Vascular aspect of SSc

Small blood vessels’ involvement contributes significantly to SSc morbidity and mortality. Dysfunction of peripheral microvasculature is reflected in RP attacks. Intermittent vasoconstriction will progress to blood vessels narrowing, obliteration and insufficient blood supply. Ischaemia and reperfusion attenuate oxidative stress with excessive production of reactive oxygen species that further aggravate endothelial cells damage. Activated endothelial cells secrete vasoactive mediators such as nitric oxide, prostacyclin, endothelin-1 (ET-1), platelet-activating factor and soluble adhesive molecules (vascular cellular adhesive molecules (sVCAM-1) and sE-selectin). Interaction between endothelial cells and T lymphocytes is accomplished via lymphocyte function-associated antigen-1, very late antigen-4 and L-selectin expressed on lymphocytes, while their counter-receptors, intercellular adhesive molecule (ICAM-1), VCAM-1 and CD34/endoglycan, are expressed on endothelial cells. Endothelial cells induce local inflammatory cell activation with TNF-α, TGF-β, IL-1α, interferon-γ (IFN-γ) and chemokines release . The production of antibodies to endothelial cells has been reported in SSc, and higher levels of anti-endothelial cells antibodies correlated with parameters reflecting microangiopathy: carbon monoxide diffusing capacity (DLCO), pulmonary arterial hypertension (PAH), digital ulcers and capillaroscopic abnormalities . Anti-endothelial cell antibodies from SSc patients could induce endothelial cells apoptosis. High titers of anti-endothelial cell antibodies correlated with apoptotic endothelial progenitor cells in bone marrow . A variety of alterations was demonstrated on nail-fold capillaroscopy in SSc: sac-like, giant and bushy capillaries, microhaemorrhages and capillary loss . Reduced capillary density was demonstrated in SSc patients, most prominent in patients with SSc-related PAH . Abnormalities in skin, pulmonary and renal microvasculature in patients with digital ulcers, PAH and scleroderma renal crisis are similar and reflect defective angiogenesis in SSc. Concentric intimal proliferation and luminal obstruction in small- and medium-sized lung vessels and myocardial tissue were demonstrated on autopsy of a SSc patient who died from PAH . Microangiopathy in SSc is a spread phenomenon and assumed to be responsible for life-threatening organ involvement, such as PAH, scleroderma renal crisis, cardiomyopathy, vascular ectasia and atrophy in the gastro-intestinal tract. Neo-vascularisation is a complex process that requires both mobilisation of endothelial progenitor cells from bone marrow and proliferation and differentiation of resident cells (endothelial cells and pericytes). In normal situations, tissue hypoxia is followed by the expression of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), ET-1, TGF-β and MCP-1, which then trigger the migration of endothelial cells from pre-existing vessels, endothelial cells proliferation and new vessels formation. In SSc patients, neo-angiogenesis is impaired despite elevated levels of VEGF and its receptors. Up-regulation of VEGF is thought to be responsible for the appearance of giant capillaries-telangiectasias . Vasculogenesis describes the formation of new vessels by circulating endothelial progenitor cells, which have to refill damaged blood vessels. In SSc, the numbers and function of bone marrow-derived CD34+/endothelial progenitor cells are depleted . Ischaemia stimulates production of TGF-β and CTGF followed by fibroblast activation and excessive extracellular matrix production. Microvascular pericytes contribute to neo-vascularisation. Pericytes may become activated and transform to collagen-producing myofibroblasts , ET-1 induced endothelial cells proliferation, smooth muscle hypertrophy and irreversible vascular obliteration. Elevated levels of ET-1 correlated with severity of RP, digital ulcers, PAH and renal failure in SSc patients . ET-1 participates in the fibrotic cascade by stimulation of fibroblast collagen production and inhibition of matrix metalloproteinase-1 activity. There are two types of receptors to ET-1: ET-A and ET-B. ET-A receptors are expressed by vascular smooth muscle cells and mediate vasoconstriction, smooth muscle cell proliferation and fibrosis. ET-B receptors are mainly expressed on endothelial cells and mediate vasodilation with release of nitric oxide. In SSc patients, ET-B receptors are down- regulated, which may displace the balance towards vasoconstriction and fibrosis .

