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Immune responses after acute ischemic stroke or myocardial infarction
International Journal of Cardiology 155 (2012) 372–377
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International Journal of Cardiology 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 / i j c a r d
Immune responses after acute ischemic stroke or myocardial infarction☆ Karl Georg Haeusler a,b,⁎, Wolf U.H. Schmidt a,b, Fabian Foehring a, Christian Meisel c, Christoph Guenther d, Peter Brunecker b, Claudia Kunze b, Thomas Helms a, Ulrich Dirnagl b, Hans-Dieter Volk c, Arno Villringer b,e,f a
Department of Neurology, Charité University Medicine Berlin, Germany Center for Stroke Research, Charité, Berlin, Germany Department of Medical Immunology, Charité University Medicine Berlin, Germany d Department of Cardiology, Charité University Medicine Berlin, Germany e Max-Planck Institute and University Hospital, Leipzig, Germany f Berlin School of Mind and Brain, Mind and Brain Institute, Humboldt-University, Berlin, Germany b c
a r t i c l e
i n f o
Article history: Received 18 August 2010 Accepted 23 October 2010 Available online 13 November 2010 Keywords: Acute ischemic stroke Acute myocardial infarction Human Immunodepression HLA-DR
a b s t r a c t Background: We recently demonstrated an immediate immunodepressive state after acute ischemic stroke in humans. Methods: In the present study, we prospectively analyzed immune responses in patients with middle cerebral artery stroke (n = 20), acute myocardial infarction (n = 20) and healthy controls (n = 20, also matched for age and gender). Results: Compared to controls, a rapid depression of monocytic HLA-DR expression and a defective lymphocytic IFN-γ production was obvious after ischemic stroke or myocardial infarction, while total counts of leukocytes and monocytes were significantly higher after myocardial infarction. A T cell-mediated lymphopenia was accentuated in patients with severe stroke, obviously predisposing these patients for nosocomial infections. Conclusions: Our data reveal an immediate and to some extent differential suppression of cell-mediated immune responses after ischemic stroke or myocardial infarction respectively. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Clinical evidence in humans [1–4] and experimental data in mice [5] demonstrated an immunodepressive state after acute ischemic stroke, as similarly reported after acute traumatic brain injury or spinal cord injury [6,7]. Secondary immunodeficiency affects the innate and adaptive peripheral immune response, mediated by an increased sympathetic activity as well as partly by the hypothalamo– pituitary–adrenal axis [5,8]. Despite an increased number of circulating monocytes [8], a rapid and prolonged functional deactivation of
Abbreviations: ACS, acute coronary syndrome; CRP, C-reactive protein; CNS, central nervous system; HLA-DR, human leukocyte antigen-DR; IFN-γ, interferon-γ; LPS, lipopolysaccharide; NK cells, natural killer cells; PAMI, patients with acute myocardial infarction; PAMI−, PAMI without nosocomial infection; SP, stroke patients; SPI−, SP without nosocomial infection; SPI+, SP with nosocomial infection; Th, T-helper cell; Th1/2, T-helper cell type 1/2; TNF-α, tumor necrosis factor α. ☆ This study was supported by the German Ministry of Education and Research (Competence Net Stroke, Berlin NeuroImaging Center, Center for Stroke Research) and the Deutsche Forschungsgemeinschaft (Klinische Forschergruppe). ⁎ Corresponding author. Department of Neurology, Charité – University Medicine Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany. Tel.: +49 30 84454244; fax: +49 30 84454264. E-mail address: [email protected] (K.G. Haeusler). 0167-5273/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2010.10.053
monocytes was obvious after acute stroke in humans, characterized by low levels of major histocompatibility class II molecule human leukocyte antigen (HLA)-DR and reduced lipopolysaccharide (LPS)induced TNF-α release ex vivo [1,2]. In addition, there was a T cellaccentuated lymphopenia within hours after stroke and an impaired mitogenic T cell response, indicated by a long-lasting suppression of lymphocytic IFN-γ release ex vivo [1,3,4,9]. As demonstrated recently, the magnitude of immunodepression correlates with clinical severity and stroke size [1,3,8], apparently predisposing these patients for nosocomial infection [1,2]. On the other hand, stroke-induced immunodepression may be beneficial in suppressing autoimmune responses to brain-specific antigens after brain injury [10]. If immunodepression is (at least partly) related to brain damage and not a general reaction of the immune system to a life threatening disease associated with stress, the immune response after myocardial infarction will be different. As similarly described in patients with acute ischemic stroke [11,12], the induction of an acute phase reaction and leukocytosis were reported in patients with acute coronary syndrome (ACS) [13,14]. Moreover, leukocytosis was correlated to the magnitude of myocardial damage and a potent predictor of death by cardiovascular disease [15–18]. The extent of monocytosis was independently and inversely related to myocardial reperfusion and systolic recovery [19]. In patients with ACS circulating monocytes showed higher mRNA expression levels of TNF-α, IL-6 and IL-10, but diminished LPS-
K.G. Haeusler et al. / International Journal of Cardiology 155 (2012) 372–377
induced TNF-α and IL-6 release ex vivo [20]. It is further known that lymphopenia is an independent predictor of mortality and atherosclerosis progression in cardiovascular diseases [16,21,22]. Accumulating evidence supports a fundamental role of a Th1/Th2 imbalance after ACS, favouring the pro-inflammatory Th1-response [23–27]. Using a highly standardized immune monitoring, we prospectively compared human immune responses after acute stroke versus myocardial infarction. To ensure comparability of the results, we used the same time-frame and study protocol. Exclusion of patients with known immunodepression or signs of acute infection on admission further improved the significance of the results. Hereby, we compared these data to age–gender-matched healthy individuals. 2. Material and methods 2.1. Study populations
2.2. Study criteria Informed consent was obtained from each patient. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki and was approved a priori by the Ethics Committee of the Charité. Exclusion criteria were TIA or (unstable) angina, coincidence of stroke and myocardial infarction, severe aphasia, known immunodepression or inflammatory disease, and any sign of acute infection on admission. Ischemic stroke was identified and located by cranial MRI on admission. SP and PAMI underwent blood sampling (C-reactive protein (CRP), procalcitonin, cortisol, differential blood count, and cytokine release) on admission, as well as on days 2 and 6 of hospitalisation. Nosocomial infection was defined as developing infection after at least 3 days of
Table 1 Baseline characteristics of 20 healthy controls (HC), as well as 20 stroke patients (SP) and 20 patients with acute myocardial infarction (PAMI).
