# Antigen-Induced Immunomodulation in the Pathogenesis of Atherosclerosis

Authors: Natalia Milioti; Alexandra Bermudez-Fajardo; Manuel L. Penichet; Ernesto Oviedo-Orta
Journal: Clinical and Developmental Immunology
 (2008)

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## Abstract

Atherosclerosis is a chronic inflammatory disorder characterised by the accumulation of monocytes/macrophages, smooth muscle cells, and lymphocytes within the arterial wall in response to the release of proinflammatory molecules. Such accumulation results in the formation of the atherosclerotic plaque, which would eventually evolve to complications such as total artery occlusion, rupture, calcification, or aneurysm. Although the molecular mechanism responsible for the development of atherosclerosis is not completely understood, it is clear that the immune system plays a key role in the development of the atherosclerotic plaque and in its complications. There are multiple antigenic stimuli that have been associated with the pathogenesis of atherosclerosis. Most of these stimuli come from modified self-molecules such as oxidised low-density lipoproteins (oxLDLs), beta2glycoprotein1 (β2GP1), lipoprotein a (LP(a)), heat shock proteins (HSPs), and protein components of the extracellular matrix such as collagen and fibrinogen in the form of advanced glycation-end (AGE) products. In addition, several foreign antigens including bacteria such as Porphyromonas gingivalis and Chlamydia pneumoniae and viruses such as enterovirus and cytomegalovirus have been associated with atherosclerosis as potentially causative or bystander participants, adding another level of complexity to the analysis of the pathophysiology of atherosclerosis. The present review summarises the most important scientific findings published within the last two decades on the importance of antigens, antigen stimulation, and adaptive immune responses in the development of atherosclerotic plaques.


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## Body

## 1. Atherosclerotic Plaque Formation

It is difficult to
identify the factors responsible for the initiation of the atheroma lesion and/or
the order in which these factors contribute to plaque formation. Nevertheless, it is known that endothelial dysfunction and high levels of circulating
cholesterol, as oxLDL, play a key role in the proinflammatory process that
triggers the first steps in the development of atherosclerotic plaques [1, 2]. Whatever the cause, these
steps are characterised by an initially reversible accumulation of lipid-laden macrophages
in the subendothelial space as a consequence of the increasing migration of
blood-derived monocytes. These cells accumulate at focal points within the
vascular wall of medium and small size arteries driven by chemokines and
adhesion molecules produced by the damaged endothelium [3–5]. Monocytes differentiate in situ into macrophages which
express membrane receptors such as Toll-like receptors and scavenger receptors
that participate in the clearance of oxLDL [6, 7]. Lymphocytes can also transmigrate
and accumulate within the arterial wall from the very earliest stages (Figure 1) [8].Figure 1
Inflammatory basis of atherosclerotic
plaque formation. Led
by inflammatory signals derived from the damaged endothelium, monocytes and
lymphocytes migrate into the vessel wall. Monocytes differentiate into
macrophages that recognise and phagocytiose oxidised LDL particles. The protein
component of the LDL particle is processed and presented in the form of
peptides by macrophages (also by dendritic cells) to T-lymphocytes in the
context of the major histocompatibility complex class II (MHC-II). Other self
or foreign antigens that may gain access to the vascular wall can also trigger
similar mechanisms. It is believed that most of these lymphocytes differentiate
in situ, under the influence of
the specific antigen stimulation, into effector T-cells, but this has yet to be
demonstrated. Upon activation, both macrophages and lymphocytes release a range
of proinflammatory molecules including chemokines which stimulate the migration
of smooth muscle cells (SMCs) from the media. SMCs contribute to foam cell
and fibrous cap formation. This process is facilitated by cytokines such as
IFNγ and TNFα secreted by proatherogenic Th1 cells and also IL-12 secreted by
macrophages and foam cells. Eventually foam cells die by apoptosis in situ leaving nondegradable
cholesterol crystals that form the lipid core of the plaque.As the
inflammatory process becomes chronic, smooth muscle cells also start to migrate
from the media into the intima layer of the vessel, in response to chemokines
and aided by the release of membrane metalloproteinases (MMPs) that enable them
to break through the elastic lamina into the subendothelial space (Figure1) [9, 10]. Persistence of inflammation
creates a vicious circle of cell migration, dedifferentiation of smooth muscle
cells, production of chemotactic and proinflammatory mediators, and cell death leading
to vascular wall remodelling and formation of a new layer called neointima (Figures 1 and 2) [11, 12].Morphological features of advanced
atherosclerotic plaques. (a) and (b) show sections from a human carotid
artery; (c) and (d) are sections from an apoE deficient mouse brachiocephalic artery.
Sections (a) and (c) have been stained with haematoxylin and eosin and sections (b) and (d) with van Gieson staining (used to demonstrate the increase of
collagen deposition and development of elastic fibres, a characteristic feature
of the atherogenic process. A positive staining is depicted by a brown colour). L:lumen of the vessel; SR: shoulder region (it is believed to
contain large numbers of proinflammatory cells including macrophages and
lymphocytes, and it is the site related with the onset of the development of
the atherosclerotic plaque); FC: fibrous cap (It also contains large
numbers of mononuclear infiltrate and smooth muscle cells that have migrated
from the media layer (M) and proliferated in response to the
local inflammatory stimuli. It is also characterised by high collagen
deposition and little or no endothelial cells); LC: lipid core (it contains mainly macrophage and smooth muscle
cell-derived foam cells, apoptotic cells, and cholesterol crystals. Older
lesions may also display signs of calcification). Contrasting differences can
be recognised in the anatomic development of atherosclerotic plaques between
human and mouse including the hypertrophy associated with the proliferation of
the smooth muscle cells in the media layer and the fibrous cap. In humans, some
lesions may also contain signs of intraplaque haemorrhage. Signs of plaque
rupture are usually best recognised in mouse (reviewed in [13, 14]).
(a)(b)(c)(d)Neointima
formation is a complex phenomenon that occurs in response to vessel wall damage
in which repair and injury mechanisms give birth to areas rich in
proinflammatory cells and collagen deposition [11, 12]. It is within these areas
where a close contact between macrophages, dendritic cells (DCs), and
lymphocytes has been reported. It has been speculated that the interaction
between lymphocytes and DCs within the neointima is responsible for the
development of local immune responses against exogenous and endogenous
atherogenic antigens. These immune responses may contribute to cell death by
apoptosis and accumulation of nondegradable cholesterol, contributing to the formation
of the lipid core of the atherosclerotic plaque [15–18]. However, the precise
mechanisms involved are not yet clear.


