TRAF-1, -2, -3, -5, and -6 Are Induced in Atherosclerotic
Plaques and Differentially Mediate Proinflammatory
Functions of CD40L in Endothelial Cells
Andreas Zirlik, Udo Bavendiek, Peter Libby, Lindsey MacFarlane, Norbert Gerdes, Joanna Jagielska,
Sandra Ernst, Masanori Aikawa, Hiroyasu Nakano, Erdyni Tsitsikov, Uwe Schönbeck
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Objective—Several lines of evidence implicate CD40 ligand (CD40L, CD154) as a mediator and marker of atherosclerosis.
This study investigated the involvement of tumor necrosis factor receptor-associated factors (TRAFs) in CD40 signaling
in endothelial cells (ECs) and their expression in atheromata and cells involved in atherogenesis.
Methods and Results—CD40L enhanced the basal expression of TRAF-1, -2, -3, and 6, but not TRAF-5 in ECs. TRAFs
associated with CD40 on ligation by CD40L. Study of ECs from TRAF-1, -2, and -5– deficient mice demonstrated
functional involvement of TRAFs in proinflammatory CD40 signaling. Whereas TRAF-1 deficiency enhanced
CD40L-induced IL-6 and MCP-1 expression, TRAF-2 and TRAF-5 deficiency inhibited CD40L-inducible IL-6 but not
MCP-1 expression. Gene silencing in human ECs further delineated functions of TRAFs in CD40 signaling. TRAF-3
silencing in ECs showed increased CD40L-induced IL-6, MCP-1, and IL-8 expression, whereas TRAF-6 silencing
increased selectively CD40L-induced MCP-1 expression. Enhanced TRAF levels in atherosclerotic lesions further
supports involvement of members of this family of signaling molecules in arterial disease.
Conclusions—These results implicate endothelial TRAF-1, -2, -3, -5, and -6 in CD40 signaling in atherogenesis,
identifying these molecules as potential targets for selective therapeutic intervention. (Arterioscler Thromb Vasc Biol.
2007;27:1101-1107.)
Key Words: atherosclerosis 䡲 CD40L 䡲 inflammation 䡲 signaling 䡲 TRAF
E
arly work on CD40/CD40L interactions focused on their
pivotal role in T-cell– dependent humoral immunity.1–3
Research during the past decade, however, revealed that
expression of the CD40/CD40L dyad extends beyond lymphocytes, to endothelial cells (ECs), smooth muscle cells
(SMCs), and macrophages, cells resident in atherosclerotic
plaques.4,5 Thus, the pathological functions of CD40/CD40L
interactions transcend B lymphocyte proliferation/differentiation and immunoglobulin class switching. Stimulation of
leukocytes and nonleukocytic cells with CD40L induces the
expression of a plethora of proinflammatory mediators, including cytokines, chemokines, adhesion molecules, matrix
degrading enzymes, and procoagulants.6 –11 In vivo studies
verified a crucial role for CD40/CD40L interactions in
numerous chronic inflammatory diseases, including rheumatoid arthritis, complications of transplantation, cancer, and
atherosclerosis.3,12–15 In accord with a pivotal role for
CD40L in atherogenesis, enhanced plasma levels of solu-
ble CD40L predict future cardiovascular events in certain
patient populations16,17 and correlate with several cardiovascular risk factors in pilot studies.18,19 Surprisingly, transplantation of either wild-type or CD40L-deficient bone marrow
into low-density lipoprotein receptor-deficient mice revealed
that CD40L from hematopoietic cells does not contribute to
atherogenesis in mice, suggesting that ECs and other resident
vascular cells contribute to CD40 signaling in arterial disease.20 However, knowledge regarding underlying signaling
pathways that mediate proatherogenic functions after CD40
engagement in ECs, a key cell type in vascular disease,
remains limited. Identification of such pathways has considerable importance, because systemic inhibition of CD40L
may have adverse consequences, given this mediator’s pivotal function in host defenses.3,12,21
Previous studies focused on CD40 signaling pathways in B
lymphocytes. Kinase pathways and tumor necrosis factor
(TNF) receptor-associated factors (TRAFs), cytoplasmic
Original received February 6, 2006; final version accepted February 7, 2007.
From Donald W. Reynolds Cardiovascular Research Center (A.Z., U.B., L.M., N.G., M.A., P.L., U.S.), Department of Medicine, Brigham and
Women’s Hospital and Harvard Medical School, Boston, Mass; Department of Cardiology (A.Z., S.E.), University of Freiburg, Germany; Department
of Cardiology (U.B., J.J.), Hannover Medical School, Hannover, Germany; Department of Immunology (H.N.), Juntendo University, School of Medicine,
Tokyo, Japan; Department of Immunology (E.T.), Children’s Hospital, Harvard Medical School, Boston, Mass; Cardiovascular Disease (U.S.),
Boehringer Ingelheim Pharmaceuticals, Inc, Ridgefield, Conn.
A.Z. and U.B. contributed equally to this work and both should be considered first authors.
Correspondence to Peter Libby, MD, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, NRB-741, Boston, MA 02115. E-mail [email protected].
harvard.edu.
© 2007 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
1101
DOI: 10.1161/ATVBAHA.107.140566
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adaptor proteins that mediate cytokine signaling of members
of the TNF-1, Toll-like-1, and IL-1 receptor superfamilies,
may participate in CD40 signaling.22,23 The role of TRAFs in
ECs remains virtually unexplored. Therefore, this study
tested the hypothesis that TRAF-1, -2, -3, -5, and -6 function
in CD40L-induced proinflammatory signaling in human and
murine primary ECs and whether such signaling functions
vary among different CD40L-induced genes in ECs and for
the same target gene even among different cell types. We also
investigated TRAF expression in murine and human atherosclerotic plaques.
