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Stress Mediators and Immune Dysfunction in Patients With Acute Cerebrovascular Diseases
Stress Mediators and Immune Dysfunction in Patients With Acute Cerebrovascular Diseases
Abstract
Background: Post-stroke immune depression contributes to the development of infections which are major
complications after stroke. Previous experimental and clinical studies suggested that humoral stress mediators
induce immune dysfunction. However, prospective clinical studies testing this concept are missing and no data
exists for other cerebrovascular diseases including intracerebral hemorrhage (ICH) and TIA.
Methods: We performed a prospective clinical study investigating 166 patients with TIA, ischemic and hemorrhagic stroke.
We measured a broad panel of stress mediators, leukocyte subpopulations, cytokines and infection markers from hospital
admission to day 7 and on follow-up after 2–3 months. Multivariate regression analyses detected independent predictors of
immune dysfunction and bacterial infections. ROC curves were used to test the diagnostic value of these parameters.
Results: Only severe ischemic strokes and ICH increased some catecholamine metabolites, ACTH and cortisol
levels. Immunodysfunction was eminent already on hospital admission after large brain lesions with
lymphocytopenia as a key feature. None of the stress mediators was an independent predictor of lymphocytopenia
or infections. However, lymphocytopenia on hospital admission was detected as an independent explanatory
variable of later infections. NIHSSS and lymphocytopenia on admission were excellent predictors of infection.
Conclusions: Our results question the present pathophysiological concept of stress-hormone mediated immunodysfunction
after stroke. Early lymphocytopenia was identified as an early independent predictor of post-stroke infections. Absence of
lymphocytopenia may serve as a negative predictive marker for stratification for early antibiotic treatment.
Citation: Liesz A, Ru¨ger H, Purrucker J, Zorn M, Dalpke A, et al. (2013) Stress Mediators and Immune Dysfunction in Patients with Acute
Cerebrovascular Diseases. PLoS ONE 8(9): e74839. doi:10.1371/journal.pone.0074839
Editor: Thiruma V. Arumugam, National University of Singapore, Singapore
Received July 9, 2013; Accepted August 5, 2013; Published September 19, 2013
Copyright: 2013 Liesz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Deutsche Forschungsgemeinschaft (VE196/3-1) and the Else Kro¨ner-Fresenius foundation
(2012-A118) and by a fellowship of the Daimler-Benz foundation to A.L. The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Arthur.Liesz@med.uni-heidelberg.de
Figure 1. Humoral stress response in patients with acute cerebrovascular diseases. Blood plasma concentrations of indicated
catecholamines and metanephrines were analyzed in (A) all patients (n = 166) or (B) only in patients not receiving catecholamine treatment
(n = 134). Urin excretion of noradrenalin, metanephrines and vanillylmandelic acid (VMA) within the first 24 h after admission was
investigated (C) in all patients with cerebrovascular events (left) or after exclusion of patients receiving catecholamine treatment during the
urine collection period (right). (D) To investigate the HPA-axis, blood plasma concentrations of ACTH and cortisol were measured at the
indicated time points after cerebrovascular events and in control patients. Group differences for all parameters were analyzed by Kruskal-
Wallis H-test and Dunn’s post hoc test. Data is presented as median and interquartile range. * indicates significant (p,0.05) difference of the
indicated group compared to the control group; horizontal bars indicate significant differences among groups at the respective time point.
doi:10.1371/journal.pone.0074839.g001
Figure 2. Alterations of the immune system after cerebrovascular events. (A) Changes of the cellular immune system were measured by the
relative lymphocyte fraction, absolute lymphocyte counts, total leukocyte number and the absolute monocyte cell counts in control patients
and at the indicated time points after cerebrovascular events. (B) Blood plasma concentrations of IL-10, IL-6 and IL-1b were determined to
analyze humoral mediators of immune function. Blood cell counts and cytokines were analyzed by analysis of variance (ANOVA) after
testing for normal distribution. Data are presented as mean+/2 standard deviation. * indicates a significant (p,0.05) difference of the
indicated group compared to control; horizontal bars indicate significant group differences at the respective time point.
