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Vol. 55, Issue 1, 133-166, March 2003
Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Leiden, The Netherlands
Abstract
I. Introduction
II. Interleukin Families in Atherosclerosis
A. Interleukin-1
B. Interleukin-2
C. The gp130 Family
D. Granulocyte-Macrophage Colony-Stimulating Factor
E. Interleukin-10
F. Chemokines
G. Interleukin-17
III. Modulation of Cytokine Function As a Therapeutic Strategy for Atherosclerosis
A. Inhibition of Expression/Translation of Interleukins and Their Receptors
B. Inhibition of Interleukin Processing
C. Neutralization of Proinflammatory Interleukins
D. Interleukin Receptor Antagonists
E. Up-Regulation of Anti-Inflammatory Interleukins
F. Inhibition of Interleukin Signaling
G. Inhibition of Interleukin-Induced Gene Expression
IV. Discussion
References
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Interleukins are considered to be key players in the chronic vascular inflammatory response that is typical of atherosclerosis. Thus, the expression of proinflammatory interleukins and their receptors has been demonstrated in atheromatous tissue, and the serum levels of several of these cytokines have been found to be positively correlated with (coronary) arterial disease and its sequelae. In vitro studies have confirmed the involvement of various interleukins in pro-atherogenic processes, such as the up-regulation of adhesion molecules on endothelial cells, the activation of macrophages, and smooth muscle cell proliferation. Furthermore, studies in mice deficient or transgenic for specific interleukins have demonstrated that, whereas some interleukins are indeed intrinsically pro-atherogenic, others may have anti-atherogenic qualities. As the roles of individual interleukins in atherosclerosis are being uncovered, novel anti-atherogenic therapies, aimed at the modulation of interleukin function, are being explored. Several approaches have produced promising results in this respect, including the transfer of anti-inflammatory interleukins and the administration of decoys and antibodies directed against proinflammatory interleukins. The chronic nature of the disease and the generally pleiotropic effects of interleukins, however, will demand high specificity of action and/or effective targeting to prevent the emergence of adverse side effects with such treatments. This may prove to be the real challenge for the development of interleukin-based anti-atherosclerotic therapies, once the mediators and their targets have been delineated.
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I. Introduction |
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Atherosclerosis remains, despite a recent decline, the most common
cause of death in the Western world. The disease course of
atherosclerosis is characterized by its chronicity, and progression in
its initial stages is particularly insidious. Chronic inflammation is
the pathological hallmark of atherosclerosis (Ross, 1986
, 1993a, 1999),
and inflammatory processes are instrumental in all stages of this
disease. Even prior to the development of detectable intimal lesions,
the expression pattern of the endothelium has been shown to be
inflammatory in nature, conforming to the response-to-injury hypothesis
as first postulated by the late Russell Ross (Ross and Glomset, 1973).
Thus, in lesion-prone sites of the arterial tree, the endothelial
expression of adhesion molecules is up-regulated, reflecting
endothelial dysfunction secondary to unfavorable hemorheology (Nakashima et al., 1998) and/or hypercholesterolemia (Rosenfeld, 1991;
Li et al., 1993; Sakai et al., 1997; Nakashima et al., 1998). In turn,
this leads to the adhesion, extravasation, and intimal accumulation of
circulating leukocytes (Nageh et al., 1997; Gerszten et al., 1998;
Nakashima et al., 1998; Ramos et al., 1999; Dong et al., 2000), and
thus to the development of the earliest detectable lesion
the fatty
streakwhich consists solely of lipid-laden macrophages and T
lymphocytes (Stary et al., 1994). These cell types are also present in
more advanced plaques, in addition to smooth muscle cells and
extracellular lipid and matrix deposits (Stary et al., 1994, 1995). The
cellular constituents of the atherosclerotic lesion are thought to
participate actively in the propagation of inflammation and,
eventually, plaque destabilization (Ross, 1999; Sukhova et al., 1999).
As well as contributing to the bulk of the lesion, plaque cells are
involved in the production and degradation of extracellular matrix and
contribute toward the formation of a necrotic lesion core by the
elaboration of toxic mediators. These cellular functions are partly
autonomous but to a large extent subject to autocrine and paracrine
control mechanisms. A plethora of mediators has been shown to be
involved in intercellular signaling in atheromatous tissue, including
small molecules such as nitric oxide (Ignarro et al., 1999; Li and
Forstermann, 2000), lipid mediators such as eicosanoids and sterols
(Hajjar and Pomerantz, 1992; Edwards and Ericsson, 1999; Schnaper et
al., 2000), and polypeptides such as cytokines (Frostegard et al.,
1999; Meager, 1999).
Whereas fatty streaks are now known to develop even in utero under the
influence of maternal hypercholesterolemia (Napoli et al., 1997),
plaques rarely give rise to symptoms before the sixth or seventh decade
of life. If primary prevention is to be the cardinal aim, the
protracted nature of lesion development will necessitate a therapeutic
strategy with a comparably prolonged duration of effectivity. In
conjunction with the as yet perfunctory levels of prognostic accuracy
for the identification of patients at risk of symptomatic
atherosclerosis, this poses stringent demands with respect to the
tolerability of any preventive intervention, including the use of
immunomodulatory therapies.
The rate of atherogenesis largely depends on the level of exposure to
major risk factors, including a positive family history, hypercholesterolemia, smoking, diabetes mellitus, and hypertension. Although the avoidance of risk factors undoubtedly constitutes the most
rewarding approach to the prevention of atherosclerosis, it has thus
far been frustrated by inadequate patient compliance and the influence
of genetic factors in determining an individual's predisposition to
atherosclerosis. This has led to the introduction of a variety of
pharmacological interventions, including the widespread use of an
extremely effective class of lipid-lowering drugs: the HMG-CoA
reductase inhibitors, or so-called statins (Braunstein et
al., 2001). Despite recent concerns regarding the induction of
rhabdomyolysis, a rare and potentially lethal side effect of statin
usage, these drugs continue to be the mainstay of most cholesterol-lowering regimens. In several clinical prevention trials
(e.g., CARE; Ridker et al., 1998), statins have also been found to
exert additional, lipid-independent, anti-inflammatory effects. These
may contribute significantly to their anti-atherogenic properties, and
this has indeed been corroborated in recent animal studies (Williams et
al., 1998). Indeed, immunomodulation could be an attractive paradigm
for the development of therapeutic alternatives to statins in
atherosclerosis prevention. This may be of particular benefit to those
whose lipid levels are (partially) unresponsive to statin therapy; as
in a substantial number of patients in the U.S. National
Cholesterol Education Program,
LDL1 cholesterol
levels cannot be attained by statin monotherapy alone (Brown et al.,
1998).
To enable rational drug design aimed at immunomodulation in
atherosclerosis, the pivotal inflammatory processes involved in this
disease need to be delineated. In this regard, extensive efforts have
been devoted to outlining the involvement of cytokines, because these
cell-regulatory proteins are known to be key players in the initiation
and control of inflammation in general. The term "cytokine" was
first coined in the 1970s and encompasses a large number of
(glyco)proteins involved in cell-to-cell signaling. Cytokines are
conventionally classified by assignment to one of six families:
interleukins, the tumor necrosis factor family, interferons,
colony-stimulating factors, growth factors, and chemokines (Henderson
and Higgs, 2000). Considerable overlap between these families exists,
however, and alternative methods of subdivision have been suggested.
Depending on the aim of classification it may be preferable to
distinguish cytokines with an essentially proinflammatory mode of
action [including tumor necrosis factor (TNF), interleukin-12 (IL-12),
IL-18, and interferon 
(IFN)] from those with largely
anti-inflammatory properties (including IL-4, IL-10, IL-13, and the
endogenous IL-1 receptor antagonist, IL-1ra) or T helper cell type I
(Th1; including IL-2, IFN, and TNF) from T helper cell type II (Th2;
including IL-3, IL-4, IL-5, IL-6, IL-10, and IL-13) cytokines.
Alternatively, it may be desirable to identify cytokines according to
their major function, such as those effecting chemoattraction
[chemokines, including monocyte chemoattractant protein-1 (MCP-1),
RANTES, macrophage inflammatory protein-1 (MIP-1), IL-8, and IL-16] or
on the basis of receptor sequence homology (e.g., those employing the
gp130 signal transduction protein, such as IL-6, IL-11, IL-12,
oncostatin M, and cardiotrophin-1). Nonetheless, a substantial degree
of pleiotropism in cytokine effector functions makes most of these
subdivisions somewhat arbitrary.
Members of each conventional cytokine family have been found to be involved in atherogenesis, and all cell types present in the atherosclerotic plaque are capable of producing and responding to cytokine mediators. It is conceivable, therefore, that intervention in cytokine signaling could provide effective prevention and/or treatment of atherosclerosis, and proof-of-principle data to this effect have been obtained in a variety of in vitro and in vivo studies, although this has not yet yielded clinically applicable protocols. In this review, we shall focus mainly on interleukins in our aim to outline the results that have been achieved to date in delineating the pathophysiological role and the therapeutic potential of cytokines in atherosclerosis. In addition, we shall discuss the potential of the modulation of cytokine activity as a therapeutic approach to the primary and secondary prevention of atherosclerosis. Following an overview of the roles ascribed to a variety of interleukins in the pathogenesis of atherosclerosis, we shall describe recent progress in this field and perceived future opportunities.
II. Interleukin Families in Atherosclerosis
By definition, interleukins are produced mainly by
leukocytes and exert their effects mainly on leukocytes.
Endothelial cells and smooth muscle cells, however, also express a
variety of interleukins and/or their respective receptors, and their
effects in atherogenesis are therefore by no means restricted to
macrophages and T cells. Thus far, more than 30 major members of the
interleukin family have been identified, and the majority of these have
been shown to play a role in atherogenesis. As applies to cytokines in
general, it is possible to subdivide the interleukins into families
according to the homology of their amino acid sequences or the homology of the receptor complexes to which they bind (Fig.
1). Of these subgroups, the gp130
receptor family comprises principally pro-atherogenic interleukins, but
most other families have both anti- and pro-atherogenic members (e.g.,
IL-1 family, IL-2 family, and c receptor family). It has not proved
feasible to pinpoint an interleukin that acts as the cardinal culprit
in the atherosclerotic process. On the contrary, it seems rather more
likely that the delicate balance between pro- and anti-inflammatory
signals that generally serves to keep inflammation in check, goes awry
in atherosclerosis, leading to a self-perpetuating mechanism of lesion
formation (Ross, 1993b; Tedgui and Mallat, 2001). Considering the
extensive interplay of soluble mediators in the atherosclerotic plaque,
however, it may prove possible to devise an anti-atherosclerotic
therapy aimed at modifying the effect of a single interleukin, provided
that due attention is paid to the mechanisms of redundancy, which have been shown to exist in cytokine signaling. In doing so, candidate interleukins cannot be identified solely by virtue of a demonstrated systemic or local modulation of their expression in the course of
atherogenesis. On the contrary, it is of paramount importance to
determine whether cytokine responses that have been observed in
relation to the development of atherosclerosis are
compensatory to, contributory to, or merely
associated with this disease. Making this distinction will
require well designed intervention studies in animal models, in which
the effect of attenuation or administration of a particular interleukin
can be evaluated. The currently favored approach entails the up- or
down-regulation of interleukin expression in atherosclerosis-prone
mouse strains by means of gene insertion ("transgenics") or gene
deletion ("knockouts"), respectively. Administration of an
interleukin or its ablation by specific antibodies/antagonists, however, can also provide valuable data regarding its role in atherogenesis. When pertinent, the results of such studies will be
discussed in the next section.
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Since the effects exerted by cytokines may differ significantly
depending on their local environment, it will also be necessary to
distinguish between the role of systemic and local variations in
cytokine levels. This type of information could in the future be
derived from cell- or organ-specific gene overexpression through the
use of specific promoters and gene deletion by means of the cre-lox
system (Perkins, 2002) or by comparison of the effects of local and
systemic administration of cytokines.
A. Interleukin-1
The IL-1 family comprises four proteins that share considerable
sequence homology and contain a 
-pleated sheet structure (Dinarello,
1997): IL-1
, IL-1, IL-1 receptor antagonist (IL-1Ra), and IL-18
(also known as IFN-inducing factor). Release of mature IL-1
requires extracellular calpain-mediated cleavage of a pro-IL-1, whereas mature IL-1 is derived proteolytically from pro-IL-1 by
intracellular IL-1-converting enzyme (ICE or caspase-1) activity. Upon binding of IL-1 or IL-1 to the IL-1 receptor type I
(IL-1RI), IL-1R accessory protein (IL-1RIAcP) is recruited by the
receptor complex, and intracellular signal transduction is triggered
through a p38 mitogen-activated protein kinase (MAPK)-activated
phosphorylation cascade. Due to extensive signal amplification, minute
amounts of IL-1 can have considerable biological activity, and as
little as 1 ng/kg intravenous IL-1 causes symptoms in humans. The
signaling cascade culminates in the nuclear translocation of the
transcription factors nuclear factor kappa B (NF-
B) and activating
protein-1 (AP-1) and the ensuing transcription of a variety of
proinflammatory genes, including autocrine amplification of IL-1
production (Suzuki et al., 1989). In addition to the IL-1RI, IL-1 may
also bind to the so-called type II interleukin-1 receptor, the
expression of which appears to be regulated by IL-4 (Colotta et al.,
1993b). Binding of IL-1 to this receptor does not result in cellular
activation, and IL-1RII is therefore presumed to act as a decoy that
negatively regulates IL-1 activity.
A further member of the IL-1 cytokine family, IFN-inducing factor,
has been termed IL-18, on the basis of its pleiotropic Th1-inducing
effects (Ushio et al., 1996). It has been assigned to the IL-1 family
on the grounds of sequence homology (26% with IL-1) and similarity
of the IL-18 receptor to IL-1R (Torigoe et al., 1997; Dinarello, 1999).
Like IL-1, IL-18 is dependent on ICE for proteolytic processing, and
on nuclear translocation of NF-B for transcriptional activation.