(Figure 1). In contrast, stimulation with interferon-␥ or
transforming growth factor-␤ did not change the expression
of any TRAF tested in these cell types (Figure 1). In
macrophages TNF-␣ stimulation only enhanced TRAF-1
expression. Basal TRAF-5 expression was most prominent in
macrophages and could be significantly enhanced by stimulation with interferon-␥. The induction of TRAF-1, -2, -3, and
-6 expression by CD40L depended on time and concentration,
requiring a minimum of 4 hour of stimulation with ⱖ0.1
␮g/mL CD40L (supplemental Figure IA and IB, available
online at http://atvb.ahajournals.org).
Materials and Methods
CD40 Ligation Triggers Association of TRAF-1, -2, -3, -5,
and -6 With CD40 in Human Vascular ECs and
Recruitment of TRAFs to the Plasma Membrane
CD40 signaling in ECs indeed utilizes TRAFs, as determined
by immunoprecipitation with an anti-CD40 antibody from
lysates of ECs cultures followed by immunoblot analysis of
the precipitates with the respective TRAF antibody. Ligation
of CD40 on ECs triggered its association with TRAF-1, -3,
and -6 from 15 to 60 minutes through 16 hours (Figure 2A)
in accord with the increased expression of these TRAFs after
CD40L stimulation. Although CD40L also triggered the
association of TRAF-2 and TRAF-5 with CD40 in ECs, this
interaction peaked at 60 minutes and declined to baseline
levels within 16 hours. Stimulation of ECs with CD40L also
enhanced TRAF expression in the plasma membrane fractions (supplemental Figure II).
Human saphenous vein ECs and human macrophages were isolated,
cultured, and stimulated with various cytokines. Expression of
TRAF1– 6 was assayed by Western blotting of whole-cell lysates and
lysates of subcellular protein fractions as described previously.24
TRAF protein expression was also quantified in lysates from normal
and diseased human carotid arteries by Western blotting and in
murine aortic sections by immunohistochemistry. Murine ECs isolated from TRAF wild-type and TRAF-deficient mice, as well
human umbilical vein ECs, silenced for the respective TRAFs were
stimulated with TNF-␣ and CD40L and assayed for IL-6, IL-8, and
MCP-1 by enzyme-linked immunosorbent assay (for detailed methods please see http://atvb.ahajournals.org).
Results
CD40L Enhances the Expression of TRAF-1, -2, -3, -5,
and -6 Differentially in Human Vascular ECs, SMCs,
and Macrophages
All cell types expressed TRAF-1, -2, -3, -5, and -6 constitutively, as determined by Western blot analysis of protein
extracts. Ligation of CD40 enhanced the total protein expression of TRAF-1, -2, -3, and -6, but not TRAF-5 in ECs. In
SMCs and macrophages, stimulation with CD40L only enhanced TRAF-1 expression (Figure 1). Notably, other proinflammatory cytokines modulated expression of these TRAFs
differently. Stimulation with IL-1␤ or TNF-␣ induced expression of TRAF-1, -3, and -6 in ECs and SMCs, whereas neither
stimulus affected the constitutive expression of TRAF-2 or -5
Enhanced Expression of TRAF-1, -2, -3, -5 and -6 in
Atherosclerotic Compared With Nondiseased
Arterial Tissue
To determine tissue expression of TRAFs, protein extracts of
nondiseased, atherosclerotic (dichotomized into fibrous or
atheromatous plaques as described previously24), or aneurysmal arteries were analyzed by Western blotting. Nondiseased tissue had barely detectable TRAF-1, -2, -3, -5, and -6
expression with the highest amounts of TRAF-6 (Figure 2B).
In contrast, diseased tissues contained all TRAFs. Compared
Figure 1. CD40L and other proinflammatory cytokines modulate TRAF-1, -2, -3,
-5, and -6 differentially in ECs, SMCs, and
macrophages. Lysates of ECs, SMCs, and
macrophages, incubated with serum-free
medium alone (None) or human recombinant IL-1␤ (10 ng/mL), TNF-␣ (50 ng/mL),
interferon-␥ (1000 U/mL), CD40L (10
␮g/mL), or transforming growth factor-␤ (20
ng/mL), were analyzed by Western blotting
using the corresponding TRAF antibody. For
control purposes, lysates of PMA-activated
Jurkat cells were applied (Jurkat).
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TRAFs and CD40 Signaling in ECs
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Figure 2. TRAF-1, -2, -3, -5, and -6 associate with CD40 in ECs and are overexpressed in atherosclerotic and aneurysmal arterial
tissue. A, Lysates of ECs incubated for the respective duration with CD40L (10 ␮g/mL) were applied either directly or after immunoprecipitation (IP) with anti-CD40 antibody to Western blot analysis with respective TRAF antibody. For control purposes, lysates of
PMA-activated Jurkat cells were applied (Jurkat). Three independent experiments yielded similar results. B, Extracts of nondiseased
(Normal), atherosclerotic (dichotomized into fibrous and atheromatous plaques), or aneurysmal (AAA) specimens were analyzed by
Western blots using the indicated TRAF antibodies. Two out of 6 individual experiments performed per group are shown (labeled as 1
and 2) as representative blots.
with fibrous plaques, atheromatous plaques contained significantly more TRAF-2 and TRAF-3 protein (supplemental
Figure III). However, TRAF-5 abounded in fibrous lesions.