## 2. The Immunologic Nature of the Atherogenic Process

Atherosclerosis
belongs to the group of chronic inflammatory diseases in which the cellular
components of the immune system play a key role in its development and
complications. The chronic accumulation of monocytes/macrophages, smooth muscle
cells, and T-lymphocytes in response to the accumulation and release of
proinflammatory molecules within the arterial wall constitutes the hallmark of
a developing atherosclerotic plaque [15]. Although most of the antigenic
stimuli that occur within atherosclerotic plaques come from modified self-molecules,
the immune response triggered is remarkably similar to inflammatory reactions
mounted against microbial organisms [19, 20]. The list of atherosclerosis-related
antigens range from oxidised low-density lipoproteins (oxLDL), heat shock
proteins (HSP) to protein components of the extracellular matrix such as collagen and
fibrinogen (Table 1). On the other hand, the disputed role of foreign antigens,
such as viruses and bacteria, in atherogenesis as causative or bystander
participants in its development introduces another level of complexity to the
analysis. This review summarises the most important scientific findings
published within the last 2 decades on the importance of antigens, antigen
stimulation, and adaptive immune responses in the development of
atherosclerotic plaques.Table 1
Antigens that have been reported to be
involved in atherogenesis.
AntigenReferenceAutoantigensOxidised low-density lipoprotein (LDL)[21–23]Beta2glycoprotein1 (beta2GP1)[24–26]Lipoprotein a (LP(a))[27]Lipoprotein-lipase (LPL)[28]Advanced glycation-end products (AGE)[29]Heat-shock proteins[30, 31]Collagen[32–34]Fibrinogen[35]Microbial antigensPorphyromonas gingivalis[36, 37]Chlamydia pneumoniae[38–49]Bacteroides forsynthus[36]Streptococcus mutans[36, 37, 50]Helicobacter pylori[51–64]Echerichia coli[65, 66]Enterovirus[67–69]Cytomegalovirus[22, 70–78]Viperin[79]


## 3. Atherosclerosis-Related Antigens

As stated above, both
endogenous and exogenous antigens are involved in atherogenesis. Table1
summarises some of the most studied antigens in relation to atherosclerotic
plaque formation.


### 3.1. Oxidised Low-Density Lipoprotein (oxLDL)

OxLDL remains one
of the most studied antigens in atherogenesis (Figure3). LDL does not normally
trigger immune responses in its native state; however when it gets oxidised, it
can initiate inflammation-leading to atherosclerosis. OxLDL is considered a neoantigen, a term used to describe modified
self-antigens for which normally the immune system is tolerant but which upon
modification have the potential to excerpt autoreactive immune responses. Modified
lipoprotein particles have the capacity to interact with the endothelium,
triggering their accumulation and retention in the subendothelial space. The
retained oxLDL particles induce macrophage foam cell formation, smooth muscle
cell migration and proliferation [80] and, in addition, stimulate
the secretion of inflammatory cytokines. OxLDL also induces the expression of
adhesion molecules promoting endothelial cell dysfunction and leukocyte
extravasation [81].Figure 3
Schematic representation of the low-density
lipoprotein particle (LDL). The LDL particle has a size of approximately 21–24 nm and is the
main transporter of unesterified cholesterol, cholesterol esters, and
triglycerides in the blood. It contains an outer layer composed of phospholipids
and unesterified cholesterol in which a single protein is embedded, the apolipoprotein
B-100 (apoB-100). These components are more susceptible to oxidation by free
radicals in the subendothelial space during inflammation. They are also targets
for the recognition of the LDL by scavenger receptors, proteoglycans, and low-density
lipoprotein receptor (LDLr). The core of the particle contains primarily
cholesterol esters and triglycerides. In atherogenesis, a large number of IgM
antibodies are created in response to oxidative stress-modified phospholipids, whereas
IgG antibodies and T-cell clones are generated against apoB-100.In atherosclerosis,
increased levels of oxLDL have been found in the serum of patients admitted with acute myocardial infarction, and immunohistochemical studies revealed the
presence of oxLDL within atherosclerotic plaques [82–85]. Proteoglycans, contained
within the extracellular matrix of the arterial wall, interact with oxLDL via
their carbohydrate groups, glycosamynoglycans (GAGs) as it has been described in vitro and in immunohistochemical
studies using defective LDL unable to bind to GAGs [86]. The initial interaction of
LDL with the proteoglycans leads to structural changes that expose the GAG-binding
sites contributing to LDL retention. This interaction is mediated by apolipoprotein B (ApoB), which is the main protein component of LDL particles [87]. At least two proteoglycan-binding
sequences within ApoB have been identified and shown to be necessary for retaining
LDL particles within the intimal layer [80]. The interaction between the
ApoB and the GAGs also facilitates LDL oxidation by oxygen-reactive species released
by vascular cells or as a result of enzymatic modifications driven by
endothelial cells, smooth muscle cells, or macrophages present within the
atherosclerotic plaques [88]. Cholesterol molecules also
have a high affinity for GAGs and for the LDL receptor. The accumulation of oxLDL
within atherosclerotic plaques has been demonstrated, among other methods, by
using monoclonal antibodies such as DLH3 that binds specifically to oxLDL but
non to native LDL (nLDL) [89]. DLH3 binds to the oxidised phosphatidylcholine groups (oxPC) and
to the phosphatidylcholine (PC)-apoB
adducts formed when LDL is oxidised. PC-apoB adducts are considered to be the principal
antibody targeted epitopes on the surface of the particles. Furthermore, oxPC also
promote endothelial-monocyte adhesion by inducing the secretion of monocyte
chemoattractant protein 1 (MCP-1) by endothelial cells [23], and can also be recognised
by scavenger receptors such as CD36 which are involved in the oxLDL uptake by macrophages
[90–93].The initial
interaction of proteoglycans with the LDL within the arterial wall leads lipid
modifications that expose GAG binding sites. Cholesterol, cholesteryl esters
and phospholipids are the main targets for lipid modifications and their role
in the development of the disease has been extensively evaluated. These
modifications play an important role, as stated above, in LDL retention within
the arterial wall which is also facilitated by interactions with surface
molecules such as apoE and lipoprotein lipase expressed on macrophages [94].However, most
studies have focused on the role of oxidised phospholipids (oxPLs) [95]. Peroxidation of the
phospholipids starts in the fatty acids. The decomposition of fatty acids
generates a spectrum of reactive species such as malondialdehyde (MDA),
4-hydroxynonenal, and the 1-palmitoyl-2-(5-oxovaleroyl)-glycero-3-phosphocholine
molecule (POVPC) that can oxidise apolipoprotein B (ApoB) as well as other
lipids, resulting in ox-PLs and oxidised protein-lipid adducts [96, 97].It has also been
established that oxLDL acts as a chemoattractant for monocytes allowing them to
infiltrate the lesion site. OxLDL has been shown to induce monocyte adhesion to
the endothelium by upregulating the expression of adhesion molecules on their
surface, by inducing macrophage major histocompatibility class II (MHC-II)
& LeuM3 cell surface expression and by accelerating monocyte
differentiation in to macrophages [22]. Endothelial cells are also
capable of oxidising LDL, contributing to a continuous generation of oxLDL
within the lesion site, and to attract more monocytes. Other inflammatory
mediators such as interleukin 1β (IL-1β), tumor necrosis factor alpha (TNF-α)
and monocyte colony stimulating factor (M-CSF) can induce the expression of the
oxLDL receptor (oxLDL-R) on the surface of endothelial cells, thus further
contributing to oxLDL accumulation [22, 98].Scavenger
receptors expressed by macrophages play a pivotal role in LDL accumulation; such
receptors include CD36 (a membrane glycoprotein), CD68, CXCL16, the scavenger
receptors A & B1 (SR-A & SR-B1), and the lectin-type oxidised low-density
lipoprotein receptor 1 (LOX1) [98, 99]. It is believed that the high
expression of the scavenger receptors on macrophages mediates lipid
accumulation and foam cell formation [100]. Following proteolytic
processing inside the cell, fragments of the oxidised-modified ApoB protein are
displayed on the surface of macrophages bound on MHC-II molecules. This process
also leads to the upregulation of important molecules such as toll-like
receptors TLR2 and TLR4 that induce proatherogenic immune responses [36]. There is also evidence that
products of the inflammatory process such as endogenous HSP60 and LDL oxidation
derivatives bind TLR4-CD14 complexes on
monocytes and macrophages eliciting proinflammatory responses [30, 101, 102]. This has been associated
with an enhanced production of cytokines, an
enhancement of oxLDL uptake, and an increase adhesion of these cells to
the endothelium mediated by IL-8 and NFκB synthesis [101, 103].The large size of
the LDL molecule (2×106kDa) favours the exposure of many epitopes
recognised by the antibodies generated, mainly IgM. OxLDL is known to be a very
potent immunogen and the antibodies generated in response to its modifications
are able to bind to many other similarly modified endogenous proteins [104]. It has been demonstrated
that there is a molecular mimicry between the head of the PC groups of oxLDL
and the PC groups expressed on the surface of many pathogens such as Streptococcus pneumoniae [105], which indicates that during
an infection, more autoantibodies against oxLDL might be generated. Studies using
experimental animal models have shown that epitopes generated during LDL
oxidation, such as oxPC, are also generated on the surface of bacteria and on
the surface of endothelial cells [21]. These epitopes bind to
antibodies that will mediate removal of oxLDL and apoptotic cells [106]. Some of the
oxidation-specific epitopes present on oxLDL are also presented on the surface
of apoptotic cells in the lesion site, and play a role in the clearance of the
damaged oxidised lipid molecules and of apoptotic cells generated during the
inflammatory response within plaques [21].


