Clinical anxiety, cortisol and interleukin-6: Evidence for specificity
in emotion–biology relationships
Aoife O’Donovana,b,*, Brian M. Hughesc, George M. Slavichb, Lydia Lynchd, Marie-Therese Cronine,
Cliona O’Farrellyf, Kevin M. Malonea
aDepartment of Psychiatry and Mental Health Research, St. Vincent’s University Hospital and School of Medicine and Medical Sciences, University College Dublin, Ireland
bDepartment of Psychiatry, University of California, San Francisco, USA
cCentre for Research on Occupational and Life Stress, National University of Ireland, Galway, Ireland
dEducation and Research Centre, St. Vincent’s University Hospital, Dublin, Ireland
eSt. Vincent’s University Hospital, Dublin, Ireland
fSchool of Biochemistry and Immunology, Trinity College Dublin, Ireland
a r t i c l ei n f o
Received 12 November 2009
Received in revised form 8 March 2010
Accepted 9 March 2010
Available online 18 March 2010
a b s t r a c t
Anxiety confers increased risk for inflammatory diseases, and elevated inflammatory activity in anxious
individuals may contribute to this increased risk. One complication, however, is that anxiety could be
associated with inflammatory activity either through a specific anxiety pathway or through a more gen-
eral negative emotionality pathway. To investigate, we measured levels of the stress hormone cortisol,
the pro-inflammatory cytokine interleukin-6 (IL-6), and the systemic inflammatory marker C-reactive
protein (CRP), as well as depression and neuroticism, in clinically anxious and non-anxious adults. Com-
pared with non-anxious participants, clinically anxious participants exhibited significantly lower levels of
morning cortisol and significantly higher levels of IL-6, independent of age, sex, and depressive symp-
toms. These group differences were robust when controlling for neuroticism. Conversely, the groups
had equivalent levels of CRP in all analyses. Results are indicative of anxiety-specific effects on inflamma-
tory activity, and highlight a pathway by which anxiety may increase risk for inflammatory diseases.
? 2010 Elsevier Inc. All rights reserved.
Negative emotions such as anxiety and depression confer in-
creased risk for disorders with an inflammatory etiology, and ele-
vated inflammatory activity may be an important mediator of
emotion–disease relationships (Kiecolt-Glaser et al., 2002). How-
ever, whereas a large number of studies have examined associa-
tions between depression and inflammation (Dantzer et al., 2008;
Whooley et al., 2007), little is known about the relationship be-
tween anxiety and inflammation. The paucity of research on this
topic is particularly striking because anxiety may be an even stron-
ger risk factor for inflammatory disorders than depression (Kub-
zansky and Kawachi, 2000; Roy-Byrne et al., 2008).
Specific cognitive biases towards threat-related information
may play a key role in the relationship between anxiety and
inflammation (Mogg and Bradley, 2005). A growing body of re-
search indicates that exposure to real or imagined psychological
threats activates multiple biological systems, including the hypo-
thalamic–pituitary adrenal (HPA) axis, which regulates inflamma-
tory activity, and the inflammatory response (Slavich et al., in
press). Exaggerated and sustained threat perception in anxious
individuals may result in chronic activation of the biological stress
response, leading to reduced secretion of the HPA axis hormone
cortisol during the morning period (Miller et al., 2007), and ele-
vated inflammatory activity (Kiecolt-Glaser et al., 2003).
Early research examining the effects of psychological states on
biological systems was based on a generality model, derived from
the observation that various physical and psychological stimuli eli-
cit a common pattern of biological reactions (Selye, 1956). How-
ever, more recently, an integrated specificity model has been
proposed, emphasizing that specific psychological states are asso-
ciated with specific patterns of biological responses (Kemeny,
2003; Moons et al., 2010). Anxiety and depression are highly
comorbid with one another, and both emotions overlap with neu-
roticism, or the general tendency to experience negative emotions
(Jylhä and Isometsä, 2006). As such, any observed associations be-
tween anxiety and inflammation could be due to general negative
emotionality or specific features of anxiety. However, anxiety ap-
pears to confer increased risk for CVD independent of associations
with depression (Kubzansky and Kawachi, 2000), threat-related
0889-1591/$ - see front matter ? 2010 Elsevier Inc. All rights reserved.
* Corresponding author at: Department of Psychiatry, University of California,
San Francisco, USA.
E-mail address: firstname.lastname@example.org (Aoife O’Donovan).
