ARTICLE
Auteur(s) : Toshiyuki
Yamamoto
Department of Dermatology, Fukushima Medical University,
Fukushima 960-1295, Japan
accepté le 18 Août 2008
Systemic sclerosis (SSc) is a connective tissue disease
involving fibrosis of the skin and various internal organs. It is
characterized by the excessive accumulation of extracellular matrix
(ECM) proteins in the skin and various internal organs, vascular
injury, and immunological abnormalities [1-3]. In early stages of
SSc, activated fibroblasts in the affected areas produce high
amounts of collagen. Histological analysis of the initial stage of
scleroderma reveals perivascular infiltrates of mononuclear cells
in the dermis, which is associated with increased collagen
synthesis in the surrounding fibroblasts. Although, as yet, the
pathogenesis of SSc has not been fully elucidated, a number of
studies have demonstrated the crucial role of several fibrogenic
cytokines released from immunocytes in initiating the sequence of
events leading to fibrosis. In this review, current findings on the
pathophysiology of scleroderma are discussed.
Clinical features
SSc begins with the edematous swelling of the fingers, in most
cases preceded by Raynaud’s phenomenon accompanied by a sensation
of coldness. The dorsa of the hands and forearms may then begin to
take on an edematous appearance and become progressively sclerotic.
The initial phase presenting edema may reflect increased vascular
permeability due to endothelial damage. SSc classification is based
on the criteria of American College of Rheumatology (ACR) [4].
LeRoy et al. [5] described limited and diffuse subsets (lSSc and
dSSc), and later identified 2 types of limited forms (limited SSc
(lSSc) and limited cutaneous SSc (lcSSc) [6]. SSc is classified
according to whether the skin involvement is confined to the area
proximal to the elbow (lSSc) or extends distally beyond it (dSSc).
lSSc is dominated by vascular impairment, and cutaneous and organ
fibrosis progress slowly, whereas dSSc rapidly progresses with
widespread inflammation, and is frequently accompanied by more
severe visceral involvement.
The cutaneous manifestations of SSc include abnormalities in
peripheral circulation, skin sclerosis, and hyper- and
hypo-pigmentation. Symptoms arising from abnormalities of
peripheral circulation are digital ulcers, elongation of the nail
fold with pitted bleeding, pitting scars, and so on. Digital ulcers
are painful, repetitive and refractory (figure 1A). When the
sclerosis of the skin reaches an advanced stage, the fingers cannot
be extended. Patients with severe dSSc present diffuse
hyperpigmentation with pruritus, and local hypopigmentation. Stem
cell factor (SCF) [7], a growth factor for mast cells and
melanocytes, is thought to play a part in inducing diffuse
hyperpigmentation and pruritus. Upon degranulation, mast cells
release mediators such as histamine, which may induce itching.
Another candidate for hyperpigmentation is endothelin, which also
has melanogenetic effects. Other skin manifestations include
telangiectasias, a shortened lingual frenulum, a reduction of the
fingertips due to bone absorption, keratosis of the lateral and
dorsal aspects of the fingers, calcinosis, and so on (figures 1B,C).
Genetic involvement
Genetic susceptibility is thought to play a role in the development
of SSc. At the murine level, mutations in the ECM protein,
fibrillin, are responsible for the phenotype of tight skin (Tsk-1)
mice [8]. The high incidence of scleroderma among the Choctaw
Indian population has been explained by the close association of
chromosome 15 to the fibrillin locus [9]. Other studies have
implicated a mutation in the promoter region of the collagen [10]
or transforming growth factor-β (TGF-β) gene [11]. A recent
study has demonstrated that variations in the promoter region of
the connective tissue growth factor (CTGF) gene (G-945C
polymorphism) are linked to susceptibility to SSc [12].
Microchimerism
The fact that the majority of patients develop SSc in the
post-childbearing years has lent support to the hypothesis that the
persistence of fetal cells may induce tolerance and initiate an
immune reaction. Several reports have detected large numbers of
fetal cells in the lesional skin of SSc patients [13, 14]. However,
there is still scant evidence that microchimerism is definitely
involved in the pathogenesis of scleroderma.
Immune dysfunction
T cells, macrophages and mast cells are present in increased
numbers or in an activated state in the lesional skin of SSc
patients, and are thought to play an active role in the
pathogenesis of the disease. Additionally, activated peripheral B
cells are found in abnormally large numbers in patients with SSc
[15]. B cells contribute not only to antibody production, but also
to T cell activation and differentiation and the production of
various cytokines.
Pathogenic autoantibodies in scleroderma
Circulating antibodies are present in most patients with SSc.
Although their role in the pathogenesis of scleroderma remains
unclear, the symptomology of SSc can be classified to some extent
by the presence of specific antibodies. Many patients with lSSc
have antibodies against centromeres, whereas anti-topoisomerase-1
(Scl-70) antibodies are often detected in patients with dSSc.
Anti-RNA polymerase III antibodies are associated with scleroderma
renal crisis and anti-Th/To antibodies are associated with
pulmonary fibrosis. Anti-PM-Scl and anti-U1-RNP antibodies are
associated with myositis and overlap syndrome.