### 3.2. Immunisation Using oxLDL Confers Atheroprotection

A series of
studies have shown the beneficial side of oxLDL. These studies have
demonstrated that immune responses against this lipoprotein may protect against
the development of the disease [107–111]. The first report came from
Palinski et al. which immunised LDL receptor-deficient rabbits using homologous
MDA-LDL. This treatment induced high titres of antibodies displaying equal specificity as those risen by the native particle and significantly reduced atherosclerotic plaque
development [107]. Studies from other
laboratories confirmed these results and showed that immunisation of
hypercholesterolemic rabbits reduced T cell and oxLDL immunoreactivity within
the neointima of immunized animals [108].The effect of LDL
immunisation on atheroprotection has also been assessed using mouse models of
the disease. George et al. was the first to report the effect of MDA modified
LDL immunisation in apoE-deficient mice(apoE−/−). Immunised mice
developed high titres of anti-MDA-LDL antibodies and the treatment
significantly reduced lesion size at the aortic sinus by more than half when compared
with their control littermates immunised with PBS. However, they did not find differences
between the groups with respect to cellular composition of the atherosclerotic
plaques [109]. Later on, Freigang et al.
showed that LDL receptor-deficient mice (LDLR−/−) immunised with
homologous malondialdehyde-modified LDL (MDA-LDL) induced the synthesis of antibodies
of different classes against distinctive epitopes on oxLDL and that this
antibody response is significantly
correlated with a reduction by approximately 40% of lesion size.
However, they also showed that immunisation with MDA-LDL raised equivalent
amounts of both T helper 1 (Th1)-related IgG2a and Th2-dependent IgG1
antibodies [110]. On the other hand, an
elegant study carried out by Zhou et al. provided evidence of the involvement
and control of the production of oxLDL-induced antibodies by T cells. They immunised apoE−/− mice with homologous plaque homogenates or homologous MDA-LDL.
They found that both antigen preparations reduced lesion development. The protective
effect was associated with a specific raise of T-cell-dependent IgG antibodies
against MDA-LDL and oxidised phospholipids which are correlated with the reduction in plaque size
and circulating cholesterol levels [111]. Despite these
demonstrations, the protective role of oxLDL during physiological conditions
remains unknown and the immunological mechanisms related with it have not yet
been fully studied.


### 3.3.β2-Glycoprotein I
(β2GpI)

Rheumatic patients
suffering from the antiphospholipid syndrome produce large amounts of antiphospholipid
antibodies. The standard phospholipid used to detect antiphospholipid
antibodies is cardiolipin, which is prone to peroxidation and is also an
important component of the oxLDL molecule [21]. A cofactor involved in
anticardiolipin binding is the β2-glycoprotein I (β2GpI), a positively charged
plasma protein circulating in the blood and also present in platelets and endothelial
cells in  atherosclerotic plaques [25]. Binding to the aPL
antibodies requires a structural change in β2GpI which occurs when the protein
binds to negatively charged phospholipids present in the atherosclerotic
plaques. When transgenic animals are immunised with β2GpI, the atherosclerosis process
is accelerated [26]. It has been reported that β2GpI
can function as a scavenger receptor to mediate lipid engulfment by
macrophages. Furthermore, histological studies showed that β2GpI is located in
the subendothelial space in areas rich in CD4+ T cells [24]. A recent study showed that
the adoptive transfer of β2GpI reactive T cells can promote the generation of
fatty streaks in LDL−/− mice, indicating that cellular autoimmunity
is involved in the pathogenesis of atherosclerosis [25].


### 3.4. Lipoprotein(a) [Lp(a)]

Lp(a) is an antigen
of relevance to atherosclerosis development [27]. Lp(a) is associated with
apolipoprotein(a) (Apo-A), another glycoprotein.
Lp(a) is present in the atherosclerotic plaques bound to fibrin. Furthermore, it
may be internalised by macrophages within the plaques and induce the expression
and secretion of chemoattractants from endothelial cells, thus triggering the attraction
of monocytes in to atherosclerotic plaques. This effect is specifically
attributed to the Apo-A component and suggests that in the presence of high
levels of Apo-A, monocyte recruitment in to the
vascular wall is favoured. The precise nature of the chemoattractant involved
is not known yet, but GM-CSF and MCP-1 have already been discarded [27].


### 3.5. Lipoprotein-Lipase (LPL)

A further self-antigen
involved in lupus-related atherosclerosis is LPL [28]. It is a member of the
lipase family that hydrolyses triglyceride molecules on lipoprotein molecules.
LPL activity is significantly decreased with the progression of the disease due
to the generation of anti-LPL antibodies [112, 113]. The hypothesis that has
been formulated is that these antibodies might bind to LPL molecules on the surface
of endothelial cells and obstruct lipid degradation by LPL, thus promoting
lipid accumulation in the atherosclerotic plaques [113].


### 3.6. Advanced Glycation End (AGE) Products

A recent study
suggests a possible role of AGE as facilitators of antigenic stimulation in
atherosclerosis by promoting the maturation of dendritic cells (DCs) [29]. AGE products stimulate the upregulation of
costimulatory and antigen presenting molecules on DCs which in turn causes T
cell proliferation through the secretion of proinflammatory cytokines. This
activation is mediated, at least in part by the upregulation of the receptors
for AGE (RAGE) and the scavenger receptor A (SR-A), which is responsible for
regulating cholesterol accumulation on DCs through the Jnk signaling pathway [29]. Immunohistological studies
have confirmed the expression of AGE, as well as AGE receptors within
atherosclerotic lesions. Cells expressing high levels of RAGE have been found located
close to AGE, where normal
RAGE is expressed in low levels in the endothelium [114, 115]. SR-A knock-out mice have
decreased atherosclerotic lesions, therefore suggesting an indirect link
between AGE stimulation and the development of atherosclerosis in these animals
[116].


### 3.7. Heat Shock Proteins (HSPs)

HSPs are released
from stressed endothelial cells and can act as chaperones in the process of
denaturation of other proteins. They can induce the production of specific
antibodies which usually accelerate atherosclerotic plaque development when
used to immunise experimental animals [26]. Human and microbial HSP60
activate vascular endothelial cells and macrophages directly through CD14 and p38 mitogen-activated protein kinase
signalling pathway in a similar
manner as bacterial lipopolysaccharide (LPS) [30], leading to IL-6 and TNF-α
secretion and promotion of atherosclerosis. HSPs are highly conserved among
different species. Antibodies involved in the atherosclerotic development
recognise both human and microbial HSPs [31].