Prostacyclin is the principal arachidonic acid metabolite of endothelial cells and vascular smooth muscle cells that express vasodilator properties and inhibit platelet adhesion. The ability of endothelial cells to synthesise and release prostacyclin is reduced in patients with RP and SSc. Production of thromboxan A ₂ potential vasoconstrictor and platelets activator is enhanced in SSc patients. Abnormal balance between prostanoids on behalf of thromboxan A ₂ may contribute to vasoconstriction in SSc, vessels obliteration and in situ thrombosis . The role of nitric oxide in SSc is complex: scleroderma endothelial cells express reduced endothelial nitric oxide synthase and increase inducible nitric oxide synthase activity, resulting in a vasoconstriction, inflammation and tissue damage. Recently, the progressive disappearance of lymphatic vessels has been reported in scleroderma .

The immune system in SSc

Both innate and adaptive autoimmunity are involved in SSc. Toll-like receptors (TLRs) control immune responses by detecting common molecular motifs such as RNA, DNA, lipopolysaccharide or endotoxin. DNA and RNA are situated inside the cell and may be recognised after cell and nuclear damage with binding through ligands: TLR7 (by RNA) or TLR9 (by DNA). Dendritic cells, mononuclear cells and B lymphocytes activated through TLRs produce IFN, IL-1, TNF-α and IL-6. The TLR2 Pro631His gene variant was robustly associated with anti-topo I, diffuse scleroderma and PAH . TLRs participate in production of autoantibodies to nucleic acid-binding proteins and increase expression of IFN-responsive genes by peripheral blood mononuclear cells. SSc sera containing anti-topo I stimulated IFN-α production by peripheral blood mononuclear cells . TLR l ligands can directly promote fibroblast differentiation into collagen-producing myofibroblasts.

Specific autoantibodies in SSc may indicate different disease subsets and organ target damage. The rate of anti-topo I and ACA was different: 60.8% versus 23.4% in DcSSc and 6.0% versus 46.7% in LcSSc. In the European League Against Rheumatism (EULAR) Scleroderma Trials and Research (EUSTAR) patients cohort (3550 SSc patients), the rates of higher modified Rodnan skin score (mRSS), joint and muscle involvement, intestinal involvement, pulmonary fibrosis, cardiomyopathy, hypertension and proteinuria were significantly higher in patients with anti-topo I compared to those with ACA . In patients with RP and abnormal nail-fold capillaroscopy, the presence of ACA, anti-Th/To, anti-topo I and anti-RNAP-III autoantibodies independently predicted progression to definite SSc in 79.5% of patients . Anti-topo I antibodies may bind DNA or RNA, induce over-production of IL-6, TGF-β1 and IL-17, and impair release of anti-inflammatory cytokine IL-10 . In a patient with early DcSSc, the levels of anti-topo I gradually reduced and finally disappeared under corticosteroids treatment in parallel with reduction in mRSS. Discontinuation of corticosteroids triggered the re-emergence of anti-topo I and skin sclerosis; skin sclerosis improved and anti-topo I levels reduced with re-start of prednisone . ACA target a variety of centromere proteins in SSc. The presence of ACA correlated with long-standing RP and SSc and has been associated with the calcinosis, RP, oesophageal dysmotility, sclerodactyly, telangectasia (CREST) variant of SSc. The absence of ACA and the presence of anti-topo I have been associated with pulmonary fibrosis, while the presence of ACA was associated with a lower likelihood of SSc-related pulmonary fibrosis . Anti-nucleolar antibodies target a subclass of small nucleolar ribonuclear proteins including U3-RNP and fibrillarin. Anti-fibrillin-1 antibodies activate fibroblasts and stimulate release of TGF-β; they are predictive of severe skin and systemic involvement and greater mortality. By contrast, anti-nucleolar pattern and anti-Th/To antibodies index mild and limited disease. The prevalence of anti-RNAP-III did not extend 10–20% in various SSc patients cohorts, but their presence was definitely associated with severe skin disease and scleroderma renal crisis . The association between anti-RNAP-III autoantibodies, co-temporal cancer and scleroderma diagnosis, and unique nucleolar RNAP-III expression pattern in malignant tissue may indicate the link between scleroderma and cancer .

Functional antibodies were demonstrated in SSc. Anti-angiotensin receptor and anti-ET-A receptor autoantibodies have been shown to bind respective receptors on endothelial cells, increase TGF-β gene expression by endothelial cells and correlate with SSc severity and mortality . Functional antibodies to methionine sulfoxide reductase A in SSc patients correlated with cardiac, lung and renal involvement and inhibited methionine sulfoxide reductase A activity in SSc sera . Stimulatory antibodies to the PDGF receptor in SSc patients induced tyrosine phosphorylation, reactive oxygen species accumulation, type I collagen-gene expression and myofibroblast phenotype in normal fibroblasts .

