scleroderma- Pathophysiology

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					Review article                                                                               Eur J Dermatol 2009; 19 (1): 14-24


Toshiyuki YAMAMOTO                           Scleroderma – Pathophysiology
Department of Dermatology,
Fukushima Medical University,
Fukushima 960-1295, Japan                    Scleroderma is a fibrotic condition characterized by immunological
                                             abnormalities, vascular injury and increased accumulation of extracel-
Reprints: T. Yamamoto                        lular matrix proteins in the skin. Although the etiology of scleroderma
<toyamade@fmu.ac.jp>                         has not yet been fully elucidated, a growing body of evidence suggests
                                             that extracellular matrix overproduction by activated fibroblasts results
                                             from complex interactions among endothelial cells, lymphocytes,
                                             macrophages, and fibroblasts via a number of mediators, such as cyto-
                                             kines, chemokines and growth factors. There is also likely to be a
                                             genetic susceptibility to the disease. Recent investigations have further
                                             suggested that reactive oxygen species (ROS) and apoptosis are
                                             involved in scleroderma. Animal models are indispensable tools for
                                             understanding the complex pathophysiology of scleroderma. In this
                                             review, current findings on the pathophysiology of human, as well as
                                             animal models of scleroderma are described, which may strengthen
                                             our understanding of the pathogenesis of, and assist in exploring new
                                             treatments for, scleroderma.
Article accepted on 18/8/2008                Key words: scleroderma, pathogenesis, fibroblast, animal model




S
         ystemic sclerosis (SSc) is a connective tissue dis-     (dSSc). lSSc is dominated by vascular impairment, and
         ease involving fibrosis of the skin and various         cutaneous and organ fibrosis progress slowly, whereas
         internal organs. It is characterized by the excessive   dSSc rapidly progresses with widespread inflammation,
accumulation of extracellular matrix (ECM) proteins in the       and is frequently accompanied by more severe visceral
skin and various internal organs, vascular injury, and           involvement.
immunological abnormalities [1-3]. In early stages of            The cutaneous manifestations of SSc include abnormali-
SSc, activated fibroblasts in the affected areas produce         ties in peripheral circulation, skin sclerosis, and hyper-
high amounts of collagen. Histological analysis of the ini-      and hypo-pigmentation. Symptoms arising from abnor-
tial stage of scleroderma reveals perivascular infiltrates of    malities of peripheral circulation are digital ulcers,
mononuclear cells in the dermis, which is associated with        elongation of the nail fold with pitted bleeding, pitting
increased collagen synthesis in the surrounding fibroblasts.     scars, and so on. Digital ulcers are painful, repetitive
Although, as yet, the pathogenesis of SSc has not been           and refractory (figure 1A). When the sclerosis of the
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      A                               B
pathophysiology of scleroderma are discussed.

Clinical features
SSc begins with the edematous swelling of the fingers, in
most cases preceded by Raynaud’s phenomenon accom-
panied 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                                             C
permeability due to endothelial damage. SSc classification
is based on the criteria of American College of Rheuma-
tology (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        Figure 1. Cutaneous manifestation of fingers associated with
whether the skin involvement is confined to the area prox-       dSSc. A) Multiple digital ulcers. B) Shortening of the tip of
imal to the elbow (lSSc) or extends distally beyond it           the fingers. C) Keratosis of the lateral aspects of the fingers.

14                                                                                            EJD, vol. 19, n° 1, January-February 2009
skin reaches an advanced stage, the fingers cannot be           polymerase III antibodies are associated with scleroderma
extended. Patients with severe dSSc present diffuse hyper-      renal crisis and anti-Th/To antibodies are associated with
pigmentation with pruritus, and local hypopigmentation.         pulmonary fibrosis. Anti-PM-Scl and anti-U1-RNP anti-
Stem cell factor (SCF) [7], a growth factor for mast cells      bodies are associated with myositis and overlap syn-
and melanocytes, is thought to play a part in inducing          drome.
diffuse hyperpigmentation and pruritus. Upon degranula-         Recently, circulating antibodies to PDGF receptors, which
tion, mast cells release mediators such as histamine,           stimulate reactive oxygen species (ROS) and collagen
which may induce itching. Another candidate for hyper-          [16], have been identified in patients with SSc. The
pigmentation is endothelin, which also has melanogenetic        ROS-Ras-ERK1/2 cascade results in fibroblast activation
effects. Other skin manifestations include telangiectasias,     and the formation of a myofibroblastic phenotype.
