Angiostatic and Angiogenic Chemokines in Systemic Sclerosis: An Overview

Abstract

In systemic sclerosis (SSc), the dysregulation of several molecular pathways seem to have a role in the disease pathogenesis. Either angiogenesis and vasculogenesis are disturbed and impaired, and an imbalance between angiogenic and angiostatic factors may be involved in the genesis and maintenance of vasculopathy. Aberrant immune system activation and function involves both B and T cells, as well as many different chemokines and cytokines. Particularly, chemokines are central to the initiation and maintenance of inflammatory responses as well as angiogenesis and fibrosis. Increased expression of several chemokines as CXCL4 (platelet factor 4), CXCL8 (IL8), CXCL5 (ENA-78), CCL5 (RANTS), CXCL9 (MIG), CCL24, CXCL10 IP-10), CXCL12, CXCL16 (SRPSDX), CCL2 (MCP-1), CCL19 (MIP-3β/ELC), CCL24 (Eotaxin 2), suggests a complex mechanism by which many immune cell types, including T cells, macrophages and neutrophils are recruited to the skin in SSc patients. Many of these chemokines have redundant roles, possibly to ensure recruitment of specific cell types. Several studies have shown a synergistic effect of combinations of these chemokines in cell recruitment, emphasizing the importance of understanding global chemokine expressions. urthermore, chemokines can be detected in peripheral blood compared with cytokines or growth factors. The utility of cytokines as biomarkers has been investigated but longitudinal studies are necessary to clarify their clinical utility for the evaluation of disease activity, therapeutic effects on skin sclerosis or interstitial lung disease and risk stratification of SSc patients. An effective therapeutic agent, able to interfere with complex chemokine networks, is warranted to attenuate perivascular inflammation, dysregulated angiogenesis and the evolution of skin and internal organ fibrosis, is the most ambitious goal for the scientific research of the future.

Keywords

Angiogenesis Chemokines Systemic sclerosis

Introduction

Cell to cell and cell to extracellular matrix (EMC) communication are regulated by several molecules such as cytokines, chemokines and adhesion molecules that guarantee a dynamic equilibrium in physiological state. Dysregulation of this tightly controlled system could be responsible for inflammatory (arthritis), proliferative (cancers) and anti-proliferative (autoimmune) diseases (1, 2).

Fig. 1

Angiogenic chemokines.

Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrosis of the skin and of internal organs, cellular and humoral immunity abnormalities and pronounced alterations in the microvasculature (3). In SSc, many molecular pathways are dysregulated and have a role in its complex pathogenesis (4, 5). Either angiogenesis and vasculogenesis are disturbed and impaired, and an imbalance between angiogenic and angiostatic factors may be involved in the pathogenetic mechanisms leading to SSc vasculopathy (4, 5). In SSc, the vascular injury occurs very early in the disease, even before the onset of fibrosis (4, 5). In the early phases of the disease when tissue inflammation is predominant, it has been suggested that vessel permeability and injury coincide with the infiltration of immune cells, mainly consisting of T cells and macrophages localized at perivascular regions (6–7–8). The immune cell recruitment is facilitated by vascular endothelial cells which, upon injury, upregulate adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and E-selectin, ultimately leading to immune activation, and tissue damage (9). Aberrant immune system activation and function involves both B and T cells, as well as many different chemokines and cytokines. Excessive activation and polarization to Th2 cells is observed with consequent cytokine overproduction (interleukin [IL] 4, IL13, IL6) that promote fibroblast proliferation, collagen deposition and increase profibrotic transforming growth factor beta (TGFβ) (10–11–12). Additional, pro-inflammatory (IL1, tumor necrosis factor alpha [TNFα], IL 17A and IL 10) (13, 14) and pro-fibrotic (IL13, IL25, IL33) cytokines are upregulated in SSc patients (15–16–17). Chemokines, deﬁned as cytokines inducing chemotaxis in nearby responsive cells, are central to the initiation and maintenance of inflammatory responses as well as angiogenesis (13). Consequently, the fine tuning of their expression at inflamed sites contributes to the resolution of the process, by promoting wound healing and tissue repair (13).

In SSc skin, several chemokines show upregulated expression: CXCL9 (MIG) and CXCL10 (IP-10), which recruit T cells (18–19–20); CCL2 (MCP-1), which recruit macrophages (21–22–23) and other chemokines such as CCL5 (RANTS), CXCL8 (IL8), CXCL5 (ENA-78) and CCL24 (Eotaxin 2), which recruit many immune cell types, including T cells, macrophages and neutrophils and up-regulate adhesion molecules expression, thus allowing diapedesis (9, 21, 24, 25). In this review, we report the literature on chemokines found in SSc and their position in the disease pathogenesis.

Chemokines and Systemic Sclerosis

Chemokines are small soluble proteins, not bigger than 8-10 kDa, that induce chemotactic activity in various cells, especially cells of the immune system, organizing T cell motility and migration in physiological immune responses as well as in pathological processes such as in chronic inflammatory and autoimmune conditions. This action is evident after binding to their specific receptors constitutively expressed on cells belonging to the immune system. Their target cells express respective and sometimes overlapping receptors (26). Inﬂammatory chemokines can be induced by cells exposition to pro-inﬂammatory cytokines or tissue damage or infection. In inﬂamed contexts, interaction of inﬂammatory chemokines with their receptors on leucocytes leads to inﬂammatory cell inﬂux and accumulation at damaged or infected sites. Membership in family of chemokines is classified on the basis of the presence of variations of a conserved cysteine motif in the mature proteins. According to this classification, chemokines can be divided into subfamilies: C (one cysteine residue is missing), CC (first two cysteine residues are immediately adjacent in the primary structure), CXC (first two cysteine residues are separated by an amino acid) and CX3C (separation involves three amino acids) chemokines. This system is crucial in several biological functions, although its key role is in regulating tissue-speciﬁc leucocyte migration under both inﬂammatory and homeostatic conditions. Functionally, chemokines attract leukocytes into tissues and contribute to regulate angiogenesis, vascular proliferation and fibrosis contributing to manifestations of scleroderma. Together, the CC and CXC subfamilies constitute the majority of chemokines, whereas the C and CX3C chemokines represent a limited subpopulation of unconventional chemokines (27). The CXC chemokines can be subdivided into two groups according to the presence of the enzyme-linked receptor (ELR) motif or Glu-Leu-Arg. The ELR+ CXC chemokines such as CXCL16 and CXCL5 are potent promoters of angiogenesis (Tab. I), by binding the unique receptor CXCR6, as well as potent activators and chemoattractants for neutrophils, whereas the ELR-chemokines, such as CXCL4, CXCL9 and CXCL10, have angiostatic (Tab. II) properties by binding the CXCR3 receptor without exhibiting neutrophil chemoattractant activities (28). These different functions appear to be due to the presence of three different splice variants of CXCR3: CXCR3-A, CXCR3-B, and CXCR3-alt (29) and, in particular, the angiostatic role is mediated solely by CXCR3-B isoform (30, 31) (Tab. I, Fig. 1).