Cellular immunity plays an important role in SSc pathogenesis. Dermal mononuclear cell infiltrates consisted mostly of activated T lymphocytes with a mean CD4+/CD8+ ratio of ∼2.4. T-cell infiltration was a prominent finding in lung specimens from patients with SSc. T cells in gastric biopsies from SSc patients robustly expressed lymphocyte function-associated antigen-1, very late antigen-4 and ICAM-1. Endothelial cells showed corresponding strong expression of VCAM-1 and ICAM-1 . Activated CD4 + T-cells in the skin demonstrated enhanced transendothelial migration and expressed elevated levels of TGF-β, CTGF, VEGF, fibroblast growth factor, IL-2, IL-4, IL-6, IL-8, IL-13, IL-17, IL-27, MCP-1, TNF-α and IFN-α in both the circulation and the affected organs (skin and lung) . ‘Classically activated’ monocytes are activated by the Th1 cytokine IFN-γ, whereas alternatively activated macrophages are activated by Th2 cytokines IL-4 and IL-13. Alternatively, activated macrophages stimulate secretion of PDGF and TGF-β. IL-13 is elevated in the serum of patients with early diffuse scleroderma and in alveolar macrophages from SSc patients with pulmonary fibrosis. In patients with LcSSc, levels of IL-13 correlated with PAH-related mortality . IL-10 cooperates with Th1 cytokines and the IL-13 receptor to suppress collagen deposition. Decreased IL-10 levels were reported in peripheral blood mononuclear cells of SSc patients. Increased IFN-inducible protein-10, MCP-1 and serum IFN-α levels were associated with pulmonary fibrosis, cardiac involvement, PAH and digital loss .

Altered relationships between Th17 and T-regulatory cells (T-regs) have been reported in SSc patients. Different peripheral blood mononuclear cell stimulations raised the frequency of Th17 clones in SSc patients. Both T-regs (CD4+CD25+ and CD8+CD28) showed quantitative and qualitative alteration in the peripheral blood mononuclear cells and skin of SSc patients. The suppressor activity of CD4+CD25 + T-regs was lower in SSc and corresponded to higher mRSS .

B-cells are involved in antigen presentation and autoantibodies production in SSc; there was an abounded presence of CD20 + B-cells in skin and lung biopsies of patients with SSc-associated interstitial lung disease . The expression of CD19 on the circulating B-cells and the expression of CD80 and CD86 on memory B-cells of SSc patients may be increased . In a tight-skin (TSK/+) mouse, a genetic model for human SSc, B-cell activation resulted in amplified CD19 signalling followed by skin sclerosis; CD19-deficient TSK/+ mouse demonstrated inhibited IL-6 production. Treatment of TSK/+ mouse with anti-CD20 monoclonal antibodies reduced skin fibrosis . Elevated serum levels of B-lymphocyte stimulator (BAFF) correlated with skin fibrosis in SSc patients. Under BAFF, stimulation SSc B-cells exhibited an enhanced production of IgG and IL-6 . IL-6, a powerful B-cell activator, is elevated in the serum and involved skin of scleroderma patients, and in scleroderma dermal fibroblasts culture. B-cell depletion using rituximab (anti CD20 monoclonal antibodies) in patients with severe skin involvement resulted in prolonged improvement in mRSS and decreased serum IL-6 concentration . IL-6 stimulates fibroblasts extracellular matrix production and tissue inhibitor of metalloproteinases-1 synthesis via up-regulation of MCP-1 and VEGF. Blockade of IL-6 with tocilizumab (anti-IL-6 soluble receptor monoclonal antibodies) in two patients with DcSSc resulted in reduction in mRSS and reduced collagen skin content .