Mouse atherosclerotic lesions also contain TRAF-1, -2, -3, -5,
and -6 as shown by immunohistochemical study of longitudinal sections of aortas from low-density lipoprotein
receptor-deficient mice fed a high-cholesterol diet for 16
weeks (Figure 3). Sections from aortas from mice of similar
age fed a regular low-fat diet showed little or no TRAF
staining (data not shown). All TRAFs colocalized with both
ECs and macrophages as assessed by immunohistochemical
study of Murine atheromata (supplemental Figure IV).
Distinct Roles for TRAF-1, -2, and -5 in CD40L-Induced
IL-6 and MCP-1 Expression in Primary Murine ECs
To examine the functional relevance of TRAF-1 for proinflammatory CD40 signaling, we isolated ECs from TRAF-1
wild-type and TRAF-1– deficient mice and analyzed supernatants for an inflammatory response to CD40L. Exposure of
CD40L enhanced the constitutive production of IL-6 and
MCP-1 protein (Figure 4A and 4B). Relative levels of IL-6
and MCP-1 release in TRAF-1– deficient ECs exceeded those
from wild-type ECs, indicating that TRAF-1 limits CD40
signaling.25 Stimulation with TNF-␣ also resulted in a relative
increase of cytokine expression in TRAF-1– deficient ECs
compared with wild-type cells (Figure 4A and 4B).
In contrast to TRAF-1, deficiency of either TRAF-2- or
TRAF-5 diminished CD40L-inducible and TNF-␣–inducible
IL-6 expression (Figure 4C). TRAF-2/-5 compound deficiency did not result in a significantly greater reduction in
CD40L-inducible or TNF-␣–induced IL-6 expression compared with single gene deficiency (Figure 4C). TRAF-2
and/or TRAF-5 deficiency did not modulate CD40L-induced
or TNF-␣–induced MCP-1 expression (Figure 4D), demonstrating distinct regulation of CD40 signaling for different
target genes.
Because previous reports implicated TRAF-1 particularly in the regulation of apoptosis, we investigated
whether TRAF-dependent modulation of cytokine expression depends on apoptosis and cell viability.25 Caspase-3/7
expression did not differ between TRAF-1– deficient cells
and wild-type controls. Supernatants from TNF-␣–stimulated TRAF-5– deficient cells had even lower caspase-3/7
expression than in corresponding controls, suggesting lack
of dependence of expression of these mediators of apoptosis in these cells (supplemental Figure VA). Similarly,
TRAF-deficient and wild-type Murine cells had comparable cell viability as assessed by lactate dehydrogenase
(LDH) release into the supernatant. Only TNF-␣–stimulated TRAF-2/-5 double-deficient cells showed an increased rate of cytotoxicity (supplemental Figure VB). To
ensure that the results obtained in ECs from various tissues
can represent those from arterial tissue, we verified some
of our findings in ECs isolated from 8 pooled aortas per
group (supplemental Figure VIA to VID).
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and IL-8 but not MCP-1 compared with anti-lamin–transfected controls, corroborating the concept that this molecule
can limit certain proinflammatory aspects of CD40 signaling
in ECs. ECs treated with TRAF-5 siRNA showed decreased
IL-6 but increased IL-8 expression (supplemental Figure
VII). Silencing of TRAF-3 in ECs supported an inhibitory
role of TRAF-3 in CD40L-induced proinflammatory gene
expression, because cells treated with anti-TRAF-3 siRNA
showed an increased basal and CD40L-stimulated expression
of all 3 proteins. Endothelial cells silenced for TRAF-6
released similar amounts of IL-6 and IL-8 and an increased
amount of MCP-1 (Figure 5).
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TRAF-1, -2, and -5 Differentially Mediate
Proinflammatory Functions of CD40L in Various
Cell Types
To test the hypothesis that proinflammatory functions of
CD40L not only show target gene selectivity but also differ
for the same target gene in various cell types, we isolated
peritoneal macrophages from TRAF wild-type and TRAFdeficient animals. Similar to the observations in ECs, TRAF1-deficient macrophages expressed higher levels of IL-6 and
MCP-1 on stimulation with CD40L compared with corresponding wild-type controls. Also, neither TRAF-2 nor
TRAF-5 affected CD40L-inducible MCP-1 expression. However, in contrast to ECs, TRAF-5 deficiency did not affect
CD40L-induced or TNF-␣–induced IL-6 expression, whereas
TRAF-5 deficiency combined with TRAF-2 heterozygosity
effectively reduced CD40L-induced and TNF-␣–induced
IL-6 expression compared with wild-type controls (supplemental Figure VIII).
Discussion
Figure 3. TRAF-1, -2, -3, -5, and -6 are overexpressed in
murine atherosclerotic lesions. Longitudinal sections of aortic
arches from low-density lipoprotein receptor-deficient mice fed
a high-cholesterol diet for 16 weeks are shown at 10⫻ and 40⫻
magnification. Immunohistochemical analysis employed respective TRAF antibodies and counterstaining with hematoxylin.
Three independent experiments yielded similar results.