#
p b 0.05 vs. SP.
hospitalisation [30]. The same diagnostic criteria for infection were used as in our previous report [1]. 2.3. MRI analysis Serial MRI examinations (on days 0, 2, and 6 after admission) were performed on a 1.5-T system (Siemens Vision, Germany) as described earlier [1]. Location and extent of ischemic stroke were evaluated by two blinded observers using Analyze 5.0 (Mayo Clinic, USA). 2.4. Laboratory measurements Serum concentration of cytokines (TNF-α, IL-6, and IL-10), cortisol and procalcitonin (normal b 500 pg/ml), expression of human leukocyte antigen-DR (HLA-DR) on monocytes as well as lipopolysaccharide (LPS)-induced monocytic TNF-α secretion ex vivo and Concanavalin A (ConA)-induced IFN-γ, IL-4, and IL-5 release were determined as earlier described [1]. 2.5. Statistical analysis
Between April 2002 and September 2003 20 patients with acute myocardial infarction (PAMI), 52 patients with ischemic stroke (SP) and 30 healthy controls were enrolled into the prospective ISAS-study [1]. “Acute” was defined as enrolment within 24 h after onset of symptoms. Matching for age and gender, 20 healthy controls as well as 20 patients with stroke affecting the left (n = 12) or right (n = 8) middle cerebral artery (MCA) were chosen for this report. Five of these SP (25%) received thrombolysis and one stroke patient needed mechanical ventilation. The median National Institute of Health Stroke Scale (NIHSS) score [28] was 6 (range 2–17) points on admission, 4 (1– 24) on day 2, and 3 (0–13) points on day 6 after stroke. According to the TOAST criteria [29] the aetiology of stroke was distributed as follows: 40% cardioembolism, 40% largeartery atherosclerosis, 10% artery dissection, and 10% unknown. According to ECG on admission, 75% of PAMI had ST-segment elevation myocardial infarction (STEMI) and 25% non-ST-segment elevation myocardial infarction (NSTEMI). Acute myocardial infarction was due to right (35%) or left (65%) coronary artery occlusion. Coronary heart disease was proven by angiography in three coronary arteries in 20%, in two coronary arteries in 55% or in one coronary artery in 25%, respectively. On admission, troponin I and CK levels were elevated in all PAMI. Left ventricular ejection fraction in PAMI was 51.3 ± 10.5% (range 34–67%). Twenty age- and gender-matched volunteers without prior cerebrovascular disease were chosen from the ISAS-study [1]. Epidemiological data are summarized in Table 1. Within hospital stay, 6 out of 20 SP and 2 out of 20 PAMI developed nosocomial infection. Two SP suffered from pneumonia, 2 from urinary tract infection (UTI) and 2 from both. In PAMI, there was one patient with pneumonia and one with UTI. Seven patients in both groups (35%) had a urinary bladder catheter. All patients with UTI had a urinary bladder catheter. SP with nosocomial infection (SPI) had significantly higher NIHSS scores on admission and greater MRI lesion volumes compared to SP without infection, as demonstrated before [1,3].
Age (years) [median ± range] Gender (female) Arterial hypertension Diabetes mellitus Dyslipoproteinaemia Prior myocardial infarction Prior stroke Statin therapy Beta-blocker therapy Acetylsalicylic acid Clopidogrel Oral anticoagulation
373
HC [n = 20]
SP [n = 20]
PAMI [n = 20]
59.5 ± 24 25% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
64 ± 40 25% 80% 30% 25% 35% 10% 15% 25% 25% 0% 0%
63 ± 34 25% 65% 10% 65%# 10% 5% 10% 30% 20% 5% 0%
Matched-pair analysis was done with respect to age and gender. The Mann– Whitney U-test was applied to detect differences of metrical, not normally distributed data. A p-value of b 0.05 was considered significant. In this preliminary study we aimed at hypothesis generation and to prepare for a larger trial. We therefore did not correct for multiple comparisons, which limits the implications of our results. Statistical analyses were performed using the SPSS® 14.0 software.
3. Results 3.1. Early laboratory findings after acute ischemic stroke or myocardial infarction Comparing data from healthy controls (HC) to stroke patients (SP) and patients with acute myocardial infarction (PAMI), CRP serum levels (normal b0.5 mg/dl) were significantly elevated in SP (p b 0.01) and PAMI (p = 0.02) on hospital admission, while body temperature, serum procalcitonin and cortisol concentration were similar (Table 2). Total counts of leukocytes and granulocytes were significantly (p b 0.001) raised in SP and PAMI (Table 2; Fig. 1). In addition, leukocytosis was more pronounced in PAMI compared to SP (p = 0.03). Compared to HC and PAMI, there was a trend towards suppression of lymphocyte counts in SP (p = 0.11), primary CD3+ T cell accentuated (p = 0.17 for HC; p = 0.31 for PAMI). Total numbers of CD8+ T cells, CD16+ natural killer (NK) cells and CD19+ B cells did not differ significantly between HC, SP and PAMI. Monocyte counts were significantly higher in PAMI compared to SP (p = 0.02) and HC (p b 0.01). Table 2 Immune markers of healthy controls (HC), stroke patients (SP) and patients with acute myocardial infarction (PAMI) on hospital admission (b24 h after onset of clinical symptoms, 8 h in average). Values are expressed as median ± range. HC [n = 20] Body temperature (°C) CRP (mg/dl) Procalcitonin (pg/ml) Cortisol (nmol/l) IL-6 in vivo (pg/ml) [n N2 pg/ml] Leukocytes (1/nl) Granulocytes (1/nl) Lymphocytes (1/nl) CD3+ T cells (1/nl) CD4+ T cells (1/nl) CD8+ T cells (1/nl) CD16+ NK cells (1/nl) CD19+ B cells (1/nl) Monocytes (1/nl) HLA-DR (antigen/monocyte) LPS-ind. TNF-α (pg/monocyte) ⁎ p b 0.05 vs. HC. # p b 0.05 vs. SP.