Fibrosis

Fibrosis is a form of tissue scarring with excess deposition of extracellular matrix as the end result of inflammation or damage. The key cellular moderator of fibrosis is the collagen-producing myofibroblasts. Myofibroblasts may be generated from mesenchymal and epithelial cells, endothelial cells and from bone-marrow-derived circulating fibrocytes. Myofibroblasts are activated by paracrine and autocrine signals and through TLRs on fibroblasts. Fibrosis is driven by multiple mediators, such as TGF-β1, PDGF, VEGF, ET-1, IL-13, IL-21, MCP-1, macrophage inflammatory protein, peroxisome proliferator-activated receptors, serum acute phase proteins, caspases and rennin–angiotensin–aldosterone system. Abnormal balance between matrix metalloproteinases and tissue inhibitor of metalloproteinases results in excess synthesis of the extracellular matrix, impaired extracellular matrix catabolism, and leads to collagen accumulation. Scleroderma fibroblasts obtained from involved skin or lungs express an activated myofibroblast-like phenotype that is maintained by TGF-β signal, TGF-β/Smad3-independent mechanisms and CTGF. The epithelium is a major cover of the skin and a mucosal barrier of the oral cavity, gastro-intestinal and respiratory tract; it plays an important role in re-surfacing injured tissue. Under ischaemic conditions, epithelial cells may lose cell–cell attachment and transform into mesenchymal or collagen-producing myofibroblasts. Scleroderma epithelial cells stimulated normal fibroblasts to express CTGF, IL-1α, ET-1 and TGF-β . Epithelial injury plays a pivotal role in SSc-interstitial lung disease .

TGF-β is one of the central pro-fibrotic cytokines. Peripheral blood mononuclear cells and tissue macrophages produce and respond to TGF-β, especially to TGF-β1 isoform. In macrophages, activation of latent TGF-β1 depends on cathepsins, plasmin, calpain, thrombospondin-1, integrin- α v β 6 and matrix metalloproteinases. TGF-β1 triggers signalling through Smad proteins that, in turn, control procollagen I and III gene transcription . Thrombospondin-1 is overexpressed by scleroderma fibroblasts; blockade of thrombospondin-1 reduced the expression of key fibrogenic proteins and prevented PDGF-induced matrix contraction by normal and scleroderma fibroblasts . Early growth response-2 (Egr-2) protein is a transcription factor that is necessary for TGF-β-induced fibrosis and is abnormally expressed in scleroderma skin and in a murine model of scleroderma . The nuclear peroxisome proliferator-activated receptor -γ signalling regulates TGF-β-dependent fibrogenesis. Peroxisome proliferator-activated receptor -γ levels were reduced in skin and lung biopsies from SSc patients and in scleroderma dermal fibroblasts . Angiotensin II, produced locally by activated macrophages and fibroblasts, stimulated TGF-β1 production, fibroblast proliferation and their differentiation into collagen-producing myofibroblasts. Angiotensin II may be produced by myofibroblasts and enhances TGF-β1 signalling by increasing Smad2/Smad3 levels along with mitogen-activated protein kinases and Egr-1. Fibrosis in animal models, SSc and idiopathic pulmonary fibrosis are accompanied by Egr-1 overexpression. Egr-1-null mice are protected from fibrosis . PDGF is secreted by a variety of cells in response to injury and effects replication, survival and migration of myofibroblasts via receptors PDGF-R-α and PDGF-R-β. TGF-β and PDGF pro-fibrotic effects may be independent of Smad2/Smad3 pathway but dependent on intracellular tyrosine kinases (c-abl- and Src-kinases). TGF-β and PDGF activated Src-kinase signalling in scleroderma and normal dermal fibroblasts. Inhibition of Src signalling reduced the synthesis of messenger RNA for collagen and fibronectin-1, reduced dermal collagen content and decreased the number of myofibroblasts . c-abl is implicated in chronic myelogeous leukaemia and appears to be activated in SSc patients. Treatment of TSK-1 mice with imatinib mesylate (c-abl-kinase inhibitor) reduced dermal thickening and prevented the differentiation of fibroblasts into collagen-producing myofibroblasts. In the model of pre-established dermal fibrosis, imatinib induced regression of pre-existing dermal fibrosis .

Chemokines (including CCL3 (MIP-1 α ) and CCL2 (MCP-1)) contribute to fibrosis by recruiting myofibroblasts, macrophages and peripheral blood mononuclear cells to sites of tissue injury. Macrophages and epithelial cells are believed to be the main sources of CCL3. Anti-CCL3 antibodies reduced the development of fibrosis in the bleomycin model of pulmonary fibrosis . Th2 cytokines including IL-4, IL-5, IL-13 and IL-21 cooperate with TGF-β to induce fibrosis. IL-13 plays an important role in latent TGF-β1 production in macrophages and stimulates TGF-β1 activation via matrix metalloproteinases and cathepsin-based proteolytic pathways. TGF-β signalling in fibroblasts regulates the oncogenic potential of epithelial cells . In a recent EUSTAR report, cancer-related death was reported in 31% among 234 non-SSc-related mortality causes (41% of all mortality cases) .