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).   Cytokines and chemokines in scleroderma
                                                                TGF-β
Genetic involvement                                             TGF-β, which occurs abundantly in platelets and is
                                                                released by activated macrophages or lymphocytes, is a
                                                                strong chemoattractant for fibroblasts. TGF-β increases
Genetic susceptibility is thought to play a role in the
                                                                the synthesis of ECM, such as collagen type I and type
development of SSc. At the murine level, mutations in
the ECM protein, fibrillin, are responsible for the pheno-      III, or fibronectin by fibroblasts, modulates cell-matrix
type of tight skin (Tsk-1) mice [8]. The high incidence of      adhesion protein receptors, and regulates the production
scleroderma among the Choctaw Indian population has             of proteins such as plasminogen activator, an inhibitor of
been explained by the close association of chromosome           plasminogen, or procollagenase, which can modify the
15 to the fibrillin locus [9]. Other studies have implicated    ECM by proteolytic action [17]. In addition, TGF-β is
a mutation in the promoter region of the collagen [10] or       capable of stimulating its own synthesis by fibroblasts
transforming growth factor-β (TGF-β) gene [11]. A recent        through autoinduction [18]. TGF-β increases TGF-β
study has demonstrated that variations in the promoter          receptor (TGF-βR) levels in fibroblasts [19], and thus
region of the connective tissue growth factor (CTGF)            the maintenance of increased TGF-β production may
gene (G-945C polymorphism) are linked to susceptibility         lead to the progressive deposition of ECM, resulting in
to SSc [12].                                                    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,
Microchimerism                                                  which is regulated at the transcriptional level [24], is rec-
                                                                ognized in fibroblasts in the skin of scleroderma patients
The fact that the majority of patients develop SSc in the       [25]. Blocking endogenous TGF-β signaling eradicates
post-childbearing years has lent support to the hypothesis      the scleroderma phenotype [26]. Thus, TGF-β plays a
that the persistence of fetal cells may induce tolerance and    key role via autocrine signaling in the pathogenesis of
initiate an immune reaction. Several reports have detected      scleroderma.
large numbers of fetal cells in the lesional skin of SSc        Signaling by TGF-β elicits potent profibrotic responses in
patients [13, 14]. However, there is still scant evidence       fibroblasts. TGF-β binds to the type II receptor, thereby
that microchimerism is definitely involved in the patho-        activating the type I receptor. Signaling occurs predomi-
genesis of scleroderma.                                         nantly by phosphorylation of cytoplasmic mediators
                                                                belonging to the Smad family. Three families of Smads
                                                                have been identified: Receptor-regulated Smad2 and -3
Immune dysfunction                                              (R-Smads), common partner Smad4 (Co-Smad), and
                                                                inhibitory Smad6 and -7 (I-Smads). In scleroderma fibro-
T cells, macrophages and mast cells are present in              blasts, phosphorylation and nuclear translocation of
increased numbers or in an activated state in the lesional      Smad2/3 are increased, suggesting activation of the
skin of SSc patients, and are thought to play an active role    Smad pathway [27]. Smad7 is shown to act as an intracel-
in the pathogenesis of the disease. Additionally, activated     lular antagonist of TGF-β signaling, and an inhibitor of
peripheral B cells are found in abnormally large numbers        TGF-β-induced transcriptional responses. In scleroderma
in patients with SSc [15]. B cells contribute not only to       skin and cultured scleroderma fibroblasts, the basal level
antibody production, but also to T cell activation and dif-     and the TGF-β-inducible expression of Smad7 are selec-
ferentiation and the production of various cytokines.           tively decreased, whereas Smad3 expression is increased
                                                                [28]. On the other hand, Smad7 expression levels in
Pathogenic autoantibodies in scleroderma                        scleroderma     fibroblasts    are    uncertain.    Smad7-
Circulating antibodies are present in most patients with        Smurf-mediated negative regulation of TGF-β signaling
SSc. Although their role in the pathogenesis of sclero-         is impaired in scleroderma fibroblasts [29]. Other signal-
derma remains unclear, the symptomology of SSc can be           ing pathways besides the Smad proteins, such as the p38
classified to some extent by the presence of specific anti-     mitogen-activated protein kinase (MAPK), phosphatidyli-
bodies. Many patients with lSSc have antibodies against         nositol 3-kinase (PI3K), c-Myb, Ets, and Egr pathways,
centromeres, whereas anti-topoisomerase-1 (Scl-70) anti-        have also been shown to mediate TGF-β signaling in
bodies are often detected in patients with dSSc. Anti-RNA       scleroderma fibroblasts.

EJD, vol. 19, n° 1, January-February 2009                                                                                15
CTGF                                                           strated increased expression of CCL2 in patients with SSc
CTGF is selectively induced in fibroblasts after activation    [47-51]. Serum levels and spontaneous production levels
by the active form of TGF-β. Recombinant CTGF protein          of CCL2 by peripheral blood mononuclear cells are ele-
was found to stimulate DNA synthesis and upregulate col-       vated in patients with SSc, compared with normal con-
lagen, fibronectin, and integrin expression in fibroblasts     trols, and are correlated with pulmonary fibrosis [49].