Recently, circulating antibodies to PDGF receptors, which
stimulate reactive oxygen species (ROS) and collagen [16], have
been identified in patients with SSc. The ROS-Ras-ERK1/2 cascade
results in fibroblast activation and the formation of a
myofibroblastic phenotype.
Cytokines and chemokines in scleroderma
TGF-β
TGF-β, which occurs abundantly in platelets and is released by
activated macrophages or lymphocytes, is a strong chemoattractant
for fibroblasts. TGF-β increases the synthesis of ECM, such as
collagen type I and type III, or fibronectin by fibroblasts,
modulates cell-matrix adhesion protein receptors, and regulates the
production of proteins such as plasminogen activator, an inhibitor
of plasminogen, or procollagenase, which can modify the ECM by
proteolytic action [17]. In addition, TGF-β is capable of
stimulating its own synthesis by fibroblasts through autoinduction
[18]. TGF-β increases TGF-β receptor (TGF-βR) levels in fibroblasts
[19], and thus the maintenance of increased TGF-β production may
lead to the progressive deposition of ECM, resulting in fibrosis.
Indeed, TGF-β mRNA levels are elevated in the lesional skin of SSc
[20-22], and shown to co-localize with type I collagen [23].
Overexpression of TGF-βR, which is regulated at the transcriptional
level [24], is recognized in fibroblasts in the skin of scleroderma
patients [25]. Blocking endogenous TGF-β signaling eradicates the
scleroderma phenotype [26]. Thus, TGF-β plays a key role via
autocrine signaling in the pathogenesis of scleroderma.
Signaling by TGF-β elicits potent profibrotic responses in
fibroblasts. TGF-β binds to the type II receptor, thereby
activating the type I receptor. Signaling occurs predominantly by
phosphorylation of cytoplasmic mediators belonging to the Smad
family. Three families of Smads have been identified:
Receptor-regulated Smad2 and -3 (R-Smads), common partner Smad4
(Co-Smad), and inhibitory Smad6 and -7 (I-Smads). In scleroderma
fibroblasts, phosphorylation and nuclear translocation of Smad2/3
are increased, suggesting activation of the Smad pathway [27].
Smad7 is shown to act as an intracellular antagonist of TGF-β
signaling, and an inhibitor of TGF-β-induced transcriptional
responses. In scleroderma skin and cultured scleroderma
fibroblasts, the basal level and the TGF-β-inducible expression of
Smad7 are selectively decreased, whereas Smad3 expression is
increased [28]. On the other hand, Smad7 expression levels in
scleroderma fibroblasts are uncertain. Smad7-Smurf-mediated
negative regulation of TGF-β signaling is impaired in scleroderma
fibroblasts [29]. Other signaling pathways besides the Smad
proteins, such as the p38 mitogen-activated protein kinase (MAPK),
phosphatidylinositol 3-kinase (PI3K), c-Myb, Ets, and Egr pathways,
have also been shown to mediate TGF-β signaling in scleroderma
fibroblasts.
CTGF
CTGF is selectively induced in fibroblasts after activation by the
active form of TGF-β. Recombinant CTGF protein was found to
stimulate DNA synthesis and upregulate collagen, fibronectin, and
integrin expression in fibroblasts [30]. A TGF-β response
element is found in the CTGF promoter, which is not present in the
promoters of other TGF-β-regulated genes, suggesting that CTGF
functions as a downstream mediator of TGF-β, and may coordinate the
action of TGF-β, such as fibroblast proliferation, adhesion, and
ECM production [31].
Overexpression of CTGF is known to occur in cultured scleroderma
fibroblasts [32, 33]. The constitutive overexpression of CTGF in
scleroderma fibroblasts is independent of TGF-β signaling but
dependent on Sp1 [34]. Moreover, serum levels of CTGF are elevated
in patients with SSc [35]. Dermal fibroblasts exposed to hypoxia
(1% O2) or CoCl2 (1-100 μM) enhance
expression of CTGF mRNA [36]. Skin fibroblasts transfected with
hypoxia-inducible factor (HIF)-1α show increased levels of CTGF
protein and mRNA, as well as nuclear staining of HIF-1α, which was
enhanced further by treatment with CoCl2. These data may
suggests that hypoxia, caused possibly by microvascular
alterations, upregulates CTGF expression through the activation of
HIF-1α in dermal fibroblasts of SSc patients, and thereby
contributes to the progression of skin fibrosis.
IL-13
An imbalance exists between the type 1 and type 2 cytokine
responses in the pathogenesis of scleroderma. Interleukin-13
(IL-13) is a pleiotropic cytokine, elaborated in significant
quantities by appropriately stimulated type 2 cells. IL-13 has the
ability to suppress proinflammatory cytokine production in
monocytes/macrophages, and is known to enhance the growth and
differentiation of B cells and to promote immunoglobulin synthesis.
In addition, in vitro studies demonstrate that IL-13 is a potent
stimulator of fibroblast proliferation and collagen production
[37-39]. The profibrotic effect of IL-13 is thought to involve
irreversible fibroblast activation, triggered either directly [40]
or indirectly through TGF-β [39, 41]. Serum levels of IL-13 are
elevated in patients with SSc, correlated with the number of plaque
lesions [42] or nailfold capillaroscopic features [43].