Vascular aspect of SSc

Small blood vessels’ involvement contributes significantly to SSc morbidity and mortality. Dysfunction of peripheral microvasculature is reflected in RP attacks. Intermittent vasoconstriction will progress to blood vessels narrowing, obliteration and insufficient blood supply. Ischaemia and reperfusion attenuate oxidative stress with excessive production of reactive oxygen species that further aggravate endothelial cells damage. Activated endothelial cells secrete vasoactive mediators such as nitric oxide, prostacyclin, endothelin-1 (ET-1), platelet-activating factor and soluble adhesive molecules (vascular cellular adhesive molecules (sVCAM-1) and sE-selectin). Interaction between endothelial cells and T lymphocytes is accomplished via lymphocyte function-associated antigen-1, very late antigen-4 and L-selectin expressed on lymphocytes, while their counter-receptors, intercellular adhesive molecule (ICAM-1), VCAM-1 and CD34/endoglycan, are expressed on endothelial cells. Endothelial cells induce local inflammatory cell activation with TNF-α, TGF-β, IL-1α, interferon-γ (IFN-γ) and chemokines release . The production of antibodies to endothelial cells has been reported in SSc, and higher levels of anti-endothelial cells antibodies correlated with parameters reflecting microangiopathy: carbon monoxide diffusing capacity (DLCO), pulmonary arterial hypertension (PAH), digital ulcers and capillaroscopic abnormalities . Anti-endothelial cell antibodies from SSc patients could induce endothelial cells apoptosis. High titers of anti-endothelial cell antibodies correlated with apoptotic endothelial progenitor cells in bone marrow . A variety of alterations was demonstrated on nail-fold capillaroscopy in SSc: sac-like, giant and bushy capillaries, microhaemorrhages and capillary loss . Reduced capillary density was demonstrated in SSc patients, most prominent in patients with SSc-related PAH . Abnormalities in skin, pulmonary and renal microvasculature in patients with digital ulcers, PAH and scleroderma renal crisis are similar and reflect defective angiogenesis in SSc. Concentric intimal proliferation and luminal obstruction in small- and medium-sized lung vessels and myocardial tissue were demonstrated on autopsy of a SSc patient who died from PAH . Microangiopathy in SSc is a spread phenomenon and assumed to be responsible for life-threatening organ involvement, such as PAH, scleroderma renal crisis, cardiomyopathy, vascular ectasia and atrophy in the gastro-intestinal tract. Neo-vascularisation is a complex process that requires both mobilisation of endothelial progenitor cells from bone marrow and proliferation and differentiation of resident cells (endothelial cells and pericytes). In normal situations, tissue hypoxia is followed by the expression of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), ET-1, TGF-β and MCP-1, which then trigger the migration of endothelial cells from pre-existing vessels, endothelial cells proliferation and new vessels formation. In SSc patients, neo-angiogenesis is impaired despite elevated levels of VEGF and its receptors. Up-regulation of VEGF is thought to be responsible for the appearance of giant capillaries-telangiectasias . Vasculogenesis describes the formation of new vessels by circulating endothelial progenitor cells, which have to refill damaged blood vessels. In SSc, the numbers and function of bone marrow-derived CD34+/endothelial progenitor cells are depleted . Ischaemia stimulates production of TGF-β and CTGF followed by fibroblast activation and excessive extracellular matrix production. Microvascular pericytes contribute to neo-vascularisation. Pericytes may become activated and transform to collagen-producing myofibroblasts , ET-1 induced endothelial cells proliferation, smooth muscle hypertrophy and irreversible vascular obliteration. Elevated levels of ET-1 correlated with severity of RP, digital ulcers, PAH and renal failure in SSc patients . ET-1 participates in the fibrotic cascade by stimulation of fibroblast collagen production and inhibition of matrix metalloproteinase-1 activity. There are two types of receptors to ET-1: ET-A and ET-B. ET-A receptors are expressed by vascular smooth muscle cells and mediate vasoconstriction, smooth muscle cell proliferation and fibrosis. ET-B receptors are mainly expressed on endothelial cells and mediate vasodilation with release of nitric oxide. In SSc patients, ET-B receptors are down- regulated, which may displace the balance towards vasoconstriction and fibrosis .