[30]. A TGF-β response element is found in the CTGF            Increased expression of CCL2 is demonstrated in sclero-
promoter, which is not present in the promoters of other       derma skin [47, 49, 51], and scleroderma fibroblasts
TGF-β-regulated genes, suggesting that CTGF functions          express increased levels of CCL2 mRNA and protein
as a downstream mediator of TGF-β, and may coordinate          [49, 51]. Stimulation with PDGF results in a significant
the action of TGF-β, such as fibroblast proliferation, adhe-   increase in CCL2 mRNA and protein [47]. Furthermore,
sion, and ECM production [31].                                 the autoinduction of CCL2 is observed in scleroderma
Overexpression of CTGF is known to occur in cultured           fibroblasts, but not in normal fibroblasts [50]. CCL2
scleroderma fibroblasts [32, 33]. The constitutive over-       levels may also be increased by IL-13, a potent stimulator
expression of CTGF in scleroderma fibroblasts is indepen-      of CCL2 [52]. These in vivo and in vitro results suggest an
dent of TGF-β signaling but dependent on Sp1 [34].             important involvement of CCL2 in the pathogenesis of
Moreover, serum levels of CTGF are elevated in patients        scleroderma.
with SSc [35]. Dermal fibroblasts exposed to hypoxia (1%       Increased numbers of mast cells are noted in scleroderma
O2) or CoCl2 (1-100 μM) enhance expression of CTGF             skin. CCL2 also recruits mast cells, in addition to mono-
mRNA [36]. Skin fibroblasts transfected with hypoxia-          cytes [53]. Human mast cells are shown to be a rich
inducible factor (HIF)-1α show increased levels of             source of chemokines, including CCL2, CCL3/macro-
CTGF protein and mRNA, as well as nuclear staining of          phage inflammatory protein-1α (MIP-1α), CCL4/MIP-1β
HIF-1α, which was enhanced further by treatment with           and CCL5/RANTES [54], as well as a number of cyto-
CoCl2. These data may suggests that hypoxia, caused pos-       kines/growth factors and mediators capable of activating
sibly by microvascular alterations, upregulates CTGF           fibroblasts or endothelial cells. Expression of SCF is upre-
expression through the activation of HIF-1α in dermal          gulated in scleroderma fibroblasts [55], and is thought to
fibroblasts of SSc patients, and thereby contributes to        contribute to the increase of mast cells in scleroderma.
the progression of skin fibrosis.                              SCF enhances CCL2 expression in human mast cells
IL-13                                                          [56]. Because CCL2 enhances type I collagen mRNA
An imbalance exists between the type 1 and type 2 cyto-        expression in skin fibroblasts, the interaction between
kine responses in the pathogenesis of scleroderma.             mast cells and fibroblasts via SCF/CCL2 may play an
Interleukin-13 (IL-13) is a pleiotropic cytokine, elaborated   important role in the development of fibrosis. CCR2 is a
in significant quantities by appropriately stimulated type 2   major CCL2 receptor. CCR2 upregulation in vascular
cells. IL-13 has the ability to suppress proinflammatory       structures, perivascular inflammatory infiltrates, and fibro-
cytokine production in monocytes/macrophages, and is           blasts has recently been demonstrated in SSc [57]. In par-
known to enhance the growth and differentiation of B           ticular, CCR2-positive fibroblasts in early-stage dSSc
cells and to promote immunoglobulin synthesis. In addi-        showed a profibrotic phenotype, with overexpression of
tion, in vitro studies demonstrate that IL-13 is a potent      α-smooth muscle actin (α-SMA), CTGF and CCL2 [57].
stimulator of fibroblast proliferation and collagen produc-    Their results suggest potential autocrine regulation of key
tion [37-39]. The profibrotic effect of IL-13 is thought to    fibrotic properties via the CCL2/CCR2 loop in the early
involve irreversible fibroblast activation, triggered either   phases of scleroderma.
directly [40] or indirectly through TGF-β [39, 41].            A novel protein, MCPIP (MCP-induced protein), upregu-
Serum levels of IL-13 are elevated in patients with SSc,       lates members of the apoptotic gene family involved in
correlated with the number of plaque lesions [42] or nail-     the induction of cell death [58], and may provide a
fold capillaroscopic features [43].                            novel molecular pathway by which CCL2/CCR2 signal
                                                               transduction is linked to transcriptional gene regulation
Chemokines                                                     leading to apoptosis. CCL2 promoter polymorphism is
Recent studies have shown that an increase in proinflam-       associated with SSc [59]. CCL2 may contribute to the
matory chemokines has been associated with the initiation      induction of dermal sclerosis directly, via its upregulation
and/or development of skin fibrosis/sclerosis, suggesting      of mRNA expression of ECM on fibroblasts, as well as
that chemokines and their receptors may be important           indirectly through the mediation of a number of cytokines
mediators of inflammation and fibrosis in scleroderma          released from immunocytes recruited into the lesional
[44]. CCL2/monocyte chemoattractant protein-1 (MCP-            skin.