Table I

Chemokines with angiogenic activity in the pathogenesis of systemic sclerosis

Chemokine	Receptors	Function	Levels	Ref
CCL 2 (MCP-1)	CCR2	Angiogenic	Overexpressed in serum, skin	42, 45
CXCL5 (ENA-78)	CXCR2	Angiogenic	Overexpressed in serum	36
CCL24 (EOTAXIN 2)	CCR3	Angiogenic	Overexpressed in serum, skin	64
CXCL 16 (SRPSDX)	CXCR6, CXCR5	Angiogenic	Overexpressed in serum, skin and BAL fluid	70, 73
CCL 19 (MIP-3β/ELC)	CCR7	Angiogenic	Overexpressed in skin	77
CXCL 8 (IL-8)	CXCR1, CXCR2	Angiogenic	Overexpressed in serum, skin	85, 87

BAL = bronchoalveolar lavage.

Table II

Chemokines with angiostatic activity in the pathogenesis of systemic sclerosis

Chemokine	Receptor	Function	Levels	Ref
CXCL 4 (PF-4)	CXCR3B	Angiostatic	Overexpressed in serum, skin and BAL fluid	98
CXCL9 (MIG)	CXCR3	Angiostatic	Overexpressed in serum	19, 29
CXCL 10 (IP10)	CXCR3, CXCR3B	Angiostatic	Overexpressed in serum	110, 120

BAL = bronchoalveolar lavage.

In SSc, the inflammatory response is activated and chemokine alterations have been described and have been implicated through human, animal and in vitro studies in its pathogenesis. Endothelial cell injury, caused by ischemia-reperfusion, may induce inflammatory cell infiltration and subsequent cytokine production leading to tissue fibrosis. During this process, chemokines mediate leukocyte and mononuclear cell chemotaxis, cell activation and induce an interaction between leukocytes and fibroblasts with release of profibrotic growth factors (32, 33), engage in the perpetuation of the chronic damage and in the induction of ﬁbrosis (21).

In particular, chemokines such as CCL2, CCL3, CCL4, CCL5, CCL7, CXCL8, CXCL12 initiate and maintain aberrant fibroblast function, which eventually leads to excessive extracellular matrix deposition (13, 15) and compound polymorphisms in CCL5 and CXCL8 seem to increase the risk of SSc (34).

Furthermore, in the course of SSc, abnormal expression of some chemokines could also influence the pathologic angiogenesis and vasculogenesis (35). Recent data suggest that upregulation of some chemokines such as CXCL9, CXCL10, CXCL16, and downregulation of others as CXCL5 may influence vasculopathy in SSc (19, 36) as well as the increase or downregulation of their receptor (19).

Angiogenic Chemokines

CCL 2 (MCP-1)

Among CC chemokines that are angiogenic, monocyte chemoattractant protein-1 (MCP-1/CCL2 and MIP-1a/CCL) that binds to CCR2, is overexpressed in SSc skin and serum of SSc patients (21, 37, 38). CCL2 is produced by macrophages, ﬁbroblasts, endothelial cells and other cells, and attracts and activates monocytes and T cells, inducing T-helper 2 cell polarization (39) and stimulates collagen production by ﬁbroblasts via speciﬁc receptors and endogenous upregulation of TGFβ expression (21, 22, 40, 41). In fact, circulating levels of CCL2 correlate in SSc with the extent of skin and pulmonary fibrosis, likely playing a role in tissue fibrosis (21, 42–43–44). SSc fibroblasts express CCR2 (45), and mice that do not express CCL2 receptor CCR2 are protected from fluorescein isothiocyanate-induced and bleomycin-induced lung fibrosis (46, 47). It has been reported also that cultured SSc dermal fibroblasts display augmented expressions of CCL2 mRNA and protein (22, 48) compared to healthy controls. Furthermore, stimulation with platelet-derived growth factor (PDGF) significantly enhanced CCL2 expression in dermal SSc fibroblasts (21, 22) as well as exogenously administered CCL2 stimulated auto-induction of CCL2 mRNA by scleroderma, but not normal, dermal fibroblasts (22). Anti-CCL2 antibody therapy reduces mononuclear phagocyte accumulation in bleomycin-challenged mice (49). Further work suggests a role for CCL2 in matrix turnover by upregulating matrix-degrading enzymes and their inhibitors such as tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) (50).

Hasegawa et al (51) reported that serum CCL2 levels declined with disease progression, along with improvement of skin thickness assessed by modified Rodnan Skin Score (mRSS). They confirmed the correlation between CCL2 and lung fibrosis: moreover, they found that when the progression of interstitial fibrosis was evaluated by variations of % of vital capacity (VC), the variations of %VC during the 3 years were inversely associated with the changes of CCL 2 circulating levels (51).

Luzina et al (52) reported that expression of CCL2 mRNA was most augmented among 4507 genes in bronchoalveolar lavage (BAL) cells from SSc inﬂammatory lungs as well as protein levels of CCL2 that was also increased and associated with the presence of interstitial lung disease (ILD), correlating negatively with lung function parameters and positively with computed tomography scores (52).

Interestingly, CCL2 inhibits alveolar epithelial cell-derived prostaglandin E2 (PGE2) synthesis and a reduction of PGE2 levels enhanced proliferation (53). Thus, an important profibrotic mechanism of MCP-1 interactions with its main receptor, CCR2, is to limit prostaglandin E2 production in alveolar epithelial cells after injury, thus promoting fibrosis (53).

High CCL2 levels have also been described in early phases of SSc and in patients with higher frequencies of cardiac, vascular or kidney involvement (13, 37, 45, 54), and an up-regulation of CCL2 production has been demonstrated in animal models of crescentic nephritis (55). In a rat model of monocrotaline-induced pulmonary hypertension, gene transfer of a dominant negative inhibitor of the CCL2 gene inhibited the progression of CCL2-induced pulmonary hypertension and mononuclear cell infiltration into the lung (56). This suggests that early inhibition of MCP-1 signaling may prevent the recruitment of monocytes/macrophages into perivascular tissues, preventing the immune-mediated development of pulmonary hypertension in this model (56).

In the early phase of SSc, in animal models, all CCL5, CCL2 CCL3 molecules are up-regulated (57), but while MCP1/CCL2 and MIP1α/CCL3 remained increased during the time, RANTES/CCL5, after an initial marked peak, rapidly decreased, suggesting a more relevant role for MCP1/CCL2 and MIP1α/CCL3 (57).

It was thus suggested that CCL2 levels could be a useful biomarker to follow-up skin and lung fibrosis and to assess therapeutic efficacy.

CCL5 (RANTES)

Levels of “regulated upon activation, normal T cell expressed and secreted” (RANTES/CCL5) and macrophage inflammatory protein-1α (MIP-1 α/CCL3) are increased in serum and BAL fluids from SSc patients (38, 58). Increased expression of RANTES and MIP-1α precedes the development of dermal and pulmonary fibrosis in a murine model of scleroderma (59). Expression of abundant CCL5 mRNA has been demonstrated in situ in the skin of SSc patients, but not in the skin of healthy control (60) and also in lung samples from patients with severe pulmonary arterial hypertension (PAH), in comparison with controls (61). Endothelial cells seem to be the major source of CCL5 within the pulmonary artery walls of SSc patients and associated with CD45+ inflammatory cell infiltrates (61).

CCL5 levels may be a potential useful biomarker of PAH in SSc patients.

CCL24 (Eotaxin 2)

The chemokine CCL24 is a potent chemoattractant for inflammatory cells, predominantly eosinophils, basophils and T helper type 2 (Th2) lymphocytes through its receptor CCR3. It is produced in various cell types including: activated T lymphocytes, macrophages, endothelial cell, epithelial cells and dermal fibroblasts (62–63–64).