Prostacyclin is the principal arachidonic acid metabolite of endothelial cells and vascular smooth muscle cells that express vasodilator properties and inhibit platelet adhesion. The ability of endothelial cells to synthesise and release prostacyclin is reduced in patients with RP and SSc. Production of thromboxan A ₂ potential vasoconstrictor and platelets activator is enhanced in SSc patients. Abnormal balance between prostanoids on behalf of thromboxan A ₂ may contribute to vasoconstriction in SSc, vessels obliteration and in situ thrombosis . The role of nitric oxide in SSc is complex: scleroderma endothelial cells express reduced endothelial nitric oxide synthase and increase inducible nitric oxide synthase activity, resulting in a vasoconstriction, inflammation and tissue damage. Recently, the progressive disappearance of lymphatic vessels has been reported in scleroderma .

The immune system in SSc

Both innate and adaptive autoimmunity are involved in SSc. Toll-like receptors (TLRs) control immune responses by detecting common molecular motifs such as RNA, DNA, lipopolysaccharide or endotoxin. DNA and RNA are situated inside the cell and may be recognised after cell and nuclear damage with binding through ligands: TLR7 (by RNA) or TLR9 (by DNA). Dendritic cells, mononuclear cells and B lymphocytes activated through TLRs produce IFN, IL-1, TNF-α and IL-6. The TLR2 Pro631His gene variant was robustly associated with anti-topo I, diffuse scleroderma and PAH . TLRs participate in production of autoantibodies to nucleic acid-binding proteins and increase expression of IFN-responsive genes by peripheral blood mononuclear cells. SSc sera containing anti-topo I stimulated IFN-α production by peripheral blood mononuclear cells . TLR l ligands can directly promote fibroblast differentiation into collagen-producing myofibroblasts.

Specific autoantibodies in SSc may indicate different disease subsets and organ target damage. The rate of anti-topo I and ACA was different: 60.8% versus 23.4% in DcSSc and 6.0% versus 46.7% in LcSSc. In the European League Against Rheumatism (EULAR) Scleroderma Trials and Research (EUSTAR) patients cohort (3550 SSc patients), the rates of higher modified Rodnan skin score (mRSS), joint and muscle involvement, intestinal involvement, pulmonary fibrosis, cardiomyopathy, hypertension and proteinuria were significantly higher in patients with anti-topo I compared to those with ACA . In patients with RP and abnormal nail-fold capillaroscopy, the presence of ACA, anti-Th/To, anti-topo I and anti-RNAP-III autoantibodies independently predicted progression to definite SSc in 79.5% of patients . Anti-topo I antibodies may bind DNA or RNA, induce over-production of IL-6, TGF-β1 and IL-17, and impair release of anti-inflammatory cytokine IL-10 . In a patient with early DcSSc, the levels of anti-topo I gradually reduced and finally disappeared under corticosteroids treatment in parallel with reduction in mRSS. Discontinuation of corticosteroids triggered the re-emergence of anti-topo I and skin sclerosis; skin sclerosis improved and anti-topo I levels reduced with re-start of prednisone . ACA target a variety of centromere proteins in SSc. The presence of ACA correlated with long-standing RP and SSc and has been associated with the calcinosis, RP, oesophageal dysmotility, sclerodactyly, telangectasia (CREST) variant of SSc. The absence of ACA and the presence of anti-topo I have been associated with pulmonary fibrosis, while the presence of ACA was associated with a lower likelihood of SSc-related pulmonary fibrosis . Anti-nucleolar antibodies target a subclass of small nucleolar ribonuclear proteins including U3-RNP and fibrillarin. Anti-fibrillin-1 antibodies activate fibroblasts and stimulate release of TGF-β; they are predictive of severe skin and systemic involvement and greater mortality. By contrast, anti-nucleolar pattern and anti-Th/To antibodies index mild and limited disease. The prevalence of anti-RNAP-III did not extend 10–20% in various SSc patients cohorts, but their presence was definitely associated with severe skin disease and scleroderma renal crisis . The association between anti-RNAP-III autoantibodies, co-temporal cancer and scleroderma diagnosis, and unique nucleolar RNAP-III expression pattern in malignant tissue may indicate the link between scleroderma and cancer .

Functional antibodies were demonstrated in SSc. Anti-angiotensin receptor and anti-ET-A receptor autoantibodies have been shown to bind respective receptors on endothelial cells, increase TGF-β gene expression by endothelial cells and correlate with SSc severity and mortality . Functional antibodies to methionine sulfoxide reductase A in SSc patients correlated with cardiac, lung and renal involvement and inhibited methionine sulfoxide reductase A activity in SSc sera . Stimulatory antibodies to the PDGF receptor in SSc patients induced tyrosine phosphorylation, reactive oxygen species accumulation, type I collagen-gene expression and myofibroblast phenotype in normal fibroblasts .

