Transsignaling of Interleukin-6 Crucially Contributes to

Transsignaling of Interleukin-6 Crucially Contributes to
Atherosclerosis in Mice
Harald Schuett, René Oestreich, Georg H. Waetzig, Wijtske Annema, Maren Luchtefeld, Anja Hillmer,
Udo Bavendiek, Johann von Felden, Dimitar Divchev, Tibor Kempf, Kai C. Wollert, Dirk Seegert,
Stefan Rose-John, Uwe J.F. Tietge, Bernhard Schieffer, Karsten Grote
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Objective—Transsignaling of interleukin (IL)-6 is a central pathway in the pathogenesis of disorders associated with
chronic inflammation, such as Crohn disease, rheumatoid arthritis, and inflammatory colon cancer. Notably, IL-6 also
represents an independent risk factor for coronary artery disease (CAD) in humans and is crucially involved in vascular
inflammatory processes.
Methods and Results—In the present study, we showed that treatment with a fusion protein of the natural IL-6
transsignaling inhibitor soluble glycoprotein 130 (sgp130) and IgG1-Fc (sgp130Fc) dramatically reduced atherosclerosis
in hypercholesterolemic Ldlr⫺/⫺ mice without affecting weight gain and serum lipid levels. Moreover, sgp130Fc
treatment even led to a significant regression of advanced atherosclerosis. Mechanistically, endothelial activation and
intimal smooth muscle cell infiltration were decreased in sgp130Fc-treated mice, resulting in a marked reduction of
monocyte recruitment and subsequent atherosclerotic plaque progression. Of note, patients with CAD exhibited
significantly lower plasma levels of endogenous sgp130, suggesting that a compromised counterbalancing of IL-6
transsignaling may contribute to atherogenesis in humans.
Conclusion—These data clarify, for the first time, the critical involvement of, in particular, the transsignaling of IL-6 in
CAD and warrant further investigation of sgp130Fc as a novel therapeutic for the treatment of CAD and related diseases.
(Arterioscler Thromb Vasc Biol. 2012;32:281-290.)
Key Words: atherosclerosis 䡲 cardiovascular diseases 䡲 interleukins 䡲 pathophysiology 䡲 signal transduction
A
vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1.8,9 Thereby, recruitment
and infiltration of inflammatory cells into the subendothelium are initiated. Furthermore, cytokines trigger the
release of matrix degrading proteases, chemokines, and
reactive oxygen species, which accelerate atherosclerotic
lesion progression.4,10 –12
IL-6 is an important proinflammatory cytokine that has
been identified as an independent risk factor for CAD13,14 and
for which expression in human atherosclerotic plaques has
already been demonstrated.15 Sustained IL-6 production is
involved in chronic low-level inflammation associated with
obesity, which gives rise to insulin resistance and type 2
diabetes16 and further promotes atherosclerosis. Moreover,
convincing evidence from experimental studies points to a
broad potential of IL-6 as a direct proatherosclerotic mediator.17,18 Accordingly, wild-type and atherosclerosis-prone
Apoe⫺/⫺ mice exhibit a dramatically increased lesion size
therosclerotic cardiovascular disease with its complications, such as acute myocardial infarction and stroke, is
a major cause of morbidity and mortality in Western countries. It is well accepted that atherosclerosis is a chronic
vascular inflammatory disease, mediated by a concerted
action of the innate and the adaptive immunity.1– 4 Inflammation defines the majority of mechanisms involved in atherosclerotic plaque development.5 However, 70% of clinical
events in patients with coronary artery disease (CAD) still
cannot be prevented with modern drug therapy, including
statins, emphasizing an urgent need for supplementary antiinflammatory treatment strategies.6
Proinflammatory cytokines are central mediators of the
chronic inflammatory process within the vascular wall. These
cytokines, especially members of the interleukin (IL)
family, contribute to endothelial activation by the induction of chemoattractant proteins, such as monocyte chemotactic protein-1,7 and adhesion molecules, including
Received on: June 19, 2011; final version accepted on: October 19, 2011.
From the Department of Cardiology and Angiology, Hannover Medical School, Hannover, Germany (H.S., R.O., M.L., A.H., U.B., J.v.F., D.D., T.K.,
K.C.W., B.S., K.G.); Department of Pediatrics, Center for Liver, Digestive, and Metabolic Diseases, University Medical Center Groningen, University
of Groningen, Groningen, The Netherlands (W.A., U.J.F.T.); CONARIS Research Institute AG, Kiel, Germany (G.H.W., D.S.); Institute of Biochemistry,
Christian-Albrechts-University, Kiel, Germany (S.R.-J.).
Drs Schieffer and Grote are equally contributing senior authors.
Correspondence to Karsten Grote, PhD, Department of Cardiology and Angiology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625
Hannover, Germany. E-mail [email protected]
© 2011 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
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DOI: 10.1161/ATVBAHA.111.229435
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when treated with recombinant IL-6 in addition to a high-fat
diet.19 To the contrary, complete IL-6-deficiency seems to be
rather atheroprotective in both diet- and pathogen-associated
atherosclerosis.20 –23 Thus, the role of IL-6 in atherogenesis
still remains to be determined.
As the signaling mechanism of IL-6 is quite complex, a
systemic IL-6 deficiency might therefore not represent the
optimal tool to unravel the diverse contributions of IL-6 to
atherosclerosis. The so-called classic IL-6 signaling is initiated on binding of IL-6 to the membrane-bound IL-6 receptor
(IL-6R) followed by subsequent complexation with the ubiquitously expressed coreceptor and signal transducer glycoprotein (gp) 130.24 In addition, a process termed transsignaling
has increasingly attracted attention in recent years. In IL-6
transsignaling, IL-6 binds to soluble forms of IL-6R (sIL-6R)
and activates gp130. Whereas classic signaling is confined to
restricted cell populations expressing the membrane-bound
IL-6R, such as hepatocytes and certain leukocytes, transsignaling dramatically expands the range of potential target cells
for IL-6 signaling.17 Remarkably, recent studies revealed that
IL-6 transsignaling, in particular, is crucially involved in
chronic diseases with a clear inflammatory component, such
as Crohn disease, rheumatoid arthritis, and inflammatory
colon cancer.25,26
Soluble forms of gp130 (sgp130) are the natural inhibitors of IL-6 transsignaling and are present in molar excess
in human plasma to prevent global transsignaling. To test
the hypothesis that targeting IL-6 transsignaling can curtail
the chronic vascular inflammation as pathophysiological
basis of atherosclerosis, we used an optimized fusion
protein of the natural sgp130 and IgG1-Fc.27 This therapeutic fusion protein, called sgp130Fc, selectively inhibits
IL-6 transsignaling by neutralizing IL-6/sIL-6R complexes
without affecting classic signaling via the membranebound IL-6R.27,28
Our present study demonstrates that sgp130Fc treatment of
atherosclerosis-prone Ldlr⫺/⫺ mice on a high-fat, high-cholesterol diet resulted in a dramatic reduction of the atherosclerotic lesion formation and even led to a significant
regression of advanced atherosclerosis. Moreover, patients
with CAD exhibited significantly lower plasma levels of
endogenous sgp130. These results underscore that IL-6 transsignaling, in particular, paves the way for atherogenesis and
imply new therapeutic strategies for the treatment of this
major health burden.
Methods
Animals
Male C57BL/6J Ldlr⫺/⫺ (B6.129S7-Ldlrtm1Her/J) and C57BL/6J
mice were obtained from the Jackson Laboratory (Bar Harbor, ME)
and maintained under pathogen-free conditions in the Central Animal Facility at Hannover Medical School. All experiments were
approved by the governmental animal ethics committee and performed according to the guidelines of the Federation of European
Animal Science Associations.
Treatment
The study was subdivided in 2 sections: (1) IL-6 transsignaling and
atherogenesis and (2) IL-6 transsignaling and established atherosclerosis. (1) For the analysis of IL-6 transsignaling and atherogenesis
(see Figure 2), Ldlr⫺/⫺ mice at the age of 10 to 12 weeks were
randomly assigned to 4 groups. All groups were fed a high-fat,
high-cholesterol diet (D12108, Research Diets, New Brunswick,
NJ); 2 group were intraperitoneally injected twice weekly with
sgp130Fc (0.5 mg/kg body weight, CONARIS Research Institute,
Kiel, Germany), whereas the control groups received only the
vehicle phosphate-buffered saline (PBS, PAA Laboratories, Coelbe,
Germany). Tissue and fasted plasma were collected from euthanized
mice of the treatment and control group after 8 (n⫽5–7) and 16
(n⫽6 –9) weeks of diet, respectively. (2) For the analysis of IL-6
transsignaling and established atherosclerosis (see Figure 4, with a
graphical scheme of the study outline), Ldlr⫺/⫺ mice at the age of 10
to 12 weeks were randomly assigned to 3 groups. All groups were
fed the high-fat, high-cholesterol diet. Starting after 12 weeks of
feeding, 1 group was intraperitoneally injected twice weekly with
sgp130Fc (0.5 mg/kg body weight), whereas the control group
received only the vehicle (PBS). Tissue and fasted plasma were
collected from euthanized mice after 12 weeks of diet (baseline,
n⫽7) and 24 weeks of diet, including 12 weeks with sgp130Fc
injection (sgp130Fc, n⫽10) or with vehicle injection (control,
n⫽8).