Silencing of TRAFs by siRNA Implicates TRAF-1, -2, -3,
-5, and -6 in CD40 Signaling in Human ECs
Primary human umbilical vein ECs transfected with TRAF1– directed siRNA released more IL-6 and MCP-1 but significantly less IL-8 on stimulation with CD40L than those
transfected with lamin-directed siRNA, demonstrating that
TRAF functions vary for different target genes (supplemental
Figure VII). Transfection with TRAF-2– directed siRNA
significantly reduced CD40L-stimulated expression of IL-6
This study demonstrates that CD40L and other proinflammatory cytokines differentially modulate TRAF-1, -2, -3, -5, and
-6 expression in ECs, SMCs, and macrophages, key cell types
in arteries and arterial disease. The results also establish the
functional relevance of these TRAFs for proinflammatory
signaling events in ECs, and hence in inflammatory vascular
diseases such as atherosclerosis.
To date, analysis of TRAF functions after CD40 ligation
focused on lymphoid cells and generated inconsistent results,3,22,25 caused in part by diverse methods and cell typespecific and target gene-specific differences in signal transduction mediated by CD40L. Indeed, CD40 signaling may
even induce different pathways in the same cell type depending on the stage of differentiation.23,26 The present data
demonstrate that CD40L uses TRAF-1, -2, -3, -5, and -6
differentially for IL-6, IL-8, and MCP-1 expression in ECs,
supporting the concept of target gene-dependent CD40 signaling. Furthermore, TRAF-associated signaling induced by
CD40L differs from activation pathways used by other
proinflammatory cytokines, as indicated by the distinct modulation of TRAF expression by CD40L compared with IL-1␤,
TNF-␣, transforming growth factor-␤, and interferon-␥. In
that context, our group recently described that ligation of
CD40 on ECs activates Egr-1, a transcription factor not
altered by TNF-␣ and IL-1␤ in this cell type.27 Our observations in macrophages, which contrast in some respects with
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Figure 4. TRAF-1, -2, and -5 differentially
mediate CD40L and TNF-␣–induced IL-6
and MCP-1 expression in Murine ECs. A to
D, Supernatants from ECs from TRAF-1–
competent (traf1⫹⫹) and TRAF-2/-5– competent (traf2⫹⫹5⫹⫹), as well as TRAF-1– deficient (traf1⫺), TRAF-5– deficient (traf2⫹⫹5⫺),
TRAF-2– deficient (traf2⫺5⫹⫹), and TRAF-2/
-5– double-deficient (traf2⫺5⫺) mice incubated for 24 hours with medium alone
(hatched bar), recombinant murine CD40L
(5 ␮g/mL, black bar), or recombinant
murine TNF-␣ (10 ng/mL, gray bar) were
analyzed by enzyme-linked immunosorbent
assay for IL-6 (left) and MCP-1 (right) protein expression. Data are presented as
means⫾SEM of at least 6 independent
experiments per group. #Pⱕ0.05 when
compared with CD40L-induced value of
respective TRAF-gene competent groups.
§Pⱕ0.05 when compared with TNF-␣–
induced value of respective TRAF genecompetent groups.
the findings in ECs, corroborate the notion that CD40
signaling differs not only between different target genes
within the same cell type but also for the same target gene in
different cell types.
The inducible expression of TRAF-1 by CD40L, IL-1␤,
and TNF-␣ in ECs, SMCs, and macrophages observed here
agrees with previous reports in B cells and freshly isolated
monocytes.28,29 The physiological role of TRAF-1 remains
controversial. Conflicting reports identify TRAF-1 as either a
promoter or an inhibitor of signals triggered by CD40L and
TNF-␣ in various cell types. Similarly, others have suggested
TRAF-1 as cofactor and inhibitor of TRAF-2– dependent
NF-␬B and JNK activation.30 –32 Our data demonstrate that
TRAF-1 may indeed promote and at the same time inhibit
certain proinflammatory functions of CD40L. Thus, TRAF-1
deficiency enhances both CD40L- and TNF-␣–induced IL-6
and MCP-1 expression in ECs, suggesting that TRAF-1
negatively regulates these cytokines in this cell type. Our
siRNA studies in human umbilical vein ECs parallel those
findings for IL-6 and MCP-1, while suggesting a positive role
for TRAF-1 in CD40L-induced IL-8 expression.
In contrast to TRAF-1 and TRAF-5, earlier studies have
shown that overexpression of TRAF-2 in cell lines suffices to
activate NF-␬B and JNK.25,33,34 Thus, TRAF-2 likely stimulates signaling by TNF receptor family members, including
CD40, in this cell type. However, several reports also
described inhibitory signaling functions of TRAF-2 in lymphoid and other cells.35,36 The present report identifies
TRAF-2 as an activator of CD40L-induced and TNF-␣–
induced IL-6 and IL-8 expression in ECs. However, TRAF-2
does not participate in MCP-1 expression initiated by the
same cytokines in this cell type.
Similar to TRAF-2 deficiency, TRAF-5– deficient ECs
showed significantly reduced CD40L-induced and TNF-␣–
induced IL-6 expression compared with wild-type controls.
However, ECs treated with TRAF-5 siRNA released significantly more IL-8 protein into the supernatant, suggesting that
TRAF-5 mediates and inhibits certain proinflammatory functions of these cytokines. Our data agree with previous reports
in B cells, which implicated the participation of TRAF-5 in
CD40 and TNF receptor signaling.34,37,38 In contrast to the
observations in ECs, TRAF-5 does not mediate CD40Linduced or TNF-␣–induced IL-6 expression in M⌽, an
illustration of cell type-selective signaling by these mediators.