Brain, Behavior, and Immunity 24 (2010) 1074–1077
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attentional biases are not observed in depression (Mogg and Brad-
ley, 2005). Although neuroticism has been associated with exag-
associations between neuroticism and inflammation is mixed
(Pitsavos et al., 2006; Toker et al., 2005). Thus, we hypothesize that
clinically anxious participants will have lower morning cortisol
and higher levels of inflammatory markers compared with non-
anxious participants, independent of comorbid depressive symp-
toms and neuroticism.
Participants were recruited via posters, flyers and emails dis-
tributed in a large suburban area in Dublin, Ireland. Promotional
materials stated that the study was recruiting individuals currently
experiencing high or low levels of psychological distress, and that
participants would receive a €10 book token. The sample included
27 participants with Hospital Anxiety and Depression Scale Anxi-
ety (HADS-A) scores in the clinical range (anxious group
[scores P 8]:
HADS-A = 10.04,
SD = 8.04) and 29 participants with low scores on the HADS-A
(non-anxious group [scores < 8]: M HADS-A = 5.17, SD = 1.49; M
age = 30.97, SD = 10.21; Zigmond and Snaith, 1983). All partici-
pants were white and 68% were female. Exclusion criteria included
the presence of chronic illness, acute illness within the previous
two weeks, possible current infection, alcoholism, use of medica-
tion (apart from the oral contraceptive pill), anesthesia in the pre-
vious three months, and night shift work in the previous two
weeks. The study was approved by the local institutional ethics
SD = 1.91;
age = 33.52,
2.2. Materials and measures
2.2.1. Anxiety and depression
The Hospital Anxiety and Depression Scale (HADS) is a widely
used and well-validated measure that assesses non-somatic symp-
toms of anxiety and depression (Zigmond and Snaith, 1983). The
HADS includes a 7-item anxiety scale (HADS-A) and a 7-item
depression scale (HADS-D). A review of 747 studies confirmed
the validity of the HADS in assessing severity of anxiety and
depression, and determined that the HADS has sensitivity and
specificity of approximately 80% in defining caseness for anxiety
and depressive disorders based on a clinical cutoff score of 8 (Bjel-
land et al., 2002; Crawford et al., 2001). Internal consistency for the
HADS-A (a = .72) and HADS-D (a = .63) were acceptable.
Neuroticism was assessed with the 12-item Eysenck Personality
Questionnaire-Revised, Short Form, which has reliability and valid-
ity comparable with the full-length EPQ (Barrett and Eysenck,
1992). Internal consistency for the neuroticism scale was accept-
able (a = .80).
2.2.3. Health status
Self-reported physical health measures included: self-ratings of
physical health ‘‘right now” as ‘‘excellent”, ‘‘very good”, ‘‘good”,
‘‘fair” or ‘‘poor”; acute illness (e.g., cold or influenza) during the
previous three months (cold/flu incidence); and past and current
Salivettes were used to obtain saliva samples (Sartstedt Inc.,
Germany). Each salivette comprised a cotton dental roll contained
within two sterile plastic tubes. Participants were instructed to
place the cotton roll in their mouth for a minimum of 90 s to sat-
urate it with saliva before returning it to the plastic tube, which
was then capped tightly and stored at ?20 ?C. When all samples
were ready for analysis, tubes were centrifuged for 10 min at
3000RPM for separation of supernatant. Salivary cortisol analyses
were performed in duplicate using a commercially available high
sensitivity salivary cortisol enzyme immunoassay (Salimetrics
LLC, USA). Intra- and inter-assay coefficients of variation were <7%.
2.2.5. IL-6 and hsCRP
Blood was collected in clot activator vacutainer tubes (Becton,
Dickinson & Company, USA) for the measurement of IL-6 and high
sensitivity CRP (hsCRP). Serum samples were obtained following
centrifugation at 2000RPM for 10 min, and stored at ?20 ?C until
analysis. Analyses for IL-6 were performed in duplicate using a
commercially available high sensitivity IL-6 ELISA (R&D Systems,
USA). Intra- and inter-assay coefficients of variation were <10%.
High sensitivity tests of CRP permit the examination of levels with-
in the low-normal range (hsCRP, Ridker, 2001). Analyses for hsCRP
were performed by means of particle enhanced immunonephelom-
etry using CardioPhase hsCRP reagents on the BN System (Dade
Behring, USA). The intra-assay coefficient of variation for hsCRP
was <6%, and all samples were analysed on a single run. Due to
insufficient availability of serum for four participants, CRP results
are based on 52 participants.