Chemokines
Recent studies have shown that an increase in proinflammatory
chemokines has been associated with the initiation and/or
development of skin fibrosis/sclerosis, suggesting that chemokines
and their receptors may be important mediators of inflammation and
fibrosis in scleroderma [44]. CCL2/monocyte chemoattractant
protein-1 (MCP-1) belongs to a C-C chemokine superfamily, and
numerous types of cells are capable of expressing CCL2 in the
presence of serum or specific stimuli. A growing body of
evidence has demonstrated that CCL2 gene expression is upregulated
in human fibrosis, as well as in animal models of fibrosis. In
vitro studies show that CCL2 upregulates type I collagen mRNA
expression in rat fibroblasts, which is indirectly mediated by
endogenous upregulation of TGF-β gene expression [45]. CCL2
enhances expression of matrix metalloproteinase-1 (MMP-1), MMP-2 as
well as tissue inhibitor of metalloproteinase-1 (TIMP-1) in
cultured skin fibroblasts [46]. Recent studies have demonstrated
increased expression of CCL2 in patients with SSc [47-51]. Serum
levels and spontaneous production levels of CCL2 by peripheral
blood mononuclear cells are elevated in patients with SSc, compared
with normal controls, and are correlated with pulmonary fibrosis
[49]. Increased expression of CCL2 is demonstrated in scleroderma
skin [47, 49, 51], and scleroderma fibroblasts express increased
levels of CCL2 mRNA and protein [49, 51]. Stimulation with PDGF
results in a significant increase in CCL2 mRNA and protein [47].
Furthermore, the autoinduction of CCL2 is observed in scleroderma
fibroblasts, but not in normal fibroblasts [50]. CCL2 levels may
also be increased by IL-13, a potent stimulator of CCL2 [52]. These
in vivo and in vitro results suggest an important involvement of
CCL2 in the pathogenesis of scleroderma.
Increased numbers of mast cells are noted in scleroderma skin.
CCL2 also recruits mast cells, in addition to monocytes [53]. Human
mast cells are shown to be a rich source of chemokines, including
CCL2, CCL3/macrophage inflammatory protein-1α (MIP-1α), CCL4/MIP-1β
and CCL5/RANTES [54], as well as a number of cytokines/growth
factors and mediators capable of activating fibroblasts or
endothelial cells. Expression of SCF is upregulated in scleroderma
fibroblasts [55], and is thought to contribute to the increase of
mast cells in scleroderma. SCF enhances CCL2 expression in human
mast cells [56]. Because CCL2 enhances type I collagen mRNA
expression in skin fibroblasts, the interaction between mast cells
and fibroblasts via SCF/CCL2 may play an important role in the
development of fibrosis. CCR2 is a major CCL2 receptor. CCR2
upregulation in vascular structures, perivascular inflammatory
infiltrates, and fibroblasts has recently been demonstrated in SSc
[57]. In particular, CCR2-positive fibroblasts in early-stage dSSc
showed a profibrotic phenotype, with overexpression of α-smooth
muscle actin (α-SMA), CTGF and CCL2 [57]. Their results suggest
potential autocrine regulation of key fibrotic properties via the
CCL2/CCR2 loop in the early phases of scleroderma.
A novel protein, MCPIP (MCP-induced protein), upregulates
members of the apoptotic gene family involved in the induction of
cell death [58], and may provide a novel molecular pathway by which
CCL2/CCR2 signal transduction is linked to transcriptional gene
regulation leading to apoptosis. CCL2 promoter polymorphism is
associated with SSc [59]. CCL2 may contribute to the induction of
dermal sclerosis directly, via its upregulation of mRNA expression
of ECM on fibroblasts, as well as indirectly through the mediation
of a number of cytokines released from immunocytes recruited into
the lesional skin.
Others
Platelet-derived growth factor (PDGF) has mitogenic activity for
mesenchymal cells, regulates matrix metabolism, has chemotactic and
vasoactive properties, and produces inflammatory cytokines [60].
Overexpression of PDGF has been reported in a number of fibrotic
diseases. Elevated levels of PDGF-A chain are demonstrated in
scleroderma skin [61]. In addition, TGF-β upregulates PDGF-α mRNA
and protein levels in scleroderma fibroblasts, in comparison with
the control [61]. On the other hand, increased expression of the
PDGF B-chain and β-receptor in scleroderma skin has also been
reported [62-64].
IL-4 is known to promote fibroblast proliferation, gene
expression, and synthesis of ECM proteins such as collagen and
tenascin [65]. IL-4 has been shown to upregulate TIMP-2 in dermal
fibroblasts via the MAPK pathway [66] as well as to upregulate
TGF-β production in eosinophils [67] and T cells [68]. Increased
IL-4 production is detected in the sera or in activated peripheral
blood mononuclear cells of patients with SSc [69]. Scleroderma
fibroblasts express more IL-4 receptor α and produce more collagen
after IL-4 stimulation [70].