Owing to its proinflammatory effects on endothelial cells (Jirik et
al., 1989; Loppnow and Libby, 1989a,b; Sironi et al., 1989; Suzuki et
al., 1989; Sica et al., 1990b; Bochner et al., 1991; Clinton et al.,
1992; Collins et al., 1995; Garcia et al., 2000), smooth muscle cells
(Loppnow and Libby, 1989a, 1990; Wang et al., 1991; Clinton et al.,
1992; Braun et al., 1995; Stanford et al., 2000), and macrophages (Sica
et al., 1990b), and due to its production by all of these cell types in
atherosclerotic lesions (Moyer et al., 1991; Tipping and Hancock, 1993;
Galea et al., 1996), IL-1 was one of the first cytokines to be
considered instrumental in the propagation of vessel wall inflammation
in atherosclerosis. It is thought to facilitate early lesion formation
by increasing leukocyte adhesion to endothelial cells (Bevilacqua et
al., 1985; Wang et al., 1995) and mediating leukocyte transmigration
(Moser et al., 1989; Furie anf McHugh, 1989). Subsequently, locally
produced IL-1 may serve to maintain an inflammatory milieu by autocrine and paracrine stimulation of cytokine (Jirik et al., 1989; Loppnow and
Libby, 1989a,b, 1990, 1992; Sironi et al., 1989; Sica et al., 1990a,b;
Wang et al., 1991; Clinton et al., 1992; Li et al., 1995; Taki et al.,
1999; Garcia et al., 2000; Stanford et al., 2000) and adhesion molecule
expression (Osborn et al., 1989; Bochner et al., 1991; Braun et al.,
1995; Collins et al., 1995). In the advanced plaque, IL-1-induced
up-regulation of matrix metalloproteinases may destabilize the
proteinaceous scaffold of the cap and thereby have a hand in plaque
rupture (Galis et al., 1995; Libby et al., 1995); this hypothesis is
corroborated clinically by the fact that a particular IL-1 gene
polymorphism has been found to be associated with myocardial infarction
in chlamydia pneumoniae seropositive patients (Momiyama et al., 2001),
and that pericardial fluid levels of IL-1 are raised in patients
with unstable angina pectoris (Oyama et al., 2001).
Because the IL-18 signal transduction cascade is similar to that
activated by IL-1, it is perhaps unsurprising that IL-18 has also been
found to up-regulate the expression of intercellular adhesion molecule
1 (ICAM-1) and cytokines by monocytes, including IL-1, IL-6, and
IL-8 (Dinarello, 1999), and the production of vascular cell adhesion
molecule-1 (VCAM-1) by endothelial cells (Vidal-Vanaclocha et al.,
2000). It is, therefore, entirely conceivable that IL-18 may have
pro-atherogenic properties, and Mallat et al. (2001a) have indeed
demonstrated IL-18 in atherosclerotic plaques in human carotids, which
is primarily localized to macrophages. They found the corresponding
receptor, IL-18R, to be expressed on endothelial cells and macrophages
and barely present on SMCs. These findings have subsequently been
confirmed histologically and in vitro by Gerdes et al. (2002), who also
demonstrated the functionality of the IL-18 receptor on these cells
through IL-18-mediated induction of pro-atherogenic factors, including
IL-6, IL-8, ICAM-1, and matrix metalloproteinases. In addition, the
serum level of IL-18 has recently been identified as a strong predictor
of cardiovascular death in stable and unstable angina (Blankenberg et
al., 2002). The pro-atherogenic effects of IL-18 are thought to be
mediated by IFN, since the induction of atherosclerosis by exogenous
IL-18 is abrogated by IFN deficiency in apolipoprotein E knockout
(apoE
/) mice (Whitman et al., 2002). A role for IL-18 in plaque
destabilization was suggested by the up-regulation of IL-18 mRNA levels
in symptomatic and ulcerative atherosclerotic plaques (Mallat et al.,
2001a).
In comparison with the proinflammatory reprobates of the IL-1 family,
IL-1ra appears positively angelic. IL-1ra displays affinity for the
IL-1R, but it does not induce a cellular response; it is therefore
believed to be an endogenous inhibitor of IL-1 signaling (Dinarello,
1997). IL-1ra is produced by monocytes (Arend et al., 1990),
macrophages (Janson et al., 1991), and smooth muscle cells (Beasley et
al., 1995). Recombinant intracellular IL-1ra has been shown to
counteract the IL-1-induced production of IL-6, IL-8, and monocyte
chemotactic protein by human endothelial cells (Bertini et al., 1992),
and to inhibit smooth muscle cell proliferation (Porreca et al., 1993).
Moreover, vascular inflammation is the major phenotypic characteristic
of IL-1ra-deficient mice (Nicklin et al., 2000), whereas atherogenesis
is reduced in IL-1ra transgenic mice on a high fat diet (Devlin et al.,
2002), and fatty streak formation is reduced in apoE/ mice by
IL-1ra administration (Elhage et al., 1998). Il-1ra has been found to
be present in carotid atherosclerotic plaques (Gottsater et al., 2002),
and the relevance of IL-1ra to human atherosclerosis is underscored by
the fact that certain IL-1ra alleles are associated with coronary artery disease (Francis et al., 1999) and restenosis (Kastrati et al.,
2000; Francis et al., 2001).
B. Interleukin-2
This family of cytokines encompasses a group of interleukins which
share a common receptor subunit, the "common chain" (c chain), which acts in unison with a subtype specific chain to initiate the signaling cascade. As the common receptor subunit was
initially discovered in relation to IL-2, it has also been termed the
"IL-2 receptor chain" (Takeshita et al., 1990), and the group
of cytokines that interact with this receptor has consequently been
termed the "IL-2 family" (Leonard and Lin, 2000). The members of
this interleukin family are primarily involved in T cell development and activation, and mutations of the c chain cause X-linked severe combined immunodeficiency in humans (Noguchi et al., 1993b) and lead to
thymic hypoplasia in mice (Cao et al., 1995).
In addition to IL-2, the family includes IL-4 (Russell et al., 1993),
IL-7 (Noguchi et al., 1993a), IL-9 (Russell et al., 1994), IL-15 (Giri
et al., 1994a), and IL-21 (Vosshenrich and Di Santo, 2001). All members
interact with receptor complexes consisting of an interleukin-specific
chain and the common c chain (Fig. 1). Moreover, the IL-4 chain is also a component of the IL-13 receptor complex (Zurawski et
al., 1993), and for purposes of classification, we shall include IL-13
in this interleukin family. A substantial degree of functional
redundancy is extolled by the IL-2 family members, which is
comprehensible in view of considerable overlap in their signaling
pathways. Thus, Janus kinase 1 (Jak1) and Jak3 have been found to be
activated by the subtype-specific chains and the constant c chain,
respectively (Miyazaki et al., 1994; Russell et al., 1994; Leonard and
Lin, 2000), which ultimately cascades into the activation of
transcription by the common downstream effector molecules "signal
transducer and activator of transcription" 5a (Stat5a), Stat5b, and
Stat3 (Lin et al., 1995; Lin and Leonard, 2000). IL-4 and IL-13 are somewhat distinct in activating Jak2 and Stat-6 via a c
chain-independent pathway (Palmer Crocker et al., 1996).
IL-2 (Arbustini et al., 1991; Frostegard et al., 1999) and the IL-2R
receptor (Kishikawa et al., 1993) are expressed in atheromatous tissue,
but a direct causal role for IL-2 in atherogenesis remains to be
proven. Nonetheless, serum IL-2 levels have been found to be elevated
in ischemic heart disease (Mazzone et al., 1999) and especially
unstable angina pectoris (Mizia-Stec et al., 2002), and the risk of
acute myocardial infarction is increased following IL-2 treatment for
cancer (Kragel et al., 1990). A possible explanation for the presumed
pro-atherogenic effect of IL-2 may lie in its ability to induce a T
helper cell shift toward a Th1 phenotype. T cells have been shown to be
present in atherosclerotic lesions (Hansson et al., 1988), and Th1
cells, in particular, are believed to actively promote atherogenesis
(de Boer et al., 1999; Frostegard et al., 1999; Huber et al., 2001;
Laurat et al., 2001; Song et al., 2001). In its capacity as an
autocrine stimulator of Th1 cell differentiation and proliferation
(Kurt-Jones et al., 1987; Harel-Bellan et al., 1988), IL-2 may promote
the expansion and activation of this T cell subset, and, consequently,
plaque development.
Conversely, IL-4 is known to promote Th2-type responses (partly by
autocrine activation) and to exert immunosuppressive effects on
macrophages, including the suppression of proinflammatory cytokine production and the stimulation of IL-1ra elaboration (Paul, 1991). This
cytokine is therefore considered to be potentially anti-atherogenic. The highly pleiotropic effects of IL-4, however, reserve a rather more
complicated role for IL-4 in atherosclerosis. Thus, whereas mice
deficient in Stat6, which is one of the mediators activated by IL-4,
develop larger atherosclerotic lesions than their wild-type counterparts (Huber et al., 2001), IL-4 deficient mice do not display
increased susceptibility to diet-induced atherosclerosis (George et
al., 2000a). They have even been found to be relatively resistant to
the acceleration of fatty streak formation by heat shock protein 65 or
mycobacterium tuberculosis (George et al., 2000b). Similarly,
reconstitution with IL-4-deficient bone marrow in LDLr/ mice
reduces atherosclerotic lesion formation in the aortic arch and the
thoracic aorta compared with reconstitution with wild-type bone marrow
(King et al., 2002). Although IL-4 expression in atherosclerotic
plaques appears to be limited (Uyemura et al., 1996), among the
pro-atherogenic effects of IL-4 we may count the up-regulation of
P-selectin (Khew-Goodall et al., 1999) and 15-lipoxygenase (Lee et al.,
2001b) expression by endothelial cells, VCAM-1 (Barks et al., 1997) and
matrix metalloproteinase 1 (MMP-1) (Sasaguri et al., 1998) expression
by vascular smooth muscle cells, and the augmentation of CD36 receptor
expression (Feng et al., 2000) and cholesterol esterification
(Cornicelli et al., 2000) in macrophages. On the other hand, IL-4 has
also been shown to inhibit smooth muscle cell proliferation (Vadiveloo et al., 1994; Sasaguri et al., 1998) and macrophage adhesiveness (Elliott et al., 1991). The net effect of IL-4 in atherosclerosis thus
still hangs in the balance, and it may vary with the stage of the disease.
IL-9 was initially identified as a mast cell and T cell growth factor
(Renauld et al., 1990) and has subsequently been shown to lead to
exaggerated Th2-type inflammatory responses (Godfraind et al., 1998;
McLane et al., 1998) and thymic lymphomas (Renauld et al., 1994) in
IL-9 transgenic mice. IL-9 is not entirely independent in its actions,
however, since IL-9 production by T lymphocytes requires IL-2-mediated
stimulation (Houssiau et al., 1992), and the mitogenic effect of IL-9
on T lymphocytes requires their preactivation (Uyttenhove et al.,
1988). In a murine model of Gram-negative bacterial shock, IL-9 led to
suppression of TNF, IL-12, and IFN, possibly mediated by an
induction of IL-10 expression (Grohmann et al., 2000). In agreement
with this study, IL-9 has been found to induce the expression of the
intracellular cytokine signal inhibitors cytokine-inducible
SH2-containing protein, suppressor of cytokine signaling (SOCS)-2 and
SOCS-3 (Lejeune et al., 2001). SOCS-3, in particular, may impair
signaling by pro-atherogenic cytokines that act through the gp130
receptor, including IL-6 and IL-12. Some of the activities of IL-9 may
also be mediated by its induction of IL-22 (IL-TIF), which shares 22%
sequence homology with IL-10 (Dumoutier et al., 2000). Although its
role in atherosclerosis has thus far not been elucidated, it appears that IL-9 may be potentially anti-atherogenic through a deflection of
the immune response from a Th1 to a Th2 type. Albeit that a caveat
needs to be added, as overzealous stimulation of Th2 responses may well
prove to be detrimental in the later stages of atherosclerosis. Thus,
mast cells have been identified in advanced plaques (Kaartinen et al.,
1994a; Jeziorska et al., 1997) and are presumed to promote plaque instability by the secretion of chymase (Kaartinen et al., 1994b; Kovanen, 1997) and the stimulation of calcification (Jeziorska et al., 1998). Their stimulation may promote, rather than impede, the
development of atherosclerotic complications.
IL-15 is produced by a variety of cells, including monocytes (Musso et
al., 1999) and endothelial cells (Oppenheimer-Marks et al., 1998;
Krishnaswamy et al., 1999), and has an activity profile similar to
IL-2, without sharing sequence homology (Waldmann and Tagaya, 1999).
IL-15 mediates extravasation of lymphocytes through its stimulatory and
chemotactic effects on natural killer cells (Carson et al., 1994;
Allavena et al., 1997) and T lymphocytes (Giri et al., 1995; Sancho et
al., 1999) and by the up-regulation of hyaluronan on the endothelium
(Estess et al., 1999). Recently, atherosclerotic lesions in humans and
apoE/ mice were found to contain IL-15-responsive T cells as well
as IL-15 itself, which colocalizes with oxidized LDL-positive
macrophages (Houtkamp et al., 2001, Wuttge et al., 2001). IL-15 may
therefore accelerate atherogenesis by promoting the recruitment and
antigen-independent induction of T lymphocytes.
Despite sharing only 20 to 25% sequence homology and differing from
IL-4 in lacking an effect on T cell function (Zurawski and de Vries,
1994), IL-13 is highly akin to IL-4 with respect to its
immunomodulatory properties (Opal and DePalo, 2000), which is likely to
be attributable to IL-4R-mediated Stat6 activation by both cytokines
(Hart et al., 1999). In monocytes, IL-13 attenuates the expression of a
wide range of inflammatory cytokines, including IL-1, IL-6, IL-8,
IL-10, IL-12, MIP-1, granulocyte-macrophage colony-stimulating
factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), IFN,
and TNF, while up-regulating the expression of IL-1ra (de Waal
Malefyt et al., 1993; Mijatovic et al., 1997). Nitric oxide production
is inhibited by IL-13 in macrophages (Doherty et al., 1993; Bogdan et
al., 1997) and smooth muscle cells (Ruetten and Thiemermann, 1997). The
properties of IL-13 are not exclusively anti-inflammatory, however, as
exemplified by the IL-13-mediated potentiation of IL-8 receptor
expression, 15-lipoxygenase expression, and LDL oxidation by monocytes
(Nassar et al., 1994; Folcik et al., 1997; Bonecchi et al., 2000), and
of IL-8 and MCP-1 release in response to IL-1 or TNF in SMCs
(Jordan et al., 1997). Moreover, IL-13 is known to enhance the
transmigration of leukocytes by stimulating the endothelial expression
of adhesion molecules (Bochner et al., 1995; Ying et al., 1997) and
chemotactic factors (Goebeler et al., 1997). In analogy with IL-4, the
overall effect of IL-13 in atherosclerosis is still controvertible.