Additionally, CCL24 was shown to stimulate human lung fibroblast proliferation and collagen synthesis (65). Knock out mice to CCL24 revealed significantly reduced inflammatory cell infiltration into BAL fluid in OVA-induced model of pulmonary inflammation (66). Recently, it was discovered that high CCL24 sera levels were described in limited and diffused phases of SSc and that SSc skin showed significant overexpression of both CCL24 and its cognate receptor, CCR3 (67). A blockade of CCL24, using a specific monoclonal antibody, was described in the past to result in anti-inflammatory and anti-atherosclerotic features, that are significantly relevant in the SSc pathogenesis (68, 69). Recently, it was revealed that treatment with anti-CCL24 monoclonal antibody in bleomycin-induced scleroderma mice model, exhibited reduced symptoms of the disease, including dermal thickness, immune cell infiltration to the lung and collagen deposition in the skin (70).

Over-expression of CCL24 in the skin and sera of SSc patients may suggest involvement of CCL24 in the pathogenesis of SSc.

CXCL 16 (SRPSDX)

CXCL16 is a CXC chemokine expressed on a number of different cell types, including leukocytes, endothelial cells (ECs) and keratinocytes that bind to CXCR6 (71). CXCL16 is present in membrane-bound receptors CXCR6 and CXCR5 that facilitate firm adhesion of emigrating leukocytes, and soluble forms that mediate chemotactic properties (72, 73). It is well known that this angiogenic chemokine is overexpressed in SSc patients, both in skin and serum and its receptor CXCR6 is overexpressed on ECs of the skin (19). Elevation of serum CXCL16 levels were found in SSc patients and associated with greater extent of skin fibrosis but not with the severity of pulmonary fibrosis (71). Rabquer et al (19), analyzing skin biopsies and serum samples of SSc patients, found that CXCL16 was significantly upregulated in early SSc or with PAH. CXCL16 was actively expressed in bronchial epithelial cells and lung fibroblasts, and increased concentrations can be found in BAL fluids from patients with interstitial lung disease (ILD) (74).

CXCL16 seems to play an important role in the development of skin sclerosis in patients with SSc but it is not useful as a clinical marker of pulmonary fibrosis.

CXCL5 (ENA-78)

CXCL5 is a member of CXC chemokines with glutamic acid-leucine-arginine (ELR) motif and it is also known as epithelial-derived neutrophil activating peptide 78 or ENA-78, produced by epithelial cells following stimulation by IL1 or TNFα (75). It is also expressed in eosinophil, which supports the notion that eosinophils are not mere targets for chemokines, but also have immunoregulatory properties (76). CXCL5 has been found elevated in BAL fluid from patients with idiopathic pulmonary fibrosis (IPF) (77). In SSc, Ichimura et al (36) reported that serum CXCL5 levels were significantly decreased in diffuse SSc (dcSSc) and limited SSc (lcSSc) patients compared with healthy controls, suggesting a negligible role for CXCL5 in the fibrotic process underlying SSc. Furthermore, serum CXCL5 levels were reduced in early SSc (<1 year), characterized by abnormal vascular activation, correlated with disease duration and decreased during the progression of the disease, amplifying the risk for the development of digital ulcers. This suggested that CXCL5 is associated with disease onset and with vascular aspects of disease progression (36).

Serum CXCL5 levels may serve as an inverse marker for the severity of SSc vasculopathy.

CCL19 (MIP-3β/ELC)

CCL19 is upregulated in SSc and its expression correlates strongly with markers of vascular inflammation (angiopoietin 2, Von Willebrand factor [vWF] and junctional adhesion protein 2 [JAM2]) and with macrophage markers: CD163, Siglec-1 and CCL2 The immunoﬂuorescence and in vitro data identify macrophages as an important source of CCL19 and as macrophages are present in the skin under non-inflammatory conditions, resident as well as recruited macrophages might be an important source of CCL19. A strong correlation of CCL19 with vascular inﬂammation and macrophage recruitment has been demonstrated (78).

Furthermore, CCL19 is a ligand for CCR7, which is expressed on many antigen-presenting cells and is important for their homing to lymph nodes from peripheral tissues during early- and late-stage inflammation (79, 80). This suggests that CCL19 plays a role in the recruitment of cells to the skin during SSc rather than emigration of antigen-presenting cells after activation. This is supported by the correlation of CCL19 with vascular inflammation expressed by infiltrating immune cells in SSc skin (21–22–23, 78). Overall, this study suggests that CCL19 plays a role in cutaneous inﬂammation in dcSSc (78).

CCL19 seems to be the only chemokine upregulated in SSc skin, associates with vascular injury and may be a sensitive marker for macrophage activity.

CXCL8 (IL-8)

CXCL8 (IL-8) is a chemokine often produced by macrophages and epithelial cells by mediating the inflammatory response and an angiogenic pathway (81). CXCL8 is a neutrophil chemoattractant factor and a potent proangiogenic chemokine overexpressed both in SSc serum and skin (82–83–84) that binds two receptors CXCR1 and CXCR2 even if its angiogenic properties are mediated primarily by CXCR2, and two polymorphisms of its gene have been associated with scleroderma (85). To date, CXCR2 has been shown to be upregulated on SSc dermal fibroblasts (86, 87), though no studies have investigated its expression on SSc ECs. Expression levels of CXCL8 are increased in SSc serum and skin, particularly in early SSc patients with disease of less than 1 year duration (84). A recent study demonstrated that plasma CXCL8 levels are elevated in anti-topoisomerase I-positive SSc (88). CXCL8 is also increased in BAL ﬂuid from SSc patients, suggesting a critical role for CXCL8 in SSc-ILD (58, 89). Compound polymorphisms in CCL5 and CXCL8 appear to increase the risk of SSc (34). Finally, Hasegawa et al (90) reported that baseline serum CXCL8 levels seem signiﬁcantly associated with severity of physical dysfunction at baseline and in subsequent years in SSc patients.

Literature data about the potential role of IL-8 in fibrosis are mainly centered on IPF showing greater IL-8 levels in lung (91). When IL-8 is depleted from IPF tissue specimens, tissue-derived angiogenic activity is markedly reduced (91). Studies in animal models of pulmonary fibrosis have shown the importance of chemokines in promoting angiogenesis, which is necessary for the development of pulmonary fibrosis (92).

Serum CXCL8 levels may be useful for predicting the decline in physical function in patients with progressive SSc.

The Angiostatic Chemokines

CXCL4 (platelet factor 4; PF-4)

CXCL4, also known as platelet factor-4, is a 70-amino acid, lysine-rich, 7.8-kDa protein that was first identified as a product of megakaryocytes and is chemotactic for neutrophils, monocytes and ﬁbroblasts, and may have important roles in inﬂammation and wound repair (93).

CXCL4 is generally considered one of the most potent anti-angiogenic chemokines, influencing angiogenesis through an integrin-dependent mechanism (94). Romagnani et al (95) reported that increased circulating levels of CXCL4, found in the serum of SSc patients, showed anti-angiogenic activity, and the disease was characterized by rarefaction of vessels, despite the presence of multiple proangiogenic factors.

Increased levels of CXCL4 were also found in patients with Raynaud's phenomenon, most of whom did not progress to SSc. This finding could support the hypothesis that CXCL4 may sensitize several cellular components, including fibroblasts but the presence of CXCL4 alone is not sufficient, but play an additional role in the initiation and maintenance of cellular activation.