### 3.8. Bacteria-Derived Antigens

The potential
relationship between bacterial infections and the induction of atherosclerosis
has been studied in different groups of cardiovascular patients including those
who develop the disease but that lack the conventional risk factors associated
with it such as hypercholesterolemia, high blood pressure, smoking and diabetes
[51]. It has been speculated that
bacterial infection may have a direct cytopathic effect on the vascular wall or
that could act indirectly through the induction of an autoimmune inflammatory response
involving mechanisms such as molecular mimicry and epitope spreading to
generate atherosclerosis [19]. Several microbial
components known to ligate pattern recognition receptors or heat shock proteins
and unmethylated CpG DNA have been reported as ligands for toll-like receptors
(TLRs), and therefore, have the potential to induce atherosclerosis [117]. TLRs are part of the
sensing mechanisms in response to infections but it has been suggested that they
may also play a contradictory role in inflammation leading to atherosclerosis. There
is evidence showing that endothelial cells and macrophages in atherosclerotic
lesions can upregulate TLR expression in response to microbial antigens [36]. It is known that
autoantibodies such as those binding to endogenous human HSP60 and oxidised LDL
can also activate TLR4 and induce proatherogenic immune responses. The
response involves the secretion of proinflammatory cytokines, MMPs, and other
inflammatory mediators (nitric oxide, endothelin-1) [118, 119].An example isPorphyromonas gingivalis which has been
detected within atherosclerotic plaques [37]. The inflammatory action of P. gingivalisfimbriae was shown to be mediated by ligation of TLR2, TLR4, CD14,
and beta2-integrins and also by the upregulation of nuclear factor kappa-B
(NF-κB). It has also been observed that P.
gingivalis fimbriae may promote atherosclerotic
plaque rupture by inducing the secretion of MMPs [120–122]. Other pathogens studied in
relation to atherosclerosis are Bacteroides
forsynthus, where the protein A secreted by the bacterium acts through CD14
and TLR2 ligations to induce atherosclerosis, whereas in the case of Streptococcus mutans it is the protein
AgI/II  that acts through CD14 and TLR4 [37].Similar mechanisms
have been described forChlamydophila pneumoniae (C. pneumoniae) infection where
LPS and bacterial HSP act as ligands to TLRs. C. pneumoniae is an intracellular prokaryotic pathogen that
infects humans provoking distinct forms of pneumonia, and it has been also proposed
that it may cause chronic inflammatory diseases such as atherosclerosis [38]. Chlamydial LPS has been
shown to induce macrophage foam cell formation and chlamydial HSP60 is known to
contribute to LDL oxidation in the presence of macrophages on the lesion site. The
presence of C. pneumoniae within atheroma
lesions has been detected by PCR and immunohistochemistry. However, detection
is sometimes difficult due to phases of activity and latency of the pathogen [123, 124]. The pathogen has been
located within DCs in close proximity to T cells [125] but the precise mechanisms responsible
for the induction of immune activation and atherosclerosis development remain
to be clarified. There is evidence that patients with acute myocardial
infarction have higher titres of antibodies against C. pneumoniae than control patients [126]. Moreover, C. pneumoniae has been also extracted
and cultured from atherosclerotic plaques [125, 127, 128]. Experiments carried out in
animal models demonstrated the induction of atherosclerosis by inoculation of C. pneumoniae [129]. C. pneumoniae can persistently infect epithelial cells and
macrophages within human atherosclerotic plaques causing a chronic and nonlytic
infection [128]. Immune responses against
Chamydia spp. infection mainly involve CD4+ T-helper (Th)1 cells and
antibodies, although other components such as CD8+ T cells also play
a key role [130–132]. The relative contribution
of these components to protection depends on several factors, such as the site
of infection, whether it is a primary or secondary infection, and whether the
infection is acute or persistent [133]. However, despite all the
evidence supporting the role of Chlamydia infection in the development of
atherosclerosis, its correlation with the development of complications remains
controversial. A key element to the debate is the failure of recent human
clinical trials and animal studies aiming to investigate the secondary
preventive effect of antibiotics on atherosclerosis [128, 129, 134]. In these studies,
antibiotic therapy was effective in clearing the acute infection, but failed to
influence the atherogenic properties of C.
pneumoniae unless the therapy was started early during the acute infection [134]. It has been hypothesised
that this may be due to a sequestration of the organism within atherosclerotic
plaques, that makes it inaccessible to both antibiotics and the cellular
components of the immune response.The microorganismHelicobacter pylori—a cause of
gastrointestinal infections—has been also
found to be present in atherosclerotic lesions but completely absent from
healthy arteries [52, 53]. However, immunohistochemical
studies could not detect its presence in the lesions but instead there was a
strong cross-reactivity of the antibodies to the different elements of the plaque
related to the acceleration of inflammatory events and plaque destabilisation.Cross-reactivity
has also been observed with antibodies against human HSP60 andE. coli-derived GroEL, an HSP [65]. The generation of anti-HSP
antibodies can induce autoimmune reactions binding to HSP on endothelial cells at
the lesion site where it is expressed at high levels due to shear stress
triggered by blood pressure, stimulation by oxLDL in situ or by inducing the secretion of proinflammatory
cytokines. The down stream effects are endothelial and macrophage damage and
subsequent inflammatory events that lead to the pathogenesis of atherosclerosis
[135–137].


### 3.9. Virus-Derived Antigens

Viruses have also
been postulated as promoters of atherosclerosis. One of the most closely linked
to this disease is cytomegalovirus. This virus infects the majority of the
human population by targeting SMCs and endothelial cells producing a latent
type of infection [138–142]. US28, one of the viral
proteins expressed on the cell surface of the cytomegalovirus after infection,
is a chemoattractant for SMCs. US28 and UL122 proteins were found to have an
11aa sequence homologous to human HSP60, and it is thought that antibodies
against these viral proteins can bind to human HSP60 expressed on stressed
endothelial cells [143]. The proteins also share
some homology with nonstressed endothelial cell markers such as CD151, CD49f,
and connexin 45 (Cx45). It is believed that during cytomegalovirus infection, antibodies
generated against these proteins can bind, by molecular mimicry, to the surface
markers on both nonstressed and already-stressed endothelial cells causing apoptosis
of endothelial cells, which is considered to be one of the key early events in atherosclerotic
plaque formation. It has been also suggested that endothelial cell stress
induces HSP60 expression enhancing the binding of circulating autoantibodies
and amplifying the endothelial cell damage [143]. Interesting results
relating cytomegalovirus with atherosclerosis are derived from studies investigating
the expression of the viperin gene. The human viperin gene has been suggested
as a potential marker for cytomegalovirus infection [144]. Viperin, which is highly
conserved among species, has a well-known antiviral effect and its use for the
local treatment of cytomegalovirus has been recently proposed. Viperin is
expressed by endothelial cells and SMCs in the vascular wall of disease vessels
but no expression has been detected in the normal arteries [144].Another pathogen
associated with atherosclerosis is enterovirus, especially the enterovirus
group coxsackie B virus [67]. High levels of enterovirus
antibodies have been detected in patients with myocardial infraction but it has
not yet been fully established whether the virus contributes to the
pathogenesis of the disease [67, 68, 145]. Other infectious organisms that
have been implicated in the pathogenesis of atherosclerosis involve a member of
the herpes virus family that is known to induce atherosclerosis in chickens [146]. The virus alters cellular
metabolism resulting in cholesterol accumulation which is a common mechanism
proposed for all virally-induced atherosclerosis. There is also evidence
that the virus promotes smooth muscle cells (SMCs) proliferation [147–150].