1) belongs to a C-C chemokine superfamily, and numer-
ous types of cells are capable of expressing CCL2 in the       Others
presence of serum or specific stimuli. A growing body of       Platelet-derived growth factor (PDGF) has mitogenic
evidence has demonstrated that CCL2 gene expression is         activity for mesenchymal cells, regulates matrix metabo-
upregulated in human fibrosis, as well as in animal mod-       lism, has chemotactic and vasoactive properties, and pro-
els of fibrosis. In vitro studies show that CCL2 upregu-       duces inflammatory cytokines [60]. Overexpression of
lates type I collagen mRNA expression in rat fibroblasts,      PDGF has been reported in a number of fibrotic diseases.
which is indirectly mediated by endogenous upregulation        Elevated levels of PDGF-A chain are demonstrated in
of TGF-β gene expression [45]. CCL2 enhances expres-           scleroderma skin [61]. In addition, TGF-β upregulates
sion of matrix metalloproteinase-1 (MMP-1), MMP-2 as           PDGF-α mRNA and protein levels in scleroderma fibro-
well as tissue inhibitor of metalloproteinase-1 (TIMP-1) in    blasts, in comparison with the control [61]. On the other
cultured skin fibroblasts [46]. Recent studies have demon-     hand, increased expression of the PDGF B-chain and β-

16                                                                                         EJD, vol. 19, n° 1, January-February 2009
receptor in scleroderma skin has also been reported [62-       Extracellular matrix
64].
IL-4 is known to promote fibroblast proliferation, gene        The hallmark of fibrosis is the accumulation of ECM pro-
expression, and synthesis of ECM proteins such as colla-       teins, including collagen, fibronectin, proteoglycan, and
gen and tenascin [65]. IL-4 has been shown to upregulate       elastin, in the skin. The phenotype and activation of fibro-
TIMP-2 in dermal fibroblasts via the MAPK pathway [66]         blasts is dependent on both soluble factors and ECM-
as well as to upregulate TGF-β production in eosinophils       generated signals. Fibroblasts interact with the surround-
[67] and T cells [68]. Increased IL-4 production is            ing collagens via integrins. Aberrant signaling by ECM
detected in the sera or in activated peripheral blood mono-    may disturb this interaction, thereby contributing to the
nuclear cells of patients with SSc [69]. Scleroderma fibro-    persistent modulation of fibroblasts which results in fibro-
blasts express more IL-4 receptor α and produce more           sis, as seen in the autocrine loops of cytokine production
collagen after IL-4 stimulation [70].                          and excessive deposition of ECM proteins in the skin
TGF-β can contribute to the differentiation of both regu-      [80].
latory 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. Ele-         Scleroderma fibroblasts
vated levels of IL-17 have been observed in patients
with SSc, especially in the early stages [71]. IL-17 has       Fibroblasts are stimulated by inflammatory cells, such as
been reported to induce fibroblast proliferation, but not      activated T cells, monocytes/macrophages, mast cells, and
collagen production in SSc fibroblasts [71]. The role of       eosinophils. Additionally, fibroblasts themselves are not
Th17 cells in SSc should be further investigated.              only structural elements but also part of the immune sys-
IL-21/IL-21R signaling has recently been shown to pro-         tem, and can be activated to perform new functions
mote fibrosis by facilitating the development of the CD4+      important for controlling ECM synthesis and for produc-
Th2 response [72]. IL-21 increases IL-4 and IL-13 recep-       ing various cytokines, growth factors, chemokines,
tor expression in macrophages [72], thereby possibly           growth factor receptors, integrins, and oxidants. It is
enhancing fibrosis, and is abundantly expressed in the epi-    widely accepted that human skin fibroblasts are heteroge-
dermis in SSc [73].                                            neous with regard to their synthesis of collagen, prolifer-
                                                               ative responses, and response to growth factors. Enhanced
                                                               collagen synthesis is regulated at the transcriptional level.
Vascular injury                                                Some researchers think that scleroderma fibroblasts are
                                                               the result of phenotypic changes in dermal fibroblasts
Vascular injury causes endothelial cell activation, dysfunc-   caused by soluble factors; others contend that scleroderma
tion and altered capillary permeability as a primary event.    fibroblasts are recruited from circulating or resting mesen-
These are followed by an increased expression of adhesion      chymal precursor cells as fibrocytes. Alternatively, they
molecules leading to mononuclear cell infiltrates in the       may be generated by clonal selection of high-collagen-
skin. Microvascular injury may be the result of direct or      producing fibroblasts.
indirect injury by anti-endothelial cell antibodies            Myofibroblasts represent activated and contractile pheno-
(AECAs), which are frequently detected in the sera of          types which exist in fibrotic lesions. Myofibroblasts
patients with SSc [74]. AECAs can activate endothelial         express α-SMA, and can produce various cytokines,
cells to express cell adhesion molecules which alter leuko-    growth factors and chemokines. TGF-β1 is a central regu-
cyte attachment, and can lead to endothelial cell damage       lator of the phenotypic changes of fibroblasts into myofi-
and apoptosis. Kuwana et al. [75], however, proposed that      broblasts; the modulators are mechanical tension and
insufficient vascular repair machinery, due to defective       fibronectin involving the ED-A domain. The differentia-
vasculogenesis, contributes to the microvascular abnormal-     tion into myofibroblasts is regulated by mast cell media-
ity in SSc. Although circulating concentrations of angio-      tors, of which tryptase is one of the likely candidates [81].