36.3 ± 0.7 0.07 ± 0.69 140 ± 196 313 ± 383 b 2.0 [0]
SP [n = 20] 36.5 ± 1.2 0.26 ± 1.6⁎ 201 ± 238 393 ± 711 6.9 ± 13.0 [8]
5.9 ± 6.3 3.8 ± 4.4 1.7 ± 2.1 1.2 ± 1.6 0.79 ± 1.09 0.33 ± 0.54 0.23 ± 0.38 0.18 ± 0.39 0.52 ± 0.45 29,445±31,997
8.9 ± 5.9⁎ 7.1 ± 8.4⁎ 1.5 ± 3.0 1.0 ± 2.7 0.75 ± 1.5 0.32 ± 1.3 0.17 ± 0.39 0.22 ± 0.54 0.46 ± 1.1 19,609 ± 27,069⁎
2.1 ± 1.2
1.7 ± 2.0
PAMI [n = 20] 36.4 ± 2.0 0.37 ± 1.1⁎ 211 ± 482 416 ± 843 8.1 ± 215 [15] 10.5 ± 10.8⁎,# 7.3 ± 9.2⁎ 1.8 ± 3.2 1.1 ± 3.0 0.71 ± 1.3 0.38 ± 1.4 0.21 ± 0.57 0.20 ± 2.11 0.83 ± 2.2⁎,# 14,547 ± 16,534⁎,# 1.3 ± 7.5
K.G. Haeusler et al. / International Journal of Cardiology 155 (2012) 372–377
Leukocytes [1/nl]
A
18
B *
*
15
*
*
Granulocytes [1/nl]
374
12
6
* *
*
3
* Lymphocytes [1/nl]
Monocytes [1/nl]
D
1.8
1.2
0.6
2
1
0
*
1.8
* CD4+ T-cells [1/nl]
CD3+ T-cells [1/nl]
F
3
2
1
1.2
0.6
0
H *
*
* * *
*
*
40
20
4.5
* TNF-α release [pg/cell]
HLA-DR [103 antigens/cell]
60
*
0
0
G
*
5
0
E
*
10
0
C
*
3.0
1.5
0
0
controls
stroke patients
myocardial infarction
stroke patients without infection
stroke with infection
on admission
6 days after onset
2 days after onset
Fig. 1. Changes of peripheral blood leukocyte composition and of monocytic function after stroke and myocardial infarction — relation to infectious complications after stroke. Box plots (median values, 25% and 75% percentiles as boxes, minimum and maximum within the inner fence) of counts of leukocytes (A), granulocytes (B), monocytes (C), lymphocytes (D), CD3+ T cells (E), CD4+ T-helper cells (F), HLA-DR antigen expression (G), and ex vivo LPS-induced release of TNF-α (H) in healthy controls (white bar, n = 20), stroke patients in general (gray bars, n = 20), patients with myocardial infarction, (striped bar, n = 20), stroke patients without infection (light gray bars, n = 14), and stroke patients with infection (dark gray bars, n = 6) by time (on admission, day 2, and day 6 after onset of symptoms). * p b 0.05.
K.G. Haeusler et al. / International Journal of Cardiology 155 (2012) 372–377
Quantitative monocytic HLA-DR expression was significantly lower in SP and PAMI compared to HC (p ≤ 0.001), and significantly lower in PAMI compared to SP (p = 0.02) (Fig. 1). The monocytic TNF-α release upon stimulation with LPS ex vivo showed no significant difference between HC, SP and PAMI. Serum IL-6 was detectable on admission in 75% of all PAMI and in 40% of all SP (Table 2). Levels of TNF-α or IL-10 were hardly detectable (data not shown).
Table 4 Cytokine release of stroke patients (SP) and patients with acute myocardial infarction (PAMI) after Concanavalin A-stimulation ex vivo. Values are expressed as median± range. ConA-induced
[Day]
SP [n = 20]
PAMI [n = 20]
p
IFN-γ ex vivo (pg/ml)
[0] [2] [6] [0] [2] [6] [0] [2] [6]
8288 ± 37,551 6454 ± 25,587 6395 ± 41,145 18.0 ± 140 17.4 ± 111 16.5 ± 111 14.5 ± 543 18.0 ± 406 16.0 ± 354
4463 ± 20,595 11,731 ± 30,554 12,080 ± 32,057 17.0 ± 180 16.5 ± 144 16.1 ± 155 15.0 ± 246 20.5 ± 162 19.0 ± 144
0.55 0.17 0.30 0.28 0.74 0.95 0.88 0.65 0.92
IL-4 ex vivo (pg/ml)
3.2. Laboratory findings within days after acute stroke or myocardial infarction Within hospital stay, 6 out of 20 SP and 2 out of 20 PAMI developed nosocomial infection, i.e. pneumonia or urinary tract infection. On days 2 and 6 of hospitalisation serum CRP and IL-6 levels, body temperature and serum cortisol concentration were higher in SP with nosocomial infection (SPI+) compared to SP without nosocomial infection (SPI−) (Table 3). At these time points total counts of granulocytes, monocytes and leukocytes [day 2 only] as well as IL-6 release [day 6 only] were significantly (p b 0.02) higher in SPI+ compared to SPI−, while CD3+ T cell counts, monocytic HLA-DR expression and LPS-induced release of TNF-α [day 2 only] were significantly (p ≤ 0.015) lower (Table 3; Fig. 1). In SP and PAMI without nosocomial infection (PAMI−), leukocyte, granulocyte and monocyte counts decreased over time, while HLA-DR expression increased (Table 3). Nevertheless, HLA-DR expression was significantly (p b 0.001) lower in SPI− and PAMI− compared to HC at all times measured (Fig. 1). Moreover, counts of leukocytes, granulocytes and monocytes remained significantly higher in PAMI compared to HC within the study period. Serum IL-6 levels in PAMI− decreased over time. Detectable levels of serum IL-6 were found in the minority of SPI−. Cortisol levels were almost unchanged over time in PAMI− and SPI− (Table 3). As displayed in Table 4, the stimulation of whole blood-cultures with ConA ex vivo revealed a low IFN-γ release – a common marker of
375
IL-5 ex vivo (pg/ml)
Th1 immune response – in PAMI and SP on admission, while levels of IL-4 and IL-5 – both markers of Th2 function – remained almost unchanged over time in PAMI and SP. The ConA-induced cytokine release showed no significant difference between SPI+ and SPI− (data not shown). 4. Discussion The analysis of immune responses in vascular diseases has received increasing attention over the last years. Using the same time frame and study protocol to ensure better comparability, we prospectively evaluated immune responses of patients with acute stroke or myocardial infarction, respectively. Our data indicate an acute immunosuppression for both vascular disorders. However, the observed immune responses were different to some extent. Compared to healthy controls, there was an acute phase response within hours after onset of ischemic stroke or myocardial infarction [11–14], while procalcitonin serum levels were within normal range
Table 3 Immune markers of stroke patients with nosocomial infection (SPI+), stroke patients without nosocomial infection (SPI−) and patients with acute myocardial infarction without nosocomial infection (PAMI−) on days 2 and 6 of hospitalisation. Values are expressed as median ± range.
Body temperature (° C) CRP (mg/dl) [n] Cortisol (nmol/l) IL-6 (pg/ml) [n N 2 pg/ml] Leukocytes (1/nl) Granulocytes (1/nl) Lymphocytes (1/nl) CD3+ T cells (1/nl) CD4+ T cells (1/nl) CD8+ T cells (1/nl) CD16+ NK cells (1/nl) CD19+ B cells (1/nl) Monocytes (1/nl) HLA-DR expression (antigen/monocyte) LPS-induced TNF-α (pg/monocyte) # p b 0.05 vs. SPI−. ⁎ p b 0.05 for SPI− or PAMI− vs. HC.
