress on the prevalence of NK cells (or
indeed other lymphocyte phenotypes) as a potential confounding factor in our study. However, the fact that
stress did not alter mononuclear cell IL-6 gene expression indicates that a constitutive eVect alone is unlikely
to account for our Wndings. Future studies will be aimed
at further delineating the signalling pathways and mononuclear cell phenotypes involved in the IL-1 response.
This investigation was carried out in a sample of
healthy, white, middle-aged men, and results may not
generalise to other groups. The magnitude of stressinduced elevations in blood pressure, heart rate, IL-1
gene expression, and plasma IL-6 was relatively small.
However, the challenges imposed were relatively mild
compared with the diYculties many people experience
on a day-to-day basis, and may therefore be under-representative of the true stress response. Furthermore,
although these responses may not be clinically relevant
in themselves, they could well be relevant when repeated
frequently on a long-term basis.
545
Taken together, our results indicate that psychological stress could promote the inXammatory process
underlying atherosclerosis through activation of mononuclear cell IL-1 and increases in plasma IL-6. This
mechanism may also play a role in other inXammatory
diseases exacerbated by stress.
Acknowledgments
This research was supported by the British Heart
Foundation. We are grateful to Kesson Magid, Bev
Murray, and Philip C. Strike for their involvement in
data collection and to Steve Martin for advice on PCR
analysis.
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