Statistical Analysis
All data are presented as the mean⫾SEM or median with
25th/75th percentiles. After testing for normality and equal
variance, data were compared using the 2-tailed Student t test for
independent samples or 1-way ANOVA and a multiple comparison test when more than 2 groups were examined. When data
were not normally distributed, the Mann-Whitney rank sum test
or Kruskal-Wallis 1-way analysis of variance on ranks was
applied as the nonparametric test (SigmaStat 3.0, Systat Software,
Erkrath, Germany). A probability value of less than 0.05 was
considered statistically significant.
For an expanded Methods section, see the supplemental materials,
available online at http://atvb.ahajournals.org.
Results
Inhibition of Signal Transducer and Activator of
Transcription 3 Phosphorylation by sgp130Fc
In Vitro
The ability of sgp130Fc to specifically inhibit IL-6 transsignaling but not classic IL-6 signaling is already well documented.28 However, little is known about the impact of
sgp130Fc on IL-6-dependent signaling in cells crucially
involved in atherogenesis. Therefore, we analyzed the activation of the key IL-6/gp130 signaling effector signal transducer and activator of transcription (STAT)3 in monocytic
RAW 264.7 cells and primarily isolated endothelial cells,
smooth muscle cells (SMCs), and CD3⫹ T cells. These were
compared with hepatocytes, which strongly express
membrane-bound IL-6R. IL-6 induced a robust STAT3 phosphorylation in all cell types investigated, which was significantly suppressed by sgp130Fc application in RAW 264.7
cells (50 ␮g/mL) and SMCs (10 ␮g/mL) but not in hepatocytes, which only showed a trend toward lower STAT3
activity at 50 ␮g/mL (Figure 1A, 1C, and 1E). Of note,
STAT3 phosphorylation in endothelial cells could not be
activated by IL-6. Therefore, we used the IL-6/sIL-6R
fusion protein hyper-IL-6, which exclusively activates
IL-6 transsignaling via gp13029,30 and which induced a
robust STAT3 phosphorylation (Supplemental Figure I).
However, hyper-IL-6-induced STAT3 activation in endothelial cells was already suppressed with 0.1 ␮g/mL
sgp130Fc and completely blunted with 10 ␮ g/mL
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Figure 1. Soluble glycoprotein 130 (sgp130) Fc inhibits signal transducer and activator of transcription (STAT) 3 phosphorylation in
vitro. Shown is the Western blot analysis of lysates from murine monocytic RAW 264.7 cells (A), murine endothelial cells (B), murine
smooth muscle cells (SMCs) (C), murine CD3⫹ T cells (D), and murine hepatocytes (E) after stimulation with interleukin (IL)-6 (100
ng/mL) or hyper-IL-6 (1 ng/mL) for 30 minutes and pretreatment with increasing doses of sgp130Fc (0.1–50 ␮g/mL) for 60 minutes.
Membranes were incubated with an antibody specific for phospho (p)-STAT3 and reprobed with an antibody specific for STAT3. The
mean⫾SEM from 3 to 5 independent experiments is presented.
sgp130Fc (Figure 1B). Similarly, STAT3 activation by
IL-6 transsignaling in CD3⫹ T cells was already suppressed with low doses of sgp130Fc (Figure 1D). STAT3
activation by other IL-6 cytokine family members, such as
leukemia inhibitory factor or IL-11, was not impaired by
sgp130Fc, emphasizing the specificity of sgp130Fc for
IL-6 transsignaling (Supplemental Figure IIA and IIB).
Furthermore, sgp130Fc did not influence hepatic effects of
IL-6 in vivo (Supplemental Figure IIIA and IIIB).
Inhibition of IL-6 Transsignaling Attenuates
Atherosclerotic Lesion Progression
The importance of IL-6 transsignaling for atherogenesis was
investigated by comparing the atherosclerotic plaque development of hypercholesterolemic Ldlr⫺/⫺ mice treated with
sgp130Fc and Ldlr⫺/⫺ control mice after 8 and 16 weeks of
high-fat, high-cholesterol diet. As soon as after 8 weeks of
diet, Ldlr⫺/⫺ mice treated with sgp130Fc (0.5 mg/kg IP twice
weekly) exhibited a markedly reduced lesion size in the aortic
root, whereas only a slight decrease of the aortic lipid
deposition was observed, as determined by Oil Red O
staining (Figure 2A and 2B). However, after 16 weeks of diet,
sgp130Fc treatment led to an even more pronounced
reduction of atherosclerotic plaque size in the aortic root
and virtually arrested the increasing atherosclerotic burden
in the thoracoabdominal aorta of hypercholesterolemic
Ldlr⫺/⫺ mice (Figure 2A and 2B). Of note, atherosclerosis
in sgp130Fc-treated Ldlr⫺/⫺ mice was significantly attenuated despite comparable weight gain (33.8⫾3.1 [control]
versus 34.5⫾4.2 [sgp130Fc], mean⫾SD, P⫽0.7) and similar serum cholesterol and lipid levels compared with
Ldlr⫺/⫺ control mice (Supplemental Table I). Furthermore,
sgp130Fc treatment did not exhibit adverse effects on
blood pressure regulation or cardiac function as assessed
by tail-cuff plethysmography and echocardiography (Supplemental Table II).
In addition, we investigated specifically the vascular
deposition of sgp130Fc in hypercholesterolemic mice. For
this purpose, we analyzed en face preparations of aortas 6
hours after injection of DyLight-549-labeled sgp130Fc.
We observed enrichment of the fluorescent dye not only in
advanced highly Oil Red O–positive lesions but also in
inflammation-prone areas surrounding vascular branches
exposed to turbulent flow (Figure 2C). Importantly, the
fluorescent dye moiety alone did not accumulate in the
aortic wall in a nonspecific manner (Supplemental Figure
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Figure 2. Inhibition of interleukin (IL)-6 transsignaling attenuates atherosclerotic lesion progression. Thoracoabdominal aortas and sections of aortic roots were stained with Oil Red O. A and B, Representative aortas (A) (scale bars⫽5 mm) and aortic root sections (B)
(scale bars⫽250 ␮m) (top panels) after 16 weeks on a high-fat, high-cholesterol diet, and the respective quantification (bottom panels)
of Oil Red O–positive lesion areas expressed as percentage of total surface area of the thoracoabdominal aorta or aortic root. Percentage lesion areas from individual Ldlr⫺/⫺ mice after 8 and 16 weeks on a high-fat, high-cholesterol diet treated with soluble glycoprotein
130 (sgp130) Fc (0.5 mg/kg IP twice weekly, gray circles) or vehicle (white circles) are indicated. Means are represented by horizontal
bars; n⫽5 to 9 mice per group. C, Aortas of hypercholesterolemic Ldlr⫺/⫺ mice were washed and prepared en face 6 hours after injection of DyLight-549-labeled sgp130Fc (0.5 mg/kg IP). Fluorescence images (bottom panels) were recorded in the aorta before Oil Red
O staining (top panels). Scale bars⫽2.5 mm (top panel) and 250 ␮m (middle and bottom panels). Immunohistochemical analysis of
sgp130Fc deposition with anti-human Fc antibodies (D), signal transducer and activator of transcription (STAT) 3 phosphorylation (E), and
inhibitor ␬B kinase (IKK) ␣/␤ phosphorylation (F) in the aortic root of Ldlr⫺/⫺ mice after 16 weeks on a high-fat, high-cholesterol diet treated
with sgp130Fc (0.5 mg/kg IP twice weekly) or vehicle. Representative microphotographs are shown. Scale bars⫽250 ␮m. G, Western blot
analysis of lysates from aortic tissue collected from C57Bl6/J mice 3 hours after IL-6 injection (100 ␮g/kg IP) and pretreatment with sgp130Fc
(0.5 mg/kg IP) for 24 hours. Membranes were incubated with an antibody specific for phospho (p)-STAT3 or inhibitor (I) ␬B␣ and reprobed
with an antibody specific for STAT3 or GAPDH. A representative Western blot from 3 independent experiments is shown.
IV). Using an antibody specific for the human Fc fragment
of sgp130Fc, we observed the distribution of sgp130Fc
into organs such as liver, spleen, and lymph nodes (Supplemental Figure V) but also an abundant deposition in the
vessel wall and in atherosclerotic plaques of the treatment
group after 16 weeks of diet, whereas Ldlr⫺/⫺ control mice
did not show any staining (Figure 2D). Accordingly,
STAT3 activation observed in plaques of Ldlr⫺/⫺ control
mice was clearly suppressed after 16 weeks of sgp130Fc
treatment (Figure 2E). In contrast, activation of the nuclear
factor ␬B pathway, as reflected by the activity of the
nuclear factor-␬B activator kinases inhibitor ␬B kinase ␣
and inhibitor ␬B kinase ␤, was not affected by sgp130Fc
(Figure 2F). Pretreatment of C57Bl6/J mice with sgp130Fc
inhibited short-term STAT3 phosphorylation in the aorta
following IL-6 injection. The vascular nuclear factor-␬B
system was neither activated by IL-6 nor affected by
sgp130Fc treatment (Figure 2G). Similar to in vitro experiments with primarily isolated hepatocytes (Figure 1D),
sgp130Fc did not block IL-6 induced STAT3 phosphorylation in the liver (Supplemental Figure V). In addition,
sgp130Fc did not affect plasma levels of major proinflammatory cytokines or soluble markers of endothelial activation. Neither IL-6 itself nor IL-1 ␤ , tumor necrosis
factor-␣, monocyte chemoattractant protein-1, sVCAM,
and sICAM plasma levels of Ldlr⫺/⫺ mice were significantly changed on sgp130Fc treatment for 8 and 16 weeks
(Supplemental Table III).