[Day]
SPI+ [n = 6]
[2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6]
37.5 ± 2.0 38.0 ± 2.2# 5.6 ± 12.2# 4.7 ± 11.7# 486 ± 992 411 ± 369 16.8 ± 161 [6] 42.2 ± 89 [5]# 10.3 ± 8.3# 10.8 ± 7.0 8.2 ± 9.7# 8.7 ± 6.5# 1.3 ± 0.82 0.96 ± 1.0 0.78 ± 0.90# 0.65 ± 0.44# 0.44 ± 0.77 0.48 ± 0.50 0.32 ± 0.46 0.21 ± 0.34 0.14 ± 0.33 0.14 ± 0.21 0.18 ± 0.35 0.18 ± 0.39 0.90 ± 0.71# 0.99 ± 1.1# 6084 ± 5836# 7541 ± 10,825# 0.80 ± 1.4# 0.82 ± 2.9
SPI− [n = 14] 36.4 ± 1.2 36.4 ± 1.7 0.5 ± 4.9⁎ 0.5 ± 1.8⁎ 354 ± 257 293 ± 357 6.4 ± 7.7 [5] 4.0 ± 3.0 [4] 7.3 ± 4.5 6.9 ± 7.4 5.3 ± 3.8⁎ 4.2 ± 5.3 1.65 ± 1.5 1.74 ± 2.0 1.2 ± 1.6 1.4 ± 1.9 0.71 ± 0.91 0.80 ± 1.09 0.35 ± 0.90 0.39 ± 0.96 0.20 ± 0.31 0.22 ± 0.32 0.20 ± 0.36 0.21 ± 0.48 0.51 ± 0.36 0.46 ± 0.52 19,531 ± 15,845⁎ 21,783 ± 21,614⁎ 2.4 ± 2.0 2.9 ± 3.5
PAMI− [n = 18] 36.4 ± 1.1 36.0 ± 1.6 1.8 ± 3.1 [14] – 334 ± 407 341 ± 680 6.2 ± 39 [15] 4.0 ± 5.6 [6] 8.6 ± 9.0⁎ 8.0 ± 7.1⁎ 6.0 ± 7.1⁎ 5.5 ± 5.8⁎ 2.0 ± 2.6 1.9 ± 1.7 1.4 ± 2.4 1.4 ± 1.4 0.81 ± 1.2 0.85 ± 0.96 0.47 ± 1.12 0.50 ± 0.63 0.21 ± 0.59 0.20 ± 0.41 0.25 ± 1.29 0.21 ± 0.47 0.70 ± 1.0⁎
0.68 ± 0.80⁎ 14,754 ± 22,208⁎ 17,750 ± 22,584⁎ 1.5 ± 7.6 2.0 ± 2.7
376
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[1,31] and cortisol levels – as a marker of an ongoing stress reaction – were not significantly elevated. Serum IL-6 was detectable in the majority of patients with acute myocardial infarction (PAMI) on admission, but in only 40% of all stroke patients (SP). Levels of IL-6 decreased in PAMI over time, probably indicating a stress-induced augmentation of the immune function on admission [32]. Highlighting the role of leukocytes [15–18] and monocytes [19] in patients with acute coronary syndrome (ACS), total counts of leukocytes and monocytes were significantly higher in PAMI compared to SP on admission. Compared to HC, monocytic HLA-DR expression – essential for antigen-specific T cell defence – was significantly decreased in PAMI and SP, as similarly reported in patients after neurosurgery [33], cardiopulmonary bypass surgery [34], stroke [1,2,4] or sepsis [35]. The release of TNF-α after LPS stimulation ex vivo was not significantly decreased in SP and PAMI, indicating a partial functional loss of monocytic immune competence on admission. As demonstrated before [1], a mainly CD3+ T cell-accentuated lymphopenia was evident in SP with nosocomial infection (defined by clinical onset within 3 and 6 days of hospitalisation). We observed no lymphopenia in PAMI, which is in contrast to previous reports in ACS patients and probably related to the extent of myocardial damage [16,21,22]. As indicated by the almost unchanged release of IL-4 and IL-5 after mitogen stimulation ex vivo, the Th2 function was unaltered over time, as previously described in PAMI [24] and SP [1]. A rapid deactivation of T cells, indicated by reduced mitogen-induced release of IFN-γ on admission, was observed in SP [1,4,9] and PAMI, while Pasqui et al. reported an up-regulation of LPS-induced IFN-γ release ex vivo in patients with ACS [27]. With regard to the detrimental effects of IFN-γ and T-lymphocytes on the ischemic brain [36], the stroke-induced immunodepression might be self-protective by limiting the duration of auto-inflammatory processes [10]. Using clinical diagnostic criteria for infection, we cannot rule out an impact of nosocomial infections on the ongoing immune response. Therefore the laboratory data obtained on days 2 and 6 of hospitalisation have to be interpreted carefully. As demonstrated before [1,3], immunodepression was accentuated in SP with severe stroke, obviously predisposing these patients for nosocomial infections. Compared to SP without nosocomial infection the SP with nosocomial infections had significantly higher monocyte counts, while CD3+ T cell counts, monocytic HLA-DR expression and LPS-induced release of TNF-α were significantly lower. Compared to HC, HLA-DR expression was significantly reduced in patients after stroke and myocardial infarction within the study period of 6 days, indicating a persisting immunosuppressive state, irrespective of nosocomial infection. There are several strengths and limitations of our study. Firstly, in this pilot study, the relatively low number of patients did not allow correction for multiple testing, limiting the significance of the results. Secondly, ACS and ischemic stroke are heterogeneous disease entities of different aetiological subtypes representing variations in risk factors and recent medication, probably affecting the immune response and limiting the generalisation of the results. Thirdly, the interesting question of the relevance of the sympathetic and parasympathetic nervous system for the observed immune responses could not be clarified yet, since no systematic assessment of the state of the vegetative nervous system was done. Additional prospective studies are needed to clarify this point and to further validate the results. 5. Conclusion Compared to healthy controls, a rapid reduction of monocytic HLADR expression and a defective lymphocytic IFN-γ production were obvious after stroke as well as myocardial infarction. A depression of lymphocyte counts was observed after severe ischemic stroke but not after myocardial infarction. Regarding the observed differences after stroke and myocardial infarction, an additional impact of the damaged
nervous system on the immune response seems possible. More efforts are necessary to understand the regulating mechanisms of immune response after acute stroke and myocardial infarction.
Acknowledgements We thank C. Liebenthal, C. Muselmann and K. S. Siebel for assistance. We would also like to thank Dr. Brigitte Wegner (Institute for Medical Biometry and Epidemiology, Charité University Medicine Berlin) for statistical advice. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [37].