Biological samples were collected between 7:30 and 9:30AM.
Participants fasted and abstained from smoking and caffeine from
midnight, and avoided alcohol and exercise for 24 h prior to their
appointment. Females were scheduled to participate during the
follicular phase of the menstrual cycle. Participants were in-
structed to avoid rushing if they were late, and travel expenses
were awarded so that they would not walk or bike to their appoint-
ment. Upon arrival at the laboratory, participants gave written in-
formed consent and then completed a screener questionnaire. This
process lasted approximately 10 min, during which time partici-
pants were at rest. Following this rest period, participants provided
a saliva sample and then a 15 ml venous blood sample was taken
from the antecubital fossa area. Finally, participants completed
questionnaires under the supervision of the study coordinator.
2.4. Data analysis
Pearson’s correlations were used to assess relationships be-
tween continuous variables. Differences between anxious and
non-anxious participants in cortisol, IL-6 and hsCRP were assessed
with Student’s t-tests. To test the hypothesis that between-group
differences were independent of potential covariates, we reran
our primary model controlling for age, gender, depression and neu-
roticism using analysis of covariance.
3.1. Anxiety and inflammation
All participants were in good physical health as determined by
self-report. Self-reported physical health was not associated with
cortisol, IL-6, or hsCRP levels. Differences between anxious and
non-anxious participants in study variables and potential con-
founds are presented in Table 1. While cortisol was not signifi-
cantly associated with either of the inflammatory markers, IL-6
and hsCRP were significantly associated with one another
A. O’Donovan et al./Brain, Behavior, and Immunity 24 (2010) 1074–1077
(r = .32, p = .03). Correlational analyses indicated that higher levels
of anxiety were marginally related to higher levels of IL-6 (r = .27,
p = .05) and lower levels of cortisol (r = ?.25, p = .06), but not with
levels of hsCRP (Fig. 1a and b). Depression, on the other hand, was
not associated with any of the biological variables.
Compared to non-anxious participants, clinically anxious par-
ticipants had significantly lower levels of cortisol, t(54) = 2.84,
p = .006, and significantly higher levels of IL-6, t(50) = 2.01,
p = .05 (Fig. 1a and b), but they did not differ in levels of hsCRP,
t(49) = ?0.15, p = .88.
As predicted, levels of anxiety and depression were strongly
correlated (r = .51, p < .001). Given that depression has previously
been associated with cortisol and IL-6 levels, and that anxious par-
ticipants exhibited significantly higher levels of depression than
non-anxious participants, t(54) = 2.28, p = .03, it is possible that
depression contributed to the observed differences in levels of cor-
tisol and IL-6 between anxious and non-anxious participants. How-
ever, when depression was added as a covariate in our model
together with age and sex, clinically anxious participants contin-
ued to exhibit significantly lower levels of cortisol, F(1,56) = 6.05,
p = .02,partial g2= .11,aswell
F(1,52) = 4.16, p = .047, partial g2= .08. Although anxious partici-
pants did not have significantly higher levels of neuroticism than
non-anxious participants, t(48) = 1.42, p = .16, raw anxiety and
neuroticism scores were significantly associated (r = .28, p = .04).
When we included neuroticism as an additional covariate, the ef-
fect size for the between-group difference in cortisol remained
similar (even though the finding became marginally non-signifi-
cant), F(1,44) = 3.82, p = .06, partial g2= .08, and clinically anxious
participants continued to exhibit significantly higher levels of IL-
6, F(1,40) = 4.31, p = .04, partial g2= .10.
Our data indicate that clinically anxious individuals have lower
levels of morning cortisol and higher levels of the pro-inflamma-
tory cytokine IL-6. The difference between anxious and non-anx-
ious participants in cortisol and IL-6 was present even when
controlling for both depression and neuroticism, indicating some
degree of specificity in the relationship between anxiety and IL-6.
We found no difference between anxious and non-anxious partic-
ipants in levels of CRP, another inflammatory marker. There are a
number of possible reasons for our finding that anxious partici-
pants differed from non-anxious participants on one marker of
inflammation but not another. Notably, the psychosomatic path-
way that determines stress-related production of IL-6 is likely to
differ in important ways from that leading to production of CRP.
For example, previous research suggests that, unlike CRP, IL-6
may be subject to direct influence by neural and hormonal re-
sponses to stress (Zhou et al., 1993). Overall, these data provide
support for the notion of specificity at psychological and biological
levels in the relation between negative emotional states and
inflammation (Kemeny, 2003).