Schuett et al
Inhibition of IL-6 Transsignaling Reduces
Macrophage but Not CD3ⴙ T-Cell Infiltration Into
the Plaque and Suppresses the Expression of
Endothelial Adhesion Molecules
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Next, we investigated how IL-6 transsignaling promotes
atherosclerosis in a hypercholesterolemic environment. Because vascular inflammation with subsequent infiltration of
macrophages into the vessel wall represents an initial step in
the course of atherosclerosis,5 we analyzed the effect of
sgp130Fc treatment on IL-6-mediated macrophage recruitment. Remarkably, lesions of Ldlr⫺/⫺ mice receiving
sgp130Fc (0.5 mg/kg IP twice weekly) contained 30% fewer
macrophages than Ldlr⫺/⫺ control mice after 8 and 16 weeks
of high-fat, high-cholesterol diet (Figure 3A). Consistently,
we observed an enhanced migration of macrophage-like
RAW 264.7 cells toward IL-6 in vitro, which was significantly inhibited after blocking of IL-6 transsignaling by
sgp130Fc administration (Figure 3B).
Endothelial activation with the expression of adhesion
molecules plays a crucial role in the recruitment of inflammatory cells.4 To elucidate whether sgp130Fc treatment could
attenuate endothelial activation, we examined the early expression of 2 major endothelial adhesion molecules,
VCAM-1 and ICAM-1, at atherosclerotic lesions in Ldlr⫺/⫺
mice. Immunohistochemical analysis of the exceedingly atherosclerosis-susceptible aortic arch revealed an abundant
endothelial VCAM-1 and ICAM-1 expression after 8 weeks
of diet. In contrast, inhibition of IL-6 transsignaling by
sgp130Fc treatment significantly diminished VCAM-1 and
ICAM-1 expression (Figure 3C and 3D). Correspondingly,
hyper-IL-6-induced upregulation of VCAM-1 and ICAM-1
expression was inhibited by sgp130Fc in isolated endothelial
cells (Figure 3E).
Because T cells are also identified as important protagonists in the initiation and progression of atherosclerotic
vascular disease, we likewise investigated the amount of T
cells in the atherosclerotic lesion. Contrary to macrophages,
we found significantly more T cells within and adjacent to
atherosclerotic plaques in the aortic root of sgp130Fc-treated
Ldlr⫺/⫺ mice after 16 weeks of diet (Figure 3F). Interestingly,
expression of Foxp3 mRNA—a marker of antiatherosclerotic
acting Treg cells—was significantly enhanced on sgp130Fc
treatment in the spleen, which is in line with a recent study
demonstrating that IL-6 transsignaling suppresses the induction of Treg cells (Supplemental Figure VI). However, we
could not detect Foxp3⫹ cells in the aortic roots in the
experimental animals of our study.
Inhibition of IL-6 Transsignaling Reduces
Advanced Atherosclerosis
To assess a therapeutic relevance for the inhibition of IL-6
transsignaling in preexisting atherosclerosis, we investigated
the impact of sgp130Fc on already existing atherosclerotic
plaques. To establish experimental atherosclerosis, we fed
Ldlr⫺/⫺ mice a high-fat, high-cholesterol diet for an initial
period of 12 weeks (baseline). Subsequently, we continued
this feeding for additional 12 weeks with vehicle (control) or
with sgp130Fc (sgp130Fc; 0.5 mg/kg IP twice weekly)
(graphical scheme of the study outline in Figure 4A). Aortic
IL-6 Transsignaling Mediates Atherosclerosis
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lipid deposition and lesion size was determined by Oil Red O
staining. Of note, we observed a marked reduction of thoracoabdominal lipid deposition in sgp130Fc treated mice (Figure 4B). Analysis of aortic root lesion size even revealed a
significant regression of experimental atherosclerosis under
sgp130Fc treatment (Figure 4C).
Atherosclerotic Mice and Patients With CAD Have
Lower Levels of Endogenous sgp130
As endogenous sgp130 also acts as a physiological inhibitor
of IL-6 transsignaling,28 we measured sgp130 plasma levels
in atherosclerotic Ldlr⫺/⫺ mice on a high-fat, high-cholesterol diet and in nonatherosclerotic Ldlr⫺/⫺ control mice on a
normal chow diet. We observed significantly lower levels of
endogenous sgp130 in the plasma of atherosclerotic Ldlr⫺/⫺
mice (Figure 5A). Differences in sgp130 plasma levels with
respect to different periods of high-fat, high-cholesterol diet
were not detected. In a translational approach, we measured
sgp130 in plasma samples from 155 patients undergoing
coronary angiography (for patient characteristics, see Supplemental Table IV). Remarkably, patients with stable or unstable CAD exhibited significantly lower sgp130 levels as
compared with control patients without CAD (Figure 5B). Of
note, patients with unstable CAD with a 2- or 3-vessel CAD
had even more reduced sgp130 level as compared with
patients with unstable CAD and 1-vessel disease (Figure 5C).
Especially unstable CAD patients exhibited increased IL-6
and decreased sIL-6R plasma levels. To assess the IL-6
transsignaling in particular, we calculated the IL-6/sIL-6R
ratio, which was consequently enhanced in unstable patients
(Supplemental Table V).
See supplemental materials for an expanded Results section demonstrating that (1) sgp130Fc did not influence
hepatic effects of IL-6 in vivo, and (2) inhibition of IL-6
transsignaling by sgp130Fc delayed initial SMC infiltration
in atherosclerosis-prone mice without any effects on collagen
content in advanced plaques.
Discussion
In the present study, we demonstrate for the first time that
selective pharmacological inhibition of IL-6 transsignaling
using the fusion protein sgp130Fc significantly reduces the
development and progression of atherosclerotic plaques even
when atherosclerosis is already established. The antiatherosclerotic effect of sgp130Fc treatment was associated with a
distinct decrease in the expression of endothelial adhesion
molecules and consequently reduced macrophage infiltration
into the vascular lesions. Additional mechanistic experiments
confirmed that both hepatic IL-6 signaling in vitro and
activation of the acute phase response in vivo were not
impaired by sgp130Fc. Thus, sgp130Fc counteracted the
adverse effects of hypercholesterolemia on vascular inflammation, resulting in a significant reduction of atherosclerotic
burden without compromising hepatic IL-6 effects.
As a key inflammatory cytokine, IL-6 represents an independent risk factor for CAD in humans, and treatment with
supraphysiological doses of IL-6 leads to accelerated atherosclerosis in mice.13,14,19 However, studies from different
groups using IL-6-deficient mice obtained conflicting results
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Figure 3. Inhibition of interleukin (IL)-6 transsignaling reduces macrophage but not CD3⫹ T-cell infiltration into the plaque and suppresses the expression of endothelial adhesion molecules. A, Immunohistochemical analysis of atherosclerotic lesions in the aortic root
of Ldlr⫺/⫺ mice after 8 and 16 weeks on a high-fat, high-cholesterol diet treated with soluble glycoprotein 130 (sgp130) Fc (0.5 mg/kg
IP twice weekly) or vehicle. Representative 16-week lesions from Ldlr⫺/⫺ mice stained with antibodies specific for the macrophage
marker MOMA-2 (top panels) and the quantification as relative macrophage content expressed as percentage of total plaque area (bottom panels) are shown. Scale bars⫽250 ␮m. The mean⫾SEM from 5 to 9 mice per group is presented. B, Transwell assay analyzing
the in vitro migration of macrophage-like RAW 264.7 cells toward IL-6 (200 ng/mL) with or without sgp130Fc pretreatment (50 ␮g/mL)
for 4 hours. Bar graphs indicate the relative migration expressed as percentage of control. The mean⫾SEM from 5 independent experiments performed in duplicate is presented. Inhibition of IL-6 transsignaling diminishes expression of endothelial adhesion molecules. C
and D, Immunohistochemical analysis of vascular cell adhesion molecule (VCAM)-1 (C) and intercellular adhesion molecule (ICAM)-1 (D)
expression in the aortic arch of Ldlr⫺/⫺ mice after 8 weeks on a high-fat, high-cholesterol diet treated with sgp130Fc (0.5 mg/kg IP
twice weekly) or vehicle. Representative microphotographs (top panels) and the quantification of endothelial staining expressed as percentage of total vascular surface area (bottom panels) are shown. Scale bars⫽250 ␮m. The mean⫾SEM from 5 to 6 mice per group is
presented. E, Western blot analysis of lysates from primary murine endothelial cells after stimulation with hyper-IL-6 (10 ng/mL) for 30
minutes and pretreatment of sgp130Fc (10 ␮g/mL) for 60 minutes. Membranes were incubated with an antibody specific for VCAM-1
and ICAM-1 and reprobed with an antibody specific for GAPDH. A representative Western blot from 3 independent experiments is
shown. F, Immunohistochemical analysis of atherosclerotic lesions in the aortic root of Ldlr⫺/⫺ mice after 16 weeks on a high-fat, highcholesterol diet treated with sgp130Fc (0.5 mg/kg IP twice weekly) or vehicle. Representative lesions from Ldlr⫺/⫺ mice stained with
antibodies specific for the T-cell marker CD3 (top panels) and the quantification of CD3⫹ cells per aortic root section (bottom panels)
are shown. CD3⫹ cells are indicated by white arrowheads. Scale bars⫽50 ␮m. P indicates plaque. The mean⫾SEM from 5 to 9 mice
per group is presented.
regarding the pathophysiological contribution of IL-6 to the
course of atherosclerosis.31 One major drawback of all these
studies is the investigation of atherogenesis in a context of
systemic, lifetime IL-6 deficiency, eliminating both classic
IL-6 signaling and IL-6 transsignaling.