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Contents lists available at ScienceDirect
International Journal of Cardiology 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 / i j c a r d
Immune responses after acute ischemic stroke or myocardial infarction☆ Karl Georg Haeusler a,b,⁎, Wolf U.H. Schmidt a,b, Fabian Foehring a, Christian Meisel c, Christoph Guenther d, Peter Brunecker b, Claudia Kunze b, Thomas Helms a, Ulrich Dirnagl b, Hans-Dieter Volk c, Arno Villringer b,e,f a
Department of Neurology, Charité University Medicine Berlin, Germany Center for Stroke Research, Charité, Berlin, Germany Department of Medical Immunology, Charité University Medicine Berlin, Germany d Department of Cardiology, Charité University Medicine Berlin, Germany e Max-Planck Institute and University Hospital, Leipzig, Germany f Berlin School of Mind and Brain, Mind and Brain Institute, Humboldt-University, Berlin, Germany b c
a r t i c l e
i n f o
Article history: Received 18 August 2010 Accepted 23 October 2010 Available online 13 November 2010 Keywords: Acute ischemic stroke Acute myocardial infarction Human Immunodepression HLA-DR
a b s t r a c t Background: We recently demonstrated an immediate immunodepressive state after acute ischemic stroke in humans. Methods: In the present study, we prospectively analyzed immune responses in patients with middle cerebral artery stroke (n = 20), acute myocardial infarction (n = 20) and healthy controls (n = 20, also matched for age and gender). Results: Compared to controls, a rapid depression of monocytic HLA-DR expression and a defective lymphocytic IFN-γ production was obvious after ischemic stroke or myocardial infarction, while total counts of leukocytes and monocytes were significantly higher after myocardial infarction. A T cell-mediated lymphopenia was accentuated in patients with severe stroke, obviously predisposing these patients for nosocomial infections. Conclusions: Our data reveal an immediate and to some extent differential suppression of cell-mediated immune responses after ischemic stroke or myocardial infarction respectively. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Clinical evidence in humans [1–4] and experimental data in mice [5] demonstrated an immunodepressive state after acute ischemic stroke, as similarly reported after acute traumatic brain injury or spinal cord injury [6,7]. Secondary immunodeficiency affects the innate and adaptive peripheral immune response, mediated by an increased sympathetic activity as well as partly by the hypothalamo– pituitary–adrenal axis [5,8]. Despite an increased number of circulating monocytes [8], a rapid and prolonged functional deactivation of
Abbreviations: ACS, acute coronary syndrome; CRP, C-reactive protein; CNS, central nervous system; HLA-DR, human leukocyte antigen-DR; IFN-γ, interferon-γ; LPS, lipopolysaccharide; NK cells, natural killer cells; PAMI, patients with acute myocardial infarction; PAMI−, PAMI without nosocomial infection; SP, stroke patients; SPI−, SP without nosocomial infection; SPI+, SP with nosocomial infection; Th, T-helper cell; Th1/2, T-helper cell type 1/2; TNF-α, tumor necrosis factor α. ☆ This study was supported by the German Ministry of Education and Research (Competence Net Stroke, Berlin NeuroImaging Center, Center for Stroke Research) and the Deutsche Forschungsgemeinschaft (Klinische Forschergruppe). ⁎ Corresponding author. Department of Neurology, Charité – University Medicine Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany. Tel.: +49 30 84454244; fax: +49 30 84454264. E-mail address: [email protected] (K.G. Haeusler). 0167-5273/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2010.10.053
monocytes was obvious after acute stroke in humans, characterized by low levels of major histocompatibility class II molecule human leukocyte antigen (HLA)-DR and reduced lipopolysaccharide (LPS)induced TNF-α release ex vivo [1,2]. In addition, there was a T cellaccentuated lymphopenia within hours after stroke and an impaired mitogenic T cell response, indicated by a long-lasting suppression of lymphocytic IFN-γ release ex vivo [1,3,4,9]. As demonstrated recently, the magnitude of immunodepression correlates with clinical severity and stroke size [1,3,8], apparently predisposing these patients for nosocomial infection [1,2]. On the other hand, stroke-induced immunodepression may be beneficial in suppressing autoimmune responses to brain-specific antigens after brain injury [10]. If immunodepression is (at least partly) related to brain damage and not a general reaction of the immune system to a life threatening disease associated with stress, the immune response after myocardial infarction will be different. As similarly described in patients with acute ischemic stroke [11,12], the induction of an acute phase reaction and leukocytosis were reported in patients with acute coronary syndrome (ACS) [13,14]. Moreover, leukocytosis was correlated to the magnitude of myocardial damage and a potent predictor of death by cardiovascular disease [15–18]. The extent of monocytosis was independently and inversely related to myocardial reperfusion and systolic recovery [19]. In patients with ACS circulating monocytes showed higher mRNA expression levels of TNF-α, IL-6 and IL-10, but diminished LPS-
K.G. Haeusler et al. / International Journal of Cardiology 155 (2012) 372–377
induced TNF-α and IL-6 release ex vivo [20]. It is further known that lymphopenia is an independent predictor of mortality and atherosclerosis progression in cardiovascular diseases [16,21,22]. Accumulating evidence supports a fundamental role of a Th1/Th2 imbalance after ACS, favouring the pro-inflammatory Th1-response [23–27]. Using a highly standardized immune monitoring, we prospectively compared human immune responses after acute stroke versus myocardial infarction. To ensure comparability of the results, we used the same time-frame and study protocol. Exclusion of patients with known immunodepression or signs of acute infection on admission further improved the significance of the results. Hereby, we compared these data to age–gender-matched healthy individuals. 2. Material and methods 2.1. Study populations
2.2. Study criteria Informed consent was obtained from each patient. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki and was approved a priori by the Ethics Committee of the Charité. Exclusion criteria were TIA or (unstable) angina, coincidence of stroke and myocardial infarction, severe aphasia, known immunodepression or inflammatory disease, and any sign of acute infection on admission. Ischemic stroke was identified and located by cranial MRI on admission. SP and PAMI underwent blood sampling (C-reactive protein (CRP), procalcitonin, cortisol, differential blood count, and cytokine release) on admission, as well as on days 2 and 6 of hospitalisation. Nosocomial infection was defined as developing infection after at least 3 days of
Table 1 Baseline characteristics of 20 healthy controls (HC), as well as 20 stroke patients (SP) and 20 patients with acute myocardial infarction (PAMI).