In addition, CXCL4 induces the expression of thrombospondin 1 and attenuates the effects of vascular endothelial growth factor A (VEFG-A), one of the major proangiogenic regulators that largely mediates angiogenesis by primarily binding vascular endothelial growth factor receptor 2 (VEGFR2) and that is markedly increased in serum and different cell types both in the epidermis and dermis of patients with SSc (96–97–98).

These observations might explain the absence of ‘appropriate’ neovascularization in SSc, despite the presence of high levels of VEGF and VEGFR2 which is actually upregulated on SSc ECs (99). Interestingly, Manetti et al (100) reported further data to explain this paradox. They demonstrated that endogenous antiangiogenic VEGF165b splice variant is selectively upregulated in SSc skin and circulation and provided evidence that profibrotic TGFβ 1 and SRp55 splicing factor may contribute to the switch from proangiogenic to antiangiogenic VEGF isoforms and that constitutive overexpression of antiangiogenic VEGF165b leads to impaired VEGFR-2 downstream signaling and capillary morphogenesis in SSc microvascular endothelial cells (MVECs) (100).

CXCL4 is also a profibrotic chemokine, inhibiting the expression of the antifibrotic cytokine interferon-γ (from type 1 helper T cells), up-regulating profibrotic cytokines interleukin-4 and interleukin-13 (from type 2 helper T cells) and stimulating the proliferation of regulatory T cells with impaired suppressive function (101).

A recent multicenter study used proteome-wide analysis to demonstrate that CXCL4 was the predominant protein secreted by plasmacytoid dendritic cells in SSc skin and plasma (99). Furthermore, levels of CXCL4 seem to correlate with the development of skin and pulmonary fibrosis (PF) and PAH (99), but not with extent and progression of PF as measured by change in diffusing capacity of the lungs for carbon monoxide (DLCO) (102). CXCL4 levels have also been found elevated in the BAL fluid of patients with SSc-ILD (103).

The identification of CXCL4 as a marker for fibrosis and PAH may be helpful in early diagnosis and risk assessment of disease progression and an important guide in patients who require a more aggressive treatment since the earliest phase of disease.

CXCL 10 (IP-10)

Interferon-γ-inducible protein 10 (CXCL10, also called IP-10) is a 10 kDa protein and it is functionally categorized as an ‘inflammatory’ chemokine. It was initially identified as a chemokine that is induced by IFN-γ and, under the influence of cytokines, is secreted by various cell type, such as neutrophils, keratinocyte, endothelial cells, fibroblasts, monocytes, mesenchymal cells, dendritic cells, and astrocytes (104). CXCL10 lacking its ELR motif, suppresses neovascularization and functions as an ‘angiostatic’ chemokine (105) and upon binding the receptor CXCR3, it regulates immune responses by promoting recruitment of leukocytes, such as T cells, eosinophils, monocytes, and natural killer (NK) cells (106). In bleomycin-induced pulmonary fibrosis, levels of the CXCL10 are decreased. This chemokine inhibits fibroblast proliferation and deposition of extracellular matrix by regulating angiogenesis (107).

The presence of high CXCL10 levels in peripheral liquids could thus stand as a marker of the host's immune response, especially T helper (Th) 1-orientated T-cells (108).

A recent study proposed a composite chemokine score of plasma levels of CXCL10 (IFN-γ-inducible protein 10; IP-10) and CXCL11 (IFN-γ-inducible T-cell-a chemoattractant; I-TAC) was elevated in SSc patients and correlated with the Medsger severity index, particularly with the severity of lung, skin and muscle involvement (109).

It has been observed that increased CXCL10 levels were associated with cardiac involvement, signs of PAH, elevated ESR, and although only present in few patients, with digital loss and renal crises (44). Moreover, CXCL10 is specifically increased in SSc complicated by ILD (110), and could serve as a disease activity marker (51).

IFN-γ-inducible chemokine score that may be a promising stable serologic marker of a more severe form of SSc and may be useful for risk stratification of patients.

CXCL 9 (MIG)

Chemokine (C-X-C motif) ligand 9 (CXCL9) is a small cytokine belonging to the CXC chemokine family that is also known as monokine, induced by gamma interferon (MIG). CXCL9 is a T-cell chemoattractant, which is induced by IFN-γ and has antiangiogenic effects binding CXCR3 receptor. CXCL9 was found significantly elevated in SSc serum compared to normal controls, regardless the subset of disease. (19, 51, 111) even if their levels are not likely as useful as CCL2 for evaluating disease activity. Until today no relationship between CXL9 levels, variation and change in clinical parameters in SSc have been reported.

CXCL9 has antiangiogenic effects binding CXCR3 receptors, but further studies are needed to elucidate its role in SSc pathogenesis.

Chemokines and Cytokines Cross Talk in Systemic Sclerosis

In SSc, abnormal immunological regulation is considered to be a key stimulus in promoting vascular abnormalities, starting with the earliest phase of SSc (112). This applies to CD3⁺ T lymphocytes, with CD4⁺T cells predominating over CD8⁺T cells.

Mononuclear cell infiltrate is predominantly perivascular suggesting that the microvascular endothelium is one of the major targets of the immune reaction and a trigger for subsequent inflammatory changes and development of fibrosis (113).

B cells, NK cells and increased numbers of mast cells, particularly degranulated, have been detected in the skin of SSc patients in the early stages (114). Th cells differentiate from naïve cells into various subsets (Th1, Th2, Th17 and Th22) associated with a set of inducer and effector cytokines. Previously, it was thought that this differentiation was an irreversible event but recent evidence suggests that differentiated Th cells retain the flexibility to transform from one lineage to another (115–116–117). The pattern of cytokine production of skin-infiltrating T cells from SSc patients demonstrated a high concentration of interleukin-4 (IL-4) and high IL-4 mRNA expression in skin perivascular mononuclear cell infiltrates by in situ hybridization (118). IL-4 drives the production of IgE as Th2-cell-derived IL-9 contributes to the regulation of IgE production and to mast cell growth (119). T cells from peripheral blood, skin, BAL fluid, and several other tissues obtained from SSc patients have undergone oligoclonal expansion driven by one or more unidentified specific antigens (8).

In 2004, Fujii et al (111) showed that Th2 chemokines (TARC/CCL17 and MDC/CCL22) and serum CXCL10 levels were elevated in patients with diffuse and limited SSc. A few years later, Rabquer et al (19) examined CXCL9, CXCL10, and CXCL16 levels in SSc serum and skin, and their receptors in SSc skin and on ECs derived from the skin of patients with SSc. They found that while the expression of CXCL9 and CXCL10 were elevated in SSc sera, the expression of CXCR3 was reduced on SSc dermal ECs. In contrast, CXCL16 and CXCR6 were elevated in SSc sera and in SSc dermal ECs, respectively (19). These findings suggest that angiogenic chemokine receptor expression is important to promote angiogenesis in SSc since both pro- and anti-angiogenic chemokines are elevated systemically in SSc, and for this reason it is important to determine the expression of both angiogenic mediators and their receptors.

A longitudinal study demonstrated high serum levels of CXCL10 (Th1) and CCL2 (Th2) chemokines in newly diagnosed SSc. High values of CXCL10 were associated with a more severe clinical phenotype (lung and kidney involvement) and appeared to decline during the follow-up, while CCL2 levels remained unmodified, suggesting that the disease progresses from the early Th1 inflammatory condition to the advanced Th2-like stage (120).