TGF-β can contribute to the differentiation of both regulatory T
cells and inflammatory Th17 cells. IL-17 is a T cell-derived
cytokine, and functions to secrete various cytokines and chemokines
by different cell types. Elevated levels of IL-17 have been
observed in patients with SSc, especially in the early stages [71].
IL-17 has been reported to induce fibroblast proliferation, but not
collagen production in SSc fibroblasts [71]. The role of Th17 cells
in SSc should be further investigated.
IL-21/IL-21R signaling has recently been shown to promote
fibrosis by facilitating the development of the CD4+ Th2 response
[72]. IL-21 increases IL-4 and IL-13 receptor expression in
macrophages [72], thereby possibly enhancing fibrosis, and is
abundantly expressed in the epidermis in SSc [73].
Vascular injury
Vascular injury causes endothelial cell activation, dysfunction and
altered capillary permeability as a primary event. These are
followed by an increased expression of adhesion molecules leading
to mononuclear cell infiltrates in the skin. Microvascular injury
may be the result of direct or indirect injury by anti-endothelial
cell antibodies (AECAs), which are frequently detected in the sera
of patients with SSc [74]. AECAs can activate endothelial cells to
express cell adhesion molecules which alter leukocyte attachment,
and can lead to endothelial cell damage and apoptosis. Kuwana et
al. [75], however, proposed that insufficient vascular repair
machinery, due to defective vasculogenesis, contributes to the
microvascular abnormality in SSc. Although circulating
concentrations of angiogenic factors are high in SSc, the levels of
bone marrow-derived circulating endothelial precursors (CEP) are
low, suggesting a dysregulation of vasculogenesis in SSc.
Endothelin-1 (ET-1) is a prototypical endothelial cell-derived
product. Since ET-1 is a vasoconstrictive agent, loss of normal
vessel compliance and vasorelaxation may be induced by increased
levels of ET-1. ET-1 promotes fibroblast synthesis of collagen
[76], and thus provides the link between vasculopathy and fibrosis.
ET-1 can induce CTGF, and may mediate the induction of collagen
synthesis by activation of CTGF [77]. Further, ET-1 can also induce
myofibroblast differentiation in fibroblasts [78]. Circulating ET-1
levels have been observed in patients with dSSc with widespread
fibrosis and those with lSSc and hypertensive disease [79],
suggesting that soluble ET-1 levels may be a marker of fibrosis and
vascular damage. These facts underscore the importance ET-1 in
scleroderma.
Extracellular matrix
The hallmark of fibrosis is the accumulation of ECM proteins,
including collagen, fibronectin, proteoglycan, and elastin, in the
skin. The phenotype and activation of fibroblasts is dependent on
both soluble factors and ECM-generated signals. Fibroblasts
interact with the surrounding collagens via integrins. Aberrant
signaling by ECM may disturb this interaction, thereby contributing
to the persistent modulation of fibroblasts which results in
fibrosis, as seen in the autocrine loops of cytokine production and
excessive deposition of ECM proteins in the skin [80].
Scleroderma fibroblasts
Fibroblasts are stimulated by inflammatory cells, such as activated
T cells, monocytes/macrophages, mast cells, and eosinophils.
Additionally, fibroblasts themselves are not only structural
elements but also part of the immune system, and can be activated
to perform new functions important for controlling ECM synthesis
and for producing various cytokines, growth factors, chemokines,
growth factor receptors, integrins, and oxidants. It is widely
accepted that human skin fibroblasts are heterogeneous with regard
to their synthesis of collagen, proliferative responses, and
response to growth factors. Enhanced collagen synthesis is
regulated at the transcriptional level. Some researchers think that
scleroderma fibroblasts are the result of phenotypic changes in
dermal fibroblasts caused by soluble factors; others contend that
scleroderma fibroblasts are recruited from circulating or resting
mesenchymal precursor cells as fibrocytes. Alternatively, they may
be generated by clonal selection of high-collagen-producing
fibroblasts.
Myofibroblasts represent activated and contractile phenotypes
which exist in fibrotic lesions. Myofibroblasts express α-SMA, and
can produce various cytokines, growth factors and chemokines.
TGF-β1 is a central regulator of the phenotypic changes of
fibroblasts into myofibroblasts; the modulators are mechanical
tension and fibronectin involving the ED-A domain. The
differentiation into myofibroblasts is regulated by mast cell
mediators, of which tryptase is one of the likely candidates
[81].
Fibrocytes are derived from circulating monocytes (CD34+ bone
marrow-derived progenitors) and enter into the tissues. Fibrocytes
produce matrix proteins such as collagens I and III, and
participate in the remodeling process by secreting matrix
metalloproteinases [82]. Fibrocytes are also a source of
inflammatory cytokines, growth factors and chemokines. Although
fibrocytes are involved in scleroderma, their role has yet to be
fully elucidated.
Role of apoptosis
Autoreactive clones that survive the apoptotic process may lead to
increased susceptibility to autoimmune disorders. Apoptosis causes
typical cellular morphological changes including cell shrinkage,
nuclear condensation, DNA fragmentation and membrane alterations.