#
p b 0.05 vs. SP.
hospitalisation [30]. The same diagnostic criteria for infection were used as in our previous report [1]. 2.3. MRI analysis Serial MRI examinations (on days 0, 2, and 6 after admission) were performed on a 1.5-T system (Siemens Vision, Germany) as described earlier [1]. Location and extent of ischemic stroke were evaluated by two blinded observers using Analyze 5.0 (Mayo Clinic, USA). 2.4. Laboratory measurements Serum concentration of cytokines (TNF-α, IL-6, and IL-10), cortisol and procalcitonin (normal b 500 pg/ml), expression of human leukocyte antigen-DR (HLA-DR) on monocytes as well as lipopolysaccharide (LPS)-induced monocytic TNF-α secretion ex vivo and Concanavalin A (ConA)-induced IFN-γ, IL-4, and IL-5 release were determined as earlier described [1]. 2.5. Statistical analysis
Between April 2002 and September 2003 20 patients with acute myocardial infarction (PAMI), 52 patients with ischemic stroke (SP) and 30 healthy controls were enrolled into the prospective ISAS-study [1]. “Acute” was defined as enrolment within 24 h after onset of symptoms. Matching for age and gender, 20 healthy controls as well as 20 patients with stroke affecting the left (n = 12) or right (n = 8) middle cerebral artery (MCA) were chosen for this report. Five of these SP (25%) received thrombolysis and one stroke patient needed mechanical ventilation. The median National Institute of Health Stroke Scale (NIHSS) score [28] was 6 (range 2–17) points on admission, 4 (1– 24) on day 2, and 3 (0–13) points on day 6 after stroke. According to the TOAST criteria [29] the aetiology of stroke was distributed as follows: 40% cardioembolism, 40% largeartery atherosclerosis, 10% artery dissection, and 10% unknown. According to ECG on admission, 75% of PAMI had ST-segment elevation myocardial infarction (STEMI) and 25% non-ST-segment elevation myocardial infarction (NSTEMI). Acute myocardial infarction was due to right (35%) or left (65%) coronary artery occlusion. Coronary heart disease was proven by angiography in three coronary arteries in 20%, in two coronary arteries in 55% or in one coronary artery in 25%, respectively. On admission, troponin I and CK levels were elevated in all PAMI. Left ventricular ejection fraction in PAMI was 51.3 ± 10.5% (range 34–67%). Twenty age- and gender-matched volunteers without prior cerebrovascular disease were chosen from the ISAS-study [1]. Epidemiological data are summarized in Table 1. Within hospital stay, 6 out of 20 SP and 2 out of 20 PAMI developed nosocomial infection. Two SP suffered from pneumonia, 2 from urinary tract infection (UTI) and 2 from both. In PAMI, there was one patient with pneumonia and one with UTI. Seven patients in both groups (35%) had a urinary bladder catheter. All patients with UTI had a urinary bladder catheter. SP with nosocomial infection (SPI) had significantly higher NIHSS scores on admission and greater MRI lesion volumes compared to SP without infection, as demonstrated before [1,3].
Age (years) [median ± range] Gender (female) Arterial hypertension Diabetes mellitus Dyslipoproteinaemia Prior myocardial infarction Prior stroke Statin therapy Beta-blocker therapy Acetylsalicylic acid Clopidogrel Oral anticoagulation
373
HC [n = 20]
SP [n = 20]
PAMI [n = 20]
59.5 ± 24 25% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
64 ± 40 25% 80% 30% 25% 35% 10% 15% 25% 25% 0% 0%
63 ± 34 25% 65% 10% 65%# 10% 5% 10% 30% 20% 5% 0%
Matched-pair analysis was done with respect to age and gender. The Mann– Whitney U-test was applied to detect differences of metrical, not normally distributed data. A p-value of b 0.05 was considered significant. In this preliminary study we aimed at hypothesis generation and to prepare for a larger trial. We therefore did not correct for multiple comparisons, which limits the implications of our results. Statistical analyses were performed using the SPSS® 14.0 software.
3. Results 3.1. Early laboratory findings after acute ischemic stroke or myocardial infarction Comparing data from healthy controls (HC) to stroke patients (SP) and patients with acute myocardial infarction (PAMI), CRP serum levels (normal b0.5 mg/dl) were significantly elevated in SP (p b 0.01) and PAMI (p = 0.02) on hospital admission, while body temperature, serum procalcitonin and cortisol concentration were similar (Table 2). Total counts of leukocytes and granulocytes were significantly (p b 0.001) raised in SP and PAMI (Table 2; Fig. 1). In addition, leukocytosis was more pronounced in PAMI compared to SP (p = 0.03). Compared to HC and PAMI, there was a trend towards suppression of lymphocyte counts in SP (p = 0.11), primary CD3+ T cell accentuated (p = 0.17 for HC; p = 0.31 for PAMI). Total numbers of CD8+ T cells, CD16+ natural killer (NK) cells and CD19+ B cells did not differ significantly between HC, SP and PAMI. Monocyte counts were significantly higher in PAMI compared to SP (p = 0.02) and HC (p b 0.01). Table 2 Immune markers of healthy controls (HC), stroke patients (SP) and patients with acute myocardial infarction (PAMI) on hospital admission (b24 h after onset of clinical symptoms, 8 h in average). Values are expressed as median ± range. HC [n = 20] Body temperature (°C) CRP (mg/dl) Procalcitonin (pg/ml) Cortisol (nmol/l) IL-6 in vivo (pg/ml) [n N2 pg/ml] Leukocytes (1/nl) Granulocytes (1/nl) Lymphocytes (1/nl) CD3+ T cells (1/nl) CD4+ T cells (1/nl) CD8+ T cells (1/nl) CD16+ NK cells (1/nl) CD19+ B cells (1/nl) Monocytes (1/nl) HLA-DR (antigen/monocyte) LPS-ind. TNF-α (pg/monocyte) ⁎ p b 0.05 vs. HC. # p b 0.05 vs. SP.