doi:10.1371/journal.pone.0074839.g002
Figure 3. Infection markers and microbiological culture results. (A) The proportion of the positive infection markers febrile body temperature
(.38uC), CRP.5 mg/l and hsPCT .0.1 ng/ml within the study cohorts is presented as percentage of the respective group. (B) The proportion
of patients receiving antibiotic treatment is depicted for the first week after symptom onset (d2–d7, left panel) or for a new antibiotic
treatment after hospital discharge until follow-up (FU) after 60–90d (right panel). (C) Bars depict the percentage of patients of the respective
group with at least one pathological bacterial culture result during the first week after symptom onset (d2–d7) (cp. methods for definition).
Data was tested using chi-squared tests and is presented as proportions per group. Horizontal lines indicate significant (p,0.05) group
differences (chi-squared test). doi:10.1371/journal.pone.0074839.g003
Table 2. Results of univariate binary logistic an early significant increase of plasma ACTH and cortisol
regression analysis for lymphocytes ,13% on day 1. concentration after large ischemic stroke and ICH (Fig. 1D).
Table 3. Results of univariate binary logistic regression analysis for bacterial infection d2–d7.
B: regression coefficient; OR: odds ratio; CI: confidence interval; Std. OR: odds ratio standardized for standard deviation for continuous predictors (eB*SD).
Total patients n = 136 (control patients excluded); patients without catecholamine treatment (w/o Norepi.) n = 104 (during first week).
doi:10.1371/journal.pone.0074839.t003
nia,13% on day 1 (admission) included only predictive variables correlated with lymphocytopenia. In the univariate logistic regression
on admission (Table 2). Interestingly, among stress markers only model for bacterial infections within the first week after admission all
cortisol and noradrenalin levels in all patients had a significant p values obtained during the first 7d were included (Table 3). Markers
value (,0.1) while catecholamine concentrations in patients of disease severity (NIHSSS, lesion volume,
without catecholamine treatment (n = 109) were not significantly
Table 6. Results of multivariate binary logistic regression analyses for independent predictors of bacterial infections during
d2–d7 excluding obvious infection markers.
Bacterial infection d2–d7 (excluding the infection markers fever, CRP and hsPCT)
Variables in the model: NIHSSS, volume, catecholamine treatment, noradrenalin, normetanephrin, lymphocytes ,13%, mechan. ventilation; method: forward
(likelihood ration), p(in) = 0.05, p(out) = 0.10.
doi:10.1371/journal.pone.0074839.t006
catecholamine treatment, mechanical ventilation) were all signif- increase after cerebrovascular events. (4) Catecholamine and
icantly correlated with development of bacterial infections. steroid levels were neither independent predictors of lymphocy-
Lymphocytopenia, IL-10 and IL-6 concentrations on day 1 as topenia nor of bacterial infection. (5) Disease severity (NIHSSS)
well as noradrenalin and normetanephrin levels were highly was the only significant independent predictor of lymphocytope-
correlated to the subsequent development of bacterial infections nia. (6) Lymphocytopenia on admission was an independent
(Table 3). predictor of subsequent bacterial infections.
Multivariate binary regression analysis detected NIHSSS and The activation of the SNS and the HPA axis and the
age as independent predictors of lymphocytopenia,13%, but only modulation of the peripheral immune system are both well-known
NIHSSS had a significant p value ,0.05 (Table 4). Mechanical features of stroke [9,14,20–22]. This is the largest study to date
ventilation .24 h and hsPCT values .0.1 ng/ml were found as investigating a wide panel of hormonal stress mediators. It also
independent predictors of bacterial infections (Table 5). We studied the previously uncharacterized categories of ICH and
performed an additional analysis for independent predictors of TIA. We detected only a relatively minor increase of plasma
bacterial infections excluding the obviously correlated infection catecholamine whereas other pathways such as the acute phase
markers fever, CRP and hsPCT from the model (Table 6) and reaction or cytokine levels were profoundly activated in the same
detected mechanical ventilation and lymphocytopenia on admis- patients. ACTH and cortisol were already increased on the first
sion as independent predictors. day after admission after large ICH and ischemia. A stroke-
induced immunosuppression syndrome after brain ischemia was
Diagnostic Performance of Predictors for previously characterized by the pathophysiological hallmarks
Lymphocytopenia and Bacterial Infections lymphocytopenia, cellular and humoral immune dysfunction and
susceptibility to bacterial infections [10,11,16]. The key features
We further performed receiver operating characteristic (ROC)
of this syndrome also presented with a similar pattern of immuno-
curve analysis for the performance of the detected independent
modulation in patients with extensive brain ischemia and
predictors as diagnostic criteria (Fig. 4). Analyzing the indepen-
hemorrhage. In contrast no significant immune changes were
dent predictors of lymphocytopenia on admission, NIHSSS had
observed in TIA patients.