The complex actions of IL-2 family members in the vascular wall are depicted in Fig. 2.
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C. The gp130 Family
The common receptor subunit shared by the members of this family
of cytokines, gp130, was first discovered as a signal transducer for
IL-6 (Hibi et al., 1990). The other factors to employ this receptor
subunit in combination with their own specific subunit, are IL-11 (Yin
et al., 1993), IL-12 (Chua et al., 1994), leukemia inhibitory factor
(Gearing et al., 1991), oncostatin M (Gearing et al., 1992),
cardiotrophin-1 (Ip et al., 1992), ciliary neurotrophic factor (Pennica
et al., 1995), and neurotrophin-1/B cell-stimulating factor-3 (Senaldi
et al., 1999) (Fig. 1). Following gp130 binding, the Janus kinases
Jak1, Jak3, and Tyk2 and the transcription factors Stat1 and Stat3 are
phosphorylated (Heinrich et al., 1998). In this review, we shall
restrict the discussion to the interleukin members of the gp130 family.
In addition, two novel heterodimeric interleukins with an activity
profile similar to IL-12 have recently been identified. IL-23 is
composed of a p19 subunit and the p40 subunit of IL-12 (Oppmann et al.,
2000), and this cytokine acts through a receptor composed of IL-12R1
and a novel cytokine receptor subunit, IL-23R (Parham et al., 2002).
IL-27 is made up of an IL-12 p40-related and an IL-12 p35-related
protein and binds to the gp130-related receptor WSX-1/TCCR (Pflanz et
al., 2002).
Endothelial cells, smooth muscle cells, and macrophages are capable of
elaborating IL-6, and its expression has been observed in
atherosclerotic lesions in humans, hypercholesterolemic rabbits, and
apoE-deficient mice (Ikeda et al., 1992; Kishikawa et al., 1993; Seino
et al., 1994; Rus et al., 1996; Sukovich et al., 1998; Schieffer et
al., 2000). Although the endothelium is largely unresponsive to IL-6
(Podor et al., 1989), addition of the soluble IL-6R subunit (sIL-6R)
enables endothelial cells to mount an inflammatory response to IL-6, by
interacting with membrane-bound gp130 (Jones et al., 2001). This
process has been termed "trans-signaling", and it may
lead to increased endothelial cell adhesiveness by the up-regulation of
E-selectin, ICAM-1, and VCAM-1, and the release of inflammatory mediators, including MCP-1, IL-8, and IL-6 itself (Modur et al., 1997;
Romano et al., 1997). Thus, sIL-6R present in serum and/or elaborated
locally by cells in the intima may serve to augment endothelial
adhesion and extravasation of leukocytes into the atherosclerotic
plaque. Monocytes and macrophages, on the other hand, produce IL-6R
autonomously and therefore do not depend on ambient sIL-6R levels for
IL-6-mediated modulation of gene expression (Akira and Kishimoto,
1996). The effector functions of IL-6 in cells of the
monocyte/macrophage lineage include the differentiation of monocytes to
macrophages (Chomarat et al., 2000), the up-regulation of acute phase
response gene expression in hepatocytes and macrophages (Perlmutter,
1989), and the priming of macrophages for enhanced TNF production in
response to lipopolysaccharide (LPS) administration (Cochran and
Finch-Arietta, 1992). In smooth muscle cells, IL-6 induces
proliferation directly (Nabata et al., 1990; Ikeda et al., 1991) and
indirectly through the initiation of an autocrine loop mediated by the
up-regulation of gp130 (Klouche et al., 1999). In addition, smooth
muscle cells are stimulated by IL-6 to express ICAM-1 (Ikeda et al.,
1993) and to evolve into foam cells (Klouche et al., 2000).
Whereas homozygous deletion of gp130 in mice leads to intrauterine
death due to myocardial hypoplasia (Yoshida et al., 1996), IL-6-deficient mice develop normally despite an attenuated acute phase
response and impaired cellular immunity to virus infection (Kopf et
al., 1994). This is a reflection of the functional redundancy in
gp130-mediated signaling and thus of the extent to which the other
members of the gp130 family can take over IL-6-mediated functions. IL-6
was initially described as a lymphocyte stimulatory factor but has
since been found to exert a plethora of inflammatory effects (Hirano et
al., 1990). With the possible exception of IL-1, IL-6 is the cytokine
with the most extensively studied pro-atherogenic profile. Causality
has been established through the exacerbation of early atherosclerosis
by recombinant IL-6 in various atherosclerosis-prone murine models
(Huber et al., 1999). Interestingly, the progression of atherosclerotic
lesions to an advanced phenotype appears to be inhibited by IL-6 in
apoE-deficient mice, uncovering a potentially biphasic mode of action
in atherogenesis (Elhage et al., 2001), which is perhaps partly
explained by its observed anti-inflammatory properties (Barton et al.,
1996; Xing et al., 1998) and its inhibition of macrophage class A
scavenger receptor expression (Liao et al., 1999). Nonetheless,
inhibition of IL-6 signaling may be considered to constitute an
attractive therapeutic strategy for the prevention of coronary heart
disease (Stein and Kung Sutherland, 1998; Yudkin et al., 2000).
Clinically, elevated levels of IL-6 and its hepatic by-product
C-reactive protein (Verma et al., 2002) are associated with increased
risks of coronary and peripheral atherosclerosis (Erren et al., 1999;
Mazzone et al., 1999; Flex et al., 2002; Bermudez et al., 2002; Kato et
al., 2002; Stenvinkel et al., 2002; van der Meer et al., 2002),
myocardial infarction (Ridker et al., 2000b; Ikeda et al., 2001), and
the risk of death of patients with cardiovascular disease (Volpato et
al., 2001), and IL-6 has been suggested to mediate the pro-atherogenic
properties of cytomegalovirus (Blankenberg et al., 2001). In a large
multicenter study, IL-6 gene polymorphisms were found to correlate with
the severity of coronary artery disease and the risk of myocardial
infarction (Georges et al., 2001), and carotid atherosclerosis has been
shown to be independently linked with an IL-6 promoter polymorphism (Rauramaa et al., 2000; Rundek et al., 2002), as has the risk of
coronary artery disease (Humphries et al., 2001). In addition, lower
levels of soluble IL-6 receptor, a naturally occurring IL-6 antagonist,
are linked with the risk of myocardial infarction (Ueda et al., 1999).
Although these clinical findings do not establish causality, they have
identified a strong association between IL-6 levels and atherosclerosis.
Despite sharing considerable redundancy with IL-6 with respect to its
signaling and effector functions, IL-11 has been judged to be a more
anti-inflammatory member of the gp130 family of cytokines based on the
net effect of its pleiotropic actions (Schwertschlag et al., 1999; Taki
et al., 1999). In macrophages, recombinant IL-11 has been found to
attenuate macrophage expression of TNF, IL-1, IL-12, and nitric
oxide following an LPS challenge (Trepicchio et al., 1996; Leng and
Elias et al., 1997). These effects are direct and mediated by NF-B
down-regulation (Trepicchio et al., 1997), as is IL-11-mediated
attenuation of smooth muscle cell proliferation and cytokine production
(Zimmerman et al., 2002). In endothelial cells, IL-11 provides
protection against immune-mediated injury (Mahboubi et al., 2000), and
inhibits apoptosis through up-regulation of survivin (Mahboubi et al.,
2001). In CD4+ lymphocytes, IL-11 has been found to induce a shift from
a Th1 to a Th2 phenotype (Bozza et al., 2001). This effect has been put
to use in immunomodulatory treatment employing IL-11 in psoriasis
(Trepicchio et al., 1999) and Crohn's disease (Sands et al., 1999),
and it may also offer therapeutic possibilities in the setting of atherosclerosis.
Activated monocytes are the primary source of IL-12 (D'Andrea et al.,
1992), a cytokine that induces proliferation (Gately et al., 1991) and
a shift toward a Th1 expression pattern in lymphocytes (Hsieh et al.,
1993). IL-12 was originally implicated in atherosclerosis by Uyemura et
al. (1996), who observed an abundance of p40 mRNA and IL-12 p70 protein
in atherosclerotic lesions, and up-regulation of IL-12 production by
monocytes following the addition of highly oxidized LDL. Subsequently,
atherosclerotic lesions in apoE-deficient mice were found to contain
IL-12, and their progression to be accelerated by daily injections of
recombinant IL-12 (Lee et al., 1999). Conversely, a selective defect of
macrophage IL-12 synthesis due to 12/15-lipoxygenase deficiency reduces
lesion formation in atherosclerosis-prone Apobec-1//ApoE/ mice
(Zhao et al., 2002). In clinical practice, raised serum levels of IL-12
have been found to be associated with acute myocardial infarction (Zhou et al., 2001a).
D. Granulocyte-Macrophage Colony-Stimulating Factor
The genes encoding the members of this familyIL-3, IL-5, and
GM-CSFare clustered on the human chromosome 5 (van Leeuwen et al.,
1989) (Fig. 1). Their products bind to receptor complexes consisting of
a common chain (c) and a cytokine-specific chain (Hayashida
et al., 1990; Kitamura et al., 1991), resulting in the activation of
JAK/STAT, the ras/MAPK, and the phosphatidylinositol-3 kinase pathway
(Guthridge et al., 1998). The primary effector functions to be
identified for this family are the promotion of hematopoietic
proliferation, survival, and differentiation, which is confirmed by the
invariable occurrence of a myeloproliferative disorder in human common
chain transgenic mice (Nishinakamura et al., 1995). Since mice
deficient for IL-3, IL-5, or GM-CSF suffer from pulmonary alveolar
proteinosis, signaling via receptors involving the common chain is
thought to exert additional pleiotropic actions on mature cells of the
monocyte/macrophage lineage (D'Andrea et al., 1998).
Indeed, IL-3 has been found to stimulate adhesion (Elliott et al.,
1990) and c-jun expression in monocytes (Mufson et al., 1992). It is
elaborated by activated T lymphocytes in atheromatous tissue and acts
on smooth muscle cells to increase migration and proliferation (Brizzi
et al., 2001). Moreover, receptors for IL-3 are also present on
endothelial cells (Colotta et al., 1993a), which respond to the
binding of IL-3 by increased proliferation, by augmented adhesion
molecule, major histocompatibility complex II, and cytokine production,
and by participating in angiogenesis in vivo (Brizzi et al., 1993;
Korpelainen et al., 1995; Dentelli et al., 1999). IL-3 is thus believed
to play a role in the early stages of atherogenesis by facilitating
leukocyte extravasation and in advanced lesions by augmenting
macrophage activation, smooth muscle cell accumulation, and
neovascularization of the plaque.
The involvement of IL-5 in the stimulation of B cell and eosinophil
responses has been meticulously documented, with the aid of,
inter aliter, IL-5 transgenic (Tominaga et al., 1991), and IL-5-deficient mouse models (Kopf et al., 1996). Its role in
atherosclerosis remains uncharted territory, however. IL-5 is produced
by endothelial cells (Krishnaswamy et al., 1999), but their expression
of the IL-5 receptor subunit is limited (Colotta et al., 1993a).
IL-5 expression appears to be scanty in advanced human atherosclerotic plaques, and is associated with the presence of eosinophils (Frostegard et al., 1999). Because IL-5 is an archetypal Th2 lymphokine, it may
activate mast cells in the atherosclerotic plaque, which have been
associated with the development of unstable lesions and plaque rupture
(Kaartinen, 1994a,b, 1996a,b, 1998; Kovanen et al., 1995). Notwithstanding its low prevalence, the significance of locally produced IL-5 may thus increase in importance with the age of the
lesion, and this could lead to destabilization of the atheroma.
E. Interleukin-10
This family comprises a sizeable array of mammalian and viral
molecules that possess a considerable degree of sequence similarity with its founder member, IL-10 (Rich and Kupper, 2001; Volk et al.,
2001). These include IL-19, IL-20, IL-22, IL-24/MDA-7, IL-26/AK155, and
the IL-10 homologs encoded by Epstein-Barr virus, cytomegalovirus, herpesvirus papio, and Yaba-like disease virus (Fickenscher et al.,
2002; Wolk et al., 2002; Fig. 1).
IL-10 was initially identified as a cytokine synthesis inhibitory
factor (CSIF) (Fiorentino et al., 1989), but has subsequently been
found to be a pleiotropic immunoregulatory cytokine that is secreted by
a wide variety of cells, including lymphocytes and
monocytes/macrophages (Lalani et al., 1997b; Moore et al., 2001). IL-10
signaling is mediated by Jak1 and Stat3 and entails the down-regulation
of NF-B activity (Schottelius et al., 1999). Its effector functions
include induction of a shift of T cell cytokine expression from a Th1
to a Th2 profile (Fiorentino et al., 1989), and attenuation of the
production of proinflammatory cytokines by macrophages (Bogdan et al.,
1991; de Waal Malefyt et al., 1991a; Lang et al., 2002) and
polymorphonuclear neutrophils (Cassatella et al., 1993). In addition,
IL-10 effects differentiation of monocytes to macrophages (Allavena et
al., 1998), suppression of antigen-presenting activity (de Waal Malefyt
et al., 1991b), a decline in the release of reactive nitrogen and
oxygen intermediates (Gazzinelli et al., 1992; Mallat et al., 1999a;
Haddad and Fahlman, 2002), and inhibition of ICAM-1 expression (Song et
al., 1997). Monocyte adhesion to endothelial cells is attenuated by
IL-10 through modulation of monocyte CD18 and CD62-L expression
(Mtairag et al., 2001) and attenuation of ICAM-1 and VCAM-1 expression on endothelial cells (Krakauer, 1995; Lindner et al., 1997). IL-10 has
been found to be present in mature plaques (Uyemura et al., 1996;
Mallat et al., 1999a) and is thought to play an active role in curbing
the inflammatory milieu of the vessel wall (Tedgui and Mallat, 2001).
This is supported by the observation that IL-10 knockout (IL-10/)
mice suffer from accelerated atherosclerosis, whereas IL-10 transgenic
mice are relatively protected (Pinderski-Oslund et al., 1999). Clinical
poignancy is added by the fact that a hypoactive allele of the IL-10
promoter sequence increases the risk of cardiovascular events in
hemodialysis patients (Girndt et al., 2002), whereas serum levels of
IL-10 have been found to be decreased in patients with unstable angina
compared with patients with chronic stable angina (Smith et al., 2001).
