Download Decrease of serum levels of the anti-inflammatory

Clinical Science (2003) 105, 45–50 (Printed in Great Britain)
Decrease of serum levels of the
anti-inflammatory cytokine interleukin-10 in
patients with advanced chronic heart failure
Christian STUMPF, Christoph LEHNER, Atilla YILMAZ, Werner G. DANIEL,
and Christoph D. GARLICHS
Department of Cardiology, University of Erlangen-Nuremberg, Ulmenweg 18, 91054 Erlangen, Germany
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Inflammation plays a significant contributory role in the pathogenesis of chronic heart failure
(CHF). Many studies have shown enhanced plasma levels of proinflammatory cytokines [i.e.
tumour necrosis factor-α (TNF-α) and interleukin (IL)-6] in patients with CHF. However, there
are only few reports on the regulation of anti-inflammatory cytokines such as IL-10. IL-10 has
potent deactivating properties in macrophages and T-cells and thus acts as a down-regulator
of cell-mediated immune responses. The aim of the present study was to assess whether serum
concentrations of IL-10 significantly differ between patients with CHF and healthy control subjects.
Patients with CHF [n = 50; 66.9 +
− 12.6 years; mean ejection fraction, 22.1 +
− 9.2 %; New York
Heart Association (NYHA) class II–IV] and 25 healthy controls (63.6 +
10.2
years)
were examined.
−
Of the 50 patients with CHF, 32 patients were taking aspirin (100 mg/day) and 33 patients had
lipid-lowering therapy with a statin. Serum IL-10 as well as TNF-α concentrations were measured
using commercially available immunoassays. Patients with CHF showed significantly lower IL-10
concentrations (2.3 +
− 1.9 compared with 5.2 +
− 2.3 pg/ml; P < 0.001). Patients with advanced
CHF (NYHA class III and IV) had the lowest IL-10 plasma levels. Aspirin and statin therapy did
not significantly influence serum levels of IL-10. The ratio of TNF-α to IL-10 was significantly
higher in patients with advanced CHF (NYHA class III and IV, ratio 3.2 +
− 1.2 and 3.1 +
− 1.1
respectively, compared with control 0.4 +
0.2;
P
<
0.01).
Our
present
study
demonstrates
−
significantly decreased serum levels of IL-10 in patients with advanced CHF. Since IL-10 is known as
a potent anti-inflammatory cytokine, its decrease in advanced CHF may favour the inflammatory
milieu in CHF.
INTRODUCTION
Over the past decade there has been increasing interest in
the potential role of inflammatory mediators in cardiac
diseases, such as chronic heart failure (CHF). Elevated
circulating levels of cytokines, in particular tumour
necrosis factor-α (TNF-α) and interleukin (IL)-6, have
consistently been identified in patients with CHF [1,2].
Several studies [2a,2b] suggest that the extent of cytokine
production directly correlates with the severity of the
disease process. Furthermore, studies such as the Studies
of the Left Ventricular Dysfunction database showed
an increased rate of mortality with increasing levels of
TNF-α in patients with CHF [3]. Interestingly, many
aspects of the syndrome of CHF can be explained
by the known biological effects of these inflammatory
Key words: chronic heart failure, cytokines, immunology, inflammation.
Abbreviations: ACE, angiotensin-converting enzyme; CHF, chronic heart failure; dCM, dilated cardiomyopathy; EF, ejection
fraction; (hs)CRP, (high sensitivity) C-reactive peptide; iCM, ischaemic cardiomyopathy; IL, interleukin; LV, left ventricular;
NYHA, New York Heart Association; TGF-β1, transforming growth factor-β1; TNF-α, tumour necrosis factor-α; sTNFR,
soluble TNF-receptor.
Correspondence: Dr Christian Stumpf (e-mail [email protected]).