Cellular immunity plays an important role in SSc pathogenesis. Dermal mononuclear cell infiltrates consisted mostly of activated T lymphocytes with a mean CD4+/CD8+ ratio of ∼2.4. T-cell infiltration was a prominent finding in lung specimens from patients with SSc. T cells in gastric biopsies from SSc patients robustly expressed lymphocyte function-associated antigen-1, very late antigen-4 and ICAM-1. Endothelial cells showed corresponding strong expression of VCAM-1 and ICAM-1 . Activated CD4 + T-cells in the skin demonstrated enhanced transendothelial migration and expressed elevated levels of TGF-β, CTGF, VEGF, fibroblast growth factor, IL-2, IL-4, IL-6, IL-8, IL-13, IL-17, IL-27, MCP-1, TNF-α and IFN-α in both the circulation and the affected organs (skin and lung) . ‘Classically activated’ monocytes are activated by the Th1 cytokine IFN-γ, whereas alternatively activated macrophages are activated by Th2 cytokines IL-4 and IL-13. Alternatively, activated macrophages stimulate secretion of PDGF and TGF-β. IL-13 is elevated in the serum of patients with early diffuse scleroderma and in alveolar macrophages from SSc patients with pulmonary fibrosis. In patients with LcSSc, levels of IL-13 correlated with PAH-related mortality . IL-10 cooperates with Th1 cytokines and the IL-13 receptor to suppress collagen deposition. Decreased IL-10 levels were reported in peripheral blood mononuclear cells of SSc patients. Increased IFN-inducible protein-10, MCP-1 and serum IFN-α levels were associated with pulmonary fibrosis, cardiac involvement, PAH and digital loss .

Altered relationships between Th17 and T-regulatory cells (T-regs) have been reported in SSc patients. Different peripheral blood mononuclear cell stimulations raised the frequency of Th17 clones in SSc patients. Both T-regs (CD4+CD25+ and CD8+CD28) showed quantitative and qualitative alteration in the peripheral blood mononuclear cells and skin of SSc patients. The suppressor activity of CD4+CD25 + T-regs was lower in SSc and corresponded to higher mRSS .

B-cells are involved in antigen presentation and autoantibodies production in SSc; there was an abounded presence of CD20 + B-cells in skin and lung biopsies of patients with SSc-associated interstitial lung disease . The expression of CD19 on the circulating B-cells and the expression of CD80 and CD86 on memory B-cells of SSc patients may be increased . In a tight-skin (TSK/+) mouse, a genetic model for human SSc, B-cell activation resulted in amplified CD19 signalling followed by skin sclerosis; CD19-deficient TSK/+ mouse demonstrated inhibited IL-6 production. Treatment of TSK/+ mouse with anti-CD20 monoclonal antibodies reduced skin fibrosis . Elevated serum levels of B-lymphocyte stimulator (BAFF) correlated with skin fibrosis in SSc patients. Under BAFF, stimulation SSc B-cells exhibited an enhanced production of IgG and IL-6 . IL-6, a powerful B-cell activator, is elevated in the serum and involved skin of scleroderma patients, and in scleroderma dermal fibroblasts culture. B-cell depletion using rituximab (anti CD20 monoclonal antibodies) in patients with severe skin involvement resulted in prolonged improvement in mRSS and decreased serum IL-6 concentration . IL-6 stimulates fibroblasts extracellular matrix production and tissue inhibitor of metalloproteinases-1 synthesis via up-regulation of MCP-1 and VEGF. Blockade of IL-6 with tocilizumab (anti-IL-6 soluble receptor monoclonal antibodies) in two patients with DcSSc resulted in reduction in mRSS and reduced collagen skin content .