In B cells, TRAF-2 and TRAF-5 exhibit overlapping functions in CD40 signaling.
Xu et al39 observed that B cells from TRAF-3–null mice
show augmented CD23 and proliferate normally on stimulation with CD40L, suggesting that TRAF-3 does not require
CD40 signaling. In contrast, our data demonstrate that ECs
treated with siRNA targeting TRAF-3 released significantly
more basal and CD40L-induced IL-6, IL-8, and MCP-1 into
the supernatant than appropriate control cells, suggesting an
inhibitory role for TRAF-3 in CD40 signaling. Our data agree
with a previous report by Urbich et al40 that demonstrated that
activation of TRAF-3 by shear stress abrogates CD40Lmediated endothelial activation.
Previous studies on B lymphocytes derived from TRAF6 – deficient mice suggested that TRAF-6 is instrumental in
CD40L-induced proinflammatory cytokine production and B
cell maturation.41 Similarly, Andrade et al and others42,43
implicated TRAF-6 as important mediator of CD40 signals in
monocytes and macrophages. In contrast to these findings,
our siRNA studies suggest that in ECs, TRAF-6, if anything,
inhibits CD40L-induced proinflammatory protein expression,
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TRAF-1, expression levels of TRAF-2 and -3 were significantly greater in atheromatous lesions compared with fibrous
lesions or aneurysmal arteries, whereas fibrous lesions expressed significantly more TRAF-5. Atherosclerotic lesions
overexpress CD40L, a mediator that can trigger mechanisms
associated with plaque progression and thrombosis.6,9,14,15,45
Therefore, high levels of TRAF-2 expression may promote
inflammatory signaling by CD40L and other cytokines,
potentially linking TRAF-2 not only to atherogenesis but also
to plaque complications. Because the same TRAFs may exert
opposing functions for different target genes, the net effects
of TRAF overexpression on inflammatory activity and atherosclerosis will require future study.
In sum, the present study provides new functional insights
into signaling mechanisms initiated by the proatherogenic
CD40/CD40L dyad in vascular cells. The results demonstrate
that both CD40L and TNF-␣ differentially use TRAF-1, -2,
-3, -5, and -6 for proinflammatory signaling depending on the
target gene and cell type investigated and directly implicate
TRAFs in vascular disease. Manipulation of TRAFs may
permit selective modulation of proatherogenic functions of
CD40L and other proinflammatory cytokines of the TNF
receptor-like and IL-1/Toll-like receptor superfamily.
Acknowledgments
The authors thank R. Reynolds, E. Shvartz, and E. Simon-Morrissey
for skillful technical assistance, and G. Sukhova for technical advice
(all Brigham & Women’s Hospital). The authors thank T.W. Mak,
the originator of the TRAF-2– deficient mice, for his permission to
use them in this study (University of Toronto, Toronto, Canada).
Sources of Funding
Figure 5. Inhibition by siRNA shows distinct roles of TRAF-3
and -6 in CD40 signaling in human umbilical vein ECs. Human
umbilical vein ECs were transfected with indicated TRAFdirected siRNA (250 nM final concentration), plated for 12 hours
in growth medium, starved for 12 hours in serum-reduced
medium, and incubated with either medium (hatched bar) or 10
␮g/mL of recombinant human CD40L (black bar) for 24 hours.
Supernatants were assayed for IL-6, IL-8, and MCP-1 by
enzyme-linked immunosorbent assay. Data are presented as
means⫾SEM of at least 4 experiments per group. Representative blots are shown. Cell lysates were subjected to Western
blot analysis with the antibodies indicated. Cells transfected
with scrambled siRNA served as internal control.
highlighting once again cell-type specific differences of
TRAF functions.
Previous reports implicated TRAF-1 in particular, but also
other TRAFs in apoptosis.44 Our data suggest that TRAFdependent protein expression does not depend on apoptosis
and cell viability. We found extensive expression of TRAF-1,
-2, -3, -5, and -6 in sections of murine atherosclerotic aortic
arches. Co-localization studies confirmed TRAF expression
by both ECs and macrophages in situ. Furthermore, lysates of
human atherosclerotic and aneurysmal arteries overexpressed
TRAFs compared with lysates from apparently normal arteries. These data further support a role for TRAFs in the
pathogenesis of atherosclerosis. Interestingly, in contrast to
This work was supported by grants from the Fondation Leducq and
NIH (HL-34636) to P.L.; NIH (HL-66086) to U.S. and M.A.; the
Ernst Schering Research Foundation to N.G.; and the MD/PhD
program of the Medical School Hannover to J.J. and the Deutsche
Forschungsgemeinschaft to U.B. (BA 1997/1-1 and 3-1) and A.Z.
(ZI 743/1-1 and 3-1).
Disclosure
None.