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mediators: serum concentrations of TNF-α frequently
found in CHF can induce progressive left ventricular (LV)
dysfunction, LV remodelling, fetal gene expression and
cardiomyopathy [4]. Thus, analogous to the well known
elevation of neurohormones in CHF, TNF-α can predict
the functional class and the clinical outcome in patients
with CHF. The emerging link between inflammatory
cytokines in the pathogenesis and progression of CHF
has already resulted in the development of anti-cytokine
strategies that might be used as adjunctive therapy in
patients with CHF. Although inflammatory mediators
have moved to the centre of interest in leading heart
failure trials, little is known about the role of antiinflammatory cytokines in this disease setting. IL-10,
which is produced by various inflammatory cells, especially macrophages [5] and T-cells, is a major inhibitor
of cytokine synthesis, suppresses macrophage function
and inhibits the production of proinflammatory cytokines [6] as well as matrix metalloproteinases, which
have already been described as playing an important
role in CHF [7]. Recently, Bolger et al. [8] have shown
that IL-10 inhibited TNF-α release from peripheral
blood mononuclear cells (‘PBMC’) isolated from patients
with CHF. In other chronic inflammatory disease states,
e.g. atherosclerosis, IL-10 has already been described
as having protective properties in delaying disease progression with high levels of expression being associated
with significantly decreased cell death and inducible nitric
oxide synthase expression [9]. Recently, decreased serum
levels of IL-10 were measured in patients with unstable
angina suggesting further its protective role in chronic
inflammatory disease conditions [10].
Because of its anti-inflammatory potential, the therapeutic use of IL-10 has also been tested in many
chronic inflammatory diseases where TNF-α is believed
to play a prominent role (e.g. rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and cardiac
allograft rejection) [11]. In addition, an interesting report
was made by Gullestad et al. [12] in which they found
that the intravenous administration of immunoglobulin
to patients with CHF increases IL-10 levels and improves
LV ejection fraction (EF).
Although, there are some interesting data on the antiinflammatory potential of IL-10 in chronic inflammatory
diseases, only little is known about its role in the pathophysiology of CHF. In the present study, we examined
the IL-10 plasma concentrations in patients suffering
CHF.
METHODS
Patients and controls
Patients (n = 50, 38 men and 12 women; mean age
66.9 +
− 12.6 years; 40 inpatients and 10 outpatients) with
symptomatic CHF, defined as dyspnea or fatigue at
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2003 The Biochemical Society
Table 1 Baseline characteristics of CHF patients and ageand sex-matched control subjects
Values are means +
− S.D. AT1, angiotensin-II-receptor-1.
Parameters
Controls
CHF patients
P value
Age (years)
Sex (male/female)
NYHA functional class (II/III/IV)
Inpatients/outpatients
Cause of CHF
Coronary artery disease
Idiopathic dCM
LVEF (%)
Mean duration of CHF (years)
Medication
ACE inhibitors
β-Blockers
Diuretics
Digitalis
AT1 blockers
Aspirin
63.6 +
− 10.2
18/7
–
–
66.9 +
− 12.6
38/12
11/31/8
40/10
P = 0.48
P = 0.56
–
–
–
61.3 +
− 7.1
–
39
11
22.1 +
− 9.2
1.9 +
− 0.4
–
P < 0.001
–
–
–
–
–
–
–
40
34
38
17
8
32
–
–
–
–
–
–
rest or on exertion for more than 3 months, were
studied (Table 1). The severity of the CHF ranged
from New York Heart Association (NYHA) functional
class II to IV (Table 1). The underlying cause of
CHF was classified as coronary artery disease (n = 39)
or idiopathic dilated cardiomyopathy (dCM; n = 11),
based on disease history and coronary angiography.
All patients underwent echocardiography. Mean EF
measured was 22.2 +
− 9.2 %. None of the patients was
acutely decompensated. Their clinical and haemodynamic
situations were stable, with no changes in medication
during the last month before study entrance. None of the
patients had haemodynamic support devices. The main
reason for admission of inpatients was elective coronary
angiography.
Blood was taken after admission before any change
of medication or any diagnostical or interventional procedure was done which could have provoked an inflammatory reaction.