36.3 ± 0.7 0.07 ± 0.69 140 ± 196 313 ± 383 b 2.0 [0]
SP [n = 20] 36.5 ± 1.2 0.26 ± 1.6⁎ 201 ± 238 393 ± 711 6.9 ± 13.0 [8]
5.9 ± 6.3 3.8 ± 4.4 1.7 ± 2.1 1.2 ± 1.6 0.79 ± 1.09 0.33 ± 0.54 0.23 ± 0.38 0.18 ± 0.39 0.52 ± 0.45 29,445±31,997
8.9 ± 5.9⁎ 7.1 ± 8.4⁎ 1.5 ± 3.0 1.0 ± 2.7 0.75 ± 1.5 0.32 ± 1.3 0.17 ± 0.39 0.22 ± 0.54 0.46 ± 1.1 19,609 ± 27,069⁎
2.1 ± 1.2
1.7 ± 2.0
PAMI [n = 20] 36.4 ± 2.0 0.37 ± 1.1⁎ 211 ± 482 416 ± 843 8.1 ± 215 [15] 10.5 ± 10.8⁎,# 7.3 ± 9.2⁎ 1.8 ± 3.2 1.1 ± 3.0 0.71 ± 1.3 0.38 ± 1.4 0.21 ± 0.57 0.20 ± 2.11 0.83 ± 2.2⁎,# 14,547 ± 16,534⁎,# 1.3 ± 7.5
K.G. Haeusler et al. / International Journal of Cardiology 155 (2012) 372–377
Leukocytes [1/nl]
A
18
B *
*
15
*
*
Granulocytes [1/nl]
374
12
6
* *
*
3
* Lymphocytes [1/nl]
Monocytes [1/nl]
D
1.8
1.2
0.6
2
1
0
*
1.8
* CD4+ T-cells [1/nl]
CD3+ T-cells [1/nl]
F
3
2
1
1.2
0.6
0
H *
*
* * *
*
*
40
20
4.5
* TNF-α release [pg/cell]
HLA-DR [103 antigens/cell]
60
*
0
0
G
*
5
0
E
*
10
0
C
*
3.0
1.5
0
0
controls
stroke patients
myocardial infarction
stroke patients without infection
stroke with infection
on admission
6 days after onset
2 days after onset
Fig. 1. Changes of peripheral blood leukocyte composition and of monocytic function after stroke and myocardial infarction — relation to infectious complications after stroke. Box plots (median values, 25% and 75% percentiles as boxes, minimum and maximum within the inner fence) of counts of leukocytes (A), granulocytes (B), monocytes (C), lymphocytes (D), CD3+ T cells (E), CD4+ T-helper cells (F), HLA-DR antigen expression (G), and ex vivo LPS-induced release of TNF-α (H) in healthy controls (white bar, n = 20), stroke patients in general (gray bars, n = 20), patients with myocardial infarction, (striped bar, n = 20), stroke patients without infection (light gray bars, n = 14), and stroke patients with infection (dark gray bars, n = 6) by time (on admission, day 2, and day 6 after onset of symptoms). * p b 0.05.
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Quantitative monocytic HLA-DR expression was significantly lower in SP and PAMI compared to HC (p ≤ 0.001), and significantly lower in PAMI compared to SP (p = 0.02) (Fig. 1). The monocytic TNF-α release upon stimulation with LPS ex vivo showed no significant difference between HC, SP and PAMI. Serum IL-6 was detectable on admission in 75% of all PAMI and in 40% of all SP (Table 2). Levels of TNF-α or IL-10 were hardly detectable (data not shown).
Table 4 Cytokine release of stroke patients (SP) and patients with acute myocardial infarction (PAMI) after Concanavalin A-stimulation ex vivo. Values are expressed as median± range. ConA-induced
[Day]
SP [n = 20]
PAMI [n = 20]
p
IFN-γ ex vivo (pg/ml)
[0] [2] [6] [0] [2] [6] [0] [2] [6]
8288 ± 37,551 6454 ± 25,587 6395 ± 41,145 18.0 ± 140 17.4 ± 111 16.5 ± 111 14.5 ± 543 18.0 ± 406 16.0 ± 354
4463 ± 20,595 11,731 ± 30,554 12,080 ± 32,057 17.0 ± 180 16.5 ± 144 16.1 ± 155 15.0 ± 246 20.5 ± 162 19.0 ± 144
0.55 0.17 0.30 0.28 0.74 0.95 0.88 0.65 0.92
IL-4 ex vivo (pg/ml)
3.2. Laboratory findings within days after acute stroke or myocardial infarction Within hospital stay, 6 out of 20 SP and 2 out of 20 PAMI developed nosocomial infection, i.e. pneumonia or urinary tract infection. On days 2 and 6 of hospitalisation serum CRP and IL-6 levels, body temperature and serum cortisol concentration were higher in SP with nosocomial infection (SPI+) compared to SP without nosocomial infection (SPI−) (Table 3). At these time points total counts of granulocytes, monocytes and leukocytes [day 2 only] as well as IL-6 release [day 6 only] were significantly (p b 0.02) higher in SPI+ compared to SPI−, while CD3+ T cell counts, monocytic HLA-DR expression and LPS-induced release of TNF-α [day 2 only] were significantly (p ≤ 0.015) lower (Table 3; Fig. 1). In SP and PAMI without nosocomial infection (PAMI−), leukocyte, granulocyte and monocyte counts decreased over time, while HLA-DR expression increased (Table 3). Nevertheless, HLA-DR expression was significantly (p b 0.001) lower in SPI− and PAMI− compared to HC at all times measured (Fig. 1). Moreover, counts of leukocytes, granulocytes and monocytes remained significantly higher in PAMI compared to HC within the study period. Serum IL-6 levels in PAMI− decreased over time. Detectable levels of serum IL-6 were found in the minority of SPI−. Cortisol levels were almost unchanged over time in PAMI− and SPI− (Table 3). As displayed in Table 4, the stimulation of whole blood-cultures with ConA ex vivo revealed a low IFN-γ release – a common marker of
375
IL-5 ex vivo (pg/ml)
Th1 immune response – in PAMI and SP on admission, while levels of IL-4 and IL-5 – both markers of Th2 function – remained almost unchanged over time in PAMI and SP. The ConA-induced cytokine release showed no significant difference between SPI+ and SPI− (data not shown). 4. Discussion The analysis of immune responses in vascular diseases has received increasing attention over the last years. Using the same time frame and study protocol to ensure better comparability, we prospectively evaluated immune responses of patients with acute stroke or myocardial infarction, respectively. Our data indicate an acute immunosuppression for both vascular disorders. However, the observed immune responses were different to some extent. Compared to healthy controls, there was an acute phase response within hours after onset of ischemic stroke or myocardial infarction [11–14], while procalcitonin serum levels were within normal range
Table 3 Immune markers of stroke patients with nosocomial infection (SPI+), stroke patients without nosocomial infection (SPI−) and patients with acute myocardial infarction without nosocomial infection (PAMI−) on days 2 and 6 of hospitalisation. Values are expressed as median ± range.
Body temperature (° C) CRP (mg/dl) [n] Cortisol (nmol/l) IL-6 (pg/ml) [n N 2 pg/ml] Leukocytes (1/nl) Granulocytes (1/nl) Lymphocytes (1/nl) CD3+ T cells (1/nl) CD4+ T cells (1/nl) CD8+ T cells (1/nl) CD16+ NK cells (1/nl) CD19+ B cells (1/nl) Monocytes (1/nl) HLA-DR expression (antigen/monocyte) LPS-induced TNF-α (pg/monocyte) # p b 0.05 vs. SPI−. ⁎ p b 0.05 for SPI− or PAMI− vs. HC.