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TRAF-1, -2, -3, -5, and -6 Are Induced in Atherosclerotic Plaques and Differentially
Mediate Proinflammatory Functions of CD40L in Endothelial Cells
Andreas Zirlik, Udo Bavendiek, Peter Libby, Lindsey MacFarlane, Norbert Gerdes, Joanna
Jagielska, Sandra Ernst, Masanori Aikawa, Hiroyasu Nakano, Erdyni Tsitsikov and Uwe
Schönbeck
Arterioscler Thromb Vasc Biol. 2007;27:1101-1107; originally published online March 1, 2007;
doi: 10.1161/ATVBAHA.107.140566
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright © 2007 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636
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ATVB/2007/140566
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Online supplement:
Expanded Materials and Methods:
Reagents – TRAF-1, -2, -3, -5, and -6 antibodies were obtained from Santa Cruz, anti-TRAF-2 also
from Cell Signaling, anti-TRAF-5 also from ebioscience, and anti-Lamin antibody was purchased
from Novocastra. Unless stated differently, all antibodies used for FACS were from BD Pharmingen
and all ELISA antibodies from Pierce Endogen. Recombinant proteins were from R&D with exception
of human CD40L from Leinco and human IL-6 and IL-8 from Pierce Endogen. All siRNA
oligonucleotides were designed and purchased from Qiagen. All human experiments employed human
CD40L (Leinco), all Murine experiments Murine CD40L (R&D).
Mice – TRAF-1 deficient mice were kindly provided by their originator Dr. Tsitsikov, TRAF-2 and
TRAF-2/-5 deficient mice from Dr. Nakano. TRAF-2 deficient single knock out mice were also
provided by Dr. Nakano with permission of their originator, Dr. T.W. Mak (Toronto, Canada). TRAF1 deficient mice were pure C57/BL6, TRAF-2, -5 and -2/-5 deficient mice were Balb/c.
Cell isolation and culture - Human saphenous vein EC (HSVEC), human umbilical vein EC
(HUVEC), and human MΦ were isolated and cultured as previously described 1. For isolation of
murine EC, corresponding TRAF-deficient and control mice were euthanized with CO2, and lungs,
heart, brain, and liver were harvested employing sterile technique, minced with a razor blade, and
digested in 0.2% collagenase type-1/1% BSA (Worthington, Lakewood, NJ and Sigma, St. Louis,
MO) for 90 min at 37°C. After washing with 0.1% BSA and filtering through a 70µm nylon mesh (BD
Biosciences, San Jose, CA), cells were resuspended in 0.1% BSA and incubated with an anti-mouse
CD31 antibody conjugated to sheep anti-rat Dynabeads (Dynal Biotech, Oslo, Norway) for 10 min at
room temperature. Cells were then separated and washed three times using a magnetic particle
concentrator (Dynal Biotech) and seeded into gelatin-coated plates. After they reached confluence, a
second magnetic sorting was performed with a rat anti-mouse ICAM-2 antibody (BD Pharmingen).
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Cells were grown in DMEM high glucose (Cambrex) supplemented with 20% fetal bovine serum
(FBS), 1% sodium pyruvate, 1% heparin, 1% bovine endothelial growth factor, 0.6% non-essential
amino acids, and 1% penicillin/streptomycin. Cells were maintained in M-199 supplemented with
0.1% FBS 24h prior to experiments. Since TRAF-2- and TRAF-2/-5-deficient mice only survive 3-4
weeks, mice were genotyped at 10-14 days of age and EC were isolated subsequently.
For isolation of peritoneal MΦ, 2 ml of 4% thioglycollate (Sigma) was injected
intraperitoneally in mice of desired genotype. After three days, the peritoneal cavity was flushed with
5ml of RPMI 1640. After lysing the erythrocytes with 0.155 M NH4Cl, filtering through a 70µm nylon
mesh, and washing with 0.2% BSA/PBS, cells were plated and maintained in RPMI 1640
supplemented with 5% FBS and 1% penicillin/ streptomycin. Before experiments, cells were
maintained in RPMI 1640 lacking FBS for 24 h.
Preparation of protein extracts - Frozen non-atherosclerotic human carotids (n=6), atherosclerotic
human carotids (n=12), dichotomized a priori into fibrous (stable; n=6) and atheromatous (n=6)
plaques by morphological criteria, as well as abdominal aortic aneurysm tissue (n=6) were
homogenized (IKA-Labortechnik, Ultra-turrax T 25) and lysed as described previously 1.
Immunoprecipitation and Western blotting- Confluent cell cultures were scraped in lysis buffer (50
mM HEPES pH 7.4, 1 % Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 μg/ml aprotinin,
leupeptin and pepstatin A), incubated 30 min at 4˚C, and clarified (14,000 x g, 10 min). Lysates were
precleared with Protein-G-Sepharose (30 min, 4˚C, Amersham-Pharmacia), incubated (2 h, 4˚C) with
the specific goat or mouse anti-human CD40 antibody (10 μg/ml, Sigma; or Ancell), followed by
incubation with Protein-G-Sepharose (1 h, 4˚C). The beads were washed (4X) in lysis buffer and
finally subjected to Western blot analysis, as described previously 1, 2.
Preparation of plasma membrane fraction – Cell lysates were centrifuged at 3,500 x g for 5min.
Plasma membrane fractions were pelleted from the supernatants by centrifugation at 100,000 x g for
90min.
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Immunohistochemistry – Serial cryostat sections (6 µm) of Murine aortic arches from LDLR-deficient
mice fed a high cholesterol diet (1.25 cholesterol, 0% cholate; Research Diets) for 16 weeks were
fixed in acetone (-20°C, 5 minutes), air-dried, and stained by the avidin-biotin-peroxidase method as
described previously 1.
siRNA Transfections – HUVEC were grown to 80% confluency, transfected at 250nM final
concentration of specified TRAF-specific siRNA employing the Amaxa Nucleofector device (Amaxa),
the HUVEC specific Amaxa Nucleofector kit, and program V-01, according to the manufacturer’s
instructions. Subsequently, cells were cultured for 24h for attachment and stimulated for 24 h with the
indicated concentrations of human CD40L. Cell lysates and supernatants were collected. Cells
transfected with Lamin-directed or scrambled siRNA served as internal control. Transfection efficacy
was >75% assessed by FACS analysis of cells transfected with FITC-labeled siRNA (Fig.I, online
supplement).