Medical treatment consisted of angiotensinconverting-enzyme (ACE) inhibitors (80 %), β-blockers
(68 %), diuretics (76 %), digitalis (34 %) and angiotensinII-receptor-1 blockers (16 %). Of the patients with CHF,
32 were taking aspirin and 18 were without aspirin
therapy. All patients had serum creatinine levels lower
than 150 µmol/l, and none had any concomitant diseases such as fever, infection, myocarditis, malignancies,
collagen vascular disease, pulmonary disease or thyreotoxicosis. Control subjects were 25 healthy sex- and agematched blood donors (18 men and 7 women; mean
age 63.6 +
− 10.2 years) without any history of cardiac
disease. The study was approved by the Local Ethics
Interleukin-10 and chronic heart failure
Committee and informed consent for participation in
the study was obtained from all individuals.
Table 2 Plasma cytokine levels in CHF patients and control
subjects
Blood sampling protocol
Cytokine
Controls
CHF patients
P value
For serum sampling, blood was drawn into pyrogenfree blood collection tubes without additives (Becton
Dickinson, Heidelberg, Germany). Tubes were immediately immersed in melting ice and centrifuged within
15 min (1000 g for 15 min at 4 ◦ C). Serum and plasma
was stored at − 80 ◦ C in multiple aliquots until analysis.
Samples were frozen and thawed only once.
IL-10 (pg/ml)
TNF-α (pg/ml)
sTNFR1 (pg/ml)
sTNFR2 (pg/ml)
TNF-α/IL-10
TGF-β1 (pg/ml)
hsCRP (mg/l)
5.2 +
− 2.3
2.5 +
− 1.8
860 +
− 279
1750 +
− 620
0.4 +
− 0.2
1745 +
− 322
2.93 +
− 4.1
2.3 +
− 1.9
6.5 +
− 2.9
1760 +
− 783
2740 +
− 746
3.2 +
− 1.2
1662 +
− 280
13.8 +
− 15.2
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P < 0.01
P = 0.34 (ns)
P < 0.001
Values are means +
− S.D. ns, non significant.
Laboratory analysis
Serum levels of IL-10, transforming growth factorβ1 (TGF-β1), TNF-α, soluble TNF-receptor (sTNFR)
1 and sTNFR2 as well as high-sensitivity C-reactive
protein (hsCRP) were measured using commercially
available ELISA kits [R&D Systems, Minneapolis, MN,
U.S.A.; and Biocheck Inc., Burlingame, CA, U.S.A. (for
hsCRP)], according to the manufacturers’ instructions.
For measuring levels of TGF-β1, serum samples had to
be acidified to activate latent immunoreactive TGF-β1,
detectable by the Quantikine TGF-β1 immunoassay.
All kits had the following sandwich ELISA format:
microtitre plates already precoated with a murine monoclonal antibody against the human cytokine being
measured. Standards of the analyte and serum samples
were added in duplicate, along with a second antibody
against another epitope of the analyte conjugated to
horseradish peroxidase (for TGF-β1, TNF-α, sTNFR1
and sTNFR2) or alkaline phosphatase (for IL-10; high
sensitivity assay kit). The samples were incubated for 2 h
(3 h for TGF-β1 measurements). Finally, the chromogen
tetramethyl benzidine was added and incubated for
20 min (1 h for TGF-β1) in the dark. After the addition of
1 M H2 SO4 , the absorbance at 450 nm (490 nm for TGFβ1; reference filter 570 nm and 650 nm for TGF-β1) were
read and standard curves were plotted in a Spectramax
Plus microplate reader (Molecular Devices, Munich,
Germany). Due to the high sensitivity of the assay kit
by using an amplification system, the IL-10 ELISA has
a lower detection limit of 0.5 pg/ml. The sensitivities for
the other assay kits were: TGF-β1, 7.0 pg/ml; TNF-α,
4.4 pg/ml; sTNFR1, 3.0 pg/ml; sTNFR2, 1.0 pg/ml; and
hsCRP, 0.1 mg/l. All assays were conducted by staff
who were blinded to the clinical status of the individual subjects. The intra- and inter-assay coefficients of
variation in our laboratory were below 9 %.
Statistical analysis
The data were analysed by non-parametric methods
to avoid assumptions about the distribution of the
measured variables. Comparisons between groups were
made with the Mann-Whitney U test. The association
Figure 1 Relation of plasma IL-10 levels to NYHA functional
class
Values are means +
− S.D.
of measurements with other biochemical parameters was
assessed by the Spearman rank correlation test. All values
are reported as means +
− S.D. Statistical significance was
considered to be indicated by a value of P < 0.05.