## 3.1. Oxidised Low-Density Lipoprotein (oxLDL)

OxLDL remains one
of the most studied antigens in atherogenesis (Figure3). LDL does not normally
trigger immune responses in its native state; however when it gets oxidised, it
can initiate inflammation-leading to atherosclerosis. OxLDL is considered a neoantigen, a term used to describe modified
self-antigens for which normally the immune system is tolerant but which upon
modification have the potential to excerpt autoreactive immune responses. Modified
lipoprotein particles have the capacity to interact with the endothelium,
triggering their accumulation and retention in the subendothelial space. The
retained oxLDL particles induce macrophage foam cell formation, smooth muscle
cell migration and proliferation [80] and, in addition, stimulate
the secretion of inflammatory cytokines. OxLDL also induces the expression of
adhesion molecules promoting endothelial cell dysfunction and leukocyte
extravasation [81].Figure 3
Schematic representation of the low-density
lipoprotein particle (LDL). The LDL particle has a size of approximately 21–24 nm and is the
main transporter of unesterified cholesterol, cholesterol esters, and
triglycerides in the blood. It contains an outer layer composed of phospholipids
and unesterified cholesterol in which a single protein is embedded, the apolipoprotein
B-100 (apoB-100). These components are more susceptible to oxidation by free
radicals in the subendothelial space during inflammation. They are also targets
for the recognition of the LDL by scavenger receptors, proteoglycans, and low-density
lipoprotein receptor (LDLr). The core of the particle contains primarily
cholesterol esters and triglycerides. In atherogenesis, a large number of IgM
antibodies are created in response to oxidative stress-modified phospholipids, whereas
IgG antibodies and T-cell clones are generated against apoB-100.In atherosclerosis,
increased levels of oxLDL have been found in the serum of patients admitted with acute myocardial infarction, and immunohistochemical studies revealed the
presence of oxLDL within atherosclerotic plaques [82–85]. Proteoglycans, contained
within the extracellular matrix of the arterial wall, interact with oxLDL via
their carbohydrate groups, glycosamynoglycans (GAGs) as it has been described in vitro and in immunohistochemical
studies using defective LDL unable to bind to GAGs [86]. The initial interaction of
LDL with the proteoglycans leads to structural changes that expose the GAG-binding
sites contributing to LDL retention. This interaction is mediated by apolipoprotein B (ApoB), which is the main protein component of LDL particles [87]. At least two proteoglycan-binding
sequences within ApoB have been identified and shown to be necessary for retaining
LDL particles within the intimal layer [80]. The interaction between the
ApoB and the GAGs also facilitates LDL oxidation by oxygen-reactive species released
by vascular cells or as a result of enzymatic modifications driven by
endothelial cells, smooth muscle cells, or macrophages present within the
atherosclerotic plaques [88]. Cholesterol molecules also
have a high affinity for GAGs and for the LDL receptor. The accumulation of oxLDL
within atherosclerotic plaques has been demonstrated, among other methods, by
using monoclonal antibodies such as DLH3 that binds specifically to oxLDL but
non to native LDL (nLDL) [89]. DLH3 binds to the oxidised phosphatidylcholine groups (oxPC) and
to the phosphatidylcholine (PC)-apoB
adducts formed when LDL is oxidised. PC-apoB adducts are considered to be the principal
antibody targeted epitopes on the surface of the particles. Furthermore, oxPC also
promote endothelial-monocyte adhesion by inducing the secretion of monocyte
chemoattractant protein 1 (MCP-1) by endothelial cells [23], and can also be recognised
by scavenger receptors such as CD36 which are involved in the oxLDL uptake by macrophages
[90–93].The initial
interaction of proteoglycans with the LDL within the arterial wall leads lipid
modifications that expose GAG binding sites. Cholesterol, cholesteryl esters
and phospholipids are the main targets for lipid modifications and their role
in the development of the disease has been extensively evaluated. These
modifications play an important role, as stated above, in LDL retention within
the arterial wall which is also facilitated by interactions with surface
molecules such as apoE and lipoprotein lipase expressed on macrophages [94].However, most
studies have focused on the role of oxidised phospholipids (oxPLs) [95]. Peroxidation of the
phospholipids starts in the fatty acids. The decomposition of fatty acids
generates a spectrum of reactive species such as malondialdehyde (MDA),
4-hydroxynonenal, and the 1-palmitoyl-2-(5-oxovaleroyl)-glycero-3-phosphocholine
molecule (POVPC) that can oxidise apolipoprotein B (ApoB) as well as other
lipids, resulting in ox-PLs and oxidised protein-lipid adducts [96, 97].It has also been
established that oxLDL acts as a chemoattractant for monocytes allowing them to
infiltrate the lesion site. OxLDL has been shown to induce monocyte adhesion to
the endothelium by upregulating the expression of adhesion molecules on their
surface, by inducing macrophage major histocompatibility class II (MHC-II)
& LeuM3 cell surface expression and by accelerating monocyte
differentiation in to macrophages [22]. Endothelial cells are also
capable of oxidising LDL, contributing to a continuous generation of oxLDL
within the lesion site, and to attract more monocytes. Other inflammatory
mediators such as interleukin 1β (IL-1β), tumor necrosis factor alpha (TNF-α)
and monocyte colony stimulating factor (M-CSF) can induce the expression of the
oxLDL receptor (oxLDL-R) on the surface of endothelial cells, thus further
contributing to oxLDL accumulation [22, 98].Scavenger
receptors expressed by macrophages play a pivotal role in LDL accumulation; such
receptors include CD36 (a membrane glycoprotein), CD68, CXCL16, the scavenger
receptors A & B1 (SR-A & SR-B1), and the lectin-type oxidised low-density
lipoprotein receptor 1 (LOX1) [98, 99]. It is believed that the high
expression of the scavenger receptors on macrophages mediates lipid
accumulation and foam cell formation [100]. Following proteolytic
processing inside the cell, fragments of the oxidised-modified ApoB protein are
displayed on the surface of macrophages bound on MHC-II molecules. This process
also leads to the upregulation of important molecules such as toll-like
receptors TLR2 and TLR4 that induce proatherogenic immune responses [36]. There is also evidence that
products of the inflammatory process such as endogenous HSP60 and LDL oxidation
derivatives bind TLR4-CD14 complexes on
monocytes and macrophages eliciting proinflammatory responses [30, 101, 102]. This has been associated
with an enhanced production of cytokines, an
enhancement of oxLDL uptake, and an increase adhesion of these cells to
the endothelium mediated by IL-8 and NFκB synthesis [101, 103].The large size of
the LDL molecule (2×106kDa) favours the exposure of many epitopes
recognised by the antibodies generated, mainly IgM. OxLDL is known to be a very
potent immunogen and the antibodies generated in response to its modifications
are able to bind to many other similarly modified endogenous proteins [104]. It has been demonstrated
that there is a molecular mimicry between the head of the PC groups of oxLDL
and the PC groups expressed on the surface of many pathogens such as Streptococcus pneumoniae [105], which indicates that during
an infection, more autoantibodies against oxLDL might be generated. Studies using
experimental animal models have shown that epitopes generated during LDL
oxidation, such as oxPC, are also generated on the surface of bacteria and on
the surface of endothelial cells [21]. These epitopes bind to
antibodies that will mediate removal of oxLDL and apoptotic cells [106]. Some of the
oxidation-specific epitopes present on oxLDL are also presented on the surface
of apoptotic cells in the lesion site, and play a role in the clearance of the
damaged oxidised lipid molecules and of apoptotic cells generated during the
inflammatory response within plaques [21].