Indeed, as IL-10 is known to down-regulate MMP-9 production and
up-regulate tissue inhibitor of metalloproteinase-1 (TIMP-1) expression
in macrophages (Lacraz et al., 1995), IL-10 may have a direct
stabilizing influence on advanced plaques. Moreover, the combined
weight of these data has led to extensive speculation about the
therapeutic applicability of IL-10 in atherosclerosis (Terkeltaub,
1999).
F. Chemokines
On the basis of their chemoattractant activity for leukocytes, the
interleukins IL-8 and IL-16 have been classified as chemokines (Center
and Cruikshank, 1982; Mukaida et al., 1989). IL-16 has not been
scrutinized in an atherosclerotic context, and any potential influence
is likely to be mediated mainly by its effects on lymphocyte function,
which include stimulation of migration, proliferation, and cytokine
production (Cruikshank et al., 2000). IL-8, on the other hand, is well
established as a pro-atherogenic factor (Reape and Groot, 1999). Its
expression is induced in monocytes and macrophages following the
addition of oxidized LDL and cholesterol, respectively (Terkeltaub et
al., 1994; Wang et al., 1996). Atheromatous tissue has been found to
contain IL-8, most of which is thought to be derived from intimal
macrophages (Apostolopoulos et al., 1996; Wang et al., 1996). In
addition, cytokine-stimulated vascular smooth muscle cells elaborate
IL-8 (Wang et al., 1991), and endothelial cells respond to cyclic
stretch by up-regulation of IL-8 production (Okada et al., 1998).
Boisvert et al. (1998) have discovered an important role for
macrophage-derived IL-8 in atherosclerotic lesion development, as
transplantation of IL-8/ bone marrow to irradiated and atherogenic
diet-fed LDLr/ mice resulted in less extensive intimal macrophage
accumulation than transplantation using IL-8+/+ donors. IL-8 is
presumed to accelerate atherogenesis by increasing the endothelial
adhesiveness for monocytes (Gerszten et al., 1999), by its mitogenic
and chemoattractant actions on smooth muscle cells (Yue et al., 1994),
and by mediating angiogenesis in the atherosclerotic plaque (Simonini
et al., 2000). Furthermore, IL-8 may cause destabilization of advanced
plaques through its inhibitory effect on TIMP-1 expression in
macrophages and an ensuing increase in metalloproteinase activity
(Moreau et al., 1999). Interestingly, IL-8 levels have been found to be
elevated in peripheral blood monocytes from hypercholesterolemic
patients (Porreca et al., 1999), and serum IL-8 levels to be associated
with unstable angina pectoris and acute myocardial infarction (Zhou et
al., 2001a), reflecting the potential clinical relevance of
IL-8-mediated functions in atherosclerosis.
G. Interleukin-17
The term IL-17 harbors a family of proinflammatory cytokines, of
which the founder member was found to be an ortholog of murine CTLA-8
(Rouvier et al., 1993) and its gene to have been captured by the T
lymphotropic herpesvirus saimiri (Rouvier et al., 1993, Yao et al.,
1995a,b; Fossiez et al., 1998). It is primarily produced by activated
memory T cells and Th1/Th0 cells (Aarvak et al., 1999) and binds to a
ubiquitously expressed receptor (Yao et al., 1995a). More recently, the
variants IL-17B, IL-17C, IL-17E, IL-17F, and IL-25 have been cloned,
which are considered to signal through subtype-specific receptors (Li
et al., 2000b; Hymowitz et al., 2001; Lee et al., 2001a; Hurst et al.,
2002). IL-17 induces the expression of proinflammatory mediators by a
variety of cells, including the production of IL-6 and IL-8 by stromal
cells (Yao et al., 1995a,b), ICAM-1 by fibroblasts and keratinocytes
(Yao et al., 1995b; Albanesi et al., 1999), as well as IL-1, IL-1ra, IL-6, IL-10, TNF, prostaglandin E2, MMP-3, and
MMP-9 by macrophages (Jovanovic et al., 1998, 2001). Binding of IL-17
to its receptor results in an increase in Ca2+
influx, a decrease of intracellular cAMP levels, activation of mitogen-activated protein kinases, and stimulation of NF-B activity (Jovanovic et al., 1998; Awane et al., 1999). The activity profiles of
IL-17B and IL-17C differ from that of IL-17 in that they fail to induce
IL-6 in fibroblasts but are capable of stimulating the release of
TNF and IL-1 from the monocytic cell line THP-1 (Li et al.,
2000b). IL-17E has been shown to stimulate NF-B activity and the
production of IL-8 in TK-10 cells (Lee et al., 2001a). The IL-17 family
has not yet been implicated in atherogenesis, but its proinflammatory
effects on macrophages, the stimulation of endothelial IL-2 and MCP-1
elaboration by IL-17F (Starnes et al., 2001), the production of IL-17
by activated T cells, and the widespread expression of the IL-17
receptor make this interleukin family a potential pro-atherogenic candidate.
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III. Modulation of Cytokine Function As a Therapeutic Strategy for Atherosclerosis |
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From the preceding discussion it will have become evident that, despite having been thoroughly researched with respect to their basic immunological functions, many of the interleukins identified to date have yet to be typecast on the atherosclerotic stage (Table 1). When classified according to their perceived role in atherogenesis, a large number thus remain in the "unknown" category. A similarly sizable group has been found to be pro-atherogenic, and only a small subset has been adjudicated to possess an equivocal (IL-4, IL-13) or anti-atherogenic (IL-1ra, IL-9, IL-10, IL-11) propensity. It therefore appears that the most rewarding strategies of interleukin modulation for the prevention of atherosclerosis are likely to involve the down-regulation of signaling mediated by proinflammatory cytokines. Nonetheless, due attention also needs to be paid to the intriguing therapeutic possibility of harnessing the anti-atherogenic potential of anti-inflammatory interleukins. The modulation of (patho)physiological effects exerted by cytokines that have thus far been adjudicated to have either an overtly pro- or an anti-atherogenic role on the evidence of animal intervention studies are, in the short term, the most likely candidates for the development of such strategies (Table 2; Fig. 3).
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The function of interleukins is tightly regulated at a number of levels in their production, processing, and signaling cascades. Interleukins being proteins, the first step in their production necessitates the binding of nuclear transcription factors to enable gene transcription. Following mRNA translation, the production of mature molecules requires additional proteolytic processing for a number of interleukins and interleukin receptors. The ambient concentration of some interleukins is known to be negatively regulated following exposure on the cell surface or release into the surrounding extracellular space. This may involve neutralization of interleukins by binding to a specific antibody or to a soluble form of its corresponding receptor.
Interleukin molecules that escape endogenous regulation mechanisms can
bind to their target receptor and thus initiate a signaling sequence.
The abundance of the membrane-bound form of interleukin receptors may
be controlled by endocytosis and degradation via the
ubiquitin-proteasome system. The signaling cascade is frequently rather
complex and often shares redundancy with those activated by other
members of a particular interleukin family. A varied array of pathways
has been found to convey interleukin signaling to the nucleus,
frequently involving receptor-mediated activation of kinases (including
Jaks, Tyks, and MAPKs) and subsequent activation of nuclear
transcription factors (including STATs, NF-B, and AP-1) (Fig. 1).
Intracellular signal transduction is negatively controlled by specific
inhibitors of the Jak-STAT pathway that regulate its components by
dephosphorylation, degradation by the ubiquitin-proteasome pathway, and
binding of dominant-negative STATs. Signaling eventually culminates in
the transcriptional activation of a cytokine-specific set of genes, the
products of which mediate the biological functions of the cytokine in
question by intracellular, autocrine, paracrine and endocrine mechanisms.
In theory, any step in the production and effector pathways of a
particular interleukin may be considered to represent a potential target for therapies aimed at modulating its biological activity (Fig.
4). In practice, various approaches are
not yet feasible due to a lack of detailed understanding of the
mechanisms involved. Moreover, the specificity of such interventions is
frequently limited by considerable redundancy in interleukin
processing and signaling pathways. Although this may be desirable if
the goal of the intervention is a general reduction of proinflammatory signaling, a more subtle change of cellular functions may require direct alteration of extracellular interleukin levels or
interleukin-receptor interaction. In the specific case of
atherosclerosis, the more difficult hurdles on the course to the
clinical use of cytokine modulation therapy are hidden in the insidious
and chronic nature of the atherosclerotic process (Ross 1986, 1993a,
1999). Inherent in this observation is the need for any strategy aimed
at primary prevention to be comparably chronic in its
duration of action. In view of the high prevalence of the disease and
its still poorly predictable course, such a strategy would also need to
be safe, effective, and affordable. Most of the interleukin-based
treatments that have been conceived thus far do not answer these
demands. In the meantime, it may be more realistic to focus on a remedy that is capable of effecting secondary or
tertiary prevention. An example of the latter is the
phenotypic stabilization of unstable atherosclerotic atheromata to
avert the risk of plaque rupture and fatal thrombosis. This may be
achievable by the use of a short, and possibly localized, course of
anti-interleukin therapy.
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In this review, we discuss examples of techniques directed at modulating each of the steps described above. We shall pay particular attention to methods that have been shown to hold promise for the prevention of atherosclerosis or those that interfere with the function of interleukins thought to be involved in atherogenesis.
A. Inhibition of Expression/Translation of Interleukins and Their Receptors
The foremost approach to the specific inhibition of interleukin
(receptor) expression and translation has been the use of short strands
of (modified) nucleotides that are complimentary to stretches of mRNA
encoding the target protein. This is thought to lead to formation of
DNA:RNA duplexes and subsequent degradation of the mRNA sequence by
RNaseH. The advent of this oligonucleotide-based "antisense"
therapy was hailed as the dawn of a new era of highly specific and
effective treatments for a variety of diseases, ranging from cancer to
hypertension (Raizada et al., 2000; Lebedeva and Stein, 2001). This
unbridled optimism has been somewhat deflated in recent years, however,
as it has transpired that the mechanism of action of antisense
molecules is frequently less specific and far more complex than
originally conceived (Lebedeva and Stein, 2001). Moreover, unmodified
oligonucleotides are rapidly degraded in vivo, and efficient
transfection of target cells with antisense constructs has proved
difficult. Nonetheless, several studies describing the
antisense-mediated down-regulation of interleukin production have been
reported (Crooke, 2000).
IL-1 is known to inhibit endothelial cell proliferation, and thereby
to promote the type of endothelial injury that is thought to
precipitate atherogenesis (Ross, 1986). Furthermore, IL-1 is an
autocrine stimulator of adhesion molecule expression, including ICAM-1
and E-selectin, and the up-regulation of these molecules by hypoxic
endothelial cells has been found to be mediated by IL-1 (Shreeniwas
et al., 1992). Antisense oligodeoxynucleotides (ODNs) directed against
IL-1 have been found to prevent endothelial cell senescence, to
prolong their life span, and to hinder adhesion molecule production in
vitro (Maier et al., 1990; Maier and Ragnotti, 1993). Moreover, the
IL-1 -mediated up-regulation of cyclooxygenase expression in
endothelial cells has been shown to be limited by the addition of ODNs
directed against protein kinase C (PKC), which is a mediator in the
signal transduction pathway that leads to IL-1 induction (Hsu et
al., 1999). Because interleukin-1 also affects smooth muscle cell
function, Hsu et al. (1999) transfected vascular smooth muscle cells in
vitro with an Epstein-Barr virus-derived vector expressing IL-1
antisense transcripts, which repressed the expression of matrix genes
such as type I collagen and fibronectin by smooth muscle cells and
prolonged their life span. In macrophages, more specifically the
macrophage-like cell line U937, the expression of IL-1 can also be
down-regulated by means of antisense techniques employing
phosphorothioate oligonucleotides (Yahata et al., 1996).
The platelet-derived growth factor (PDGF)-mediated up-regulation of
IL-6 in smooth muscle cells can be attenuated by antisense ODNs
directed against this pro-atherogenic interleukin (Roth et al., 1995).
This has been shown to inhibit cell division, and has thus established
IL-6 as a mediator of PDGF-induced smooth muscle cell proliferation.
The feasibility of antisense-mediated inhibition of IL-6 expression in
the vessel wall has been demonstrated by ex vivo pressure-mediated
transfection of naked oligonucleotides into human saphenous vein
explants, which resulted in 70 to 75% inhibition of IL-6 expression,
as measured 2 h after the transfection procedure (Mann et al.,
1999).
Chemokine function has also been successfully repressed by antisense
techniques. Thus, the role of IL-8 as a monocyte-derived angiogenic
factor was revealed in vitro by the inhibition of monocyte-induced angiogenic activity following the administration of an IL-8 antisense oligonucleotide (Koch et al., 1992), and pretreatment of human pulmonary artery endothelial cells with antisense against MCP-1 has
been shown to reduce TNF-induced trans-endothelial
monocyte migration (Maus et al., 2000).
Rather than inhibiting the production of interleukins themselves,
antisense strategies could also be deployed against interleukin signaling by altering the expression of the relevant receptor. Indeed,
ODNs directed against the IL-1 receptor have been shown to diminish
IL-1-stimulated prostaglandin E2 synthesis in
murine and human fibroblasts (Burch and Mahan, 1991), and in vivo
applicability was confirmed by the finding that subcutaneous injection
of IL-1 receptor antisense in mice decreased neutrophil accumulation at sites of IL-1 injection.
Intriguingly, ODNs containing cytidine phosphate guanosine motifs have
been identified as potent stimulators of Th1 type responses, and this
type of aspecific effect needs to be taken into account during the
design of anti-inflammatory antisense sequences (Chu et al., 1997). In
a drive to enhance the specificity as well as the efficacy,
tolerability, and duration of action of antisense-mediated mRNA
cleavage, the attention has turned to the use of ribozymes. These are
RNA molecules with intrinsic endonuclease activity, which bind to
target RNA in a base pair-specific fashion, and subsequently catalyze
the cleavage of this RNA strand by facilitating the hydrolysis of
phosphodiester bonds (Zaug et al., 1986; James and Gibson, 1998).