RESULTS
Circulating levels of anti-inflammatory
cytokines in patients with CHF
Patients with CHF had significantly decreased levels of
IL-10 compared with healthy control subjects (2.3 +
− 1.9
compared with 5.2 +
− 2.3 pg/ml respectively; P < 0.01;
Table 2). In particular, patients suffering advanced CHF,
as classified as NYHA class III and IV, showed the lowest
IL-10 levels (Figure 1).
Another important cytokine that exerts antiinflammatory effects is TGF-β1. Levels of TGF-β1 were
not significantly decreased in our group of CHF patients
(1662 +
− 280 compared with 1745 +
− 322 pg/ml respectively; P = 0.34; Table 2).
We next examined whether the IL-10 levels were
related to the cause of heart failure and compared the
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Figure 2 Relation of plasma TNF-α (A), sTNFR1 (B), sTNFR2 (C) and hsCRP (D) to NYHA functional class in all subjects
Values are means +
− S.D. NS, not significant.
plasma levels in patients suffering dCM with those having
ischaemic cardiomyopathy (iCM). We found significantly
decreased IL-10 levels in both patient groups. However,
there was no significant difference between iCM and
dCM patients (2.1 +
− 1.8 compared with 2.4 +
− 1.9 pg/ml
respectively; P = 0.28).
Next, we evaluated if there is a relationship between
plasma IL-10 levels and medications taken by CHF
patients, especially those with known anti-inflammatory
actions, e.g. aspirin and statins. We found no association
between serum concentrations of IL-10 and the use of
aspirin or statins (aspirin, 2.25 +
− 1.2 compared with
2.34 +
1.4
pg/ml,
P
=
0.48;
statin,
2.39 +
−
− 1.2 compared
with 2.5 +
1.4
pg/ml,
P
=
0.42).
−
Circulating levels of inflammatory
cytokines in patients with CHF
Plasma TNF-α levels were higher in patients with CHF
than in healthy control subjects (6.5 +
− 2.9 compared with
2.5 +
− 1.8 pg/ml respectively; P < 0.01; Figure 2A) and
increased as NYHA functional class increased. Levels of
the sTNFR1 as well as sTNFR2 followed a similar pattern
(Figures 2B and 2C).
Another sign of persistent immunactivation in CHF
patients is mirrored by an increased level of hsCRP.
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2003 The Biochemical Society
Figure 3 hsCRP levels in patients with dCM and iCM as well
as healthy control subjects
Values are means +
− S.D.
hsCRP levels were significantly elevated in our group
of CHF patients, with the highest levels found in
NYHA class IV patients (2.93 +
− 4.1 compared with
13.8 +
− 15.2 mg/l; Figure 2D). However, plasma levels of
hsCRP were found to be significantly higher in iCM
patients compared with the dCM group (16.7 +
− 8.9
compared with 7.3 +
− 3.8 mg/l; P < 0.01; Figure 3).
Interleukin-10 and chronic heart failure
Next, we compared the inflammatory to the antiinflammatory ‘profile’ in our group of CHF patients
given as the ratio of TNF-α to IL-10. This ratio was found
to be significantly higher in the CHF group, especially
in NYHA class III and class IV (NYHA class III and IV,
3.2 +
− 1.2 and 3.1 +
− 1.1 respectively; control, 0.4 +
− 0.2;
P < 0.01).
DISCUSSION
CHF is one of the major health care problems in
developed countries and one of the most frequent reason
for patients being admitted to hospital. Despite significant
advances in its treatment, the prognosis of CHF remains
still poor. Over the past decade there has been increasing
interest in the potential role of inflammatory mediators
in CHF. This interest was fostered further by the
observation that many aspects of the CHF syndrome can
be explained by the known biological effects of these
molecules [3]. Consequently, there is a growing body
of evidence that anti-cytokine therapy may represent
an additional approach for the treatment of patients
with CHF. Despite the growing number of studies
examining the role of proinflammatory mediators in the
pathogenesis of CHF, there are only a few reports on
the potential role of anti-inflammatory cytokines such as
IL-10 or TGF-β1.