## 3.2. Immunisation Using oxLDL Confers Atheroprotection

A series of
studies have shown the beneficial side of oxLDL. These studies have
demonstrated that immune responses against this lipoprotein may protect against
the development of the disease [107–111]. The first report came from
Palinski et al. which immunised LDL receptor-deficient rabbits using homologous
MDA-LDL. This treatment induced high titres of antibodies displaying equal specificity as those risen by the native particle and significantly reduced atherosclerotic plaque
development [107]. Studies from other
laboratories confirmed these results and showed that immunisation of
hypercholesterolemic rabbits reduced T cell and oxLDL immunoreactivity within
the neointima of immunized animals [108].The effect of LDL
immunisation on atheroprotection has also been assessed using mouse models of
the disease. George et al. was the first to report the effect of MDA modified
LDL immunisation in apoE-deficient mice(apoE−/−). Immunised mice
developed high titres of anti-MDA-LDL antibodies and the treatment
significantly reduced lesion size at the aortic sinus by more than half when compared
with their control littermates immunised with PBS. However, they did not find differences
between the groups with respect to cellular composition of the atherosclerotic
plaques [109]. Later on, Freigang et al.
showed that LDL receptor-deficient mice (LDLR−/−) immunised with
homologous malondialdehyde-modified LDL (MDA-LDL) induced the synthesis of antibodies
of different classes against distinctive epitopes on oxLDL and that this
antibody response is significantly
correlated with a reduction by approximately 40% of lesion size.
However, they also showed that immunisation with MDA-LDL raised equivalent
amounts of both T helper 1 (Th1)-related IgG2a and Th2-dependent IgG1
antibodies [110]. On the other hand, an
elegant study carried out by Zhou et al. provided evidence of the involvement
and control of the production of oxLDL-induced antibodies by T cells. They immunised apoE−/− mice with homologous plaque homogenates or homologous MDA-LDL.
They found that both antigen preparations reduced lesion development. The protective
effect was associated with a specific raise of T-cell-dependent IgG antibodies
against MDA-LDL and oxidised phospholipids which are correlated with the reduction in plaque size
and circulating cholesterol levels [111]. Despite these
demonstrations, the protective role of oxLDL during physiological conditions
remains unknown and the immunological mechanisms related with it have not yet
been fully studied.


## 3.3.β2-Glycoprotein I
(β2GpI)

Rheumatic patients
suffering from the antiphospholipid syndrome produce large amounts of antiphospholipid
antibodies. The standard phospholipid used to detect antiphospholipid
antibodies is cardiolipin, which is prone to peroxidation and is also an
important component of the oxLDL molecule [21]. A cofactor involved in
anticardiolipin binding is the β2-glycoprotein I (β2GpI), a positively charged
plasma protein circulating in the blood and also present in platelets and endothelial
cells in  atherosclerotic plaques [25]. Binding to the aPL
antibodies requires a structural change in β2GpI which occurs when the protein
binds to negatively charged phospholipids present in the atherosclerotic
plaques. When transgenic animals are immunised with β2GpI, the atherosclerosis process
is accelerated [26]. It has been reported that β2GpI
can function as a scavenger receptor to mediate lipid engulfment by
macrophages. Furthermore, histological studies showed that β2GpI is located in
the subendothelial space in areas rich in CD4+ T cells [24]. A recent study showed that
the adoptive transfer of β2GpI reactive T cells can promote the generation of
fatty streaks in LDL−/− mice, indicating that cellular autoimmunity
is involved in the pathogenesis of atherosclerosis [25].


## 3.4. Lipoprotein(a) [Lp(a)]

Lp(a) is an antigen
of relevance to atherosclerosis development [27]. Lp(a) is associated with
apolipoprotein(a) (Apo-A), another glycoprotein.
Lp(a) is present in the atherosclerotic plaques bound to fibrin. Furthermore, it
may be internalised by macrophages within the plaques and induce the expression
and secretion of chemoattractants from endothelial cells, thus triggering the attraction
of monocytes in to atherosclerotic plaques. This effect is specifically
attributed to the Apo-A component and suggests that in the presence of high
levels of Apo-A, monocyte recruitment in to the
vascular wall is favoured. The precise nature of the chemoattractant involved
is not known yet, but GM-CSF and MCP-1 have already been discarded [27].


## 3.5. Lipoprotein-Lipase (LPL)

A further self-antigen
involved in lupus-related atherosclerosis is LPL [28]. It is a member of the
lipase family that hydrolyses triglyceride molecules on lipoprotein molecules.
LPL activity is significantly decreased with the progression of the disease due
to the generation of anti-LPL antibodies [112, 113]. The hypothesis that has
been formulated is that these antibodies might bind to LPL molecules on the surface
of endothelial cells and obstruct lipid degradation by LPL, thus promoting
lipid accumulation in the atherosclerotic plaques [113].


## 3.6. Advanced Glycation End (AGE) Products

A recent study
suggests a possible role of AGE as facilitators of antigenic stimulation in
atherosclerosis by promoting the maturation of dendritic cells (DCs) [29]. AGE products stimulate the upregulation of
costimulatory and antigen presenting molecules on DCs which in turn causes T
cell proliferation through the secretion of proinflammatory cytokines. This
activation is mediated, at least in part by the upregulation of the receptors
for AGE (RAGE) and the scavenger receptor A (SR-A), which is responsible for
regulating cholesterol accumulation on DCs through the Jnk signaling pathway [29]. Immunohistological studies
have confirmed the expression of AGE, as well as AGE receptors within
atherosclerotic lesions. Cells expressing high levels of RAGE have been found located
close to AGE, where normal
RAGE is expressed in low levels in the endothelium [114, 115]. SR-A knock-out mice have
decreased atherosclerotic lesions, therefore suggesting an indirect link
between AGE stimulation and the development of atherosclerosis in these animals
[116].


## 3.7. Heat Shock Proteins (HSPs)

HSPs are released
from stressed endothelial cells and can act as chaperones in the process of
denaturation of other proteins. They can induce the production of specific
antibodies which usually accelerate atherosclerotic plaque development when
used to immunise experimental animals [26]. Human and microbial HSP60
activate vascular endothelial cells and macrophages directly through CD14 and p38 mitogen-activated protein kinase
signalling pathway in a similar
manner as bacterial lipopolysaccharide (LPS) [30], leading to IL-6 and TNF-α
secretion and promotion of atherosclerosis. HSPs are highly conserved among
different species. Antibodies involved in the atherosclerotic development
recognise both human and microbial HSPs [31].