Cognitive biases towards threatening information may lead to
exaggerated threat perception and consequently, more frequent
and prolonged activation of the biological stress response in anx-
Characteristics of clinically anxious and non-anxious participants.
(n = 27)
(n = 29)
Age: M (SD)
Females: n (%)
Fear of needles: n (%)
Cold/Flu (past 3 months): n (%)
Health complaints (past
3 months): n (%)
OCP (females): n (%)
16 (80) 11 (61.1).20
Highest level of education: n (%)
Marital status: n (%)
Living with partner
Self-rated physical health: n (%)
Notes: Group differences were calculated using t-tests, Chi-square tests and Mann
Whitney U tests as appropriate. M = mean; SD = standard deviation; n = sample
size; OCP = oral contraceptive pill.
Fig. 1. (a and b) Scatterplot illustrating between and within group differences in cortisol and IL-6. Correlational analyses indicated that increasing levels of anxiety tended to
be associated with higher levels of IL-6 (r = .27, p = .05) and lower levels of cortisol (r = ?.25, p = .06). Furthermore, clinically anxious participants exhibited significantly lower
levels of morning cortisol (t = 2.84, p = .006) and significantly higher levels of interleukin-6 (IL-6; t = 2.01, p = .05) than non-anxious participants.
A. O’Donovan et al./Brain, Behavior, and Immunity 24 (2010) 1074–1077
ious individuals. Given that anxiety disorders tend to have an early Download full-text
onset and a chronic course (Kessler et al., 2005; Thyer et al., 1985),
exaggerated threat perception across many years could manifest as
chronic exposure to biological stress reactivity. In fact, the pattern
of lower morning cortisol and elevated IL-6 that anxious partici-
pants exhibited in the present study resembles the pattern previ-
ously observed in association with chronic stress (Kiecolt-Glaser
et al., 2003; Miller et al., 2007). Further research is necessary to
examine if chronic exaggerated threat perception mediates this
In contrast with the large body of research examining the asso-
ciation between depression and inflammation (Dantzer et al.,
2008), evidence regarding the association between anxiety and
inflammatory activity is limited. Furthermore, the limited research
on state anxiety and inflammation has yielded a mixed pattern of
results (Chandrashekara et al., 2007; Maes et al., 1998). Different
degrees of depression and neuroticism within anxious individuals,
differences in severity of anxious symptoms, and variation in spe-
cific inflammatory proteins measured across studies could account
for this mixed pattern of findings. The present data highlight that it
is inappropriate to hypothesize simple emotion–inflammation
relationships. Rather, investigators must pay attention to charac-
teristics of the specific negative emotion of interest, the level at
which that emotion is experienced, and to the complex network
of proteins involved in the inflammatory response.
4.1. Limitations and future directions
The present findings must be interpreted in light of several lim-
itations. First, although our protocol involved collecting all biolog-
ical samples during a two-hour period, it is possible that
differences between anxious and non-anxious participants in sleep
disruption or time since awakening contributed to our findings.
Second, the cross-sectional design of the present research does
not permit us to determine causal direction in the relations ob-
served between anxiety, cortisol, and IL-6. However, pro-inflam-
matory cytokines appear to selectively promote symptoms of
depression without influencing levels of anxiety (Capuron et al.,
2001; Lieb et al., 2006), supporting the formulation that the direc-
tion of effect would be from anxiety to inflammation and not vice
versa. Finally, the present small study was a first step towards
examining anxiety-specific effects on inflammatory activity. IL-6
is an important target for psychoneuroimmunological study be-
cause of the key role this cytokine plays in regulating normal
inflammatory responses and in the pathophysiology of numerous
inflammatory diseases. However, a more complete understanding
of anxiety effects on specific aspects of the inflammatory response
would require the measurement of multiple pro- and anti-inflam-
In sum, the present data indicate that clinically anxious individ-
uals have lower morning cortisol and elevated IL-6 compared with
non-anxious individuals, highlighting a potential pathway by
which anxiety may increase risk for inflammatory diseases. Impor-
tantly, our findings suggest that anxiety may be associated with
inflammatory activity independent of depression and neuroticism,
thus indicating specificity in relationships between negative emo-
tions and biological responses. Consequently, they support the
adoption of an integrated specificity model to elucidate relation-
ships between negative emotions and inflammation.
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