Of note, IL-6 has also physiological properties (eg, the
acute phase response, regeneration of the epithelium in the
intestine, and metabolic control in the liver) that are mediated
predominantly by the classical IL-6 signaling via the
membrane-bound IL-6R.18,32–34 In contrast, recent studies
Schuett et al
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Figure 4. Inhibition of interleukin (IL)-6 transsignaling reduces advanced atherosclerosis. A, Schematic outline of the study on advanced
atherosclerosis. Thoracoabdominal aortas and sections of aortic roots were stained with Oil Red O. B and C, Representative aortas (B)
(scale bars⫽5 mm) and aortic root sections (C) (scale bars⫽250 ␮m) (top panels) of Ldlr⫺/⫺ mice after 12 weeks on a high-fat, highcholesterol diet and followed by another 12 weeks on a high-fat, high-cholesterol diet combined with soluble glycoprotein 130 (sgp130)
Fc treatment (0.5 mg/kg IP twice weekly) or vehicle and the respective quantification (bottom panels) of Oil Red O–positive lesion areas
expressed as percentage of total surface area of the thoracoabdominal aorta or aortic root are shown. Means are represented by horizontal bars; n⫽5 to 9 mice per group.
underscored that in all cases tested, proinflammatory functions of IL-6, such as recruitment of mononuclear cells or
inhibition of regulatory T-cell differentiation, could be inhibited by sgp130Fc, which does not affect IL-6 responses via
the membrane-bound IL-6R.26
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Thus, unspecific targeting of the IL-6 pathway in the
therapy of chronic inflammatory diseases carries the risk of
provoking serious side effects by suppressing not only the
deleterious effects of IL-6, depending especially on the IL-6
transsignaling, but also the beneficial IL-6 functions mediated
C
Figure 5. Atherosclerotic mice and patients with coronary artery disease (CAD) have lower levels of endogenous soluble glycoprotein
(sgp) 130. A, Plasma samples of Ldlr⫺/⫺ mice after feeding a high-fat, high-cholesterol diet (n⫽23) or a normal chow diet (n⫽15) were
analyzed for sgp130 by ELISA. The median and 25th to 75th percentile is presented. B, Plasma samples from 155 patients undergoing
coronary angiography were analyzed for sgp130 by ELISA. Patients were classified as controls without CAD (n⫽28), stable CAD
(n⫽40), and unstable CAD exhibiting unstable angina pectoris (AP) (n⫽39) or myocardial infarction (MI) (n⫽48). C, Subgroup analysis of
control patients without CAD (n⫽28) and patients with unstable CAD subdivided in 1-vessel (CAD 1, n⫽27), 2-vessel (CAD 2, n⫽29),
and 3-vessel (CAD 3, n⫽31) CAD. The median and 25th to 75th percentiles are presented. Patient characteristics are summarized in
Supplemental Table IV.
288
Arterioscler Thromb Vasc Biol
February 2012
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by the classical signaling.35–37 Especially, the observed increase in serum cholesterol levels is a serious disadvantage in
anti-inflammatory therapy (eg, for rheumatoid arthritis) with
anti-IL-6R antibodies (tocilizumab), which enhances the
cardiovascular risk and thereby renders this strategy ineligible for the long-term treatment of cardiovascular diseases.38,39
In contrast, sgp130Fc as the selective inhibitor of IL-6
transsignaling neither impeded acute phase response nor
resulted in deteriorated lipid metabolism, thereby offering a
drug profile more appropriate for the therapy of CAD.
Furthermore, the inhibitory potency of sgp130Fc was very
specific for IL-6 transsignaling, as the activity of other IL-6
cytokine family members, such as leukemia inhibitory factor
and IL-11, was not inhibited by sgp130Fc.
The onset of atherosclerosis is characterized by a loss of
endothelial integrity accompanied by an inflammatory activation recruiting monocytes, in particular, into the subintimal
space, which significantly contributes to the consecutive
atherosclerotic lesion formation.1,4,5 Recent studies have provided broad evidence that IL-6 is a central mediator of
vascular inflammation: IL-6 promotes the expression of
endothelial VCAM-1 and ICAM-1, as well as the secretion
of chemoattractant proteins, such as monocyte chemoattractant protein-1, and it acts as a chemoattractant itself.17,18,40,41
We demonstrated a markedly attenuated endothelial expression of VCAM-1 and ICAM-1 in atherosclerotic Ldlr⫺/⫺
mice treated with sgp130Fc. In addition, IL-6-induced upregulation of VCAM-1 and ICAM-1 was completely abolished by sgp130Fc in isolated endothelial cells. Moreover, the
recruitment of monocytes into the vessel wall with subsequent differentiation into lipid-laden macrophage foam cells
was consequently diminished under sgp130Fc therapy. This
latter finding might be attributable not only to the reduced
expression of endothelial adhesion molecules but also to the
interference of sgp130Fc with the IL-6 induced migration of
monocytic cells41 that we observed in vitro.
With regard to the adaptive immunity, it has been revealed
that IL-6 transsignaling effectively inhibits the induction of
regulatory T cells, which are known to exert atheroprotective
properties.42,43 In line with these data, we could demonstrate
that the IL-6 transsignaling in T cells was markedly inhibited
by sgp130Fc in vitro. Consequently, the extent of Foxp3
expression in the spleen as a central organ of the adaptive
immunity was significantly increased on treatment with
sgp130Fc in atherosclerotic mice. Therefore, it is tempting to
speculate that atheroprotective and atheroregressive effects of
sgp130Fc might also be mediated by a preserved induction of
regulatory T cells. However, because of the low amount
of this rare T-cell population, we were not able to detect
Foxp3⫹ cells in the aortic root of the experimental groups
after 16 weeks of a high-cholesterol diet.
In addition to our results regarding vascular monocyte
recruitment, SMCs likewise emerged to be sensitive to
sgp130Fc treatment. Thus, IL-6-induced migration and proliferation of SMCs18— both processes that are assumed to be
critical in early atherosclerosis44—were distinctly attenuated
on transsignaling blockade by sgp130Fc in vitro. In vivo, we
observed a significant reduction of SMCs within early atherosclerotic lesions in contrast to the initial increase of SMCs
in the lesions of Ldlr⫺/⫺ mice without sgp130Fc treatment.
However, SMC content and collagen deposition in advanced
lesions are not affected by sgp130Fc treatment (Supplemental
Results and Supplemental Figure VI). The fate of SMCs in
atherogenesis, though, is still unsolved, and their functions
appear rather controversial—stabilization in the cap region
versus perpetuation of inflammation within the lesion.44 – 46
However, especially in the initial phase of atherogenesis,
intimal SMCs are supposed to attract monocytes and macrophages via expression of cytokines and adhesion molecules
and consecutively trap them in the vessel wall.44 Therefore,
the beneficial effects of IL-6 transsignaling blockade by
sgp130Fc might be mainly attributable to the suppression of
monocyte recruitment and macrophage retention and the
subsequent striking prevention of atherosclerotic plaque
formation.
Finally, IL-6 represents an independent risk factor for
CAD and is abundantly found in human atherosclerotic
plaques.14,15 As shown in the present study, patients with
CAD— especially those in an unstable condition— exhibited
enhanced IL-6 transsignaling and had significantly lower
plasma levels of endogenous sgp130. Interestingly, sgp130
plasma levels even declined in proportion to advancing CAD.
Because sgp130 is responsible for the adequate control of
IL-6 transsignaling35,47 a link between increased IL-6 transsignaling and atherogenesis in humans can be proposed.
sgp130Fc has been already successfully used to treat many
murine disease models of different inflammatory conditions.
In settings of high-level inflammation, sgp130Fc treatment
ameliorated experimental colitis and inflammatory arthritis
by suppression of STAT3-activation.48,49 Furthermore,
sgp130Fc treatment was reported to abrogate inflammatory
processes in a model of moderate local inflammation induced
by injection of sterile air.27 Last but not least, even chronic
low-grade airway inflammation during allergic asthma was
inhibited by sgp130Fc.50 Of note, an optimized sgp130Fc
variant is heading toward the first human studies in 2012. All
this makes sgp130Fc a promising candidate for the treatment
of experimental and severe human atherosclerosis.