an area under the curve (AUC) of 0.81, indicating a good
discriminative power (Fig. 4A). Interestingly, analyzing The current concept of post-stroke immunosuppression makes a
predictors of bacterial infections during the first week revealed a causal link between the two phenomena hormonal stress response and
similar ROC performance for NIHSSS on admission and hsPCT immune dysfunction after stroke. Accordingly, stress hor-mones,
results during the first week as well as for lymphocytopenia on particularly catecholamines, would be signaling from the injured
admission and mechanical ventilation during the first week (Fig. brain to immune cells [8,10,11,16]. By now, 11 clinical studies
4B). Using contingency table analysis for lymphocytopenia,13% [12,14,19,21,23–29] have investigated aspects of post-stroke
we found a test sensitivity of 75% and specificity of 67% and for immunosuppression in ischemic stroke of which only 5 studies were
NIHSSS.15 a sensitivity of 65% and specificity of 76% (both on prospectively designed. Only one trial detected a correlation between
day 1) for detection of subsequent bacterial infections during the stress mediators and lymphocyte number [28]. No clinical study has
first week. We therefore performed ROC analysis of combined distinctly identified catecholamines as independent predictors of
explanatory and independent variables lymphocyte dysfunction. Notably, neither the experimental landmark
[lymphocytes,13%6NIHSS] and [lymphocytes,13%6NIHSS.15] publication by Prass et al. [8] nor any other experimental study in the
revealing an excellent ROC performance (AUC 0.83) (Fig. 4C). field of post-stroke immunity measured catecholamine levels after
Combining both predictors resulted in a low test sensitivity (51%) stroke. Pharmaco-logical blockade of beta-adrenoreceptors partially
compared to the individual predictors, however, particularly the prevented the immune changes after severe experimental stroke in
test specificity of 85% and negative predictive value of 82% of Prass’ study8. However, sympathetic denervation did not affect
the combined predictor were exceptionally good. splenic cellularity in another report [22]. In view of these limited and
partially controversial findings the causal relationship between the
Discussion stress response, immune dysfunction and infectious complications
which has been advocated in numerous reviews of the subject appears
The major findings of our study are that (1) only large ischemic doubtful[7,9–11,16,30,31]. Indeed, our detailed and extensive study
and hemorrhagic strokes lead to increased plasma levels of some challenges such a causal relationship as we did not detect an
catecholamine metabolites and cortisol. (2) Extensive lesions are independent correlation of stress mediator plasma levels on one hand
associated with pronounced lymphocytopenia, increased cytokine and systemic immune modulation and infectious complica-tions on
secretion and a strong acute phase reaction. (3) Signs of bacterial the other hand even in our large study cohort.
infection including positive culture results or elevated hsPCT
Supporting Information
Figure S1 Dopamin plasma levels in patients with cerebrovascular
diseases and control patients. Plasma levels of dopamin were
measured in control patients and in patients after TIA, ICH or stroke
at the indicated time points. A significant reduction was observed
only after small infarcts compared to TIA patients and controls at d1
after stroke. Data is presented as median and interquartile range. *
indicates significant (p,0.05) difference of the indicated group
compared to the control group; horizontal bar indicates significant
Figure 4. ROC curve analysis of independent predictors for difference between respective groups.
lymphocytopenia and bacterial infections. (A) ROC curve performance
of the independent variables age and NIHSSS for relative
(TIF)
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