Fibrosis

Fibrosis is a form of tissue scarring with excess deposition of extracellular matrix as the end result of inflammation or damage. The key cellular moderator of fibrosis is the collagen-producing myofibroblasts. Myofibroblasts may be generated from mesenchymal and epithelial cells, endothelial cells and from bone-marrow-derived circulating fibrocytes. Myofibroblasts are activated by paracrine and autocrine signals and through TLRs on fibroblasts. Fibrosis is driven by multiple mediators, such as TGF-β1, PDGF, VEGF, ET-1, IL-13, IL-21, MCP-1, macrophage inflammatory protein, peroxisome proliferator-activated receptors, serum acute phase proteins, caspases and rennin–angiotensin–aldosterone system. Abnormal balance between matrix metalloproteinases and tissue inhibitor of metalloproteinases results in excess synthesis of the extracellular matrix, impaired extracellular matrix catabolism, and leads to collagen accumulation. Scleroderma fibroblasts obtained from involved skin or lungs express an activated myofibroblast-like phenotype that is maintained by TGF-β signal, TGF-β/Smad3-independent mechanisms and CTGF. The epithelium is a major cover of the skin and a mucosal barrier of the oral cavity, gastro-intestinal and respiratory tract; it plays an important role in re-surfacing injured tissue. Under ischaemic conditions, epithelial cells may lose cell–cell attachment and transform into mesenchymal or collagen-producing myofibroblasts. Scleroderma epithelial cells stimulated normal fibroblasts to express CTGF, IL-1α, ET-1 and TGF-β . Epithelial injury plays a pivotal role in SSc-interstitial lung disease .

TGF-β is one of the central pro-fibrotic cytokines. Peripheral blood mononuclear cells and tissue macrophages produce and respond to TGF-β, especially to TGF-β1 isoform. In macrophages, activation of latent TGF-β1 depends on cathepsins, plasmin, calpain, thrombospondin-1, integrin- α v β 6 and matrix metalloproteinases. TGF-β1 triggers signalling through Smad proteins that, in turn, control procollagen I and III gene transcription . Thrombospondin-1 is overexpressed by scleroderma fibroblasts; blockade of thrombospondin-1 reduced the expression of key fibrogenic proteins and prevented PDGF-induced matrix contraction by normal and scleroderma fibroblasts . Early growth response-2 (Egr-2) protein is a transcription factor that is necessary for TGF-β-induced fibrosis and is abnormally expressed in scleroderma skin and in a murine model of scleroderma . The nuclear peroxisome proliferator-activated receptor -γ signalling regulates TGF-β-dependent fibrogenesis. Peroxisome proliferator-activated receptor -γ levels were reduced in skin and lung biopsies from SSc patients and in scleroderma dermal fibroblasts . Angiotensin II, produced locally by activated macrophages and fibroblasts, stimulated TGF-β1 production, fibroblast proliferation and their differentiation into collagen-producing myofibroblasts. Angiotensin II may be produced by myofibroblasts and enhances TGF-β1 signalling by increasing Smad2/Smad3 levels along with mitogen-activated protein kinases and Egr-1. Fibrosis in animal models, SSc and idiopathic pulmonary fibrosis are accompanied by Egr-1 overexpression. Egr-1-null mice are protected from fibrosis . PDGF is secreted by a variety of cells in response to injury and effects replication, survival and migration of myofibroblasts via receptors PDGF-R-α and PDGF-R-β. TGF-β and PDGF pro-fibrotic effects may be independent of Smad2/Smad3 pathway but dependent on intracellular tyrosine kinases (c-abl- and Src-kinases). TGF-β and PDGF activated Src-kinase signalling in scleroderma and normal dermal fibroblasts. Inhibition of Src signalling reduced the synthesis of messenger RNA for collagen and fibronectin-1, reduced dermal collagen content and decreased the number of myofibroblasts . c-abl is implicated in chronic myelogeous leukaemia and appears to be activated in SSc patients. Treatment of TSK-1 mice with imatinib mesylate (c-abl-kinase inhibitor) reduced dermal thickening and prevented the differentiation of fibroblasts into collagen-producing myofibroblasts. In the model of pre-established dermal fibrosis, imatinib induced regression of pre-existing dermal fibrosis .

Chemokines (including CCL3 (MIP-1 α ) and CCL2 (MCP-1)) contribute to fibrosis by recruiting myofibroblasts, macrophages and peripheral blood mononuclear cells to sites of tissue injury. Macrophages and epithelial cells are believed to be the main sources of CCL3. Anti-CCL3 antibodies reduced the development of fibrosis in the bleomycin model of pulmonary fibrosis . Th2 cytokines including IL-4, IL-5, IL-13 and IL-21 cooperate with TGF-β to induce fibrosis. IL-13 plays an important role in latent TGF-β1 production in macrophages and stimulates TGF-β1 activation via matrix metalloproteinases and cathepsin-based proteolytic pathways. TGF-β signalling in fibroblasts regulates the oncogenic potential of epithelial cells . In a recent EUSTAR report, cancer-related death was reported in 31% among 234 non-SSc-related mortality causes (41% of all mortality cases) .