In summary, our study demonstrates that IL-6 transsignaling is
causally involved in the pathogenesis of atherosclerosis in
mice and moreover potentially linked to atherogenesis in
humans. Selective inhibition of IL-6 transsignaling by
sgp130Fc resulted in an attenuated endothelial activation and
a distinct decrease of atherosclerotic burden with lesions
containing fewer macrophages and SMCs. Importantly, all
this could be achieved without affecting the acute phase
response, weight gain, or blood cholesterol levels (Supplemental Figure VIII). Because these effects were independent
of the prevailing hypercholesterolemia, we could underscore
the importance of vascular inflammation in this context.
Thus, targeting IL-6 transsignaling with sgp130Fc represents
a promising approach for the treatment of atherosclerosis and
prevention of life-threatening CAD.
Acknowledgments
We are grateful for excellent assistance by Silke Pretzer, Mirja
Sirisko, Nathalie Stonka, Ivonne Marquardt, and Aleksandra
Bayat-Graw.
Schuett et al
IL-6 Transsignaling Mediates Atherosclerosis
Sources of Funding
This work was supported by grants from the German Research
Foundation (DFG, SFB415-B5) and the Cluster of Excellence
“Inflammation at Interfaces” (to S.R.J.), from the Netherlands
Organization for Scientific Research (VIDI Grant 917-56-358) (to
U.J.F.T.), from the Deutsche Forschungsgemeinschaft (SFB566/3B9) (to B.S.), and from the Federal Ministry of Education and
Research (01GU0711) (to K.G.).
21.
22.
23.
Disclosures
G.H.W. and D.S. are employed by CONARIS Research Institute AG
(Kiel, Germany) and are inventors on patents owned by
CONARIS. S.R.-J. has stock ownership in CONARIS and is an
inventor on patents owned by CONARIS.
24.
25.
26.
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Transsignaling of Interleukin-6 Crucially Contributes to Atherosclerosis in Mice
Harald Schuett, René Oestreich, Georg H. Waetzig, Wijtske Annema, Maren Luchtefeld, Anja
Hillmer, Udo Bavendiek, Johann von Felden, Dimitar Divchev, Tibor Kempf, Kai C. Wollert,
Dirk Seegert, Stefan Rose-John, Uwe J.F. Tietge, Bernhard Schieffer and Karsten Grote
Arterioscler Thromb Vasc Biol. 2012;32:281-290; originally published online November 10,
2011;
doi: 10.1161/ATVBAHA.111.229435
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SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
SUPPLEMENTAL MATERIAL
Supplemental Methods
Hemodynamic monitoring
Systolic blood pressure from Ldlr−/− and C57BL/6J mice was continuously monitored by tailcuff plethysmography (BP-2000, VisiTech Systems, Apex, NC, USA). Mice were adapted to
this procedure over a 7-day period prior to the experimental diet and systolic blood pressure
was further recorded weekly during the entire experiment. Furthermore, echocardiography
was performed at the end of the experiments in anesthetized mice (2% isoflurane, Hoechst,
Unterschleißheim, Germany) using a high-resolution echocardiography system (Vevo 770,
VisualSonics, Amsterdam, The Netherlands) equipped with a cardiovascular scanhead (RMV
707B, VisualSonics) as described recently.1
Tissue preparation
Preparation of mouse tissues was performed as described previously.2,
3
Briefly, after
withdrawal of blood from the right ventricle, heart and aorta were removed after perfusion
with PBS. The thoraco-abdominal aorta was fixed in 3.7% formalin for the measurement of
atherosclerotic burden. The heart, including the aortic root, and the remaining aortic arch,
liver, spleen and intestinal lymphnodes were embedded and snap-frozen in Tissue Tek OCT
(Sakura Finetek, Staufen, Germany) for histochemistry and immunohistochemistry.
Fluorescence Labeling of sgp130Fc
In order to analyze the vascular distribution of sgp130Fc, we labeled the substance using the
DyLight™ 549 Microscale Antibody Labeling Kit according to the manufacturer’s protocol
(Thermo Scientific, Rockford, IL, USA). Briefly, one vial of DyLight™ 549 Reagent was
1
SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
dissolved in 100 µg sgp130Fc solution (in PBS; PAA Laboratories, Colbe, Germany) and
purified with a resin spin column to remove excess fluor. Hypercholesterolemic Ldlr−/− mice
at the age of 16 wks were injected with fluorescence-labeled sgp130Fc (0.5 mg/kg i.p.) and
sacrificed 6 hours after injection. Subsequently, the thoraco-abdominal aorta was removed,
opened longitudinally, pinned on a black silicone-covered dish and photographed under an
epi-fluorescence microscope using appropriate filter sets (DM4000 B microscope with a
10¯/0.3 objective, DFC350 FX camera and QWin software, all Leica Microsystems, Wetzlar,
Germany).
Histochemistry and immunohistochemistry
Atherosclerotic burden of the thoracoabdominal aorta was quantified by en face oil red Ostaining, using an approach modified from Jagavelu et al.3 and Lichtman et al.4. Briefly, the
formalin-fixed thoracoabdominal aorta was stained with propylenglycol-dissolved oil red O (2
hours at room temperature) and the remaining fat was removed under a stereomicroscope
(Stemi DV4, Carl Zeiss Microimaging, Jena, Germany). Subsequently, the aorta was opened
longitudinally, pinned on a black silicone-covered dish and photographed under PBS
immersion using a CCD-camera-equipped stereomicroscope (AxioCam MRm, Carl Zeiss
Microimaging and SZ61, Olympus, Hamburg, Germany). Pictures were analysed by using
QWin imaging software (Leica Microsystems) and the percent surface area occupied by oil
red O-stained lesions was determined.
Within the aortic root, serial cryostat sections (6 µm, CM3050S, Leica Microsystems) at the
level of all 3 cusps were prepared and atherosclerotic lesions were analysed at three locations,
each separated by 120 µm. Some of these sections were stained with oil red O for the
measurement of atherosclerotic plaque size. The remaining sections were used for
immunohistochemical analysis of the atherosclerotic plaque composition. Frozen sections (5
2
SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
µm) of liver, spleen and lymphnodes were likewise prepared. Air-dried sections were fixed in
ice-cold acetone and stained with the respective antibody: anti-human IgG Fc (Rockland,
Gilbertsville, PA, USA), anti-phospho-STAT3 antibody (Tyr705, Cell Signaling Technology,
Frankfurt am Main, Germany), anti-phospho-IKKα/β (Ser176/180, Santa Cruz Santa Cruz,
Santa Cruz, CA, USA), anti-mouse macrophage/monocyte (MOMA-2, Acris Antibodies,
Herford, Germany), anti-VCAM-1 (BD Pharmingen, Heidelberg, Germany), anti-ICAM-1
(BioLegend, Uithorn, The Netherlands), anti CD3 (Abcam, Cambridge, UK), anti-Foxp3
(Frankfurt/Main, Germany) and anti-alpha-smooth muscle actin (alpha-SMA; alkaline
phosphatase conjugated, Sigma-Aldrich, Seelze, Germany). For human IgG, phospho-STAT3,
phospho-IKKα/β, MOMA-2, VCAM-1 and ICAM-1, sections were incubated with the
appropriate biotinylated secondary antibody, following incubation with horseradishperoxidase-conjugated streptavidin (both from Vector Laboratories, Burlingame, CA, USA)
and visualisation using AEC substrate chromogen (DAKO, Hamburg, Germany). For alphaSMA, visualisation was carried out with the VECTOR Red substrate kit (Vector
Laboratories). All sections were then counterstained with Mayer’s hematoxylin (Carl Roth,
Karlsruhe, Germany). Sirius red staining was used to visualize collagen as described before.5
Briefly tissue sections were incubated with 0.1% Sirius red F3BA (Sigma-Aldrich) in
saturated picric acid for 1 hour and then rinsed with 0.01 N HCl for 1 minute twice. The
sections were then dehydrated with 70% ethanol for 30 seconds. Total collagen appeared red
in bright field microscopy whereas structural mature type I collagen appeared bright orangered in polarized light microscopy.
Morphometric data were obtained using a light microscope and post-processing these pictures
with the appropriate imaging software (DM4000 B microscope with a 5¯/0.15 objective,
DFC320 camera and QWin software, all Leica Microsystems). The atherosclerotic plaque size
was determined by calculating the percentage of oil red O-positive area to the total cross-
3
SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
sectional vessel wall area. Similarly, accumulation of collagen (Sirius red-positive area),
macrophages (MOMA-2-positive area), T-Cells (CD3-positive cells) and SMCs (alpha-SMApositive area) was determined. In each case, the average value of two sections at all three
cutting sites was used for analysis. Additionally, the percentage of endothelial surface area
stained by anti-VCAM-1 and anti-ICAM-1 in the aortic arch was determined (DM4000 B
microscope, 20¯/0.5 objective and DFC320 camera, Leica Microsystems and ImageJ
software, NIH). The aortic arch was defined as from the ostium to 1 mm caudal from the left
subclavian artery branch point.