genic factors are high in SSc, the levels of bone marrow-      Fibrocytes are derived from circulating monocytes
derived circulating endothelial precursors (CEP) are low,      (CD34+ bone marrow-derived progenitors) and enter into
suggesting a dysregulation of vasculogenesis in SSc.           the tissues. Fibrocytes produce matrix proteins such as col-
Endothelin-1 (ET-1) is a prototypical endothelial cell-        lagens I and III, and participate in the remodeling process
derived product. Since ET-1 is a vasoconstrictive agent,       by secreting matrix metalloproteinases [82]. Fibrocytes are
loss of normal vessel compliance and vasorelaxation            also a source of inflammatory cytokines, growth factors
may be induced by increased levels of ET-1. ET-1 pro-          and chemokines. Although fibrocytes are involved in
motes fibroblast synthesis of collagen [76], and thus pro-     scleroderma, their role has yet to be fully elucidated.
vides the link between vasculopathy and fibrosis. ET-1
can induce CTGF, and may mediate the induction of col-
lagen synthesis by activation of CTGF [77]. Further, ET-1      Role of apoptosis
can also induce myofibroblast differentiation in fibroblasts
[78]. Circulating ET-1 levels have been observed in            Autoreactive clones that survive the apoptotic process
patients with dSSc with widespread fibrosis and those          may lead to increased susceptibility to autoimmune disor-
with lSSc and hypertensive disease [79], suggesting that       ders. Apoptosis causes typical cellular morphological
soluble ET-1 levels may be a marker of fibrosis and vas-       changes including cell shrinkage, nuclear condensation,
cular damage. These facts underscore the importance ET-1       DNA fragmentation and membrane alterations. This may
in scleroderma.                                                in turn cause apoptotic cells to become a possible source

EJD, vol. 19, n° 1, January-February 2009                                                                               17
of autoantigens [83]. Scleroderma fibroblasts are thought        damage to DNA. ROS can cause several abnormalities
to escape apoptosis because cultured scleroderma fibro-          such as endothelial cell damage or enhanced platelet acti-
blasts are resistant to Fas-induced apoptosis [84, 85],          vation, leading to upregulation of the expression of adhe-
and apoptosis of fibroblasts in SSc skin lesions has not         sion molecules or secretion of inflammatory or fibrogenic
been observed [85]. TGF-β protects myofibroblasts from           cytokines including PDGF and TGF-β; excessive oxida-
undergoing apoptosis. Serum-starved rat lung fibroblasts         tive stress has been implicated in the pathogenesis of
treated with IL-1 result in apoptosis which can be reduced       scleroderma [95]. Indeed, scleroderma fibroblasts produce
by concomitant treatment with TGF-β [86]. Also, α-               ROS constitutively [96]. Other effects of oxygen radicals
SMA-positive myofibroblasts increase in number follow-           include the stimulation of skin fibroblast proliferation at
ing stimulation by TGF-β, which protects these myofibro-         low concentrations [97] and the production of increased
blasts against apoptosis induction. Other studies have           amounts of collagen [98], suggesting that low oxygen ten-
shown that pretreatment with TGF-β significantly reduced         sion may contribute to the increased fibrogenic properties
apoptosis caused by serum starvation in myofibroblasts,          of scleroderma fibroblasts. Furthermore, several of the
whereas this was not the case with non-myofibroblasts            autoantigens targeted by scleroderma autoantibodies frag-
[85]. Thus TGF-β1 may play a role in inducing                    ment in the presence of ROS and specific metals such as
apoptosis-resistant fibroblast populations in SSc. In            iron or copper [99]. The authors suggest that tissue ische-
scleroderma fibroblasts, the Bcl-2 level is significantly        mia generates ROS, which in turn induces the fragmenta-
higher, whereas the Bax level significantly lower [85].          tion of specific autoantigens. On the other hand, oxidative
On the other hand, endothelial cell apoptosis is thought to      stress transiently induces CCL2 mRNA and protein
occur early in the pathogenesis of scleroderma. Endothe-         expression in cultured skin fibroblasts [100], suggesting
lial cell apoptosis was first noted in the UCD-200/206           that ROS may play a regulatory role in inflammation by
chickens, which develop hereditary systemic connective           modulating monocyte chemotactic activity.
tissue disease resembling human SSc [87]. This phenom-
enon occurs before perivascular mononuclear cell infiltra-
tion. Also, terminal deoxynucleotidyl transferase (TdT)-         Animal models of scleroderma
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            Animal models are useful in providing clues for under-
apoptosis of endothelial cells induces resistance to apopto-     standing various human diseases and for testing new
sis in fibroblasts largely through PI3K-dependent mechan-        methods of treatment. Although animal models which
isms [88]. Furthermore, fibroblasts exposed to a medium          exhibit all the aspects of SSc are not currently available,
conditioned by apoptotic endothelial cells present myofi-        several experimental animal models, such as bleomycin-
broblast changes [88].                                           induced murine scleroderma, tight skin (Tsk) mouse, Tsk2
The serum soluble Fas (sFas) levels are higher in patients       mouse, sclerodermatous graft-versus-host disease (Scl-
with SSc [89-91]. Untreated SSc patients have signifi-           GvHD) mouse, University of California at Davis line 200
cantly higher serum sFas levels than the treated SSc             (UCD-200) chicken, and exogenous injections of TGF-
patients and healthy controls [92]. It has been suggested        β/CTGF-induced murine fibrosis model, etc., have been
that increased sFas levels in the serum of SSc patients can      examined so far.