This may in turn cause apoptotic cells to become a possible source
of autoantigens [83]. Scleroderma fibroblasts are thought to escape
apoptosis because cultured scleroderma fibroblasts are resistant to
Fas-induced apoptosis [84, 85], and apoptosis of fibroblasts in SSc
skin lesions has not been observed [85]. TGF-β protects
myofibroblasts from undergoing apoptosis. Serum-starved rat lung
fibroblasts treated with IL-1 result in apoptosis which can be
reduced by concomitant treatment with TGF-β [86]. Also,
α-SMA-positive myofibroblasts increase in number following
stimulation by TGF-β, which protects these myofibroblasts against
apoptosis induction. Other studies have shown that pretreatment
with TGF-β significantly reduced apoptosis caused by serum
starvation in myofibroblasts, whereas this was not the case with
non-myofibroblasts [85]. Thus TGF-β1 may play a role in inducing
apoptosis-resistant fibroblast populations in SSc. In scleroderma
fibroblasts, the Bcl-2 level is significantly higher, whereas the
Bax level significantly lower [85].
On the other hand, endothelial cell apoptosis is thought to
occur early in the pathogenesis of scleroderma. Endothelial cell
apoptosis was first noted in the UCD-200/206 chickens, which
develop hereditary systemic connective tissue disease resembling
human SSc [87]. This phenomenon occurs before perivascular
mononuclear cell infiltration. Also, terminal deoxynucleotidyl
transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) is
shown to be positive on the endothelial cells in human scleroderma
skin [87]. Recent studies have shown that apoptosis of endothelial
cells induces resistance to apoptosis in fibroblasts largely
through PI3K-dependent mechanisms [88]. Furthermore, fibroblasts
exposed to a medium conditioned by apoptotic endothelial cells
present myofibroblast changes [88].
The serum soluble Fas (sFas) levels are higher in patients with
SSc [89-91]. Untreated SSc patients have significantly higher serum
sFas levels than the treated SSc patients and healthy controls
[92]. It has been suggested that increased sFas levels in the serum
of SSc patients can protect autoreactive T cells from FasL-induced
apoptosis [91]. Spontaneous apoptosis of CD8+ T cells in the
peripheral blood is significantly higher in patients with SSc
compared with normal controls, while spontaneous apoptosis in CD4+
T cells occur at similar rates in both SSc and controls [93].
Enhanced helper T cell function, resulting in the reduction CD8+ T
cells, may lead to autoimmunity by modifying the immune
balance.
Akt is one of the key enzymes inhibiting both spontaneous and
stress-induced apoptosis. 3’-phosphorylated phosphoinositides bind
to the pleckstrin domain of Akt. Akt activity may result in the
inhibition of pro-apoptotic Bad, Bax, Bik, and caspase-9 by
phosphorylation. It has recently been reported that Akt is active
in scleroderma fibroblasts. Cultured scleroderma fibroblasts
exhibited high levels of p-Akt, in comparison to control
fibroblasts [94]. TGF-β can activate Akt in fibroblasts, and by
doing so, may also induce apoptosis resistance in scleroderma
fibroblasts. These findings point to a potential role for Akt in
the resistance of scleroderma fibroblasts to apoptosis.
Oxidant stress
ROS generated during various metabolic and biochemical reactions
have multifarious effects that include oxidative damage to DNA. ROS
can cause several abnormalities such as endothelial cell damage or
enhanced platelet activation, leading to upregulation of the
expression of adhesion molecules or secretion of inflammatory or
fibrogenic cytokines including PDGF and TGF-β; excessive oxidative
stress has been implicated in the pathogenesis of scleroderma [95].
Indeed, scleroderma fibroblasts produce ROS constitutively [96].
Other effects of oxygen radicals include the stimulation of skin
fibroblast proliferation at low concentrations [97] and the
production of increased amounts of collagen [98], suggesting that
low oxygen tension may contribute to the increased fibrogenic
properties of scleroderma fibroblasts. Furthermore, several of the
autoantigens targeted by scleroderma autoantibodies fragment in the
presence of ROS and specific metals such as iron or copper [99].
The authors suggest that tissue ischemia generates ROS, which in
turn induces the fragmentation of specific autoantigens. On the
other hand, oxidative stress transiently induces CCL2 mRNA and
protein expression in cultured skin fibroblasts [100], suggesting
that ROS may play a regulatory role in inflammation by modulating
monocyte chemotactic activity.
Animal models of scleroderma
Animal models are useful in providing clues for understanding
various human diseases and for testing new methods of treatment.
Although animal models which exhibit all the aspects of SSc are not
currently available, several experimental animal models, such as
bleomycin-induced murine scleroderma, tight skin (Tsk) mouse, Tsk2
mouse, sclerodermatous graft-versus-host disease (Scl-GvHD) mouse,
University of California at Davis line 200 (UCD-200) chicken, and
exogenous injections of TGF-β/CTGF-induced murine fibrosis model,
etc., have been examined so far.