Enzyme-linked immunoabsorbent assay (ELISA) - Murine and human IL-6, IL-8, and MCP-1 were
quantified in the supernatant of cultures by ELISA.
Apoptosis and Cytotoxicity Assays – To assess apoptosis and cytotoxicity in Murine cells, Capsase-3/7
and LDH protein concentrations were quantified in supernatants employing commercially available
assays (ApoOne Homogeneous Caspase-3/7 Assay, CytotoxOne Assay, Promega) according to the
protocol of the manufacturer.
Data analysis - Western blots were analyzed densitometrically using ImageJ (NIH software). Data of
at least three experiments were pooled and presented as mean ± SEM. Statistics employed the
ATVB/2007/140566
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Student’s two-tailed t test for paired or unpaired values (where appropriate). A p value <0.05 was
considered statistically significant.
References
1.
Sukhova GK, Schonbeck U, Rabkin E, Schoen FJ, Poole AR, Billinghurst RC, Libby P.
Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable
human atheromatous plaques. Circulation. 1999;99:2503-2509.
2.
Bavendiek U, Libby P, Kilbride M, Reynolds R, Mackman N, Schonbeck U. Induction of
tissue factor expression in human endothelial cells by CD40 ligand is mediated via activator
protein 1, nuclear factor kappa B, and Egr-1. J Biol Chem. 2002;277:25032-25039.
ATVB/2007/140566
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Online Figure Legends:
Fig. I: Time- and concentration-dependent induction of TRAF-1, -2, -3, and -6 by CD40L in EC. A,
Lysates of EC incubated for the indicated time periods with serum-free medium alone (None) or
CD40L (10µg/ml) were analyzed by Western blotting employing various TRAF antibodies. B, Lysates
of EC incubated for 24 h with serum-free medium alone (None) or respective concentrations of
CD40L, were analyzed by Western blotting employing respective TRAF antibodies.
Fig. II: CD40L recruits TRAF-1, -2, -3, -5, and -6 to the plasma membrane in human EC. Plasma
membrane fractions of lysates of EC incubated for the respective duration with CD40L (10µg/ml)
were applied to Western blot analysis with respective TRAF antibodies. For control purposes, lysates
of PMA-activated Jurkat cells were applied.
Fig. III: TRAF-1, -2, -3, -5, and -6 are overexpressed in atherosclerotic and aneurysmal arterial tissue.
Extracts of non-diseased (Normal), atherosclerotic (dichotomized into fibrous and atheromatous
plaques), or aneurysmal (AAA) specimens were analysed by Western blots employing the indicated
TRAF antibodies. Pooled densitometric values of at least six individual experiments are shown as
graphs. Data represent means ± SEM. § = P≤0.05 when compared to values of the non-diseased group.
Fig. IV: TRAF-1, -2, -3, -5, and -6 colocalize with endothelial cells and macrophages in murine
atherosclerotic sections. Sections of aortic arches from LDLR-deficient mice fed a high cholesterol
diet for 16 weeks were double stained with the indicated TRAF antibodies and an endothelial (CD31)
and macrophage marker (Mac-3) and shown at 40x magnification. Red staining signifies TRAF
expression, blue staining the respective cell marker. Three independent experiments yielded similar
results.
Fig. V: TRAFs, apoptosis, and cytoxicity in murine EC. A, TRAF-1- and TRAF5- deficient cells and
respective controls were stimulated with medium alone (hatched bar), CD40L (5µg/ml, black bar), and
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TNFα (10ng/ml, grey bar) and Caspase 3/7 release into the supernatant was quantified employing a
commercially available assay. Data are presented as means ± SEM of at least four independent
experiments per group. § = P≤0.05 when compared to TNFα-induced value of respective TRAF-gene
competent groups. B, Supernatants from cells treated as described under Fig. 4 A-D were assayed for
LDH employing a commercially available assay. Data are presented as means ± SEM of at least four
independent experiments per group. § = P≤0.05 when compared to TNFα-induced value of respective
TRAF-gene competent groups.
Fig. VI: TRAF-1 and -5-dependent pro-inflammatory gene expression confirmed in arterial murine
EC. Supernatants from endothelial cells (EC) isolated only from aortas from a pool of 8 TRAF-1
competent- (traf1++) and TRAF-5-competent (traf2++5++) as well as TRAF-1-deficient (traf1--), TRAF5-deficient (traf2++5--) mice, respectively, incubated for 24h with medium alone (hatched bar),
recombinant murine CD40L (5µg/ml, black bar), or recombinant murine TNFα (10ng/ml, grey bar)
were analyzed by ELISA for IL-6 (left) and MCP-1 (right) protein expression. Data are presented as
means ± SD of one experiment assayed in triplicate. # = P≤0.05 when compared to CD40L-induced
value of respective TRAF-gene competent groups. § = P≤0.05 when compared to TNFα-induced value
of respective TRAF-gene competent groups.