Indeed, stable expression of ribozymes aimed at IL-1 and ICE can
effect a dramatic decrease in the steady-state levels of their target
mRNAs in the monocytic cell line THP-1 (Leavitt et al., 2000) and
minimized hammerhead ribozymes have been shown to be active against
IL-2 (Sioud et al., 1997). In vivo efficacy and in vitro reduction of
TNF-induced IL-6 production has been demonstrated for IL-6 ribozymes
(Mahieu et al., 1994). The first cardiovascular target to have been
successfully inhibited by ribozyme therapy directed against cytokine
expression is transforming growth factor (TGF) production in
smooth muscle cells (Su et al., 2000). In vivo, TGF ribozymes have
been shown to reduce neointima formation in a rat model of vascular
injury (Yamamoto et al., 2000).
Although ribozymes may prove to be more effective and specific than
antisense oligonucleotides due to their enzymatic mode of action, they
share similar limitations to their biological activity. Thus, efficient
cellular transfection is difficult to achieve, and once it has
occurred, the duration of action is curtailed by a short intracellular
half-life. Both problems have been extensively addressed, to varying
degrees of success. Cellular uptake has been increased by the use of
lipid, peptide, and polymer delivery systems, and nuclease-mediated
degradation has been inhibited by chemical modifications of the
oligonucleotide backbone (Morishita et al., 1994; Hughes et al., 2001;
Lebedeva and Stein, 2001). Circumventing both disadvantages in a single
approach may be possible by cloning ribozymes into an expression vector
that affords avid transfection of target cells in addition to an
extended duration of expression. These are characteristics of viral
vectorsretroviruses, adenoviruses, and adeno-associated viruses
(AAVs) being the main protagonists. These viral vectors have all been
used as a carrier for ribozymes, but AAVs are particularly promising as
they combine the main advantage of adenoviruses, i.e., high efficiency
of transduction, with the prolonged expression due to integration of
transgenes in the genome that is typical of retroviruses (Monahan and
Samulski, 2000). AAVs have been shown to be capable of transducing
endothelial and vascular smooth muscle cells in vitro, but expression
in vivo is confined to the adventitia (Lynch et al., 1997). Therefore, to constitute a useful gene transfer vehicle in the prevention of
atherosclerosis, the issue of targeting accuracy needs to be addressed,
for instance by selecting the most suitable virus serotype, or by using
an adenovirus/AAV hybrid system (Recchia et al., 1999; Monahan and
Samulski, 2000).
B. Inhibition of Interleukin Processing
As mentioned, the production of mature forms of various cytokines
requires proteolytic processing of inactive precursors. This is
exemplified by the conversion of pro-IL-1 and pro-IL-18 by
ICE/caspase-1 (Wilson et al., 1994; Tone et al., 1997), and the
cleavage of membrane-bound TNF by TNF-converting enzyme (Black et
al., 1997). Interestingly, the shedding of the soluble form of several
interleukin receptors has also been found to require metalloproteinase
activity, including IL-1R, IL-2R, and IL-6R (Mullberg et al., 1995,
1997). With respect to atherosclerosis, interference with
proteinase-dependent processing may constitute an attractive strategy
to attenuate the release of active IL-1 or to inhibit
sIL-6R-mediated transfer of IL-6 sensitivity to cells that do not
themselves express IL-6R (Jones et al., 2001).
Several naturally occurring ICE inhibitors have been described
(Croucher et al., 2000). Thus, cowpox virus protein A (CrmA) protects
cells infected by cowpox from immunological clearance by preventing the
release of IL-1 (Ray et al., 1992), whereas the baculovirus protein
p35 performs a similar function in baculovirus-infected cells (Bump et
al., 1995). Smooth muscle cells produce an endogenous ICE inhibitor,
which has been identified as serpin proteinase inhibitor 9 (PI-9)
(Schonbeck et al., 1997; Young et al., 2000). Interestingly, protein
levels of this enzyme have been found to be decreased in unstable
plaques in conjunction with a reciprocal up-regulation of IL-1,
suggesting an endogenous anti-inflammatory role for constitutive PI-9
expression. Consequently, inhibition of caspase-1 activity might be an
effective strategy in the prevention of lesion destabilization. In
considering the feasibility of therapeutic ICE inhibition, one might
either opt to capitalize on the potency of naturally occurring
antagonists, or one could interpret their action as a paradigm for the
development of synthetic ICE inhibitors (Livingston, 1997). Several
such compounds have been developed that display activity in vitro. This
includes the down-regulation by WIN 67694 of the LPS-induced
release of IL-1 by murine macrophages (Miller et al., 1995), and the
reduction of human myocardial ischemic dysfunction in an ex vivo organ
culture model by YVAD (Pomerantz et al., 2001). In vivo, a single
intraperitoneal dose of VE-13,045 administered after an LPS challenge
reduced murine IL-1 serum levels by 50 to 70% (Ku et al., 1996).
Prior to clinical use, however, the specificity for caspase-1 of the
compound in question needs to be warranted, in view of the degree of
conservation that has been found to exist between the active sites of
the caspase family members. Moreover, due heed should be paid to the
potentially detrimental inhibition of caspase-1-mediated apoptosis,
which could contribute toward tissue hyperplasia or even neoplasia.
C. Neutralization of Proinflammatory Interleukins
The biological activity of interleukins is partially regulated by
anti-cytokine antibodies, soluble cytokine receptors, and cytokine-binding proteins, the elaboration of which is frequently controlled by the interleukin concerned (Heaney and Golde, 1998; Slifka
and Whitton, 2000). Soluble interleukin receptors are produced by
alternative splicing of mRNA or by proteolytic cleavage of full-length
receptors. For instance, IL-1 activity is inhibited by the soluble type
II IL-1 receptor (Giri et al., 1990), which is shed from neutrophils in
response to proinflammatory stimuli, including TNF, IL-13, and
endotoxin (Colotta et al., 1994; Giri et al., 1994b). Its
pathophysiological roles are thought to include the limitation of IL-1
activity in sepsis (Giri et al., 1994b). Whereas the plasma level of
soluble IL-2R has been deemed to be a marker for T cell activation in
ischemic heart disease (Simon et al., 2001), high levels of sIL-2R
paradoxically reduce the relative risk of lesion instability, which is
known to be associated with increased inflammatory activity in the
plaque (Blum et al., 1995; Takeshita et al., 1997; Simon et al., 2001).
Moreover, in vitro studies have evidenced the inhibition of
IL-2-induced activation of peripheral mononuclear cells by sIL-2R (Zorn
et al., 1994). Soluble IL-4-binding proteins are known to occur in mice
(Fernandez-Botran and Vitetta, 1990) and humans (Fanslow et al., 1993).
The benefit of sIL-4R in preventing IL-4-mediated inflammatory
responses has been demonstrated in a murine model of asthma (Henderson
et al., 2000), and its administration has been found to be safe and to stabilize lung functions in patients with moderate asthma (Renz, 1999).
For some soluble interleukin receptors, however, the effects are rather
less clear-cut (Heaney and Golde, 1998). The trans-signaling activity conferred by sIL-6R has already been discussed (Jones et al.,
2001), as well as its role in endothelial cell activation (Modur et
al., 1997; Romano et al., 1997). By contrast, the soluble receptor
subunit for another member of the IL-6 family, IL-11, has been found to
antagonize IL-11 activity (Curtis et al., 1997). Likewise, the soluble
form of the gp130 subunit shared by the IL-6 family of receptors is
thought to inhibit IL-6 mediated signaling by binding the IL-6/sIL-6R
complex (Narazaki et al., 1993; Muller-Newen et al., 1998; Jostock et
al., 2001).
Antagonistic binding proteins have recently also been found for IL-13,
IL-18, and IL-22 (Zhang et al., 1997; Xu et al., 1998b; Novick et al.,
1999). IL-18-binding protein (IL-18bp) has been characterized as a
modulator of the Th1 response on the basis of its ability to inhibit
IL-18-mediated up-regulation of IFN, IL-8, NF-B, and VCAM-1
(Reznikov et al., 2000; Vidal-Vanaclocha et al., 2000). The human
IL-18bp gene encodes at least four isoforms (Kim et al., 2000b),
and its expression is increased by IFN in a range of human cell
lines (Muhl et al., 2000). Serum levels of IL-18bp are raised in septic
patients, with a concomitant decrease in free IL-18, and its roles are
therefore presumed to include the provision of negative feedback in
states of high inflammatory activity (Novick et al., 2001). Recently,
Mallat et al. (2001b) have demonstrated the anti-atherogenic potential
of IL-18bp. They have found electrotransfer of an expression plasmid
encoding murine IL-18bp to attenuate atherosclerotic lesion development
in the aorta of apoE/ mice. This treatment also resulted in changes in plaque composition, comprising a decrease in inflammatory cell content and an increase of smooth muscle cell and collagen content of
the lesion. IL-18bp therefore appears to have a beneficial effect on
plaque stability as well as plaque progression. Moreover, IL-18bp may
promote ischemia-induced neovascularization by inhibiting the
anti-angiogenic role of IL-18, and its administration could therefore
also aid postinfarction myocardial recovery (Mallat et al., 2002).
Interestingly, poxvirus proteins have been identified that share
considerable sequence homology with human IL-18bp. These inhibit virus
elimination by the host's immune system by binding IL-18, attenuating
IL-18-induced IFN production, and impairing natural killer cell
cytotoxicity (Born et al., 2000; Calderara et al., 2001) and may be
promising anti-atherogenic agents in their own right. Similar
protective functions appear to be served by the viral capture and
modification of other cytokine receptor genes (Spriggs, 1996; McFadden
et al., 1998), including the IL-1R (Spriggs et al., 1992) and IL-8R
(Rosenkilde et al., 1999), and the chemokine binding proteins M-T1 and
M-T7 (Upton et al., 1992; Graham et al., 1997; Lalani et al., 1997a).
The latter is an IFNR homolog and has been used successfully in the attenuation of angioplasty-induced neointima formation in rat carotid
arteries (Liu et al., 2000). A 38-kDa glycopeptide encoded by the
tanapox virus binds IL-2, IL-5, and IFN, and inhibits the
TNF-induced expression of E-selectin, VCAM-1, and ICAM-1 by tanapox
virus-infected primary endothelial cells (Paulose et al., 1998).
Other cytokines have also been targeted by soluble receptor therapy,
the foremost example being the antagonism of TNF. TNF has been
suggested to be pro-atherogenic by virtue of its presence in
atherosclerotic lesions and its proinflammatory effects on all cell
types involved in atherogenesis, including the up-regulation of
adhesion molecules, chemoattractants, cytokines, and growth factors
(LeBoeuf and Schreyer, 1998). Although systemic TNF levels are not
correlated with an increased propensity to atherosclerosis, the level
of TNF is an independent risk factor for the occurrence of acute
coronary events in patients with coronary artery disease (Ridker et
al., 2000a; Sack, 2002). Most importantly, however, TNF levels are
known to be raised in congestive heart failure and to exacerbate heart
failure in murine models, probably due to excessive myocardial
remodeling (Bradham et al., 2002).
TNFR-IgG fusion proteins have proven their worth in reducing the TNF
-mediated induction of proinflammatory interleukins, including IL-1
and IL-6 (Abraham et al., 1994; Kubota et al., 2000; Kadokami et al.,
2001). Two TNF blockers have recently been evaluated in clinical
trials: etanercept (Enbrel), a fusion protein of the soluble form of
the TNFR and the Fc portion of human immunoglobulin IgG1 and infliximab
(Remicade), a chimeric IgG1 monoclonal antibody that contains a murine
binding site for TNF. Despite encouraging results in early clinical
studies, in which subcutaneous etanercept administration appeared to be
safe and to result in improvement of cardiac function in patients with advanced heart failure (Bozkurt et al., 2001), a large-scale phase II/III trial (RENEWAL) has recently been prematurely discontinued due
to a lack of benefit (Louis et al., 2001). The introduction of
infliximab as a therapy for rheumatoid arthritis, on the other hand,
has been marred by the recent report of a case of sudden death in a
patient without heart failure following a single 200 mg infusion (de'
Clari et al., 2002). Moreover, a phase II clinical trial investigating
the use of infliximab in advanced congestive heart failure has been
placed on hold after the death of seven patients in the treatment group.
The experience with infliximab, in particular, may point to a
potentially protective effect of TNF in heart failure. Thus, TNF
has been found to induce protein synthesis in cardiac myocytes (Hiraoka
et al., 2001) and to lead to inflammatory autoregulation by means of
the translocation of functionally inactive NF-B p50 homodimers
(Haudek et al., 2001).
In an atherosclerotic context, blockade of TNF by administration of
soluble TNFR has been found to accelerate endothelial recovery after
balloon angioplasty of rat carotid arteries (Krasinski et al., 2001).
Because endothelial damage is thought to be an important process in
atherogenesis and atherosclerotic plaque erosion, the inhibition of
TNF-mediated impairment of endothelial function could yield
considerable merit. In an analogous approach, adenovirus-mediated
transfer of a secreted TGF type II receptor has been demonstrated to
inhibit luminal loss after percutaneous transluminal coronary
angioplasty of porcine coronary arteries (Kingston et al.,
2001). Specific targeting to inflammatory tissues may refine such gene
transfer approaches, as demonstrated by gene vectors in which the
TNFR-IgG fusion protein sequence has been placed under the control of a
serum amyloid A promoter. Serum amyloid A levels increase dramatically
in inflammatory conditions, and the plasmid-mediated expression of such
a construct has been shown to be activated in vitro by IL-1 and
TNF (Rygg et al., 2001). Nonetheless, TNF also exerts potentially
anti-atherogenic functions, including the inhibition of lipoprotein
lipase (Tengku-Muhammad et al., 1996) and the attenuation of macrophage
scavenger receptor activity (van Lenten and Fogelman, 1992; Hsu et al.,
1996; Schreyer et al., 1996). In addition, TNF deficiency has no
effect on atherogenesis in apoE/ mice (Schreyer et al., 2002),
whereas TNFR1 deficiency even predisposes to atherosclerosis (Schreyer
et al., 1996). As is the case with respect to heart failure,
controversy thus still shrouds antagonism of TNF activity as a
treatment for atherosclerosis, which is also borne out by the fact that
administration of TNF-binding protein in apoE/ mice attenuates
fatty streak formation in females, whereas it has no effect in male
mice (Elhage et al., 1998).