CXCL8 and CCL2 are the most critical chemokines for the recruitment of neutrophils and monocytes, respectively. Further, the transition from neutrophil to monocyte accumulation during inﬂammation might be linked to a shift in chemokine synthesis from CXCL8 to CCL2, probably via IL-6 signaling (121). Therefore, increased CXCL8 production may trigger initial inﬂammation while CCL2 may contribute to the subsequent chronic inﬂammation and ﬁbrosis in SSc. Thus, CXCL8 levels, rather than CCL2 levels, can likely predict the progression of SSc, despite the ﬁnding that serum levels of CXCL8 and CCL2 were associated with each other (90).

Conclusions

Increased expression of several chemokines as CXCL4, CXCL8, CXCL5, CCL5, CXCL9, CCL24, CXCL10, CXCL12, CXCL16, CCL2, CCL2, CCL19, suggests a complex mechanism by which immune cells are recruited into SSc skin. Many of these chemokines have redundant roles, possibly to ensure recruitment of specific cell types. Several studies have shown a synergistic effect of combinations of these chemokines in cell recruitment, emphasizing the importance of understanding global chemokine expressions (122, 123). In fact, chemokines act through numerous pathways and mechanisms, including attraction of inflammatory cells; direct action on target cells, such as fibroblasts and endotheliocytes; stimulation of production and activation of TGFβ, including autocrine mechanism; and imbalance intracellular signaling activated by these molecules. The effects of chemokines on endothelial cells allow angiogenesis, a process that is necessary for the influx of fibroblasts and inflammatory cells as well as epithelial cell alterations that are required for the development of fibrosis (43).

Furthermore, because chemokines can be detected easier in peripheral blood compared with cytokines or growth factors, their utility as biomarkers of SSc have been investigated. It is interesting to note that the levels of inflammatory chemokines are increased in SSc independently of the disease phenotypes and clinimetric measures. This suggests that, while chemokines are clearly associated with the pathogenesis of SSc, other factors may be critical for determining the level of disease activity and severity even if some of them could be useful as predictive biomarkers, in particular CXCL4, CXL8, CXCL5 and CCL2 (124).

In conclusion, the clinical utility of chemokines as biomarkers is not yet clarified particularly because some factors may influence the levels (disease phase, treatment, etc.). Serial measurements or longitudinal study of serum chemokines levels could be useful for the evaluation of disease activity, therapeutic effects of skin sclerosis or interstitial pneumonia and risk stratification of SSc patients. An integrative and effective therapeutic agent able to interfere with complex chemokine networks, described above, so as to attenuate perivascular inflammation, dysregulated angiogenesis and skin and internal organ fibrosis, is the most ambitious goal for the scientific research of the future.

Footnotes

Financial support: No grants or funding have been received for this study.

Conflict of interest: None of the authors has financial interest related to this study to disclose.

References

OShea

Lipsky

Cytokines and autoimmunity.

Nature Reviews. Immunology 2002, 2:3745. Luster A.D. Chemokines – chemotactic cytokines that mediate inflammation. N Engl J Med 1998;338436–445

Langer

Chavakis

Leukocyte – endothelial interactions in inflammation.

J Cell Mol Med 2009;13 (7):1211–1220

Gabrielli

Avvedimento

Krieg

Scleroderma.

N Engl J Med 2009;360 (19):1989–2003

Abraham

Varga

Scleroderma: from cell and molecular mechanisms to disease models.

Trends Immunol 2005;26 (11):587–595

Geyer

Müller-Ladner

The pathogenesis of systemic sclerosis revisited.

Clin Rev Allergy Immunol 2011;40 (2):92–103

Kahaleh

LeRoy

Autoimmunity and vascular involvement in systemic sclerosis (SSc).

Autoimmunity 1999;31 (3):195–214

Kräling

Maul

Jimenez

Mononuclear cellular infiltrates in clinically involved skin from patients with systemic sclerosis of recent onset predominantly consist of monocytes/macrophages.

Pathobiology 1995;63 (1):48–56

Sakkas

Artlett

Jimenez

Platsoucas

Oligoclonal T cell expansion in the skin of patients with systemic sclerosis.

J Immunol 2002;168 (7):3649–3659

Sato

Abnormalities of adhesion molecules and chemokines in scleroderma.

Curr Opin Rheumatol 1999;11 (6):503–507

10.

OReilly

Hügle

van Laar

T cells in systemic sclerosis: a reappraisal.

Rheumatology (Oxford) 2012;51 (9):1540–1549

11.

Sakkas

Chikanza

Platsoucas

Mechanisms of Disease: the role of immune cells in the pathogenesis of systemic sclerosis.

Nat Clin Pract Rheumatol 2006;2 (12):679–685

12.

Higashi-Kuwata

Makino

Inoue

Takeya

Ihn

Alternatively activated macrophages (M2 macrophages) in the skin of patient with localized scleroderma.

Exp Dermatol 2009;18 (8):727–729

13.

Codullo

Baldwin

Singh

et al.

An investigation of the inflammatory cytokine and chemokine network in systemic sclerosis.

Ann Rheum Dis 2011;70 (6):1115–1121

14.

Xing

Yang

et al.

IL-17A induces endothelial inflammation in systemic sclerosis via the ERK signaling pathway.

PLoS ONE 2013;8 (12):e85032

15.

Luzina

Todd

Sundararajan

Atamas

The cytokines of pulmonary fibrosis: Much learned, much more to learn.

Cytokine 2015;74 (1):88–100

16.

Hams

Armstrong

Barlow

et al.

IL-25 and type 2 innate lymphoid cells induce pulmonary fibrosis.

Proc Natl Acad Sci USA 2014;111 (1):367–372

17.

Rankin

Mumm

Murphy

et al.

IL-33 induces IL-13-dependent cutaneous fibrosis.

J Immunol 2010;184 (3):1526–1535

18.

Farina

York

Di Marzio

et al.

Poly(I:C) drives type I IFN- and TGFβ-mediated inflammation and dermal fibrosis simulating altered gene expression in systemic sclerosis.

J Invest Dermatol 2010;130 (11):2583–2593

19.

Rabquer

Tsou

Hou

et al.

Dysregulated expression of MIG/CXCL9, IP-10/CXCL10 and CXCL16 and their receptors in systemic sclerosis.

Arthritis Res Ther 2011;13 (1):R18

20.

Groom

Luster

CXCR3 ligands: redundant, collaborative and antagonistic functions.

Immunol Cell Biol 2011;89 (2):207–215

21.

Distler

Pap

Kowal-Bielecka

et al.

Overexpression of monocyte chemoattractant protein 1 in systemic sclerosis: role of platelet-derived growth factor and effects on monocyte chemotaxis and collagen synthesis.

Arthritis Rheum 2001;44 (11):2665–2678

22.

Galindo

Santiago

Rivero

Rullas

Alcami

Pablos

Chemokine expression by systemic sclerosis fibroblasts: abnormal regulation of monocyte chemoattractant protein 1 expression.

Arthritis Rheum 2001;44 (6):1382–1386

23.

Yamamoto

Eckes

Hartmann

Krieg

Expression of monocyte chemoattractant protein-1 in the lesional skin of systemic sclerosis.

J Dermatol Sci 2001;26 (2):133–139

24.