Bleomycin-induced scleroderma model
Bleomycin is an agent that can induce pulmonary fibrosis and
infrequently, scleroderma in human beings [101]. Repeated local
injections of bleomycin into the dorsal skin induces histological
dermal sclerosis resembling human scleroderma, characterized by
thickened collagen bundles, the deposition of homogenous materials,
and cellular infiltrates in the thickened dermis in various mice
strains [102-104] (figure 2). Mast cell
infiltration increases, and marked degranulation and elevated
plasma histamine levels are also evident [102]. Hydroxyproline
contents as well as mRNA levels of type I collagen significantly
increase in the sclerotic skin. α-SMA-positive myofibroblasts are
observed in the dermis, and gradually increase in tandem with the
induction of dermal sclerosis [105]. Interestingly, autoantibodies
are detectable in the serum [102].
Three considerations recommend this model: It is easy to use;
the dermal sclerosis can be induced in a relatively short time; and
the histopathological features of dermal sclerosis most closely
resemble those of human scleroderma. A recent report shows
that a one-time injection of bleomycin-poly(L-lactic acid)
microspheres can induce dermal sclerosis in mice [106].
Using this model, several studies of the pathogenesis of this
disease have been performed. TGF-β is a mediator of the fibrotic
effect of bleomycin at the transcriptional level and the TGF-β
response element is required for bleomycin stimulation of the
proα1(I) collagen promoter [107]. In the bleomycin model, TGF-β is
detected in the lesional skin, and increased expression and
synthesis of TGF-β1 is dominant in bleomycin-‘susceptible’ mice
strains [108]. Inhibition of TGF-β suppresses dermal sclerosis
[109, 110]. Fibroblasts show predominantly nuclear localization of
Smad3 and intense staining for phospho-Smad2/3, whereas expression
of Smad7 is downregulated, a fact which may account for sustained
activation of TGF-β/Smad signaling [111]. Expression and synthesis
of IL-13 as well as IL-13 receptor (IL-13R)-α2 mRNA expression are
upregulated, whereas IL-13R-α1 mRNA levels are not significantly
enhanced [112]. IL-13 may promote the progression of cutaneous
fibrosis/sclerosis in this model. Indeed, recent studies have shown
that IL-13-deficient mice failed to develop an increase in skin
sclerosis after bleomycin treatment [113]. Expression of CCL2 as
well as its major receptor, CCR2 is enhanced in the sclerotic skin
[114]. Administration of anti-CCL2 neutralizing antibody reduces
dermal sclerosis, along with collagen content as well as mRNA
expression of type I collagen in the skin. More directly,
bleomycin-induced dermal sclerosis is abrogated in MCP-1-deficient
mice [115]. These data suggest that CCL2 and CCR-2 signaling plays
an important role in the pathogenesis of bleomycin-induced
scleroderma.
Bleomycin induces apoptosis. TUNEL-positivity is prominently
detected on keratinocytes and infiltrating mononuclear cells, but
not endothelial cells and fibroblasts following bleomycin treatment
[116]. DNA fragmentation reveals laddering of the whole skin.
Expression of FasL mRNA is upregulated, whereas Fas mRNA is
continuously detected. mRNA expression as well as activity of
caspase-3 is also enhanced in the skin. Administration of
neutralizing anti-FasL antibody reduces the development of dermal
sclerosis, in association with the reduction of TUNEL-positive
mononuclear cells and the blockade of apoptosis. Caspase-3 activity
is also significantly reduced after anti-FasL treatment. Moreover,
dermal sclerosis is less induced in both Fas- and FasL-deficient
strains [117]. Excessive apoptosis, which is mediated by the
Fas/FasL pathway and caspase-3 activation, is involved in this
model. Tumor necrosis factor receptor (TNFR)p55-deficient mice
developed severe sclerotic changes of the dermis following
bleomycin exposure much earlier than the wild type [118]. Induction
of MMP-1 expression is significantly inhibited in TNFRp55-deficient
mice. Signaling mediated by TNFRp55 is thought to play an essential
role in MMP-1 expression as well as in the collagen degradation
process in the bleomycin model.
In vitro, bleomycin upregulates mRNA expression of collagen, as
well as fibrogenic cytokines such as TGF-β1 and CTGF, in human skin
fibroblasts [119]. Thus, the induction of dermal sclerosis by
bleomycin is considered to be, in part, mediated by inflammatory
and fibrogenic cytokines, as well as by the direct effect of
bleomycin on ECM synthesis in fibroblasts. Numerous therapeutic
approaches have been investigated in this model [120].
Tight skin mouse model
The Tsk mutation in the fibrillin-1 gene maps to chromosome 2 and
is inherited in an autosomal dominant fashion. Fibrillin is a large
ECM structural protein and the major component of microfibrils. Tsk
mice have excessive accumulation of collagen in the skin, as seen
in the hypodermis and superficial fascia, as well as the lung and
heart; however, vascular involvement has not been associated with
this condition [121]. There are, however, numerous biochemical and
molecular abnormalities that resemble those present in patients
with SSc. mRNA expression of TGF-β, type I, III and VI collagen are
under temporal and spatial regulation during postnatal growth and
development in the Tsk1/+ mice [122]. Collagen α1(I) and α1(III)
gene-expressing fibroblasts are increased in Tsk1/+ fibrotic
lesions.