Fig. VII: Inhibition by siRNA shows distinct roles of TRAF-1, -2, and -5 in CD40 signaling in
HUVEC. HUVEC were transfected with indicated TRAF-directed siRNA (250nM final
concentration), plated for 12 h in growth medium, starved for 12 h in serum-reduced medium, and
incubated with either medium (hatched bar) or 10 µg/ml of recombinant human CD40L (black bar) for
24 h. Supernatants were assayed for IL-6, IL-8, and MCP-1 by ELISA. Data are presented as means ±
SEM of at least four experiments per group. Representative blots are shown. Cell lysates were
subjected to Western blot analysis with the antibodies indicated. Cells transfected with anti-Lamin
siRNA served as internal control.
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Fig. VIII: TRAF-1, -2 and -5 differentially mediate CD40L and TNFα induced IL-6 and MCP-1
expression in macrophages. A-D, IL-6 and MCP-1 was measured in the conditioned media of
peritoneal macrophages isolated from TRAF-1 competent- (traf1++) and TRAF-2/-5-competent
(traf2++5++) as well as TRAF-1-deficient (traf1--), TRAF-5-deficient (traf2++5--), TRAF-2 heterozygous
/TRAF-5-deficient (traf2+-5--) mice and stimulated for 24 h with media alone (Control, hatched bar),
recombinant murine CD40L (5µg/ml, black bar), or recombinant murine TNFα (10ng/ml, grey bar).
IL-6 and MCP-1 expression was quantified by ELISA. Data are presented as means ± SEM of at least
six independent experiments per group. # = P≤0.05 when compared to CD40L-induced value of
respective TRAF-gene competent groups. § = P≤0.05 when compared to TNFα-induced value of
respective TRAF-gene competent groups.
Fig. IX.: Transfection efficacy of siRNA in HUVEC. HUVEC transfected with FITC-labeled siRNA
or non-labelled control siRNA were harvested after 24h with trypsin-EDTA and transfection efficacy
was analyzed by FACS. Representative FACS of a total of three experiments yielding similar results is
shown.
Online Fig. I
A
TRAF-1
TRAF-2
TRAF-3
TRAF-6
0 5’ 30’ 4h 12h 36h
CD40L (10 µg/ml)
B
TRAF-1
TRAF-2
TRAF-3
TNFα
IL-1β
None
TRAF-6
0.1 0.3 1 3 10 μg/ml
CD40L (24 h)
Plasma
-membrane
TRAF-1
TRAF-2
TRAF-3
TRAF-5
0 10’ 30’ 90’ 16h
Jurkat
TRAF-6
CD40L (10 µg/ml)
Online Figure II
TRAF-6 / GAPDH
E
AAA
AAA
25
0
75
AAA
75
AAA
D
Atheromatous
0
Fibrous
25
TRAF-2 / GAPDH
50
Atheromatous
§
Normal
AAA
B
Fibrous
50
TRAF-5 / GAPDH
75
Atheromatous
Fibrous
Normal
TRAF-1 / GAPDH
75
Normal
Atheromatous
0
Atheromatous
25
Fibrous
50
Fibrous
75
Normal
C
Normal
TRAF-3 / GAPDH
A
§
50
25
0
100
§
50
25
0
Online Figure III
Colocalization with endothelial cells (CD31, blue staining)
TRAF-1
TRAF-2
TRAF-3
TRAF-5
TRAF-6
Colocalization with macrophages (anti-Mac-3, blue staining)
Online Figure IV
B
A
200
§
150
LDH (%)
Caspase-3/-7 (%)
200
100
50
0
traf1++ traf1-- traf5++ traf5--
Control
§
150
100
50
0
traf1++
traf1--
CD40L 5μg/ml
traf2++5++ traf2++5-- traf2--5++ traf2--5--
TNFα 10ng/ml
Online Figure V
B
400
MCP-1 (%)
IL-6 (%)
A 500
300
200
200
D
0
traf1++
traf1--
0
D
400
400
300
300
MCP-1 (%)
IL-6 (%)
300
100
100
C
200
100
0
Online Fig. VI
400
traf1++
traf1--
traf5++
traf5--
200
100
traf5++
Control
traf5--
0
CD40L 5μg/ml
TNFα 10ng/ml
Control
CD40L 5μg/ml
400
#
200
100
IL-6 pg/ml
IL-6 pg/ml
300
30
20
10
IL-8 pg/ml
0
5000
4000
3000
#
2000
§
0
Traf-1
MCP-1 ng/ml
#
40
#
200
100
10.0
7.5
5.0
2.5
0.0
7000
6000
5000
4000
3000
2000
1000
0
Traf-2
#
§
#
Traf-5
Lamin
GAPDH
CD40L
5μg/ml
#
0
50
1000
#
IL-8 pg/ml
MCP-1 ng/ml
0
300
Lamin
- + - +
anti-lamin anti-traf1
siRNA
siRNA
GAPDH
CD40L
5μg/ml
- + - + - + - +
anti-lamin anti-traf5 anti-traf2
siRNA
siRNA
siRNA
anti-traf5/2
siRNA
Online Figure VII
A
#
800
B
§
Online Fig. VIII
500
§
MCP-1 (%)
IL-6 (%)
700
600
500
400
300
200
400
300
#
200
100
100
0
0
traf1++
traf1--
D
C
700
traf1--
300
250
500
400
300
#
200
§
MCP-1 (%)
600
Il-6 (%)
traf1++
200
150
100
50
100
0
0
traf2++5++
Control
traf2++5--
traf2-+5-CD40L 5μg/ml
traf2++5++
traf2++5--
traf2-+5--
TNFα 10ng/ml
Cell Counts
Online Fig. IX
Mock transfected ECs
ECs transfected with FITClabeled siRNA
Fluorescence