Virus-encoded interleukin and interleukin receptor homologs are also
thought to function as antigens and haptens, respectively, in the
generation of autoantibodies against a series of interleukins that have
been found to occur naturally in healthy humans and certain disease
states (Bendtzen et al., 1998), including antibodies against IL-1
(Bendtzen et al., 1989, 1994), IL-6 (Hansen et al., 1991; Bendtzen et
al., 1994), and IL-10 (Bendtzen et al., 1994). The (patho)physiological
role of these antibodies remains somewhat unclear, although most
neutralize their target interleukins in vitro (Svenson et al., 1992;
Hansen et al., 1993, 1995). Several have also been found to attenuate
interleukin activity in vivo, and anti-cytokine therapy by means of
monoclonal antibodies has also been investigated in the context of
atherosclerosis. An important role has been assigned to CD40L-CD40
interactions in the pathogenesis of atherosclerosis (Mach et al.,
1997). Accordingly, treatment with anti-CD40L antibody reduces de novo
atherogenesis in atherosclerosis-prone mice (Mach et al., 1998) and
cardiac allograft arteriopathy in a murine heterotopic cardiac
transplant model (Wang et al., 2002) and has also been found to alter
the histological appearance of pre-existing atherosclerotic lesions
toward a more stabilized phenotype (Lutgens et al., 2000; Schonbeck et
al., 2000). By contrast, antibody-mediated neutralization of TGF
signaling accelerates atherogenesis in apoE/ mice, and leads to the
development of a more inflammatory plaque phenotype (Mallat et al.,
2001c). Despite being a CD40-inducible protein (Zan et al., 1998),
TGF thus appears to have anti-atherogenic properties, and its
inhibition would therefore be undesirable in the prevention of atherogenesis.
The chronic nature of atherosclerosis and the generally rapid clearance
of administered antibodies, however, would necessitate repeated
parenteral administration to ensure prolonged efficacy. Eliciting an
endogenous antibody response by immunization with the cytokine in
question may circumvent this problem. A humoral immune response has
previously been shown to be mounted against most therapeutically
administered recombinant interleukin preparations (Revoltella, 1998),
and this observation has paved the way for the introduction of
intentional interleukin immunization. Svenson et al. (2000) have
immunized mice with recombinant murine IL-1 in conjunction with
purified protein derivative of tuberculin, which resulted in the
development of IL-1 neutralizing autoantibodies that attenuate the
expression of IL-6 in vivo. Alternatively, a synthetic interleukin
receptor antagonist may be used as an antigen for the induction of
autoimmunity against interleukins, as has been demonstrated for IL-6
(Ciapponi et al., 1997), or vaccination may be conducted with a DNA
vaccine encoding antigenic epitopes of the cytokine concerned (Youssef
et al., 1998). Thus, rats have been found to mount a protracted immune
response to Fas ligand after a course of vaccinations with FasL cDNA
(Wildbaum et al., 2000). The resulting autoantibodies inhibited the
production of TNF by cultured T lymphocytes in vitro and provided
protection against experimental autoimmune encephalomyelitis in vivo.
In considering the therapeutic scope of humoral anti-interleukin immune
response induction, however, one needs to take into account that some
anti-cytokine antibodies have been found to stabilize cytokine
functions rather than solely neutralizing their activity (Bendtzen et
al., 1990; Wendling et al., 1993). Antibodies to IL-3, IL-4, and IL-7
have thus been demonstrated to form complexes with their target
interleukins, which prolongs their in vivo half-life (Finkelman et al.,
1993). This has led to the realization that the efficacy of monoclonal
anti-interleukin therapy constitutes a balance between the
neutralization avidity and the rate of clearance of the formed complex.
These characteristics may partly depend on the specific epitope
recognized by the antibody, and meticulous preclinical assessment of
complex clearance is therefore indicated prior to clinical evaluation.
D. Interleukin Receptor Antagonists
Endogenous regulation of interleukin activity also occurs at the
level of ligand-receptor interaction. A major exponent of this type of
modulation is the control of IL-1 signaling by the endogenous IL-1
receptor antagonist, IL-1ra (Arend et al., 1998; Smith, 2000). First
discovered in the 1980s (Arend et al., 1985), this factor has been
extensively studied as a potential anti-inflammatory compound. Systemic
treatment with IL-1ra has been proven to be beneficial in the treatment
of rheumatoid arthritis in animal models and in humans, as judged by
histological and clinical improvement (Bresnihan et al., 1998; Cunnane
et al., 2001). As discussed above, IL-1ra has also been suggested as an
important protective factor in atherogenesis (Francis et al., 1999) and
restenosis (Kastrati et al., 2000), and its administration is currently
under scrutiny as a potential anti-atherogenic therapy. Elhage et al.
(1998) have demonstrated that subcutaneous injection of IL-1ra by means of an osmotic pump (25 mg/kg/day for 1 month) leads to a significant reduction in fatty streak formation in the aortic sinus of apoE/ mice on an atherogenic diet (Elhage et al., 1998). Short-term treatment
with IL-1ra has been found to be well tolerated. Due to the central
role of IL-1 in the immune response, however, long-term systemic
treatment with an inhibitor of this factor may not be desirable. It is
encouraging, therefore, that local gene therapeutic approaches
involving IL-1ra have provided promising results in the attenuation of
cerebral, pancreatic, and articular inflammation in animal models (Yang
et al., 1997; Fernandes et al., 1999; Giannoukakis et al., 1999) and
are currently awaiting evaluation in clinical trials (Del Vecchio et
al., 2001).
In lieu of naturally occurring antagonists, inhibitors of interleukin
receptors have also been developed by synthetic means. Phage display
techniques have led to the development of AF12198, a 15-mer peptide
with nanomolar affinity for the human type I IL-1 receptor, which does
not bind to the human type II receptor (Akeson et al., 1996), and
inhibits IL-1-induced ICAM-1 expression by endothelial cells in vitro.
Moreover, it down-regulates IL-6 induction in cynomolgus monkeys and is
thus considered to be the first small molecule to show IL-1 receptor
antagonist activity in vivo.
In general, the development of small interleukin receptor antagonists
has proved difficult, however, due to the complex and multipoint
high-affinity interactions between interleukin receptors and their
ligands. A more rewarding strategy has been the mutation of existing
ligands. IL-6 ligand-receptor interaction can be blocked by IL-6
variants that have been mutated to display increased affinity for IL-6R
and decreased binding to gp130 (Sun et al., 1997; Devlin et al., 1998;
Honemann et al., 2001). Due to their interference with gp130
interaction, these IL-6 receptor antagonists may also function as IL-11
antagonists (Sun et al., 1997). IL-12, in its active form, consists of
two disulfide-bonded subunits, p40 and p35, and synthetic antagonists
have been devised for human (Ling et al., 1995) and murine IL-12
(Gillessen et al., 1995) by homodimerization of the IL-12 p40 subunit.
The p40 homodimer acts as a potent IL-12 antagonist in vitro, reduces
the murine Th1 type response to endotoxin in vitro (Gately et al.,
1996), and protects mice from septic shock following LPS injection
(Mattner et al., 1997). Considering the importance that has been
assigned to Th1-mediated processes in atherosclerosis, this approach
may also hold promise in the prevention of atherogenesis.
The possibility of attenuating interleukin binding by introducing a
blocking antibody response to its receptor has also been explored. In
vitro, binding of IL-2 to IL-2R can be inhibited by the addition of
humanized antibodies that are bispecific for anti-IL-2 receptor and
(Pilson et al., 1997). In addition to its inhibitory activity on
IL-2 signaling, this antibody displays activity against IL-15, possibly
by virtue of competing for the shared IL-2 receptor subunit. In a
monkey model of autoimmune uveitis, this antibody has been demonstrated
to markedly reduce inflammation after twice-weekly intravenous
injections for 4 weeks (Guex-Crosier et al., 1997). Furthermore,
antagonism of IL-2 by means of anti-IL-2R antibodies, including the
commercial preparations basiliximab and dacluzimab, has proven an
effective addition to the immunosuppressive regimen following renal
allograft transplantation (Vincenti et al., 1998; Onrust and Wiseman,
1999). This therapeutic efficacy is believed to also be partly due to
inhibition of IL-15-mediated responses (Boelaars-van Haperen et al.,
2001). With respect to other interleukins, antibody blockade of IL-4R
and IL-6R has been found to alleviate antigen-induced airway
hyperresponsiveness and collagen-induced arthritis, respectively
(Gavett et al., 1997; Takagi et al., 1998; Mihara et al., 2001), and
antibody directed at IL-18R reduces LPS-induced inflammation and
mortality in mice (Xu et al., 1998a).
Opsonization and complement activation are believed to contribute to
the mechanism of action of interleukin-receptor antibodies, and the
ensuing elimination of cells expressing the relevant receptor may
attenuate inflammatory pathways elicited by interleukin binding. In
analogy, the specificity of interleukin binding has been employed in
devising a "Trojan horse" strategy for the targeting of cytotoxic compounds. This entails the administration of fusion proteins consisting of an interleukin and a toxic polypeptide domain, as used in
the transfer of pseudomonas exotoxin to IL-4R-expressing breast
carcinoma cells (LeMaistre et al., 1998) and of diphtheria toxin to
IL-2R-expressing lymphomas (Leland et al., 2000). Significant toxic
side effects may limit this type of therapy to acutely life-threatening and incurable diseases (Bagel et al., 1998). This would almost certainly exclude atherosclerosis as a candidate ailment, although it
could be applicable in a short-term strategy for the prevention of
restenosis following angioplasty of atherosclerotic lesions. Thus, it
is interesting to note that Miller et al. (1996) have found
atherosclerotic vascular thickening in rabbits following aortic balloon
angioplasty to be reduced by an interleukin-2 receptor-specific fusion
protein, termed DAB486-IL-2, in which the
receptor binding domain of diphtheria toxin had been replaced by a
human IL-2 sequence. DAB486-IL-2 was administered
for 10 days following angioplasty (0.1 mg/kg/day i.v.), found to be
well tolerated for the duration of the experiment, and to result in
complete inhibition of lesion formation compared with controls.
E. Up-Regulation of Anti-Inflammatory Interleukins
As has been discussed in a previous section, several interleukins
have been ascribed a putative anti-atherogenic role, including IL-9,
IL-10, IL-11, and potentially IL-4 and IL-13 (Table 1). All of these
cytokines are known to induce a Th2 type cytokine response, and have
been implicated in the pathogenesis of Th2-mediated diseases (Barnes,
2001a). Consistently, their inhibition has been suggested as a
potential treatment for these conditions, including asthma (Henderson
et al., 2000; Barnes 2001b; Zhou et al., 2001b). Overexpression
of these interleukins, on the other hand, has been speculated to
ameliorate a variety of Th1-mediated inflammatory conditions, such as
rheumatoid arthritis, septic shock, and atherosclerosis. Their
up-regulation may therefore hold promise as a therapeutic modality in
these diseases, and several studies to this effect have been reported.
Endotoxin-elicited shock has been used as a model for the evaluation of
the role and the therapeutic potential of all of these interleukins in
Th1-mediated inflammation. The protective effect of IL-4 has been
studied in a murine model of Gram-negative septic shock following
Pseudomonas aeruginosa infection (Giampietri et al., 2000).
Mortality was found to be reduced by IL-4 treatment, correlating with a
decrease in TNF elaboration. Similarly beneficial effects have been
found after prophylactic injections of recombinant IL-9 in this model
(Grohmann et al., 2000). This effect is accompanied by a reduction in
TNF, IL-12 p40, and IFN levels, and appears to be IL-9-specific,
as heat-inactivated IL-9 did not improve survival rates. Circulating
IL-10 levels were found to be markedly augmented by IL-9 injection, and
this may be partly responsible for an indirect suppression of
proinflammatory cytokine expression, as IL-10 itself also reduces
TNF production and lethality in murine endotoxemia (Gerard et al.,
1993). Conversely, IL-9 production in mast cells is greatly stimulated
by IL-10, closing a potent positive feedback loop (Stassen et al.,
2000). IL-11, on the other hand, inhibits LPS-induced up-regulation of
TNF, IL-1, and IFN by an IL-10-independent mechanism in vivo,
and has been found to result in a 60% inhibition of LPS-induced
elaboration of TNF, IL-1, IL-12 p40, and nitric oxide by murine
peritoneal macrophages in vitro (Trepicchio et al., 1997). Moreover,
IL-11 reduces lung TNF levels and neutrophil sequestration, and
improves pulmonary vasomotor function in a model of LPS-induced lung
injury (Sheridan et al., 1999). IL-13 even leads to a paradoxical
decrease in IL-10 levels following intraperitoneal LPS injection,
despite TNF, IFN, and IL-12 attenuation, and is therefore also
presumed to exert its protective effect in endotoxemic shock through an
IL-10-independent pathway (Muchamuel et al., 1997).
The chondroprotective and anti-colitic properties of anti-inflammatory
interleukins have also been evaluated. Locally applied recombinant
human IL-4 and IL-10 attenuated cartilage degradation and mononuclear
cell activity in human rheumatoid synovium that had been engrafted
subcutaneously to SCID CB17 mice. Moreover, IL-10, but not IL-4,
decreased the expression of ICAM-1 by synovial cells in this model
(Jorgensen et al., 1998). IL-11 also significantly reduced the severity
of collagen-induced arthritis in mice (Walmsley et al., 1998), and
possibly of rheumatoid arthritis in humans (Moreland et al., 2001). In
a rat model of inflammatory bowel disease, intraperitoneal adenoviral
transfer of IL-4 has been found to significantly inhibit tissue damage,
serum and colon IFN levels, and myeloperoxidase activity in the
distal colon (Hogaboam et al., 1997).
Surprisingly little is known about the atheroprotective role of these
interleukins, and IL-10 is undoubtedly the most extensively studied
candidate in this respect. A relative deficiency of IL-10 signaling has
been implicated in the pathogenesis of a variety of chronic autoimmune
conditions, including rheumatoid arthritis, Crohn's disease, multiple
sclerosis, and psoriasis. Promising results have been obtained in
studies addressing the therapeutic potential of IL-10 administration in
animal models of these diseases (Croxford et al., 1998; Kim et al.,
2000a; Lubberts et al., 2000), and the outcomes of early clinical
trials have been encouraging with respect to safety and efficacy, but
these require confirmation on a larger scale (van Deventer et al.,
1997; Asadullah et al., 1999; Colombel et al., 2001). The advantageous
potential of IL-10 in dampening the inflammatory background of
atherosclerosis is strongly suggested by several in vitro and animal
studies (Terkeltaub, 1999). Thus, atherogenesis is decreased in IL-10
transgenic mice on a high-fat diet, whereas IL-10 knockout (IL-10/)
mice display an increased atherogenic tendency (Pinderski-Oslund et
al., 1999), which is ameliorated by plasmid-mediated transfer of IL-10
(Mallat et al., 1999b). Furthermore, transfer of bone marrow from IL-10 transgenic mice to LDLr/ mice inhibits atherosclerosis by altering the phenotype of the resident lymphocyte and macrophage populations in
the atherosclerotic plaque (Pinderski et al., 2002).