Van Damme

Proost

Lenaerts

Opdenakker

Structural and functional identification of two human, tumor-derived monocyte chemotactic proteins (MCP-2 and MCP-3) belonging to the chemokine family.

J Exp Med 1992;176 (1):59–65

25.

Luster

Chemokineschemotactic cytokines that mediate inflammation.

N Engl J Med 1998;338 (7):436–445

26.

Bromley

Mempel

Luster

Orchestrating the orchestrators: chemokines in control of T cell traffic.

Nat Immunol 2008;9 (9):970–980

27.

Van Raemdonck

Van den Steen

Liekens

Van Damme

Struyf

CXCR3 ligands in disease and therapy.

Cytokine Growth Factor Rev 2015;26 (3):311–327

28.

Belperio

Keane

Arenberg

et al.

CXC chemokines in angiogenesis.

J Leukoc Biol 2000;68 (1):1–8

29.

Lasagni

Francalanci

Annunziato

et al.

An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth induced by IP-10, Mig, and I-TAC, and acts as functional receptor for platelet factor 4.

J Exp Med 2003;197 (11):1537–1549

30.

Vandercappellen

Van Damme

Struyf

The role of CXC chemokines and their receptors in cancer.

Cancer Lett 2008;267 (2):226–244

31.

Yang

Richmond

The angiostatic activity of interferon-inducible protein-10/CXCL10 in human melanoma depends on binding to CXCR3 but not to glycosaminoglycan.

Mol Ther 2004;9 (6):846–855

32.

Giuggioli

Manfredi

Colaci

Manzini

Antonelli

Ferri

Systemic sclerosis and cryoglobulinemia: our experience with overlapping syndrome of scleroderma and severe cryoglobulinemic vasculitis and review of the literature.

Autoimmun Rev 2013;12 (11):1058–1063

33.

Elhai

Avouac

Kahan

Allanore

Systemic sclerosis at the crossroad of polyautoimmunity.

Autoimmun Rev 2013;12 (11):1052–1057

34.

Lee

Zhao

Kim

Xiong

Song

Evidence of potential interaction of chemokine genes in susceptibility to systemic sclerosis.

Arthritis Rheum 2007;56 (7):2443–2448

35.

Romagnani

Lasagni

Annunziato

Serio

Romagnani

CXC chemokines: the regulatory link between inflammation and angiogenesis.

Trends Immunol 2004;25 (4):201–209

36.

Ichimura

Asano

Akamata

et al.

Fli1 deficiency contributes to the suppression of endothelial CXCL5 expression in systemic sclerosis.

Arch Dermatol Res 2014;306 (4):331–338

37.

Scala

Pallotta

Frezzolini

et al.

Cytokine and chemokine levels in systemic sclerosis: relationship with cutaneous and internal organ involvement.

Clin Exp Immunol 2004;138 (3):540–546

38.

Bandinelli

Del Rosso

Gabrielli

et al.

CCL2, CCL3 and CCL5 chemokines in systemic sclerosis: the correlation with SSc clinical features and the effect of prostaglandin E1 treatment.

Clin Exp Rheumatol 2012;30 2 Suppl 71S44–S49

39.

Tseng

Horner

Tam

Loda

Rollins

Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1.

Nature 2000;404 (6776):407–411

40.

Gharaee-Kermani

Denholm

Phan

Costimulation of fibroblast collagen and transforming growth factor beta1 gene expression by monocyte chemoattractant protein-1 via specific receptors.

J Biol Chem 1996;271 (30):17779–17784

41.

Denton

Shi-Wen

Sutton

Abraham

Black

Pearson

Scleroderma fibroblasts promote migration of mononuclear leucocytes across endothelial cell monolayers.

Clin Exp Immunol 1998;114 (2):293–300

42.

Yamamoto

Pathogenic role of CCL2/MCP-1 in scleroderma.

Front Biosci 2008;132686–2695

43.

Atamas

White

The role of chemokines in the pathogenesis of scleroderma.

Curr Opin Rheumatol 2003;15 (6):772–777

44.

Eloranta

Franck-Larsson

Lövgren

et al.

Type I interferon system activation and association with disease manifestations in systemic sclerosis.

Ann Rheum Dis 2010;69 (7):1396–1402

45.

Carulli

Handler

Coghlan

Black

Denton

Can CCL2 serum levels be used in risk stratification or to monitor treatment response in systemic sclerosis?

Ann Rheum Dis 2008;67 (1):105–109

46.

Moore

Paine

III Christensen

et al.

Protection from pulmonary fibrosis in the absence of CCR2 signaling.

J Immunol 2001;167 (8):4368–4377

47.

Gharaee-Kermani

McCullumsmith

Charo

Kunkel

Phan

CC-chemokine receptor 2 required for bleomycin-induced pulmonary fibrosis.

Cytokine 2003;24 (6):266–276

48.

Yamamoto

Eckes

Krieg

High expression and autoinduction of monocyte chemoattractant protein-1 in scleroderma fibroblasts.

Eur J Immunol 2001;31 (10):2936–2941

49.

Smith

Chemotactic cytokines mediate leukocyte recruitment in fibrotic lung disease.

Biol Signals 1996;5 (4):223–231

50.

Castelino

Varga

Emerging cellular and molecular targets in fibrosis: implications for scleroderma pathogenesis and targeted therapy.

Curr Opin Rheumatol 2014;26 (6):607–614

51.

Hasegawa

Fujimoto

Matsushita

Hamaguchi

Takehara

Sato

Serum chemokine and cytokine levels as indicators of disease activity in patients with systemic sclerosis.

Clin Rheumatol 2011;30 (2):231–237

52.

Luzina

Atamas

Wise

Wigley

Xiao

White

Gene expression in bronchoalveolar lavage cells from scleroderma patients.

Am J Respir Cell Mol Biol 2002;26 (5):549–557

53.

Moore

Peters-Golden

Christensen

et al.

Alveolar epithelial cell inhibition of fibroblast proliferation is regulated by MCP-1/CCR2 and mediated by PGE2.

Am J Physiol Lung Cell Mol Physiol 2003;284 (2):L342–L349

54.

Hasegawa

Sato

Takehara

Augmented production of chemokines (monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta) in patients with systemic sclerosis: MCP-1 and MIP-1alpha may be involved in the development of pulmonary fibrosis.

Clin Exp Immunol 1999;117 (1):159–165

55.

Hogaboam

Steinhauser

Chensue

Kunkel

Novel roles for chemokines and fibroblasts in interstitial fibrosis.

Kidney Int 1998;54 (6):2152–2159

56.

Ikeda

Yonemitsu

Kataoka

et al.

Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary hypertension in rats.

Am J Physiol Heart Circ Physiol 2002;283 (5):H2021–H2028

57.

Uguccioni

DApuzzo

Loetscher

Dewald

Baggiolini

Actions of the chemotactic cytokines MCP-1, MCP-2, MCP-3, RANTES, MIP-1 α and MIP-1 β on human monocytes.

Eur J Immunol 1995;25 (1):64–68

58.

Bolster

Ludwicka

Sutherland

Strange

Silver

Cytokine concentrations in bronchoalveolar lavage fluid of patients with systemic sclerosis.

Arthritis Rheum 1997;40 (4):743–751

59.

Zhang

McCormick

Desai

Gilliam

Murine sclerodermatous graft-versus-host disease, a model for human scleroderma: cutaneous cytokines, chemokines, and immune cell activation.