Plasma lipid analysis
Plasma samples were collected after 4 hours of fasting. Total cholesterol, triglycerides and
phospholipids were measured enzymatically on a Cobas Fara (Roche Diagnostics Systems,
Nutley, NJ, USA) using Sigma-Aldrich reagents. Cholesterol distribution among different
lipoprotein subfractions was determined following sequential tabletop ultracentrifugation as
described by Tietge et al.6
Cell culture
Hepatocytes from C57BL/6J mice were isolated as previously described.2 Briefly, 3 x 106
cells were cultured on collagen-coated 6-cm cell culture dishes (Nunc, Wiesbaden, Germany)
in DMEM (Biochrom, Berlin, Germany) containing 4.5 g/l glucose supplemented with 10%
FCS and 1% penicillin/streptomycin (both from PAA) for 24 hours. SMCs were isolated from
the aorta of C57BL/6J mice by an enzymatic dispersion method as described before.7 Cells
were cultured on collagen-coated 75 cm2 flasks (Nunc) in DMEM (Biochrom) containing 1.0
g/l glucose supplemented with 20% FCS and 1% penicillin/streptomycin, and cells at
passages 3–8 were used for successive analysis. Endothelial cells were isolated from the lung
4
SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
of C57BL/6J mice using an enzymatic dispersion method with the help of anti-CD144
antibody (VE-cadherin, BD Pharmingen) and Dynabeads (Dynal, Invitrogen) following the
manufacturer’s protocol. Cells were cultured on fibronectin-coated 25 cm2 flasks (Nunc) in
DMEM/F-12 (Gibco, Invitrogen) plus endothelial cell growth supplement containing 20%
FCS and 1% penicillin/streptomycin. Cells at passages 2–4 were used for successive analysis.
T-cells were obtained by passing spleens from C57Bl/6 WT-mice (10-12 weeks) through a
stainless mesh, following negative selection using the Pan T-cell Isolation Kit (Miltenyi
Biotech, Gladbach, Germany). The majority of these isolated cells was positive for the
common T-cell antigen CD3 (>90%). 2-3x106 cells/well were cultured in 24-well plates
(Biochrom) in RPMI (PAA) supplemented with 10% FCS, 50 µM β-mercaptoethanol and 1%
penicillin/streptomycin. As a murine monocyte/macrophage cell line RAW 264.7 cells were
used (LGC Standards, Wesel, Germany) and cultured in 75 cm2 flasks (Nunc) in RPMI (PAA)
supplemented with 10% FCS and 1% penicillin/streptomycin. All cells were starved overnight
and stimulated with 100 ng/ml IL-6 (Cell Systems, St. Katharinen, Germany) or 10 ng/ml
hyper-IL-68 for the indicated time points under serum-free conditions.
Cell migration assay
Migration of SMCs and RAW 267.4 cells was evaluated using transwell cell culture inserts
(Corning, Amsterdam, The Netherlands). Briefly, SMCs and RAW 264.7 were starved for 24
hours, and 1 x 105 (SMCs) or 2.5 x 105 (RAW 264.7) cells were placed in the upper chamber
of transwell cell culture inserts (8 µm pore size for SMCs, 5 µm pore size for RAW 264.7).
The lower chamber contained IL-6 (SMCs: 100 ng/ml; RAW 264.7: 200 ng/ml) and for some
experiments additionally sgp130Fc (SMCs: 10 µg/ml; RAW 264.7: 50 µg/ml, CONARIS
Research Institute AG, Kiel, Germany). Migration of SMCs was carried out for 48 hours at
37°C, 5% CO2. SMCs which had migrated into the lower chamber were quantified by
5
SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
counting in a Neubauer chamber. Migration of RAW 264.7 was carried out for 4 h at 37°C
and 5% CO2. Migrated RAW 264.7 cells were quantified by DAPI staining of the formalinfixed transwell inserts and counting of 15 high power fields (DM4000 B microscope with a
40¯/0.75 objective, DFC350 FX camera and QWin software, all Leica Microsystems).
Cell proliferation assay
5 x 103 SMCs/well were seeded on 96-well tissue culture plates and allowed to grow for 24 h.
After starvation for another 24 hours SMCs were stimulated with IL-6 (100 ng/ml) and for
some experiments additionally with sgp130Fc (10 µg/ml; CONARIS). Cell proliferation after
48 hours at 37°C and CO2 was measured on the basis of DNA synthesis by BrdU
incorporation with a commercial colorimetric quantification kit (Roche, Mannheim,
Germany) according to the manufacturer’s protocol. Each experimental condition was
performed in triplicate. The amount of reaction product was determined by measuring the
absorbance at 450 nm using a plate reader (µQuant, BIO-TEK, Bad Friedrichshall, Germany).
Real-time Polymerase chain reaction (PCR)
Total RNA from spleen tissue was isolated using TriFast-Reagent (peqLAB, Erlangen,
Germany) and reverse-transcribed with SuperScript reverse transcriptase (Invitrogen),
oligo(dT) primers, and deoxynucleoside triphosphates. Real-time PCR was performed in
duplicates in a total volume of 25 µl using brilliant SYBR green PCR master mixture
(Stratagene, Agilent Technoligies, Waldbronn, Germany) on a 7300 Real-Time-PCR System
(Applied Biosystems, Darmstadt, Germany) in 96-well PCR plates (Applied Biosystems).
Real-time PCR was done with an initial denaturation step at 95°C for 10 min followed by 40
PCR cycles consisting of 95°C for 15 s, 57°C for 1 min, and 72°C for 1 min, and SYBR
Green fluorescence emission were monitored after each cycle. For normalization, expression
6
SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
of β-actin was determined in parallel in duplicate. Relative gene expression was calculated
using the 2–ΔΔCT method. PCR primers were obtained from MWG Biotech (Ebersberg,
Germany), Foxp3: forward primer, 5´-CAG TCA AAG AGC CCT CAC AA-3´, reverse
primer 5´-AAG GCA GGC TCT TCA TGT TT-3´ (121 bp); β-actin: forward primer: 5´-CAT
GTA CGT AGC CAT CCA GGC-3´, reverse primer 5´-CTC TTT GAT GTC ACG CAC
GAT-3´ (229 bp).
Western blot
Proteins from cellular or tissue extracts were separated by denaturing SDS-PAGE (10%), and
transferred to a PVDF membrane (GE Healthcare, Freiburg, Germany). Transferred proteins
were probed with anti-phospho-STAT3 antibody (Tyr705, Cell Signaling Technology), antiSTAT3 antibody (Cell Signaling Technology), anti-IκB-α (Santa Cruz Biotechnology), antiVCAM-1 (R&D Systems, Wiesbaden, Germany), anti-ICAM-1 (BioLegend) and antiGAPDH (Santa Cruz Biotechnology). Visualization was accomplished using an appropriate
peroxidase-conjugated secondary antibody, ECL solution, and ECL film (Hyperfilm ECL, GE
Healthcare). Films were analyzed using an image analysis system (GeneGenius, Syngene,
Cambridge, United Kingdom) and the software Quantity One (BioRad, Munich, Germany).
ELISA
Commercial ELISAs were performed according to the manufacturers’ protocols and analysed
by a plate reader (µQuant, Bio-Tek Instruments). Plasma samples obtained from C57Bl/6J
mice were analysed for SAA (Tridelta, Maynooth, Ireland) after IL-6 (100 µg/kg i.p.) or LPS
(Sigma-Aldrich, 2 mg/kg i.p.) treatment for 24 hours with or without sgp130Fc (0.5–15 µg/kg
i.p.). Plasma samples obtained from Ldlr−/− mice after 8 and 16 wks of high-fat, highcholesterol diet with or without sgp130Fc (0.5 µg/kg i.p., twice weakly) were analyzed for IL-
7
SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
6, IL-1β, TNF-α, MCP-1, sICAM and sVCAM using an immunoassay from R&D Systems.
Plasma samples from 155 patients undergoing coronary angiography at the Department of
Cardiology at Hannover Medical School were analysed for sgp130, IL-6 and sIL-6R using an
immunoassay from R&D Systems. The study was approved by the review committee of the
Hannover Medical School and patients provided written informed consent. Patient
characteristics are summarized in Supplemental Table 4.
8
SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
Supplemental Results
Sgp130Fc does not influence hepatic effects of IL-6 in vivo
We tested if the hepatic IL-6 effects – which are probably predominantly mediated by the
classic, membrane-bound IL-6R-dependent signaling – are compromised by sgp130Fc. IL-6
is one of the key cytokines for inducing the acute phase response, leading to elevated serum
levels of hepatocyte-derived acute phase proteins such as serum amyloid A (SAA).8, 9 Upon
injection of IL-6 (100 µg/kg i.p.) mice displayed a 10-fold increase in SAA serum levels.
Notably, even high doses of sgp130Fc – up to 30-fold more than used in the atherosclerosis
study (i.e. 15 mg/kg i.p.) – did not compromise IL-6-induced SAA release (Supplemental
Figure III A). Moreover, when investigating the LPS-triggered acute phase response, which is
mediated not only by IL-6 but also by tumor necrosis factor (TNF)-α and IL-1β, sgp130Fc
treatment similarly did not impair SAA release (Supplemental Figure III B).