## 3.8. Bacteria-Derived Antigens

The potential
relationship between bacterial infections and the induction of atherosclerosis
has been studied in different groups of cardiovascular patients including those
who develop the disease but that lack the conventional risk factors associated
with it such as hypercholesterolemia, high blood pressure, smoking and diabetes
[51]. It has been speculated that
bacterial infection may have a direct cytopathic effect on the vascular wall or
that could act indirectly through the induction of an autoimmune inflammatory response
involving mechanisms such as molecular mimicry and epitope spreading to
generate atherosclerosis [19]. Several microbial
components known to ligate pattern recognition receptors or heat shock proteins
and unmethylated CpG DNA have been reported as ligands for toll-like receptors
(TLRs), and therefore, have the potential to induce atherosclerosis [117]. TLRs are part of the
sensing mechanisms in response to infections but it has been suggested that they
may also play a contradictory role in inflammation leading to atherosclerosis. There
is evidence showing that endothelial cells and macrophages in atherosclerotic
lesions can upregulate TLR expression in response to microbial antigens [36]. It is known that
autoantibodies such as those binding to endogenous human HSP60 and oxidised LDL
can also activate TLR4 and induce proatherogenic immune responses. The
response involves the secretion of proinflammatory cytokines, MMPs, and other
inflammatory mediators (nitric oxide, endothelin-1) [118, 119].An example isPorphyromonas gingivalis which has been
detected within atherosclerotic plaques [37]. The inflammatory action of P. gingivalisfimbriae was shown to be mediated by ligation of TLR2, TLR4, CD14,
and beta2-integrins and also by the upregulation of nuclear factor kappa-B
(NF-κB). It has also been observed that P.
gingivalis fimbriae may promote atherosclerotic
plaque rupture by inducing the secretion of MMPs [120–122]. Other pathogens studied in
relation to atherosclerosis are Bacteroides
forsynthus, where the protein A secreted by the bacterium acts through CD14
and TLR2 ligations to induce atherosclerosis, whereas in the case of Streptococcus mutans it is the protein
AgI/II  that acts through CD14 and TLR4 [37].Similar mechanisms
have been described forChlamydophila pneumoniae (C. pneumoniae) infection where
LPS and bacterial HSP act as ligands to TLRs. C. pneumoniae is an intracellular prokaryotic pathogen that
infects humans provoking distinct forms of pneumonia, and it has been also proposed
that it may cause chronic inflammatory diseases such as atherosclerosis [38]. Chlamydial LPS has been
shown to induce macrophage foam cell formation and chlamydial HSP60 is known to
contribute to LDL oxidation in the presence of macrophages on the lesion site. The
presence of C. pneumoniae within atheroma
lesions has been detected by PCR and immunohistochemistry. However, detection
is sometimes difficult due to phases of activity and latency of the pathogen [123, 124]. The pathogen has been
located within DCs in close proximity to T cells [125] but the precise mechanisms responsible
for the induction of immune activation and atherosclerosis development remain
to be clarified. There is evidence that patients with acute myocardial
infarction have higher titres of antibodies against C. pneumoniae than control patients [126]. Moreover, C. pneumoniae has been also extracted
and cultured from atherosclerotic plaques [125, 127, 128]. Experiments carried out in
animal models demonstrated the induction of atherosclerosis by inoculation of C. pneumoniae [129]. C. pneumoniae can persistently infect epithelial cells and
macrophages within human atherosclerotic plaques causing a chronic and nonlytic
infection [128]. Immune responses against
Chamydia spp. infection mainly involve CD4+ T-helper (Th)1 cells and
antibodies, although other components such as CD8+ T cells also play
a key role [130–132]. The relative contribution
of these components to protection depends on several factors, such as the site
of infection, whether it is a primary or secondary infection, and whether the
infection is acute or persistent [133]. However, despite all the
evidence supporting the role of Chlamydia infection in the development of
atherosclerosis, its correlation with the development of complications remains
controversial. A key element to the debate is the failure of recent human
clinical trials and animal studies aiming to investigate the secondary
preventive effect of antibiotics on atherosclerosis [128, 129, 134]. In these studies,
antibiotic therapy was effective in clearing the acute infection, but failed to
influence the atherogenic properties of C.
pneumoniae unless the therapy was started early during the acute infection [134]. It has been hypothesised
that this may be due to a sequestration of the organism within atherosclerotic
plaques, that makes it inaccessible to both antibiotics and the cellular
components of the immune response.The microorganismHelicobacter pylori—a cause of
gastrointestinal infections—has been also
found to be present in atherosclerotic lesions but completely absent from
healthy arteries [52, 53]. However, immunohistochemical
studies could not detect its presence in the lesions but instead there was a
strong cross-reactivity of the antibodies to the different elements of the plaque
related to the acceleration of inflammatory events and plaque destabilisation.Cross-reactivity
has also been observed with antibodies against human HSP60 andE. coli-derived GroEL, an HSP [65]. The generation of anti-HSP
antibodies can induce autoimmune reactions binding to HSP on endothelial cells at
the lesion site where it is expressed at high levels due to shear stress
triggered by blood pressure, stimulation by oxLDL in situ or by inducing the secretion of proinflammatory
cytokines. The down stream effects are endothelial and macrophage damage and
subsequent inflammatory events that lead to the pathogenesis of atherosclerosis
[135–137].


## 3.9. Virus-Derived Antigens

Viruses have also
been postulated as promoters of atherosclerosis. One of the most closely linked
to this disease is cytomegalovirus. This virus infects the majority of the
human population by targeting SMCs and endothelial cells producing a latent
type of infection [138–142]. US28, one of the viral
proteins expressed on the cell surface of the cytomegalovirus after infection,
is a chemoattractant for SMCs. US28 and UL122 proteins were found to have an
11aa sequence homologous to human HSP60, and it is thought that antibodies
against these viral proteins can bind to human HSP60 expressed on stressed
endothelial cells [143]. The proteins also share
some homology with nonstressed endothelial cell markers such as CD151, CD49f,
and connexin 45 (Cx45). It is believed that during cytomegalovirus infection, antibodies
generated against these proteins can bind, by molecular mimicry, to the surface
markers on both nonstressed and already-stressed endothelial cells causing apoptosis
of endothelial cells, which is considered to be one of the key early events in atherosclerotic
plaque formation. It has been also suggested that endothelial cell stress
induces HSP60 expression enhancing the binding of circulating autoantibodies
and amplifying the endothelial cell damage [143]. Interesting results
relating cytomegalovirus with atherosclerosis are derived from studies investigating
the expression of the viperin gene. The human viperin gene has been suggested
as a potential marker for cytomegalovirus infection [144]. Viperin, which is highly
conserved among species, has a well-known antiviral effect and its use for the
local treatment of cytomegalovirus has been recently proposed. Viperin is
expressed by endothelial cells and SMCs in the vascular wall of disease vessels
but no expression has been detected in the normal arteries [144].Another pathogen
associated with atherosclerosis is enterovirus, especially the enterovirus
group coxsackie B virus [67]. High levels of enterovirus
antibodies have been detected in patients with myocardial infraction but it has
not yet been fully established whether the virus contributes to the
pathogenesis of the disease [67, 68, 145]. Other infectious organisms that
have been implicated in the pathogenesis of atherosclerosis involve a member of
the herpes virus family that is known to induce atherosclerosis in chickens [146]. The virus alters cellular
metabolism resulting in cholesterol accumulation which is a common mechanism
proposed for all virally-induced atherosclerosis. There is also evidence
that the virus promotes smooth muscle cells (SMCs) proliferation [147–150].