J Immunol 2002;168 (6):3088–3098

60.

Anderegg

Saalbach

Haustein

Chemokine release from activated human dermal microvascular endothelial cellsimplications for the pathophysiology of scleroderma?

Arch Dermatol Res 2000;292 (7):341–347

61.

Dorfmüller

Zarka

Durand-Gasselin

et al.

Chemokine RANTES in severe pulmonary arterial hypertension.

Am J Respir Crit Care Med 2002;165 (4):534–539

62.

Schaefer

Meyer

Pods

et al.

Endothelial and epithelial expression of eotaxin-2 (CCL24) in nasal polyps.

Int Arch Allergy Immunol 2006;140 (3):205–214

63.

Dulkys

Schramm

Kimmig

et al.

Detection of mRNA for eotaxin-2 and eotaxin-3 in human dermal fibroblasts and their distinct activation profile on human eosinophils.

J Invest Dermatol 2001;116 (4):498–505

64.

Siddiqui

Secor

Jr Silbart

Broncho-alveolar macrophages express chemokines associated with leukocyte migration in a mouse model of asthma.

Cell Immunol 2013;281 (2):159–169

65.

Kohan

Puxeddu

Reich

Levi-Schaffer

Berkman

Eotaxin-2/CCL24 and eotaxin-3/CCL26 exert differential profibrogenic effects on human lung fibroblasts.

Ann Allergy Asthma Immunol 2010;104 (1):66–72

66.

Pope

Zimmermann

Stringer

Karow

Rothenberg

The eotaxin chemokines and CCR3 are fundamental regulators of allergen-induced pulmonary eosinophilia.

J Immunol 2005;175 (8):5341–5350

67.

Matucci Cerinic

Katav

Segal-Salto

Levy

Mor

A novel antibody blocking CCL24/CCR3 reduces chemokines of immune cells and the transition of fibroblasts to myofibroblasts in systemic sclerosis “SSc”.

Abstracts from the 4th Systemic Sclerosis World Congress, Feb 18-20, 2016, Lisbon, Portugal. J Scleroderma Relat Disord. 2016;1 (1):37 Abstract CO-37

68.

Ablin

Entin-Meer

Aloush

et al.

Protective effect of eotaxin-2 inhibition in adjuvant-induced arthritis.

Clin Exp Immunol 2010;161 (2):276–283

69.

Mor

Afek

Entin-Meer

Keren

George

Anti eotaxin-2 antibodies attenuate the initiation and progression of experimental atherosclerosis.

World J Cardiovascular Dis 2013;3 04 339–346

70.

Mor

Levy

Katav

Segal-Salto

Matucci Cerinic

CM-101, a novel monoclonal antibody blocking CCL24 ameliorates experimental systemic sclerosis (SSC) and idiopathic pulmonary fibrosis (IPF).

Abstracts from the 4^th Systemic Sclerosis World Congress, Feb 18-20, 2016, Lisbon, Portugal. J Scleroderma Relat Disord 2016;137 Abstract CO-36.

71.

Yanaba

Muroi

Yoshizaki

et al.

Serum CXCL16 concentrations correlate with the extent of skin sclerosis in patients with systemic sclerosis.

J Rheumatol 2009;36 (9):1917–1923

72.

Gough

Garton

Wille

et al.

A disintegrin and metalloproteinase 10-mediated cleavage and shedding regulates the cell surface expression of CXC chemokine ligand 16.

J Immunol 2004;172 (6):3678–3685

73.

Matloubian

David

Engel

Ryan

Cyster

A transmembrane CXC chemokine is a ligand for HIV-coreceptor Bonzo.

Nat Immunol 2000;1 (4):298–304

74.

Cossu

Andracco

Santaniello

et al.

Serum levels of vascular dysfunction markers reflect disease severity and stage in systemic sclerosis patients.

Rheumatology 2016;55 (6):111211116

75.

Hesselstrand

Wildt

Bozovic

et al.

Biomarkers from bronchoalveolar lavage fluid in systemic sclerosis patients with interstitial lung disease relate to severity of lung fibrosis.

Respir Med 2013;107 (7):1079–1086

76.

Persson

Monsef

Andersson

et al.

Expression of the neutrophil-activating CXC chemokine ENA-78/CXCL5 by human eosinophils.

Clin Exp Allergy 2003;33 (4):531–537

77.

Antoniou

Tzouvelekis

Alexandrakis

et al.

Different angiogenic activity in pulmonary sarcoidosis and idiopathic pulmonary fibrosis.

Chest 2006;130 (4):982–988

78.

Mathes

Christmann

Stifano

et al.

Global chemokine expression in systemic sclerosis (SSc): CCL19 expression correlates with vascular inflammation in SSc skin.

Ann Rheum Dis 2014;73 (10):1864–1872

79.

Pickens

Chamberlain

Volin

Pope

Mandelin

II Shahrara

Characterization of CCL19 and CCL21 in rheumatoid arthritis.

Arthritis Rheum 2011;63 (4):914–922

80.

Lalor

Segal

Lymphoid chemokines in the CNS.

J Neuroimmunol 2010;224 1-256–61

81.

Wolff

Burns

Middleton

Rot

Endothelial cell “memory” of inflammatory stimulation: human venular endothelial cells store Interleukin 8 in Weibel-Palade bodies.

J Exp Med 1998;188 (9):1757–1762

82.

Furuse

Fujii

Kaburagi

et al.

Serum concentrations of the CXC chemokines interleukin 8 and growth-regulated oncogene-alpha are elevated in patients with systemic sclerosis.

J Rheumatol 2003;30 (7):1524–1528

83.

Reitamo

Remitz

Varga

et al.

Demonstration of interleukin 8 and autoantibodies to interleukin 8 in the serum of patients with systemic sclerosis and related disorders.

Arch Dermatol 1993;129 (2):189–193

84.

Koch

Kronfeld-Harrington

Szekanecz

et al.

In situ expression of cytokines and cellular adhesion molecules in the skin of patients with systemic sclerosis.

Their role in early and late disease. Pathobiology 1993;61 5-6239–246

85.

Renzoni

Lympany

Sestini

et al.

Distribution of novel polymorphisms of the interleukin-8 and CXC receptor 1 and 2 genes in systemic sclerosis and cryptogenic fibrosing alveolitis.

Arthritis Rheum 2000;43 (7):1633–1640

86.

Carulli

Ong

Ponticos

et al.

Chemokine receptor CCR2 expression by systemic sclerosis fibroblasts: evidence for autocrine regulation of myofibroblast differentiation.

Arthritis Rheum 2005;52 (12):3772–3782

87.

Kadono

Kikuchi

Ihn

Takehara

Tamaki

Increased production of interleukin 6 and interleukin 8 in scleroderma fibroblasts.

J Rheumatol 1998;25 (2):296–301

88.

Gourh

Arnett

Assassi

et al.

Plasma cytokine profiles in systemic sclerosis: associations with autoantibody subsets and clinical manifestations.

Arthritis Res Ther 2009;11 (5):R147

89.

Schmidt

Martinez-Gamboa

Meier

et al.

Bronchoalveoloar lavage fluid cytokines and chemokines as markers and predictors for the outcome of interstitial lung disease in systemic sclerosis patients.

Arthritis Res Ther 2009;11 (4):R111

90.

Hasegawa

Asano

Endo

et al.