IL-10, initially designated cytokine synthesisinhibitory factor (‘CSIF’), was originally identified as
a product of macrophages and lymphocytes of the
Th2 subtype. It inhibits many cellular processes that
have already been described to play important roles in
CHF progression such as production of matrix metalloproteinases [13] and cytokines (e.g. TNF-α) [14], activation of nuclear factor κB (‘NF-κB’) [15] as well as
apoptosis and cell death [16]. In vitro experiments
showed that the expression of IL-10 in lipopolysaccharide-stimulated monocytes is delayed relative to
that of other proinflammatory cytokines, such as TNF-α,
and coincides with their down-regulation [17]. Moreover,
in vivo studies showed that plasma TNF-α levels are
higher and remained elevated for a longer period of
time in IL-10-deficient (IL-10−/− ) mice injected with
lipopolysaccharide than in IL-10+/+ mice [18]. Another
important cytokine with anti-inflammatory potential is
TGF-β. It was first reported to be a deactivating factor
of macrophages capable of suppressing inducible nitric
oxide synthase protein expression in macrophages. TGFβ1 also has potent anti-inflammatory effects on vascular
cells by down-regulating cytokine-induced expression
of E-selectin and vascular cellular adhesion molecule-1
(‘VCAM-1’) [19,20].
In our present study, we observed significantly decreased levels of IL-10 in patients with CHF. In particu-
lar, patients suffering advanced CHF, given as NYHA
class III and IV, showed the lowest IL-10 levels. In contrast with IL-10, levels of TGF-β1 were not significantly decreased.
On the other hand, as expected, we found signs of
chronic activation of the immune system as mirrored
by an activation of proinflammatory cytokines, such as
TNF-α, sTNFR1 and sTNFR2. Additionally, we also
found significantly elevated levels of hsCRP, another
important marker of systemic inflammation with the
highest levels seen in NYHA class IV patients. Levels
of hsCRP were, however, higher in CHF due to
ischaemia, supporting the present inflammatory theory
of atherosclerosis. Recently, it was found that increased
CRP levels in CHF predict a higher hospitalization and
readmission rate within a shorter period of time in cases
of deteriorating CHF [21]. Moreover, high CRP levels
are associated with increased mortality and may also be
associated with a worse therapeutic response [22].
Thus, obviously, the ‘immunologic balance’ in our
present study group is changed in favour of a more
inflammatory milieu as seen in an increased TNF-α
to IL-10 ratio. In particular, patients suffering NYHA
class III/IV CHF showed a high TNF/low IL-10
constellation. The exact mechanism and cause of this
disturbed balance still remains unclear. The clinical importance of this constellation, however, is underscored
by a report [23] demonstrating a strong association
between the combined high TNF-α/low IL-10 constellation and early graft rejection in a population of heart
transplant recipients. The clinical importance of IL-10
is fostered further by some encouraging reports on its
therapeutical use in chronic inflammatory diseases in
which inflammatory mediators, such as TNF-α, are
believed to predominate, including psoriasis [24], chronic
hepatitis C [25] and Crohn’s disease [26]. It has
furthermore been shown that human recombinant IL10 can be safely administered and is generally well
tolerated. It was only recently that Bolger et al. [8]
demonstrated the inhibition of TNF-α production by
IL-10 in peripheral blood mononuclear cells isolated
from CHF patients. Previously, Damas et al. [27] showed
that the production of TNF-α stimulated monocyte
chemotactic protein-1 (‘MCP-1’), a chemotactic cytokine
believed to play an important role in the development
and progression of CHF, is decreased by IL-10 in rat
ventricular cardiomyocytes.
On the basis of our present results, it is tempting to
hypothesize that decreased levels of IL-10 may favour
a more inflammatory milieu and that, in particular,
patients with a high TNF-α/IL-10 ratio could benefit
from a therapy with recombinant IL-10. However, these
preliminary data about the value of IL-10 in chronic heart
failure need to be confirmed in larger patient studies and
trials of its therapeutical use should be considered.
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Received 13 December 2002/11 February 2003; accepted 14 March 2003
Published as Immediate Publication 14 March 2003, DOI 10.1042/CS20020359
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