## 4. T-Cell Antigen Immune Responses and Atherosclerotic Plaque Development

T lymphocytes are
present in atherosclerotic plaques at all stages of its development [15]. Most T cells within
atherosclerotic plaques are CD4+ and a small fraction of the
population consists of CD8+ T cells. CD4+ cells isolated
from human plaques have been found to express the αβ T cell receptor (αβ TCR) [8, 151] that recognises antigens
presented in the context of HLA-DR in the surface of APCs. The close proximity
between T lymphocytes and APCs within atherosclerotic plaques supports the view
that these lymphocytes are involved in antigen recognition and antigen-specific
proliferation in the shoulder regions of atherosclerotic lesions [81]. They are attracted to the
tissues by chemokines and adhesion molecules expressed on the surface of endothelial
cells. Although the production of IgM, also called natural auto-antibodies,
seems to be predominant in atherosclerosis, the presence in the serum of IgG
antibodies specific to oxLDL epitopes is indicative of the involvement of CD4+ T cells in the process of affinity maturation and isotype class switching of
B-cell clones specific to atherogenic antigens [21].The unbalance
between pro- and anti-inflammatory immune responses appears to be responsible
for the development of atherosclerosis. The activation of naïveCD4+ T cells generates one of the two major types of functionally different effector
T cells, the T-herper1 (Th1) or the Th2. The response of the former cells is
considered to be proinflammatory in the context of atherosclerosis and is characterised
by the secretion of IFN-γ, IL-12 and TNF-α which are all involved in macrophage
activation. IFN-γ also drives Th1 cell differentiation that can be inhibited by
IL-10 [21]. Th2 response is considered
to be anti-inflammatory due to the secretion and action of IL-10 and other
cytokines such as IL-4, IL-5, and IL-13, all linked to B cell activation and
differentiation. Th2 differentiation is favoured by IL-4 and it can be inhibited
by IFN-γ. Th1 is the predominant T cell subset found in atherosclerotic lesions
[70, 152, 153].The type of
antibody produced, driven by Th1 or Th2 immune responses, also plays a key role
in atherogenesis [21]. The synthesis of IgG1
antibodies indicates a predominant Th2 response while IgG2a is indicative of
Th1 responses [21]. The regulation of the
balance between Th1 and Th2 immune responses appears to be controlled by
another T cell subset referred to as regulatory T cells (Tregs) (reviewed in [154]). It has been suggested that
plaque size correlates with the number of Th1 cells present within the lesions [155].On the other hand,
Th2 activation and proliferation appears to be triggered by epitope-specific
stimulation [7, 156–160] or by the induction of
natural antibodies involved in the clearance of lipoprotein particles [95]. Auto-antibodies against oxLDL
have been found circulating in the plasma. There is a correlation between the concentrations
of these antibodies in plasma and lesion size [161]. Recent experimental
evidence shows that pneumococcal vaccination using an animal model of
atherosclerosis induces the production of anti-oxLDL IgM antibodies, which
inversely correlates
with the development of atherosclerotic plaques [162]. It has been also proposed
that IgM antibodies may bind to oxLDL preventing its binding and degradation by
macrophages, or even prevent the uptake of apoptotic cells by macrophages [163, 164]. However, the mechanisms
involved in the production of these antibodies or their precise role in
atherogenesis have not yet been addressed. Th2 responses are also recognised in
advanced stages of atherosclerosis, when hypercholesterolemia is prominent and
there seems to be a shift of the immune response towards a Th2 type, indicating
that in late stages the immune system is trying to overcome the
pro-inflammatory damage [152].Noticeably, the
therapeutic correction of the balance between these two types of responses has
been pivotal for the development of novel interventions, such as vaccines
against the development of the disease. Experiments carried out using inbred
stains of mice show that C57BL/6 mice are more prone to develop Th1 responses and
more atherosclerosis than BALB/c mice which are prone to develop Th2 responses and
consequently atheroresistant [165]. It has also been noted that
deletion of STAT6, a transcription factor required for the activation of Th2
responses, prone these mice to develop atherosclerosis [166]. Treatment of hypercholesteremic mice
with recombinant IFN-γ also accelerates atherosclerotic plaque development [167], an effect that is reversed when
mice receive the drug pentoxyfyllin, a potent Th1 blocker [155].Switching the
immune response to a Th2 type can be achieved by the expression of the
anti-inflammatory cytokine IL-10 which suppresses the effect of proinflammatory
cytokines such as IL-12 and IFN-γ [168, 169]. The athero-protective
effect of IL-10 was noted even in mice fed a high-fat diet. However, the
treatment failed to influence plasma cholesterol levels indicating that the
IL-10 effects are due to modulation of the immune response involved in intraplaque
inflammation mechanisms [169]. Deficiency of T-bet, a transcription
factor required for Th1 differentiation, in experimental animals significantly
reduced atherosclerotic lesions. This effect was linked to a reduction in
number of proliferating smooth muscle cells in the intima layer [170]. T-bet deficient mice have
also shown a skewed immune response towards the Th2-type when HSPs were administered
to these mice [170]. These findings suggest that
transcriptional regulation in T cell differentiation can represent a good
target to immunomodulate atherosclerosis.Just recently the
first report appeared on the possible role of Th17 cells in cardiovascular
disease [171]. Th17 cells are
characterised by IL-17
(or IL-17A), IL-17F, IL-6, TNF-α, and IL-22 expressions. Their discovery has
contributed to explain crucial regulatory mechanisms which until now the
classic control by Th1 and Th2 or Treg cell-mediated mechanisms could not
explain. Th17 cells have been suggested to play a key role in inflammation and
autoimmunity. They have also been involved in the pathogenesis of
hypersensitivity reactions. The study of their role in host defence mechanisms
has just recently started and promises to be another area of high interest in
cardiovascular biology research (see the following articles for a comprehensive
review of the recently published literature on Th17 [172–177]). Cheng et al. have
suggested that Th17/Treg balance may play a key role in controlling
inflammation, plaque destabilization, and the onset of acute coronary syndrome.
They investigated this hypothesis by assessing Th17/Treg functions through the
analysis of T cell frequencies, secretion of specific cytokines, and production
of key transcription factors in patients with acute myocardial infarction,
unstable angina and stable angina. They found that Th17 cell numbers as well as
its cytokines (IL-17, IL-6, and IL-23) and transcription factor (RORgammat)
levels were significantly higher in patients with acute coronary syndrome as
compared to controls. The study also showed a significant decrease in Treg
number, Treg-related cytokines (IL-10 and TGF-β1), and Foxp3 levels in these
patients as compared to stable angina and controls suggesting a potential role
for Th17/Treg imbalance in plaque destabilization and the onset of ACS [171].


## 5. Conclusions

The multifactorial
nature of atherosclerosis also applies to the number and quality of antigens
capable of inducing proinflammatory/proatherogenic immune responses. The
evidence accumulated so far supports the view that oxLDL is one of the most
important atherogenic antigens, by virtue of being the main trigger of
monocytes/macrophage and SMC infiltration, proliferation, and conversion in to
foam cells in the neointima layer. The key role of other self-derived antigens
such as HSPs andβ2-GpI and the presence of circulating antibodies against
them, that in most cases correlates with the clinical outcome, have been used
to justify the classification of atherosclerosis as an inflammatory disease
with an important autoimmune component. No less important is the role of
foreign antigens derived, among others, from bacteria and viruses which might play
a causal and/or at least a bystander effect contributing to the chronic
inflammatory process and its complications. Finally, the role of T lymphocytes
and the pro- and anti-inflammatory balances controlled by their different
subsets has been shown
to be crucial in the development of the disease. These responses are ultimately
driven by the nature of the initial stimuli (the antigen) and supported by a
complex cascade of events involving cytokines, components of the extracellular
matrix, and even gene expression regulators such as transcription factors. Our
current understanding of the immunopathogenic mechanisms involved in
atherosclerotic plaque development has witnessed an enormous advance in the last
decade, and some of this knowledge constitutes the foundation for the design of
the next generation of drugs to combat cardiovascular disease and reduce its devastating
consequences for the benefit of mankind.



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*Source: 723539-2008-06-09.xml*