We have recently demonstrated that de novo collar-induced atherogenesis
in LDLr/ mice (von der Thüsen et al., 2001b) is inhibited by
adenovirus-mediated overexpression of human IL-10 (hIL-10), following
both systemic and local transfer (von der Thüsen et al., 2001a)
(Fig. 5). Although we found
overexpression of hIL-10 to be immunomodulatory, as evidenced by
monocyte deactivation, it also resulted in marked serum cholesterol
lowering. The anti-atherogenic effect of systemic hIL-10 may therefore
be considered to be bipartite in this hypercholesterolemic animal
model. Local immunomodulation, however, is thought to be solely
responsible for the attenuation of atherosclerotic plaque formation
(44.9%, P < 0.05) that was observed after in vivo
endothelial hIL-10 transduction with the same vector. We have used a
similar approach in the evaluation of IL-9 as an atheroprotective agent
and found daily injections of IL-9 protein (1 µg/mouse/day i.p.) for
5 weeks to reduce carotid collar-induced atherosclerosis by 65% in
LDLr/ (P < 0.01) (Kuiper et al., 2001). An
explanation for this finding may lie in the IL-9-mediated up-regulation
of inhibitors of interleukin signaling, in addition to its enhancement
of IL-10 production (Lejeune et al., 2001).
|
The atheroprotective nature of IL-10 cannot be considered to be a
foregone conclusion, as local injection of an IL-10 expression plasmid
inhibits angiogenesis in a mouse model of hindlimb ischemia (Silvestre
et al., 2000), and administration of IL-10 protein augments arterial
disease in murine heart transplants (Furukawa et al., 1999). The effect
of IL-10 application, as a protein or following gene transfer, may
eventually be found to depend on the stage of the disease, the mode of
transfer and the dosing regimen. Furthermore, it may be possible to
tailor the pleiotropic actions of IL-10 to the use as an
anti-atherogenic agent by the use of viral IL-10 homologs. Thus, the
Epstein-Barr virus BRCF-1 gene product (vIL-10) has been found to share
84% amino acid sequence identity but only a limited number of the
pleiotropic actions of hIL-10. Perhaps most importantly, it lacks the
immunostimulatory properties of human IL-10, while sharing its
inhibitory activity with respect to cytokine synthesis and macrophage
activation (Ding et al., 2000). Furthermore, vIL-10 has been found to
lead to augmented and more prolonged expression following
adenovirus-mediated transfer in mice in comparison with its human
counterpart (Minter et al., 2001), and to effectively reduce
endothelial expression of E-selectin, P-selectin, and ICAM-1 in rats
following adenovirus-mediated transfer (Henke et al., 2000). The
application of vIL-10 may eventually prove to be preferable to hIL-10
if treatment is primarily aimed at providing an anti-inflammatory
stimulus, as is the case in the prevention of atherosclerosis.
F. Inhibition of Interleukin Signaling
The effector functions of all interleukins depend on the
activation of intracellular signaling cascades involving, inter
alia, Jaks, Tyks, and STATs (Leonard and Lin, 2000; Touw et al.,
2000). These pathways are negatively regulated by endogenous signaling inhibitors, including the SH2-containing phosphatase, SOCS, and protein
inhibitor of activated STAT families (Chung et al., 1997; Starr et al.,
1997; Liu et al., 1998; Naka et al., 1999), of which the expression is
partly controlled by interleukins themselves. These are considered to
play a pivotal role in the cross-regulation of interleukin function, as
Th2 cytokines have been found to lead to the expression of negative
regulators of Th1 cytokines, and vice versa. Moreover, interleukins may
also up-regulate inhibitors of their own signaling cascades and are
therefore subject to negative feedback loops. Thus, IL-4 activity is
controlled by SOCS-1 (SSI-1), which is elaborated in response to
interferons as well as IL-4 itself (Naka et al., 1997; Dickensheets et
al., 1999; Losman et al., 1999), and the immunosuppressive and
autoregulatory effects of IL-9, IL-10, and IL-11 are thought to be
partly mediated by the up-regulation of SOCS-3, which inhibits
STAT5-mediated signaling (Auernhammer and Melmed, 1999; Cassatella et
al., 1999; Donnelly et al., 1999; Lejeune et al., 2001).
The administration of inhibitors of cytokine signaling could have
beneficial effects in atherosclerosis. The essential role of tyrosine
kinases in cytokine signaling has prompted the evaluation of tyrosine
kinase inhibitors as therapeutic agents. In this respect, a group of
compounds called "tyrphostins" has been shown to have anti-proliferative and anti-inflammatory properties in vitro and in
vivo that are thought to be mediated by tyrosine kinase inhibition (Levitzki, 1990). Platelet-derived growth factor is among the cytokines
to be inhibited by the tyrphostins, and these have therefore been
speculated to be effective against smooth muscle cell-mediated pathological processes. The latter includes injury-induced neointima formation, and application of the tyrphostin AG-17 by means of a
perivascular controlled release implant has been found to inhibit intimal hyperplasia in injured rat carotid arteries (Golomb et al.,
1996). In addition, Huynh et al. (1998) have found ex vivo incubation
of jugular veins with AG-51 to reduce post-operative intimal
hyperplasia by 49%, following their placement as an interposition graft in rabbit carotid arteries. A relative lack of pharmacological and functional selectivity, however, may limit the applicability of
these inhibitors as immunomodulatory compounds aimed at diverting the
cytokine response from a Th1 to a Th2 expression pattern. SB 203580, for example, in addition to its originally described inhibitory
activity for p38 MAPK, also attenuates stress-activated protein kinases
and c-Jun N-terminal kinases (Cuenda et al., 1995; Clerk and Sugden,
1998). SB 203580 inhibits TNF and IL-1 expression and protects mice
from collagen-induced arthritis (Owens and Lumb, 2000) but also
attenuates IL-4, IL-5, and IL-13, and virtually blocks IL-10 production
(Koprak et al., 1999). The latter effect, in particular, is considered
undesirable in the setting of atherosclerosis. It is conceivable,
however, that specificity of protein kinase inhibition may in the
future be achieved by the transfer of endogenous inhibitors, including
SOCSs by gene therapy or protein administration. As an example,
plasmid-mediated overexpression of SOCS-1 has recently been found to
inhibit cytokine-induced CD40 expression in macrophages by blocking
IFN-mediated STAT-1 activation (Wesemann et al., 2002).
G. Inhibition of Interleukin-Induced Gene Expression
Interleukin signaling eventually culminates in the
sequence-specific binding of DNA by activated transcription factors and the ensuing up-regulation of target gene transcription. Some of these
transcription factors are considered potentially rewarding substrates
for immunomodulatory therapy, including NF-B, AP-1 and the STAT
proteins, by virtue of their capacity to integrate converging signals
from various proinflammatory cytokines and other inflammatory stimuli
(Collins, 1993; Touw et al., 2000; Tedgui and Mallat, 2001). Several
methods have been employed in the attenuation of transcription factor
activity, based on the characteristic bispecific affinity of these
molecules for regulatory proteins and DNA.
The inhibition of vascular NF-B-regulated transcription, in
particular, is presumed to hold anti-atherogenic potential. Activated NF-B has been identified in smooth muscle cells, macrophages, and
endothelial cells in the atherosclerotic lesion (Brand et al., 1996).
Functional significance for NF-B in atherogenesis has been deduced
from its colocalization with the expression of NF-B target genes in
plaques (Brand et al., 1996) and the association of its expression in
coronary atherosclerotic lesions with unstable angina (Wilson et al.,
2002). The role of NF-B as a causal mediator in atherosclerosis
remains unclear, however, which is partly due to the intrauterine
lethality associated with p65- and IB-deficiency in mice (Collins
and Cybulsky, 2001). The regulation of NF-B activity depends on the
extent of binding to its naturally occurring inhibitors, including
IB, IB, IB, and BCL3 (Ghosh and Baltimore, 1990;
Finco and Baldwin, 1995). Phosphorylation of IB by the IB kinase
(IKK) complex, containing IKK, IKK, and IKK (NEMO), leads to
IB ubiquitination and proteasome-mediated degradation. This enables
the nuclear translocation of unbound NF-B and subsequent activation
of NF-B-dependent transcription. In endothelial cells, NF-B is
thought to play an essential role in the regulation of adhesion
molecule expression in response to inflammatory stimuli, including
cytokines (Collins et al., 1995; De Caterina et al., 2001). The
endothelial NF-B/IB system is presumed to be primed in
endothelial cells in lesion-prone arterial sites, as evidenced by
increased expression of the p65 (RelA) NF-B subunit, IB, and
IB, prior to plaque development and NF-B activation (Hajra et
al., 2000). The attenuation of IB activity by IKK up-regulation has
been identified as a pivotal step in endothelial activation (Read et
al., 1994; Bennett et al., 1996; Johnson et al., 1996), whereas the
inhibition of endothelial adhesion molecule expression by nitric oxide
has been found to be mediated by IB (Spiecker et al., 1997). The
recognition of the physiological importance of this inhibitory pathway
has prompted the evaluation of the anti-atherogenic properties of
IB administration. Adenoviral transfer of IB has thus been
found to effect down-regulation of inflammatory genes in endothelial
cells, including VCAM-1, IL-1, IL-6, and IL-8 (Wrighton et al., 1996),
and to lead to inhibition of monocyte adhesion and transmigration on
TNF-activated endothelium (Weber et al., 1999). In addition,
TNF-induced endothelial expression of adhesion molecules (E-selectin
and ICAM-1) and chemokines (MCP-1) is attenuated by retrovirus-mediated
introduction of a proteolysis-resistant IB mutant, IB
N, and
the addition of pharmacological inhibitors of IB phosphorylation
and proteasome degradation (Cobb et al., 1996; Pierce et al., 1997;
Lockyer et al., 1998; Hipp et al., 2002).
NF-B may also modulate atherogenesis by regulating the transcription
of inflammatory genes in monocytes/macrophages and smooth muscle cells
(Ghosh and Baltimore, 1990; Bourcier et al., 1997). In macrophages, the
LPS-stimulated production of proinflammatory cytokines and inducible
nitric-oxide synthase (iNOS) have been found to be reduced by
adenoviral overexpression of IB and the administration of
proteasome inhibitors, respectively (Griscavage et al., 1996; Bondeson
et al., 1999). In vascular smooth muscle cells, liposomal delivery of
purified IB peptide attenuates TNF-induced proliferation
(Selzman et al., 1999), and overexpression of IB diminishes the
elaboration of the matrix metalloproteinases MMP-1, MMP-3, and MMP-9,
which may have plaque-stabilizing consequences in vivo (Bond et al.,
2001). In addition, decoy oligonucleotides to NF-B binding sites
have been used to counteract NF-B-mediated transcriptional
activation and have displayed effectivity in inhibiting graft coronary
artery disease of rat cardiac allografts following ex vivo
pressure-mediated delivery (Feeley et al., 2000). Other interleukin-activated transcription factors to have been successfully inhibited in vitro by the decoy approach include STAT1 (Ohtsubo et al.,
2000), STAT6 (Wang et al., 2000), and AP-1 (Morishita et al., 1998).
Inhibition of AP-1-mediated transcription, in particular, effectively
reduced joint destruction in a murine model of collagen-induced arthritis (Shiozawa et al., 1997).
Attenuation of NF-B activity is also presumed to constitute a
physiological feedback mechanism in inflammatory homeostasis. Thus,
several potentially anti-atherogenic interleukins reduce NF-B
activity by variably increasing IB transcription (IL-4) (Donnelly
et al., 1993; Abu-Amer, 2001), preventing IB degradation (IL-10 and
IL-13) (Lentsch et al., 1997) or increasing the expression of BCL3, a
protein with close homology to IB proteins (IL-4 and IL-9) (Richard
et al., 1999). Interestingly, NF-B inhibition has evolved as a viral
strategy of immune response evasion, exemplified by the
adenovirus-encoded E1A protein (Kalvakolanu, 1999). The up-regulation
of IL-6 by TNF and IL-1 is inhibited by E1A due to its prevention of
NF-B p65-p50 heterodimer formation; although this leaves monomeric
p50 to bind to the B element in the IL-6 promoter, this does not
induce transcription (Janaswami et al., 1992). Moreover, E1A negatively
regulates Stat1, Stat2, and Stat3 activity, and thereby attenuates
IL-6-mediated gene expression (Takeda et al., 1994). An IB homolog,
A238L, is encoded by the African swine fever virus, and this has been
shown to inhibit the production of proinflammatory cytokines in
macrophages, allowing persistent viral infection (Powell et al., 1996).
It may prove possible to exploit these anti-inflammatory traits in the
prevention of atherogenesis by overexpression or protein administration
of the interleukins or viral proteins concerned.
Synthetic compounds with inhibitory activity for NF-B have also been
described. High throughput cell-based screening has led to the
discovery of SP100030, a T cell-specific NF-B and AP-1 inhibitor
(Gerlag et al., 2000). SP100030 attenuates IL-2, IL-8, and TNF
production in T cell lines and alleviates disease progression in a
murine model of collagen-induced arthritis. Finally, it has recently
transpired that the pharmacological effects of several
anti-inflammatory compounds, including salicylates, are partly derived
from their inhibition of IB phosphorylation and degradation
(Schwenger et al., 1998; Young, 1998). This knowledge may aid the
development of derivatives of these drugs that are specifically
targeted toward the inhibition of cytokine-induced inflammation.
Once interleukin-mediated transcription of inflammatory genes has
occurred, antisense technology could be employed to interfere specifically with their translation. Interleukin-1 stimulated up-regulation of granulocyte-macrophage and granulocyte
colony-stimulating factor gene expression in endothelial cells has been
successfully inhibited by antisense ODNs (Segal et al., 1992).