Serum chemokine levels as prognostic markers in patients with early systemic sclerosis: a multicenter, prospective, observational study.

Mod Rheumatol 2013;23 (6):1076–1084

91.

Keane

Arenberg

Lynch

III et al.

The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis.

J Immunol 1997;159 (3):1437–1443

92.

Strieter

Belperio

Keane

CXC chemokines in angiogenesis related to pulmonary fibrosis.

Chest 2002;122 6 Suppl298S–301S

93.

Castelino

Varga

Emerging cellular and molecular targets in fibrosis: implications for scleroderma pathogenesis and targeted therapy.

Curr Opin Rheumatol 2014;26 (6):607–614

94.

Aidoudi

Bujakowska

Kieffer

Bikfalvi

The CXC-chemokine CXCL4 interacts with integrins implicated in angiogenesis.

PLoS ONE 2008;3 (7):e2657

95.

Romagnani

Maggi

Mazzinghi

et al.

CXCR3-mediated opposite effects of CXCL10 and CXCL4 on TH1 or TH2 cytokine production.

J Allergy Clin Immunol 2005;116 (6):1372–1379

96.

Manetti

Guiducci

Ibba-Manneschi

Matucci-Cerinic

Mechanisms in the loss of capillaries in systemic sclerosis: angiogenesis versus vasculogenesis.

J Cell Mol Med 2010;14 6a 6A1241–1254

97.

Guiducci

Giacomelli

Cerinic

Vascular complications of scleroderma.

Autoimmun Rev 2007;6 (8):520–523

98.

Distler

Scheid

et al.

Uncontrolled expression of vascular endothelial growth factor and its receptors leads to insufficient skin angiogenesis in patients with systemic sclerosis.

Circ Res 2004;95 (1):109–116

99.

van Bon

Affandi

Broen

et al.

Proteome-wide analysis and CXCL4 as a biomarker in systemic sclerosis.

N Engl J Med 2014;370 (5):433–443

100.

Manetti

Guiducci

Romano

et al.

Overexpression of VEGF165b, an inhibitory splice variant of vascular endothelial growth factor, leads to insufficient angiogenesis in patients with systemic sclerosis.

Circ Res 2011;109 (3):e14–e26

101.

Liu

Battaglia

Lee

Sun

Aster

Visentin

Platelet factor 4 differentially modulates CD4+CD25+ (regulatory) versus CD4+CD25- (nonregulatory) T cells.

J Immunol 2005;174 (5):2680–2686

102.

Volkmann

Tashkin

Roth

et al.

CXCL4 does not predict extent or progression of interstitial lung disease in systemic sclerosis [abstract].

Arthritis Rheumatol 2015;67 Suppl 10). Available from http://acrabstracts.org/abstract/cxcl4-does-not-predict-extent-or-progression-of-interstitial-lung-disease-in-systemic-sclerosis/. Accessed Dec 5, 2016.

103.

Kowal-Bielecka

Kowal

Lewszuk

Bodzenta-Lukaszyk

Walecki

Sierakowski

Beta thromboglobulin and platelet factor 4 in bronchoalveolar lavage fluid of patients with systemic sclerosis.

Ann Rheum Dis 2005;64 (3):484–486

104.

Luster

Ravetch

Biochemical characterization of a gamma interferon-inducible cytokine (IP-10).

J Exp Med 1987;166 (4):1084–1097

105.

Strieter

Polverini

Kunkel

et al.

The functional role of the ELR motif in CXC chemokine-mediated angiogenesis.

J Biol Chem 1995;270 (45):27348–27357

106.

Jinquan

Jing

Jacobi

et al.

CXCR3 expression and activation of eosinophils: role of IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma.

J Immunol 2000;165 (3):1548–1556

107.

Keane

Belperio

Arenberg

et al.

IFN-gamma-inducible protein-10 attenuates bleomycin-induced pulmonary fibrosis via inhibition of angiogenesis.

J Immunol 1999;163 (10):5686–5692

108.

Antonelli

Ferrari

Giuggioli

Ferrannini

Ferri

Fallahi

Chemokine (C-X-C motif) ligand (CXCL)10 in autoimmune diseases.

Autoimmun Rev 2014;13 (3):272–280

109.

Liu

Mayes

Tan

et al.

Correlation of interferon-inducible chemokine plasma levels with disease severity in systemic sclerosis.

Arthritis Rheum 2013;65 (1):226–235

110.

Tiev

Chatenoud

Kettaneh

Tolédano

Bach

Cabane

(Increase of CXCL10 serum level in systemic sclerosis interstitial pneumonia).

Rev Med Interne 2009;30 (11):942–946

111.

Fujii

Shimada

Hasegawa

Takehara

Sato

Serum levels of a Th1 chemoattractant IP-10 and Th2 chemoattractants, TARC and MDC, are elevated in patients with systemic sclerosis.

J Dermatol Sci 2004;35 (1):43–51

112.

Zuber

Spertini

Immunological basis of systemic sclerosis.

Rheumatology (Oxford) 2006;45 Suppl 3iii23–iii25

113.

Kong

Cheema

Keen

Wick

Gershwin

The immunobiology of systemic sclerosis.

Semin Arthritis Rheum 2008;38 (2):132–160

114.

Claman

Giorno

Seibold

Endothelial and fibroblastic activation in scleroderma.

The myth of the uninvolved skin. Arthritis Rheum 1991;34 (12):1495–1501

115.

Annunziato

Romagnani

Heterogeneity of human effector CD4+ T cells.

Arthritis Res Ther 2009;11 (6):257

116.

Talwar

Arun

Kumar

Clements

Plasticity of T helper cell subsets: Implications in periodontal disease.

J Indian Soc Periodontol 2013;17 (3):288–291

117.

Liu

Gao

et al.

Elevated levels of CD4(+)CD25(+)FoxP3(+) T cells in systemic sclerosis patients contribute to the secretion of IL-17 and immunosuppression dysfunction.

PLoS ONE 2013;8 (6):e64531

118.

Mavalia

Scaletti

Romagnani

et al.

Type 2 helper T-cell predominance and high CD30 expression in systemic sclerosis.

Am J Pathol 1997;151 (6):1751–1758

119.

Petit-Frere

Dugas

Braquet

Mencia-Huerta

Interleukin-9 potentiates the interleukin-4-induced IgE and IgG1 release from murine B lymphocytes.

Immunology 1993;79 (1):146–151

120.

Antonelli

Ferri

Fallahi

et al.

CXCL10 (alpha) and CCL2 (beta) chemokines in systemic sclerosisa longitudinal study.

Rheumatology (Oxford) 2008;47 (1):45–49

121.

Kaplanski

Marin

Montero-Julian

Mantovani

Farnarier

IL-6: a regulator of the transition from neutrophil to monocyte recruitment during inflammation.

Trends Immunol 2003;24 (1):25–29

122.

Kuscher

Danelon

Paoletti

et al.

Synergy-inducing chemokines enhance CCR2 ligand activities on monocytes.

Eur J Immunol 2009;39 (4):1118–1128

123.

Gouwy

Schiraldi

Struyf

Van Damme

Uguccioni

Possible mechanisms involved in chemokine synergy fine tuning the inflammatory response.

Immunol Lett 2012;145 1-210–14

124.

Hasegawa

Biomarkers in systemic sclerosis: Their potential to predict clinical courses.

J Dermatol 2016;43 (1):29–38