Inhibition of IL-6 transsignaling delays initial SMC infiltration without effecting SMC
and collagen content of advanced plaques
Another important aspect of atherosclerotic plaque development is the migration of SMCs
from the media into the lesion with subsequent proliferation and dedifferentiation.10 Keeping
in mind that these processes might also be modulated by IL-6, we tested if sgp130Fc could
also abrogate the detrimental influence of IL-6 on SMCs. Interestingly, in the atherosclerosisprone Ldlr−/− mice, predominantly the initial infiltration of SMCs into the plaque after 8 wks
of diet was attenuated by sgp130Fc treatment (0.5 mg/kg i.p. twice weekly), whereas no effect
of IL-6 transsignaling blockade on SMC content after 16 wks of diet in advanced plaques was
detectable (Supplemental Figure VI A). SMCs are a major source of collagen synthesis within
atherosclerotic plaques which may improve plaque stability and influence the development of
atherosclerotic complications in patients.11 However, Sirius red staining revealed no
9
SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
significant difference in total collagen content as well as in mature collagen fibres in
advanced plaques of sgp130Fc-treated mice as assessed by bright field and polarized light
microscopy, respectively (Supplemental Figure VI B). Mechanistic in vitro experiments with
SMCs provided evidence that both migration towards IL-6 and proliferation upon IL-6
application were distinctly enhanced in cultured SMCs. Both IL-6-dependent effects were in
turn significantly inhibited by pretreatment of the cells with sgp130Fc (Supplemental Figures
VI C and VI D). An effect of sgp130Fc, that is presumably responsible for reduced SMC
content in early plaques (Supplemental Figure VI A).
10
SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
Supplemental Table I. Plasma lipid analysis of Ldlr-/- mice fed a high-cholesterol diet.
mg/dl
control
sgp130Fc
P
triglycerides
569 ± 114
516 ± 87
n.s.
phospholipids
703 ± 108
779 ± 63
n.s.
total cholesterol
1310 ± 225
1310 ± 225
n.s.
VLDL* cholesterol
642 ± 110
630 ± 53
n.s.
LDL† cholesterol
314 ± 54
329 ± 28
n.s.
HDL‡ cholesterol
107 ± 18
112 ± 9
n.s.
Plasma lipids and plasma cholesterol subfractions after 16 wks of high-cholesterol diet were
not significantly different between the control and sgp130Fc group (VLDL* = very low
density lipoprotein, LDL† = low density lipoprotein, HDL‡ = high density lipoprotein). The
mean ± SEM from 6–9 mice per group is presented.
11
SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
Supplemental Table II. Hemodynamic monitoring of Ldlr-/- mice fed a high-cholesterol
diet.
control
sgp130Fc
P
systolic blood pressure (mmHg)
115.8 ± 13.3
113.9± 13.1
n.s.
heart rate (beats / min)
446.8 ± 26.1
433.8 ± 14.6
n.s.
LVEDD* (mm)
3.8 ± 0.2
3.8 ± 0.2
n.s.
LVESD† (mm)
3.0 ± 0.2
3.0 ± 0.3
n.s.
LVEDV (µl)
29.0 ± 3.8
28.5 ± 1.2
n.s.
ejection fraction (%)
44.9 ± 7.2
49.5 ± 10.7
n.s.
stroke volume (µl)
13.1 ± 2.9
14.1 ± 2.9
n.s.
cardiac output (ml / min)
5.9 ± 1.7
6.1 ± 1.2
n.s.
‡
Systolic blood pressure and echocardiographic parameters after feeding a high-cholesterol
diet were not significantly different between the control and sgp130Fc group (LVEDD* = left
ventricular enddiastolic diameter, LVESD† = left ventricular endsystolic diameter, LVEDV‡ =
left ventricular enddiastolic volume). The mean ± SEM from 6–9 mice per group is presented.
12
SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
Supplemental Table III. Cytokine plasma levels of Ldlr-/- mice fed a high-cholesterol diet.
8 wks of diet
16 wks of diet
control
sgp130Fc
P
control
sgp130Fc
P
IL-6
33.3 ± 2.3
28.7 ± 3.7
n.s.
38.7 ± 3.3
40.0 ± 3.7
n.s.
IL-1β
27.7 ± 2.2
26.2 ± 1.5
n.s.
24.1 ± 1.8
28.6 ± 3.3
n.s.
TNF-α
98.6 ± 3.7
101.1 ± 3.9
n.s.
91.6 ± 2.1
94.4 ± 1.6
n.s.
MCP-1
319.4 ± 7.3
349.0 ± 23.4
n.s.
213.3 ± 28.2
285.5 ± 23.8
n.s
sICAM
579.0 ± 28.6
587.5 ± 55.3
n.s.
560.2 ± 45.5
489.7 ± 18.4
n.s.
sVCAM
941.7 ± 38.5
999.7 ± 57.6
n.s.
1019.2 ± 90.5
987.9 ± 27.9
n.s.
pg/ml
ng/ml
Cytokine and soluble adhesion molecule plasma levels after 8 and 16 wks of high-fat, highcholesterol diet were not significantly different between the control and sgp130Fc group. The
mean ± SEM from 5–9 mice per group is presented.
13
SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
Supplemental Table IV. Patient characteristics
age
n
male / female
(median; 25th/75th percentile)
control
28
57 (53.5-66.5)
16 / 12
stable CAD
40
70 (66.75-75.5)
30 / 10
unstable CAD
39
69 (55.0-75.25)
30 / 9
48
68 (58.0-76.6)
33 / 15
- unstable AP
unstable CAD
- MI
155 patients undergoing coronary angiography were classified as controls without CAD,
stable CAD and unstable CAD exhibiting unstable angina pectoris (AP) or myocardial
infarction (MI).
14
SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
Supplemental Table V. Plasma levels of IL-6 signaling components in patients
sgp130
IL-6
sIL-6R
(ng/ml)
(pg/ml)
(ng/ml)
IL-6/sIL-6R
n
(median; 25th/75th percentile)
control
28
289.2 (274.0-305.1)
6.2 (5.0-8.7)
53.5 (42.0-66.4)
0.13 (0.08-0.17)
stable CAD
40
262.5 (244.4-277.6)**
3.2 (3.2-4.2)*
59.8 (51.2-75.1)
0.06 (0.04-0.08)*
39
258.1 (225.4-277.5)**
6.2 (3.8-8.7)#
46.0 (37.3-62.9)#
0.12 (0.09-0.2)#
- MI
48
264.0 (239.6-279.9)**
8.0 (5.0-19.4)# 49.5 (42.1-57.0)#
0.16 (0.11-0.40)#
control
28
289.2 (274.0-305.1)
6.2 (5.0-8.7)
53.5 (42.0-66.4)
0.13 (0.08-0.17)
CAD 1
27
275.5 (266.7-292.9)
7.5 (5.0-13.7)
55.7 (43.7-63.3)
0.16 (0.09-0.26)
CAD 2
29
246.8 (228.3-266.3)**,§
6.2 (5.9-22.2)
45.0 (37.1-55.5)
0.15 (0.10-0.46)
CAD 3
31
253.1 (227.3-267.0)∗*,§
7.4 (5.0-10.0)
46.3 (41.4-54.2)
0.16 (0.10-0.21)
unstable CAD
- unstable AP
unstable CAD
*P<0.05 vs. control; **P<0.05 vs. control, #P<0.05 vs. stable CAD; §P<0.001 vs. CAD 1
Plasma samples from 155 patients were analyzed for sgp130, IL-6, sIL-R. To assess IL-6
transsignaling the IL-6/sIL-6R ratio was calculated. Upper part of Supplemental Table 5:
Patients were classified as controls without CAD, stable CAD and unstable CAD exhibiting
unstable angina pectoris (AP) or myocardial infarction (MI). Lower part of Supplemental
Table 5: Patients were classified as controls without CAD or with unstable CAD with one(CAD 1), two- (CAD 2) or three-vessel CAD (CAD 3)
15
SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
Supplemental References
1.
Doerries C, Grote K, Hilfiker-Kleiner D, Luchtefeld M, Schaefer A, Holland SM,
Sorrentino S, Manes C, Schieffer B, Drexler H, Landmesser U. Critical role of the
NAD(P)H oxidase subunit p47phox for left ventricular remodeling/dysfunction and
survival after myocardial infarction. Circ Res. 2007; 100:894-903.
2.
Luchtefeld M, Schunkert H, Stoll M, Selle T, Lorier R, Grote K, Sagebiel C, Jagavelu
K, Tietge UJ, Assmus U, Streetz K, Hengstenberg C, Fischer M, Mayer B, Maresso K,
El Mokhtari NE, Schreiber S, Muller W, Bavendiek U, Grothusen C, Drexler H,
Trautwein C, Broeckel U, Schieffer B. Signal transducer of inflammation gp130
modulates atherosclerosis in mice and man. J Exp Med. 2007; 204:1935-1944.
3.
Jagavelu K, Tietge UJ, Gaestel M, Drexler H, Schieffer B, Bavendiek U. Systemic
deficiency of the MAP kinase-activated protein kinase 2 reduces atherosclerosis in
hypercholesterolemic mice. Circ Res. 2007; 101:1104-1112.
4.
Lichtman AH, Clinton SK, Iiyama K, Connelly PW, Libby P, Cybulsky MI.
Hyperlipidemia and atherosclerotic lesion development in LDL receptor-deficient
mice fed defined semipurified diets with and without cholate. Arterioscler Thromb
Vasc Biol. 1999; 19:1938-1944.
5.