protect autoreactive T cells from FasL-induced apoptosis
[91]. Spontaneous apoptosis of CD8+ T cells in the               Bleomycin-induced scleroderma model
peripheral blood is significantly higher in patients with
                                                                 Bleomycin is an agent that can induce pulmonary fibrosis
SSc compared with normal controls, while spontaneous
apoptosis in CD4+ T cells occur at similar rates in both         and infrequently, scleroderma in human beings [101].
SSc and controls [93]. Enhanced helper T cell function,          Repeated local injections of bleomycin into the dorsal
resulting in the reduction CD8+ T cells, may lead to auto-       skin induces histological dermal sclerosis resembling
immunity by modifying the immune balance.                        human scleroderma, characterized by thickened collagen
Akt is one of the key enzymes inhibiting both spontaneous        bundles, the deposition of homogenous materials, and cel-
and stress-induced apoptosis. 3’-phosphorylated phosphoi-        lular infiltrates in the thickened dermis in various mice
nositides bind to the pleckstrin domain of Akt. Akt activity     strains [102-104] (figure 2). Mast cell infiltration
may result in the inhibition of pro-apoptotic Bad, Bax,          increases, and marked degranulation and elevated plasma
Bik, and caspase-9 by phosphorylation. It has recently           histamine levels are also evident [102]. Hydroxyproline
been reported that Akt is active in scleroderma fibroblasts.     contents as well as mRNA levels of type I collagen sig-
Cultured scleroderma fibroblasts exhibited high levels of        nificantly increase in the sclerotic skin. α-SMA-positive
p-Akt, in comparison to control fibroblasts [94]. TGF-β          myofibroblasts are observed in the dermis, and gradually
can activate Akt in fibroblasts, and by doing so, may            increase in tandem with the induction of dermal sclerosis
also induce apoptosis resistance in scleroderma fibroblasts.     [105]. Interestingly, autoantibodies are detectable in the
These findings point to a potential role for Akt in the resis-   serum [102].
tance of scleroderma fibroblasts to apoptosis.                   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
Oxidant stress                                                   sclerosis most closely resemble those of human sclero-
                                                                 derma. A recent report shows that a one-time injection of
ROS generated during various metabolic and biochemical           bleomycin-poly(L-lactic acid) microspheres can induce
reactions have multifarious effects that include oxidative       dermal sclerosis in mice [106].

18                                                                                          EJD, vol. 19, n° 1, January-February 2009
      Human Scleroderma                         Murine Scleroderma   (TNFR)p55-deficient mice developed severe sclerotic
  A                                         B
                                                                     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 col-
                                                                     lagen, as well as fibrogenic cytokines such as TGF-β1 and
                                                                     CTGF, in human skin fibroblasts [119]. Thus, the induc-
                                                                     tion of dermal sclerosis by bleomycin is considered to be,
                                                                     in part, mediated by inflammatory and fibrogenic cyto-
Figure 2. Comparison of histologic features of human (A)             kines, as well as by the direct effect of bleomycin on
and bleomycin-induced murine (B) scleroderma.                        ECM synthesis in fibroblasts. Numerous therapeutic
                                                                     approaches have been investigated in this model [120].
Using this model, several studies of the pathogenesis of             Tight skin mouse model
this disease have been performed. TGF-β is a mediator of
the fibrotic effect of bleomycin at the transcriptional level        The Tsk mutation in the fibrillin-1 gene maps to chromo-
and the TGF-β response element is required for bleomycin             some 2 and is inherited in an autosomal dominant fashion.
stimulation of the proα1(I) collagen promoter [107]. In the          Fibrillin is a large ECM structural protein and the major
bleomycin model, TGF-β is detected in the lesional skin,             component of microfibrils. Tsk mice have excessive accu-
and increased expression and synthesis of TGF-β1 is                  mulation of collagen in the skin, as seen in the hypoder-
dominant in bleomycin-‘susceptible’ mice strains [108].              mis and superficial fascia, as well as the lung and heart;
Inhibition of TGF-β suppresses dermal sclerosis [109,                however, vascular involvement has not been associated
110]. Fibroblasts show predominantly nuclear localization            with this condition [121]. There are, however, numerous
of Smad3 and intense staining for phospho-Smad2/3,                   biochemical and molecular abnormalities that resemble
whereas expression of Smad7 is downregulated, a fact                 those present in patients with SSc. mRNA expression of
which may account for sustained activation of TGF-                   TGF-β, type I, III and VI collagen are under temporal and
β/Smad signaling [111]. Expression and synthesis of IL-              spatial regulation during postnatal growth and develop-
13 as well as IL-13 receptor (IL-13R)-α2 mRNA expres-                ment in the Tsk1/+ mice [122]. Collagen α1(I) and α1(III)
sion are upregulated, whereas IL-13R-α1 mRNA levels                  gene-expressing fibroblasts are increased in Tsk1/+ fibro-
are not significantly enhanced [112]. IL-13 may promote              tic lesions.