Moreover, the expression of endothelial adhesion molecules can be
inhibited by the application of phosphorothioate oligonucleotides
directed against ICAM-1, VCAM-1, and E-selectin (Bennett et al., 1994). Antisense-mediated down-regulation of endothelial ICAM-1 expression on
monocytes reduces endothelial adhesiveness for leukocytes, which may be
advantageous in atherogenesis (Steidl et al., 2000). The applicability
of ICAM-1 inhibition by means of antisense has been demonstrated in
vivo, as it has been shown to be effective in the prevention of cardiac
allograft or lung isograft failure in mice and rats, respectively
(Stepkowski et al., 1994; Toda et al., 2000). In clinical studies, the
phosphorothioate ICAM-1 antisense preparation ISIS 2302 has been found
to be well tolerated and to significantly lower the need for steroid
treatment in Crohn's disease (Yacyshyn et al., 1998).
The previously mentioned caveats that apply to antisense therapy in
general (Lebedeva and Stein, 2001) are evidently also poignant with
respect to its use in the inhibition of interleukin-induced gene
expression. The doubts that have been raised about the
sequence-specific nature of ODN-mediated effects, the poor transfection
efficiency, and the short half-life of ODNs in vivo will need to be
addressed to warrant their applicability in the prevention of atherosclerosis.
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IV. Discussion |
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TopPreviousNextReferences
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Cytokines are being increasingly recognized as a potentially
rewarding therapeutic target in a wide variety of diseases. For example, of the over 600 clinical gene therapy trials currently completed, ongoing or pending worldwide, those concerned with the
transfer of cytokine genes constitute the largest category (Gene
Therapy Clinical Trials, 2002). Most of these involve the application
of immunostimulatory cytokines for the treatment of neoplastic and
infectious diseases. Of the protocols addressing vascular diseases (51 in total), the overwhelming majority is intended to stimulate
revascularization in peripheral and coronary ischemia by cytokine
overexpression, largely employing the angiogenic growth factors
fibroblast growth factor, PDGF, and vascular endothelial growth factor.
While these hold promise for the treatment of atherosclerosis-related ischemia, it will have transpired from the preceding discussion that
cytokine-directed therapy in general, and interleukin-based treatment
specifically, is still in its infancy as a means for the prevention of
the onset and progression of atherogenesis per se. A lack of
understanding of their involvement in atherogenesis currently prevents
the use of some interleukins as targets for immunomodulation, including
the members of the IL-10 family IL-19, IL-20, and IL-22 (Table 1).
Other interleukins are overtly pleiotropic in their actions, and
attenuating or augmenting their effects may be detrimental or
beneficial, depending on the stage of atherosclerosis (IL-4, IL-13).
Yet others have been attributed primarily anti- (IL-1ra, IL-9, IL-11,
IL-10) or pro-atherogenic (including IL-1, IL-2, IL-6, IL-18)
properties, and their modulation could therefore represent the most
readily applicable approach to immunotherapy in atherosclerosis. This
type of therapy may prove to be an effective alternative to currently
used treatment protocols (e.g., lipid-lowering drugs) but could also be
useful as an adjunctive to conventional pharmacotherapy. It is possible
to conceive of several obstacles that may have impeded the development
of such immunomodulatory strategies. Some of these are related to
specific pathogenic features associated with atherosclerosis, others to
the systemic and local consequences of immunomodulation, and yet others
to purely technical aspects of interleukin therapy.
The chronic nature of atherosclerosis has doubtlessly hampered the evolution of adequate disease prevention strategies in general and also remains a significant obstacle to the preventive use of interleukin-based treatments. In considering the relative benefit of long-term use of the latter, one needs to pay attention to its cost, the practicality of its dosing regimen, and, most importantly, potential side effects.
All interleukins possess roles that are certainly not restricted to
atherosclerosis, and their actions are frequently pivotal to several
aspects of the immune system. Interleukins orchestrate defense
mechanisms against a wide range of pathogens and tumor cells, in
addition to playing a key role in various forms of nonimmune inflammation, and undiscerning diversions of the interleukin response will therefore invariably compromise one or more of these functions. For instance, whereas the inhibition of signaling by IL-2, IL-6, and
IL-12 may be beneficial in the context of atherosclerosis, these
factors have been implicated as potent antitumor agents (Maini et al.,
1997), and attenuation of IL-2 signaling, in particular, may increase
the risk of neoplasia. Conversely, Th2 cytokines are considered to have
anti-atherogenic potential, but their role in the pathogenesis of
autoimmune diseases is also well documented. Prolonged up-regulation of
these factors, although tolerable in the short-term, may have
deleterious consequences for the development or progression of
inter alia, asthma, diabetes mellitus, systemic lupus
erythematosus, and rheumatoid arthritis (Lafaille, 1998; Romagnani,
2000). Thus, whereas inhibition of atherogenesis in murine models has
been achieved by application of the IL-1 antagonist IL-1ra (Elhage et
al., 1998), the anti-inflammatory interleukin IL-10 (von der
Thüsen et al., 2001a), and the interleukin-binding protein
IL-18bp (Mallat et al., 2001b), these treatments still require
long-term toxicological evaluation before beginning clinical trials.
This type of untargeted systemic immunomodulation may eventually be
limited to short-term treatments aimed at, for instance, the induction
of regression or stabilization of existing atherosclerotic plaques.
Proof-of-principle data to this effect have been obtained in ApoE/
mice, in which the administration of antibodies to the cytokine CD40L
has been seen to result in a stabilized plaque phenotype (Lutgens et
al., 2000; Schonbeck et al., 2000). These studies indicate the
potential benefits of short-term immunomodulatory treatment, and could
serve as a paradigm for the development of similar strategies in humans.
For prolonged treatment, it may be desirable to restrict the action
radius of therapeutic compounds to the atherosclerotic lesion and/or to
ensure specificity of action for the atherosclerotic process. This will
require the identification of marker molecules and cytokine signaling
pathways, which are more or less specific for atherosclerosis, and
these efforts may be greatly aided by the advent of DNA array and phage
display technology (Faber et al., 2001; Houston et al., 2001; Monajemi
et al., 2001). Thus, employing phage display techniques, we have
recently identified a peptide sequence that specifically binds human
P-selectin (Molenaar et al., 2001). This adhesion molecule is
up-regulated on the endothelium of atherosclerosis-prone sites, and
high affinity ligands for P-selectin may therefore serve as efficient
tools for the targeting of viral and nonviral drug delivery vehicles to
the developing atherosclerotic plaque. Such techniques may eventually
also be extended to the targeting of specific cellular subsets in the atherosclerotic lesion to enhance therapeutic efficacy and reduce the
risk of bystander effects.
Alternatively, site-directed targeting could be achieved by mechanical
means. The development of local application catheters has recently been
intensified, opening up possibilities for the intravascular
instillation of therapeutic compounds. Due to the invasive nature of
such techniques, this approach will demand preparations with an
extended duration of action or therapeutics that have a lasting effect
on atherogenesis or restenosis even with a single dosage regimen. Viral
expression vectors may be used in achieving prolonged up-regulation of
anti-inflammatory interleukins, such as (v)IL-10 (Kim et al.,
2000a; Minter et al., 2001; von der Thüsen et al., 2001a),
interleukin antagonists, such as IL-1ra and soluble TNF receptor
(Giannoukakis et al., 1999; Kim et al., 2001), inhibitors of
NF-B signaling, such as IB (Wrighton et al., 1996; Bondeson et
al., 1999; Weber et al., 1999), and antisense oligonucleotides and
ribozymes directed against proinflammatory interleukins and
interleukin-induced genes. Although extended transgene expression has
been found to occur with some adenoviruses, including Ad-IL-10 (>200
days, unpublished data), the use of AAVs or Ad/AAV hybrids may be
preferable in accomplishing this goal (Lynch et al., 1997; Recchia et
al., 1999; Monahan and Samulski, 2000).
Barring the development of preparations with an extended duration of
action, however, repeated administration will continue to be required
to sustain therapeutic efficacy. This may elicit a humoral response to
the protein concerned, which could severely compromise its potency and
aggravate side effects. A further drawback of repeated administration
is the fact that most currently available preparations require
parenteral administration, which limits their tolerability and thus the
likelihood of patient compliance. Paradoxically, to reduce the need for
repeated administration, it may be possible to induce long-term
immunomodulation by deliberately opting for active immunization by
viral or nonviral means. The possibility of (DNA) vaccination as a
method of raising neutralizing antibody responses against inflammatory
interleukins has been discussed (Revoltella, 1998; Svenson et al.,
2000), as has the possibility of interleukin stabilization and
half-life extension by these antibodies (Finkelman et al., 1993). It
should be noted, however, that extended duration of effectivity could
also be regarded as a disadvantage, due to the relative irreversibility
of such therapies in case of the occurrence of deleterious side effects.
The use of smaller synthetic compounds may reduce the need for
parenteral administration and could therefore constitute a practical
alternative to the transfer of entire interleukin molecules or
anti-interleukin (receptor) antibodies. The examples discussed in this
review include inhibitors of interleukin processing (Livingston, 1997),
tyrosine kinase activity (Golomb et al., 1996; Huynh et al., 1998),
proteasome function (Bondeson et al., 1999; Richard et al., 1999), p38
MAPK (Cuenda et al., 1995; Clerk and Sugden, 1998), and NF-B (Gerlag
et al., 2000). A drawback of many of these drugs, however, is their
lack of pharmacological and functional specificity, partly due to the
involvement of their molecular targets as downstream mediators in
convergent signaling cascades, which is perhaps best exemplified by
NF-B. Careful toxicological evaluation will therefore be required
before their clinical introduction. The use of (modified) endogenous
inhibitors, including SH2-containing phosphatase, SOCS, and protein
inhibitor of activated STAT, may eventually provide the required selectivity.
The production of interleukins and most of their inhibitors is
currently a rather costly undertaking. Despite recent progress in
recombinant protein production technology and therapeutic antibody expression technology (Maini et al., 1997), this situation is unlikely
to change in the foreseeable future, making widespread prophylactic
protein treatment prohibitively expensive. With a view to these health
economic implications and the minimization of potential side effects,
it is imperative that treatment be confined to susceptible patients.
Accurate tools for the identification of patients who may benefit most
from such therapies, are therefore required. Refinement of genetic,
biochemical, and radiological markers of predisposition to
(complications of) atherosclerosis may provide important prognostic
clues. The discovery of correlations between atherosclerotic events and
interleukin-related polymorphisms, in particular, including those found
for IL-1 (Momiyama et al., 2001), IL-ra (Francis et al., 1999, 2001;
Kastrati et al., 2000), and IL-6 (Rauramaa et al., 2000; Georges et
al., 2001), may facilitate the identification of suitable patients,
whereas improved magnetic resonance imaging and ultrasound imaging of
existing plaques will provide an impetus for the noninvasive
determination of plaque "vulnerability" to rupture (Fayad and
Fuster, 2001; Choudhury et al., 2002).
In summary, the currently available methods of modulation of interleukin-mediated inflammatory pathways are not yet suited to the widespread prevention of atherosclerosis. Substantial investigative efforts are still required with respect to target identification and the definition of suitable patient populations. Technical aspects of compound specificity, duration of action, and mode of transfer await additional improvement, but the first promising signs are looming on the horizon, because several techniques have been successfully validated in animal models. The initial aims of such therapies are likely to include the lasting stabilization of pre-existing plaques by short-term cytokine immunomodulation, which possibly represents the most readily achievable objective in clinical practice in the near future.
| |
Footnotes |
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Address correspondence to: Jan H. von der Thüsen, Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, P.O. Box 9502, 2300 RA Leiden, The Netherlands. E-mail: thuesen{at}lacdr.leidenuniv.nl
DOI: 10.1124/pr.55.1.5
| |
Abbreviations |
|---|
LDL, low density lipoprotein;
TNF, tumor necrosis factor;
TNFR, TNF receptor;
IL, interleukin;
IFN, interferon ;
Th, T helper cell;
MCP-1, monocyte
chemoattractant protein-1;
RANTES, regulated on activation normal T
cell expressed and secreted;
MIP-1, macrophage inflammatory protein-1;
ICE, IL-1-converting enzyme;
MAPK, mitogen-activated protein kinase;
NF-B, nuclear factor-B;
AP-1, activating protein-1;
ICAM-1, intercellular adhesion molecule 1;
VCAM-1, vascular cell adhesion
molecule-1;
SMC, smooth muscle cell;
MMP, matrix metalloproteinase;
SOCS, suppressor of cytokine signaling;
LPS, lipopolysaccharide;
GM-CSF, granulocyte-macrophage colony-stimulating factor;
G-CSF, granulocyte-colony-stimulating factor;
Jak, Janus kinase;
STAT, signal
transducer and activator of transcription;
ODN, oligodeoxynucleotide;
PKC, protein kinase C;
PDGF, platelet-derived growth factor;
TGF, transforming growth factor ;
AAV, adeno-associated virus;
IKK, IB
kinase;
iNOS, inducible nitric-oxide synthase;
WIN 67694, Z-Val-Ala-Asp-CH2O(CO)[2,6-CI2)]Ph;
SB 203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole;
VE 13,045, carbobenzyloxy-Val-Ala-Asp(O-et)-CH2O-dichlorobenzoate.
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References |
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TopPrevious |
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friends or foes?
Immunol Today
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167-169[CrossRef][Medline].justification of methods and goals of treatment.
Chest
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979-988a potential role in atherogenesis.
Circulation
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549-554implications for atherogenesis.
Circ Res
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871-878an alternative pathway for T-cell activation in atherosclerosis?
Arterioscler Thromb Vasc Biol
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1208-1213/ mice.
Arterioscler Thromb Vasc Biol
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456-461 type II receptor inhibits luminal loss and constrictive remodeling after coronary angioplasty and enhances adventitial collagen deposition.
Circulation
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2595-2601a new effector in skin inflammation.
Curr Biol
11:
R531-R534[CrossRef][Medline].atherosclerosisan inflammatory disease.
N Engl J Med
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115-126role of cAMP.
Arterioscler Thromb Vasc Biol
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677-682the women's health and aging study.
Circulation
103:
947-953/ Mice.
FASEB J
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2730-2732
/ and LDLr/ Mice.
Circulation
103:
1164-1170/ mice through release of interferon-gamma.
Circ Res
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e34-e38potential application of ribozyme strategy to treat cardiovascular disease.
Circulation
102:
1308-1314interleukin-9 as a therapeutic target for asthma.
Resp Res
2:
80-84.
0031-6997/03/5501-133-166$7.00
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, macrophage; TC, T
cell) and exert effects that are considered to be either
pro-atherogenic (continuous line) or anti-atherogenic (dashed line).