The Tsk2 is a mutant that appeared in the offspring of a 101/H
mouse after the administration of the mutagenic agent
ethylnitrosourea [123]. Tsk2/+ mice develop a tight skin phenotype
that becomes apparent at 3-4 weeks of age. Histological examination
of skin reveals marked accumulation of collagen similar to that
observed in Tsk1/+ mice. However, in contrast to Tsk1/+ mice,
prominent mononuclear cell infiltration is present in the dermis
and adipose tissue of Tsk2/+ mice. Biochemical analysis showed that
Tsk2/+ skin had 50% more collagen than the normal mouse skin.
Collagen synthesis in Tsk2/+ cultured dermal fibroblasts is 100%
higher compared with normal fibroblasts. In neither Tsk1 nor Tsk2
mice are alterations in endothelial cell apoptosis induction
involved in the development of the disease [124].
TGF-β and IL-4 possibly play important roles in the pathogenesis
of fibrosis in Tsk mice. Fibroblasts from Tsk mice are
hyperresponsive to IL-4 and TGF-β [125]. Smad2 and Smad3 are
considered to be the primary signaling molecules involved in the
TGF-β signaling transduction pathway. Tsk fibroblasts have elevated
Smad3 transcriptional activity compared with normal fibroblasts
[126]. This may explain why Tsk fibroblasts are more responsive to
TGF-β stimulation. Previous studies concerning TGF-β mRNA
expression in Tsk mice produced inconsistent results; one group
showed increased expression in the skin of Tsk mice [127, 128],
while another detected expression in only the skin of neonate Tsk
mice [129]. Targeted mutations in either the signaling chain of the
IL-4 receptor or STAT6 prevents cutaneous hyperplasia in Tsk mice,
suggesting the importance of IL-4 [125, 130]. CD4+ T cells have
been shown to be required for the excessive accumulation of dermal
collagen in Tsk mice [131]. In Tsk mice, mast cells are abundant in
the thickened dermis and exhibit prominent degranulation [132].
Mast cells are one of major sources of IL-4. IL-4 has been shown to
induce significant levels of CCL2 production in stromal cells [133,
134]. On the other hand, CCL2 upregulates IL-4 mRNA expression and
protein production [135]. These observations have led to the
hypothesis of the mutual induction of CCL2 and IL-4. Recent studies
have shown that CCL7/MCP-3 is highly overexpressed by neonatal Tsk
fibroblasts [136]. Increased CCL7 protein secretion by Tsk
fibroblasts is observed, and CCL7 is abundantly expressed in the
dermis of Tsk mice at 10 days and 3 weeks old. Downregulation of B
cell function results in inhibition of skin fibrosis and
autoantibody production in Tsk mice [137].
Graft-versus-host disease model
In human chronic GvHD, severe cutaneous fibrosis is observed with
loss of dermal fat, atrophy of dermal appendages, mast cell
depletion, and mononuclear cell infiltration. A murine
Scl-GvHD model was produced by transplanting B10.D2 bone marrow and
spleen cells into BALB/c mice after lethal gamma irradiation of the
recipients [138]. Scl-GvHD mice exhibit remarkable skin thickening
and pulmonary fibrosis by day 21 after bone marrow transplantation,
with significant increase of type I collagen mRNA levels and
protein synthesis. TGF-β is a key regulator in this model, and
blocking of TGF-β ameliorated the skin fibrosis [138, 139]. CCL2
upregulation has also been shown in the lesional skin of this model
[140].
In addition, a modified model of GvH-induced SSc has been
developed recently [141]. Injection of spleen cells from B10.D2
mice into RAG-2 knockout mice induced dermal thickening,
progressive fibrosis of internal organs and autoantibody
generation. However, lung fibrosis was absent.
UCD-200 chicken
UCD-200 chickens spontaneously develop vascular damage, mononuclear
cell infiltrates, fibrosis of the skin and internal organs, and
polyarthritis [142, 143]. Additionally, positive AECAs, antinuclear
antibodies, anticardiolipin antibodies, and rheumatoid factors are
detected in the serum. The disease starts 1-2 weeks after hatching
with erythema and swelling of the comb, which subsequently proceeds
to a chronic stage characterized by fibrosis with excessive
accumulation of collagen. In the inflammatory phase, T cell
receptor (TCR)γ/δ+/CD3+/MHC class II− T cells prevail in the
stratum papillae, while TCR α/β+/CD3+/CD4+/MHC class II+ T cells
predominate in the deeper dermis. AECAs can induce apoptosis of
endothelial cells through antibody-dependent cell-mediated
cytotoxicity via Fas [144]; transfer of AECA-positive sera into
healthy chickens induced endothelial cell apoptosis, although this
was not followed by skin sclerosis [145]. These studies
demonstrated the in vivo apoptosis-inducing effects of AECAs.
TGF-β/CTGF induced fibrosis model
TGF-β induces rapid fibrosis and angiogenesis when injected
subcutaneously into newborn mice [146]. Takehara and colleagues
showed that TGF-β-induced subcutaneous fibrosis and subsequent CTGF
or basic fibroblast growth factor (bFGF) application caused
persistent fibrosis [147, 148]. They suggest that TGF-β plays an
important role in inducing granulation and fibrotic tissue
formation, and CTGF and bFGF are important in maintaining fibrosis
[149]. The mast cell count was significantly but transiently
increased in the early phase, while the number of macrophages
continued to rise [150]. In lesional skin, serial injections of
CTGF after TGF-β increased CCL2 mRNA expression up to 8 times in
comparison with only a single injection of TGF-β or CTGF [150].