the progression of cutaneous fibrosis/sclerosis in this              The Tsk2 is a mutant that appeared in the offspring of a
model. Indeed, recent studies have shown that IL-                    101/H mouse after the administration of the mutagenic
13-deficient mice failed to develop an increase in skin              agent ethylnitrosourea [123]. Tsk2/+ mice develop a
sclerosis after bleomycin treatment [113]. Expression of             tight skin phenotype that becomes apparent at 3-4 weeks
CCL2 as well as its major receptor, CCR2 is enhanced                 of age. Histological examination of skin reveals marked
in the sclerotic skin [114]. Administration of anti-CCL2             accumulation of collagen similar to that observed in Tsk1/
neutralizing antibody reduces dermal sclerosis, along with           + mice. However, in contrast to Tsk1/+ mice, prominent
collagen content as well as mRNA expression of type I                mononuclear cell infiltration is present in the dermis and
collagen in the skin. More directly, bleomycin-induced               adipose tissue of Tsk2/+ mice. Biochemical analysis
dermal sclerosis is abrogated in MCP-1-deficient mice                showed that Tsk2/+ skin had 50% more collagen than
[115]. These data suggest that CCL2 and CCR-2 signaling              the normal mouse skin. Collagen synthesis in Tsk2/+ cul-
plays an important role in the pathogenesis of bleomycin-            tured dermal fibroblasts is 100% higher compared with
induced scleroderma.                                                 normal fibroblasts. In neither Tsk1 nor Tsk2 mice are
Bleomycin induces apoptosis. TUNEL-positivity is prom-               alterations in endothelial cell apoptosis induction involved
inently detected on keratinocytes and infiltrating mononu-           in the development of the disease [124].
clear cells, but not endothelial cells and fibroblasts follow-       TGF-β and IL-4 possibly play important roles in the path-
ing bleomycin treatment [116]. DNA fragmentation                     ogenesis of fibrosis in Tsk mice. Fibroblasts from Tsk
reveals laddering of the whole skin. Expression of FasL              mice are hyperresponsive to IL-4 and TGF-β [125].
mRNA is upregulated, whereas Fas mRNA is continu-                    Smad2 and Smad3 are considered to be the primary sig-
ously detected. mRNA expression as well as activity of               naling molecules involved in the TGF-β signaling trans-
caspase-3 is also enhanced in the skin. Administration of            duction pathway. Tsk fibroblasts have elevated Smad3
neutralizing anti-FasL antibody reduces the development              transcriptional activity compared with normal fibroblasts
of dermal sclerosis, in association with the reduction of            [126]. This may explain why Tsk fibroblasts are more
TUNEL-positive mononuclear cells and the blockade of                 responsive to TGF-β stimulation. Previous studies con-
apoptosis. Caspase-3 activity is also significantly reduced          cerning TGF-β mRNA expression in Tsk mice produced
after anti-FasL treatment. Moreover, dermal sclerosis is             inconsistent results; one group showed increased expres-
less induced in both Fas- and FasL-deficient strains                 sion in the skin of Tsk mice [127, 128], while another
[117]. Excessive apoptosis, which is mediated by the                 detected expression in only the skin of neonate Tsk mice
Fas/FasL pathway and caspase-3 activation, is involved               [129]. Targeted mutations in either the signaling chain of
in this model. Tumor necrosis factor receptor                        the IL-4 receptor or STAT6 prevents cutaneous hyperpla-

EJD, vol. 19, n° 1, January-February 2009                                                                                    19
sia in Tsk mice, suggesting the importance of IL-4 [125,         TGF-β/CTGF induced fibrosis model
130]. CD4+ T cells have been shown to be required for            TGF-β induces rapid fibrosis and angiogenesis when
the excessive accumulation of dermal collagen in Tsk             injected subcutaneously into newborn mice [146]. Take-
mice [131]. In Tsk mice, mast cells are abundant in the          hara and colleagues showed that TGF-β-induced subcuta-
thickened dermis and exhibit prominent degranulation             neous fibrosis and subsequent CTGF or basic fibroblast
[132]. Mast cells are one of major sources of IL-4. IL-4         growth factor (bFGF) application caused persistent fibro-
has been shown to induce significant levels of CCL2 pro-         sis [147, 148]. They suggest that TGF-β plays an impor-
duction in stromal cells [133, 134]. On the other hand,          tant role in inducing granulation and fibrotic tissue forma-
CCL2 upregulates IL-4 mRNA expression and protein                tion, and CTGF and bFGF are important in maintaining
production [135]. These observations have led to the             fibrosis [149]. The mast cell count was significantly but
hypothesis of the mutual induction of CCL2 and IL-4.             transiently increased in the early phase, while the number
Recent studies have shown that CCL7/MCP-3 is highly              of macrophages continued to rise [150]. In lesional skin,
overexpressed by neonatal Tsk fibroblasts [136].                 serial injections of CTGF after TGF-β increased CCL2
Increased CCL7 protein secretion by Tsk fibroblasts is           mRNA expression up to 8 times in comparison with
observed, and CCL7 is abundantly expressed in the der-           only a single injection of TGF-β or CTGF [150]. Anti-
mis of Tsk mice at 10 days and 3 weeks old. Downregula-          CTGF reduced skin fibrosis and collagen content [151].