Junqueira LC, Cossermelli W, Brentani R. Differential staining of collagens type I, II
and III by Sirius Red and polarization microscopy. Arch Histol Jpn. 1978; 41:267-274.
6.
Tietge UJ, Maugeais C, Cain W, Grass D, Glick JM, de Beer FC, Rader DJ.
Overexpression of secretory phospholipase A(2) causes rapid catabolism and altered
tissue uptake of high density lipoprotein cholesteryl ester and apolipoprotein A-I. J
Biol Chem. 2000; 275:10077-10084.
7.
Grote K, Flach I, Luchtefeld M, Akin E, Holland SM, Drexler H, Schieffer B.
Mechanical stretch enhances mRNA expression and proenzyme release of matrix
16
SUPPLEMENTAL MATERIAL
IL-6 Transsignaling mediates Atherosclerosis
metalloproteinase-2 (MMP-2) via NAD(P)H oxidase-derived reactive oxygen species.
Circulation Research. 2003; 92:e80-86.
8.
Uhlar CM, Whitehead AS. Serum amyloid A, the major vertebrate acute-phase
reactant. Eur J Biochem. 1999; 265:501-523.
9.
Jensen LE, Whitehead AS. Regulation of serum amyloid A protein expression during
the acute-phase response. Biochem J. 1998; 334:489-503.
10.
Doran AC, Meller N, McNamara CA. Role of smooth muscle cells in the initiation
and early progression of atherosclerosis. Arterioscler Thromb Vasc Biol. 2008;
28:812-819.
11.
Adiguzel E, Ahmad PJ, Franco C, Bendeck MP. Collagens in the progression and
complications of atherosclerosis. Vasc Med. 2009; 14:73-89.
17
Supplemental Figure I
p-STAT3
STAT3
IL-6:
hyper IL-6:
-
+
-
+
Supplemental Figure I. No effect of IL-6 on STAT3 phosphorylation in endothelial cells in vitro. Western blot
analysis of lysates from murine endothelial cells after stimulation with IL-6 (100 ng/ml) and hyper-IL-6 (10 ng/ml).
Membranes were incubated with an antibody specific for p-STAT3 and reprobed with an antibody specific for
STAT3. A representative western blot from three independent experiments is shown.
Supplemental Figure II
B
A
p-STAT3
p-STAT3
STAT3
STAT3
LIF:
sgp130Fc:
-
+
-
+
+
-
IL-11:
sgp130Fc:
-
+
-
+
+
-
Supplemental Figure II. No effect of sgp130Fc on LIF- and IL-11-induced STAT3 phosphorylation in vitro. A and
B, Western blot analyses of lysates from primary murine smooth muscle cells after stimulation with LIF (A, 25
ng/ml) or IL-11 (B, 25 ng/ml) and pretreatment with sgp130Fc (10 µg/ml). Membranes were incubated with an
antibody specific for p-STAT3 and reprobed with an antibody specific for STAT3. Representative western blots
from six independent experiments are shown.
Supplemental Figure III
B
350
350
300
300
P<0.01
250
SAA [µg/mL]
SAA [µg/mL]
A
200
150
100
50
250
200
150
100
50
0
IL-6:
sgp130Fc [µg/ml]:
P<0.01
0
-
+
-
+
0.5
+
3.0
+
15
15
LPS:
sgp130Fc [µg/ml]:
-
+
-
+
0.5
+
3.0
+
15
15
Supplemental Figure III. Sgp130Fc does not influence hepatic effects of IL-6 in vivo. Plasma samples obtained
from C57Bl/6J mice after A, IL-6 (100 µg/kg i.p.) or B, LPS (2 mg/kg i.p.) treatment for 24 hours with or without
sgp130Fc (0.5–15 mg/kg i.p.) were analysed for SAA using a commercial ELISA. The mean ± SEM from six
independent experiments is presented.
Supplemental Figure IV
Dye control
oil red O
DyLightTM
Supplemental Figure IV. Dye control for sgp130Fc labeling. Aortas of hypercholesterolemic Ldlr−/− mice were
washed and prepared en face 6 h after injection of DyLight™ alone. Fluorescence images (right bottom panel)
were recorded in the aorta before oil red O staining (top panel and magnification left bottom panel). Scale bar: 2.5
mm (top panel) and 250 µm (bottom panels), respectively. One representative experiment is shown.
Supplemental Figure V
sgp130Fc
control
liver
spleen
spleen
lymph node
lymph node
anti-human Fc
anti-human Fc
anti-human Fc
liver
Supplemental Figure V. Deposition of sgp130Fc in liver, spleen and lymph node. Immunohistochemical
analysis of sgp130Fc deposition with anti-human Fc antibodies in liver, spleen and lymph node of Ldlr–/– mice
after 16 wks on a high fat, high cholesterol diet treated with sgp130Fc (0.5 mg/kg i.p. twice weekly) or vehicle.
Scale bar: 50 µm. One representative experiment is shown.
Supplemental Figure VI
liver tissue
p-STAT3
STAT3
IL-6:
sgp130Fc:
-
+
-
+
+
Supplemental Figure VI. No effect of sgp130Fc on IL-6-induced STAT-3 phosphorylation in liver tissue. Western
blot analysis of lysates from murine liver tissue 3 hours after IL-6 treatment (100 µg/kg i.p.) and pretreatment with
sgp130Fc (0.5 µg/kg i.p.) for 24 hours. Membranes were incubated with an antibody specific for p-STAT3 and
reprobed with an antibody specific for STAT3. A representative western blot from three independent experiments
is shown.
Foxp3 mRNA expression/
β-actin (2–ΔΔCT)
Supplemental Figure VII
spleen tissue
3.0
2.5
2.0
P<0.05
1.5
1.0
0.5
0
control
sgp130Fc
Supplemental Figure VII. Inhibition of IL-6 transsignaling enhances Foxp3 mRNA expression in spleen tissue.
Real-time PCR of lysates from spleen tissue of Ldlr–/– mice after 16 wks on a high fat, high cholesterol diet
treated with sgp130Fc (0.5 mg/kg i.p. twice weekly) or vehicle. The mean ± SEM from five independent
experiments is presented.
Supplemental Figure VIII
A
sgp130Fc
control
B
control
sgp130Fc
8
6
(bright field)
n.s.
60
n.s.
40
30
20
10
0
total collagen mature collagen
(bright field)
2
16 weeks
P<0.001 P<0.001
300
200
100
IL
-6
co
n
tro
l
+
I
L
sg
p1 6
30
F
sg
p1 c
30
Fc
0
smooth muscle cells
2.0
P<0.01 P<0.05
1.6
1.2
0.8
0.4
0
tro
l
+
sg ILp1 6
3
sg 0Fc
p1
30
Fc
400
D
-6
smooth muscle cells
co
n
8 weeks
(polarized light)
IL
0
control
sgp130Fc
50
4
C
migration (% of control)
Sirius red staining (%)
10
Sirius red
control
sgp130Fc
P<0.05
(polarized light)
12
BrdU-incorporation (OD450nm)
α-SMA staining (%)
α-SMA
Sirius red
70
Supplemental VIII. Inhibition of IL-6 transsignaling delays initial smooth muscle cell (SMC) infiltration without
effecting SMC and collagen content of advanced plaques. A, Immunohistochemical analysis of atherosclerotic
lesions in the aortic root of Ldlr–/– mice after 8 and 16 wks on a high fat, high cholesterol diet treated with
sgp130Fc (0.5 mg/kg i.p. twice weekly) or vehicle. Representative 8-wk lesions from Ldlr–/– mice stained with
antibodies specific for alpha-smooth muscle actin (α-SMA; top panels) and the quantification of relative SMC
content expressed as percentage of total plaque area (bottom panels) are shown. Scale bar: 250 µm. The
mean ± SEM from 5–9 mice per group is presented. B, Collagen content analysis of atherosclerotic lesions in
the root of aortic root of Ldlr–/– mice after 16 wks on a high fat, high cholesterol diet treated with sgp130Fc (0.5
mg/kg i.p. twice weekly) or vehicle. Representatives lesion stained with Sirius red and analyzed with bright field
(total collagen) or polarized light (left panels) and the quantification of relative collagen content expressed as
percentage of total plaque area (right panel) are shown. Scale bar: 250 µm, The mean ± SEM from 5–9 mice
per group is presented. C, Transwell assay analyzing the in vitro migration of smooth muscle cells towards IL-6
(100 ng/ml) with or without sgp130Fc pretreatment (10 µg/ml) for 48 hours. Bar graphs indicate the relative
migration expressed as percentage of control. The mean ± SEM from 4–8 independent experiments performed
in duplicate is presented. D, BrdU cell proliferation assay analyzing the IL-6-induced (100 ng/ml) proliferation of
smooth muscle cells in vitro with or without sgp130Fc pretreatment (10 µg/ml). Bar graphs indicate the
OD450nm. The mean ± SEM from 4–8 independent experiments is presented.
Supplemental Figure IX
IL-6
inhibition of IL-6
transsignaling
by sgp130Fc
• hepatic acute phase response
• endothelium activation
• monocyte recruitment
• SMC migration/proliferation
• serum lipid levels
• weight control
Supplemental Figure IX. Sgp130Fc inhibits pro-atherosclerotic vascular processes without affecting
physiological properties of IL-6.