Anti-CTGF reduced skin fibrosis and collagen content [151].
Kinase-deficient type II TGF-β receptor transgenic mouse
Denton et al. [152] generated transgenic mice expressing a
kinase-deficient type II TGF-β receptor selectively on fibroblasts.
These mice develop dermal and pulmonary fibrosis. Transgenic
fibroblasts proliferate more rapidly, produced more ECM, and show
increased expression of plasminogen activator inhibitor-1 (PAI-1),
CTGF, Smad3 Smad4, and Smad7. Additionally, transgenic fibroblasts
show myofibroblast differentiation [153].
Relaxin knockout mouse
Relaxin is a small peptide hormone with anti-fibrotic and
vasodilatory properties. A recent report shows that
relaxin-deficient mice present dermal fibrosis characterized by
thickening of the skin and increase in collagen content [154].
Fibroblasts derived from the skin of the null-mice produce higher
levels of collagen.
Therapeutic implications for human scleroderma
Until now, a number of therapeutic approaches have been tried with
limited success. Randomised, placebo-controlled trials revealed
that cyclophosphamide had a significantly beneficial effect on skin
sclerosis, as well as lung fibrosis [155]. Skin sclerosis fell
moderately in the cyclophosphamide-treated group (especially in
dSSc), compared with the placebo.
Interferon-γ (IFN-γ) causes potent inhibition of collagen
production, which correlates with a reduction in the corresponding
steady-state mRNA levels in cultured skin fibroblasts [156]. IFN-γ
inhibits the TGF-β-induced phosphorylation of Smad3 and the
accumulation of Smad3 in the nucleus, whereas it induces the
expression of Smad7, which prevents the interaction of Smad3 with
the TGF-β receptor [157]. A randomized, controlled trial was
carried out in 44 patients with SSc, which did not show a
significantly greater benefit from IFN-γ in improving the skin
thickness score compared with the controls [158]. IFN-γ is a
powerful type 1 inducer of cellular immunity, which may indirectly
contribute to the improvement of the imbalance in the type 2
shift.
Ultraviolet (UV) irradiation is reported to be effective for
scleroderma, in particular for the localized type. UV induces
upregulation of mRNA levels of MMPs, depletion of skin-infiltrating
T cells, and suppression of several cytokines. Also, UV reduces
CTGF mRNA expression in both normal human skin and cultured skin
fibroblasts [159]. Additionally, UV increased Smad7 mRNA levels in
healthy skin, as well as the lesional skin of localized scleroderma
[160]. These effects may contribute to the reduction of procollagen
synthesis in the skin.
Iloprost has been shown to be useful for Raynaud’s phenomenon
associated with SSc [161]. Iloprost, which is a prostacyclin
antagonist, induces prolonged vasodilation, reduces platelet
aggregation, and promotes endothelial cell lining. Additionally,
iloprost blocks the induction of CTGF and the increase in collagen
synthesis in cultured fibroblasts exposed to TGF-β [33].
A pilot study using a humanized mAb against TGF-β1 showed no
evidence of efficacy, while it was tolerant [162].
A multicenter pilot study of high-dose immunosuppressive therapy
followed by autologous stem cell replacement demonstrated dramatic
improvements in skin sclerosis and HAQ in severe SSc patients
[163]. Multicenter, randomized, clinical trials are ongoing in the
USA (Scleroderma Cyclophosphamide Or Transplant [SCOT]) and Europe
(Autologous Stem Cell Transplantation International Scleroderma
[ASTIS]).
Recent reports have shown that bosentan, an oral endothelin
receptor antagonist, reduced the number of newly formation of
digital ulcers associated with SSc [164]. New treatments include
plasma cell exchange, intravenous immunoglobulin, and biological
targeting therapies.
Perspective
Complex networks involve cell-cell and cell-matrix interactions via
mediators in the induction of cutaneous sclerosis. Activated
fibroblasts are a part of the immune system, and modulate immune
cell behavior by conditioning the local cellular and cytokine
microenvironment. Additional mechanisms such as apoptosis and
production of ROS are also thought to be involved in the induction
of scleroderma. Animal models of scleroderma are useful for
investigating the pathogenesis of this condition, and may also
serve as promising tools for the development of new therapies.
However, it must be mentioned that the animal model is a
simplification of the more complex human scleroderma. Further, the
currently suggested pathway leading to dermal sclerosis might not
suffice as the sole explanation. Nonetheless, the pathogenic
mechanism discovered in the animal model may provide novel
information, and assist in helping us to better understand the
mechanisms underlying human scleroderma. Research into the
pathogenesis of SSc has greatly progressed in recent years, and is
expected to add impetus to the development of new therapies in the
near future.
Acknowledgements
This work was supported in part by Grants-in-Aid for Research on
Intractable Diseases from the Ministry of Health, Labour and
Welfare of Japan.
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