[Day]
SPI+ [n = 6]
[2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6] [2] [6]
37.5 ± 2.0 38.0 ± 2.2# 5.6 ± 12.2# 4.7 ± 11.7# 486 ± 992 411 ± 369 16.8 ± 161 [6] 42.2 ± 89 [5]# 10.3 ± 8.3# 10.8 ± 7.0 8.2 ± 9.7# 8.7 ± 6.5# 1.3 ± 0.82 0.96 ± 1.0 0.78 ± 0.90# 0.65 ± 0.44# 0.44 ± 0.77 0.48 ± 0.50 0.32 ± 0.46 0.21 ± 0.34 0.14 ± 0.33 0.14 ± 0.21 0.18 ± 0.35 0.18 ± 0.39 0.90 ± 0.71# 0.99 ± 1.1# 6084 ± 5836# 7541 ± 10,825# 0.80 ± 1.4# 0.82 ± 2.9
SPI− [n = 14] 36.4 ± 1.2 36.4 ± 1.7 0.5 ± 4.9⁎ 0.5 ± 1.8⁎ 354 ± 257 293 ± 357 6.4 ± 7.7 [5] 4.0 ± 3.0 [4] 7.3 ± 4.5 6.9 ± 7.4 5.3 ± 3.8⁎ 4.2 ± 5.3 1.65 ± 1.5 1.74 ± 2.0 1.2 ± 1.6 1.4 ± 1.9 0.71 ± 0.91 0.80 ± 1.09 0.35 ± 0.90 0.39 ± 0.96 0.20 ± 0.31 0.22 ± 0.32 0.20 ± 0.36 0.21 ± 0.48 0.51 ± 0.36 0.46 ± 0.52 19,531 ± 15,845⁎ 21,783 ± 21,614⁎ 2.4 ± 2.0 2.9 ± 3.5
PAMI− [n = 18] 36.4 ± 1.1 36.0 ± 1.6 1.8 ± 3.1 [14] – 334 ± 407 341 ± 680 6.2 ± 39 [15] 4.0 ± 5.6 [6] 8.6 ± 9.0⁎ 8.0 ± 7.1⁎ 6.0 ± 7.1⁎ 5.5 ± 5.8⁎ 2.0 ± 2.6 1.9 ± 1.7 1.4 ± 2.4 1.4 ± 1.4 0.81 ± 1.2 0.85 ± 0.96 0.47 ± 1.12 0.50 ± 0.63 0.21 ± 0.59 0.20 ± 0.41 0.25 ± 1.29 0.21 ± 0.47 0.70 ± 1.0⁎
0.68 ± 0.80⁎ 14,754 ± 22,208⁎ 17,750 ± 22,584⁎ 1.5 ± 7.6 2.0 ± 2.7
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[1,31] and cortisol levels – as a marker of an ongoing stress reaction – were not significantly elevated. Serum IL-6 was detectable in the majority of patients with acute myocardial infarction (PAMI) on admission, but in only 40% of all stroke patients (SP). Levels of IL-6 decreased in PAMI over time, probably indicating a stress-induced augmentation of the immune function on admission [32]. Highlighting the role of leukocytes [15–18] and monocytes [19] in patients with acute coronary syndrome (ACS), total counts of leukocytes and monocytes were significantly higher in PAMI compared to SP on admission. Compared to HC, monocytic HLA-DR expression – essential for antigen-specific T cell defence – was significantly decreased in PAMI and SP, as similarly reported in patients after neurosurgery [33], cardiopulmonary bypass surgery [34], stroke [1,2,4] or sepsis [35]. The release of TNF-α after LPS stimulation ex vivo was not significantly decreased in SP and PAMI, indicating a partial functional loss of monocytic immune competence on admission. As demonstrated before [1], a mainly CD3+ T cell-accentuated lymphopenia was evident in SP with nosocomial infection (defined by clinical onset within 3 and 6 days of hospitalisation). We observed no lymphopenia in PAMI, which is in contrast to previous reports in ACS patients and probably related to the extent of myocardial damage [16,21,22]. As indicated by the almost unchanged release of IL-4 and IL-5 after mitogen stimulation ex vivo, the Th2 function was unaltered over time, as previously described in PAMI [24] and SP [1]. A rapid deactivation of T cells, indicated by reduced mitogen-induced release of IFN-γ on admission, was observed in SP [1,4,9] and PAMI, while Pasqui et al. reported an up-regulation of LPS-induced IFN-γ release ex vivo in patients with ACS [27]. With regard to the detrimental effects of IFN-γ and T-lymphocytes on the ischemic brain [36], the stroke-induced immunodepression might be self-protective by limiting the duration of auto-inflammatory processes [10]. Using clinical diagnostic criteria for infection, we cannot rule out an impact of nosocomial infections on the ongoing immune response. Therefore the laboratory data obtained on days 2 and 6 of hospitalisation have to be interpreted carefully. As demonstrated before [1,3], immunodepression was accentuated in SP with severe stroke, obviously predisposing these patients for nosocomial infections. Compared to SP without nosocomial infection the SP with nosocomial infections had significantly higher monocyte counts, while CD3+ T cell counts, monocytic HLA-DR expression and LPS-induced release of TNF-α were significantly lower. Compared to HC, HLA-DR expression was significantly reduced in patients after stroke and myocardial infarction within the study period of 6 days, indicating a persisting immunosuppressive state, irrespective of nosocomial infection. There are several strengths and limitations of our study. Firstly, in this pilot study, the relatively low number of patients did not allow correction for multiple testing, limiting the significance of the results. Secondly, ACS and ischemic stroke are heterogeneous disease entities of different aetiological subtypes representing variations in risk factors and recent medication, probably affecting the immune response and limiting the generalisation of the results. Thirdly, the interesting question of the relevance of the sympathetic and parasympathetic nervous system for the observed immune responses could not be clarified yet, since no systematic assessment of the state of the vegetative nervous system was done. Additional prospective studies are needed to clarify this point and to further validate the results. 5. Conclusion Compared to healthy controls, a rapid reduction of monocytic HLADR expression and a defective lymphocytic IFN-γ production were obvious after stroke as well as myocardial infarction. A depression of lymphocyte counts was observed after severe ischemic stroke but not after myocardial infarction. Regarding the observed differences after stroke and myocardial infarction, an additional impact of the damaged
nervous system on the immune response seems possible. More efforts are necessary to understand the regulating mechanisms of immune response after acute stroke and myocardial infarction.
Acknowledgements We thank C. Liebenthal, C. Muselmann and K. S. Siebel for assistance. We would also like to thank Dr. Brigitte Wegner (Institute for Medical Biometry and Epidemiology, Charité University Medicine Berlin) for statistical advice. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [37].
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