tion of B cell function results in inhibition of skin fibrosis
and autoantibody production in Tsk mice [137].                   Kinase-deficient type II TGF-β receptor transgenic
                                                                 mouse
Graft-versus-host disease model                                  Denton et al. [152] generated transgenic mice expressing
In human chronic GvHD, severe cutaneous fibrosis is              a kinase-deficient type II TGF-β receptor selectively on
observed with loss of dermal fat, atrophy of dermal              fibroblasts. These mice develop dermal and pulmonary
appendages, mast cell depletion, and mononuclear cell            fibrosis. Transgenic fibroblasts proliferate more rapidly,
infiltration. A murine Scl-GvHD model was produced               produced more ECM, and show increased expression of
by transplanting B10.D2 bone marrow and spleen cells             plasminogen activator inhibitor-1 (PAI-1), CTGF, Smad3
into BALB/c mice after lethal gamma irradiation of the           Smad4, and Smad7. Additionally, transgenic fibroblasts
recipients [138]. Scl-GvHD mice exhibit remarkable skin          show myofibroblast differentiation [153].
thickening and pulmonary fibrosis by day 21 after bone
marrow transplantation, with significant increase of type I      Relaxin knockout mouse
collagen mRNA levels and protein synthesis. TGF-β is a
key regulator in this model, and blocking of TGF-β ame-          Relaxin is a small peptide hormone with anti-fibrotic and
                                                                 vasodilatory properties. A recent report shows that
liorated the skin fibrosis [138, 139]. CCL2 upregulation
                                                                 relaxin-deficient mice present dermal fibrosis character-
has also been shown in the lesional skin of this model
                                                                 ized by thickening of the skin and increase in collagen
[140].
                                                                 content [154]. Fibroblasts derived from the skin of the
In addition, a modified model of GvH-induced SSc has             null-mice produce higher levels of collagen.
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       Therapeutic implications for human
and autoantibody generation. However, lung fibrosis was
absent.
                                                                 scleroderma
                                                                 Until now, a number of therapeutic approaches have been
UCD-200 chicken
                                                                 tried with limited success. Randomised, placebo-
UCD-200 chickens spontaneously develop vascular dam-             controlled trials revealed that cyclophosphamide had a
age, mononuclear cell infiltrates, fibrosis of the skin and      significantly beneficial effect on skin sclerosis, as well
internal organs, and polyarthritis [142, 143]. Additionally,     as lung fibrosis [155]. Skin sclerosis fell moderately in the
positive AECAs, antinuclear antibodies, anticardiolipin          cyclophosphamide-treated group (especially in dSSc),
antibodies, and rheumatoid factors are detected in the           compared with the placebo.
serum. The disease starts 1-2 weeks after hatching with          Interferon-γ (IFN-γ) causes potent inhibition of collagen
erythema and swelling of the comb, which subsequently            production, which correlates with a reduction in the cor-
proceeds to a chronic stage characterized by fibrosis with       responding steady-state mRNA levels in cultured skin
excessive accumulation of collagen. In the inflammatory          fibroblasts [156]. IFN-γ inhibits the TGF-β-induced phos-
phase, T cell receptor (TCR)γ/δ+/CD3+/MHC class II− T            phorylation of Smad3 and the accumulation of Smad3 in
cells prevail in the stratum papillae, while TCR α/β+/           the nucleus, whereas it induces the expression of Smad7,
CD3+/CD4+/MHC class II+ T cells predominate in the               which prevents the interaction of Smad3 with the TGF-β
deeper dermis. AECAs can induce apoptosis of endothe-            receptor [157]. A randomized, controlled trial was carried
lial cells through antibody-dependent cell-mediated cyto-        out in 44 patients with SSc, which did not show a signifi-
toxicity via Fas [144]; transfer of AECA-positive sera into      cantly greater benefit from IFN-γ in improving the skin
healthy chickens induced endothelial cell apoptosis,             thickness score compared with the controls [158]. IFN-γ
although this was not followed by skin sclerosis [145].          is a powerful type 1 inducer of cellular immunity, which
These studies demonstrated the in vivo apoptosis-                may indirectly contribute to the improvement of the
inducing effects of AECAs.                                       imbalance in the type 2 shift.

20                                                                                           EJD, vol. 19, n° 1, January-February 2009
Ultraviolet (UV) irradiation is reported to be effective for
scleroderma, in particular for the localized type. UV
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