Histopathological cutaneous alterations in systemic sclerosis: a clinicopathological study

Introduction  

The aims of the present study were to identify histopathological parameters which are linked to local clinical skin disease at two distinct anatomical sites in systemic sclerosis (SSc) patients with skin involvement (limited cutaneous systemic sclerosis (lcSSc) or diffuse cutaneous systemic sclerosis (dcSSc)) and to determine the sensitivity of SSc specific histological alterations, focusing on SSc patients without clinical skin involvement (limited SSc (lSSc)).     

B cell depletion in diffuse progressive systemic sclerosis: safety,skin score modification and IL-6 modulation in an up to thirty-six months follow-up open-label trial

Introduction  

An over-expression of CD19 has been shown in B cells of systemic sclerosis (SSc) and B cells are thought to contribute to the induction of skin fibrosis in the tight skin mouse model. The aim was to define the outcome on safety and the change in skin score after rituximab therapy in SSc patients and to correlate the clinical characteristics with the levels of interleukin (IL)-6 and with the immune cell infiltrate detected by immunohistochemistry.     

Chronic Toll-like receptor 4 stimulation in skin induces inflammation,macrophage activation,transforming growth factor beta signature gene expression,and fibrosis

Introduction

The crucial role of innate immunity in the pathogenesis of systemic sclerosis (SSc) is well established, and in the past few years the hypothesis that Toll-like receptor 4 (TLR4) activation induced by endogenous ligands is involved in fibrogenesis has been supported by several studies on skin, liver, and kidney fibrosis. These findings suggest that TLR4 activation can enhance transforming growth factor beta (TGF-β) signaling, providing a potential mechanism for TLR4/Myeloid differentiation factor 88 (MyD88)-dependent fibrosis.

Methods

The expression of TLR4, CD14 and MD2 genes was analyzed by real-time polymerase chain reaction from skin biopsies of 24 patients with diffuse cutaneous SSc. In order to investigate the effects of the chronic skin exposure to endotoxin (Lipopolysaccharide (LPS)) in vivo we examined the expression of inflammation, TGF-β signaling and cellular markers genes by nanostring. We also identified cellular subsets by immunohistochemistry and flow cytometry.

Results

We found that TLR4 and its co-receptors, MD2 and CD14, are over-expressed in lesional skin from patients with diffuse cutaneous SSc, and correlate significantly with progressive or regressive skin disease as assessed by the Delta Modified Rodnan Skin Score. In vivo, a model of chronic dermal LPS exposure showed overexpression of proinflammatory chemokines, recruitment and activation of macrophages, and upregulation of TGF-β signature genes.

Conclusions

We delineated the role of MyD88 as necessary for the induction not only for the early phase of inflammation, but also for pro-fibrotic gene expression via activation of macrophages. Chronic LPS exposure might be a model of early stage of SSc when inflammation and macrophage activation are important pathological features of the disease, supporting a role for innate immune activation in SSc skin fibrosis.     

A multiparameter approach to monitor disease activity in collagen-induced arthritis

Introduction

Recent accumulating evidence indicates a crucial involvement of macrophage lineage in the pathogenesis of systemic sclerosis (SSc). To analyze the assembly of the monocyte/macrophage population, we evaluated the expression of CD163 and CD204 and various activated macrophage markers, in the inflammatory cells of the skin and in the peripheral blood mononuclear cells (PBMCs) derived from patients with SSc.

Methods

Skin biopsy specimens from 6 healthy controls and 10 SSc patients (7 limited cutaneous SSc and 3 diffuse cutaneous SSc) were analyzed by immunohistochemistry using monoclonal antibody against CD68 (pan-macrophage marker), CD163 and CD204. Surface and/or intracellular protein expression of CD14 (marker for monocyte lineage), CD163 and CD204 was analysed by flow cytometry in PBMCs from 16 healthy controls and 41 SSc patients (26 limited cutaneous SSc and 15 diffuse cutaneous SSc). Statistical analysis was carried out using Mann-Whitney U test for comparison of means.

Results

In the skin from SSc patients, the number of CD163⁺ cells or CD204⁺ cells between the collagen fibers was significantly larger than that in healthy controls. Flow cytometry showed that the population of CD14⁺ cells was significantly greater in PBMCs from SSc patients than that in healthy controls. Further analysis of CD14⁺ cells in SSc patients revealed higher expression of CD163 and the presence of two unique peaks in the CD204 histogram. Additionally, we found that the CD163⁺ cells belong to CD14^brightCD204⁺ population.

Conclusions

This is the first report indicating CD163⁺ or CD204⁺ activated macrophages may be one of the potential fibrogenic regulators in the SSc skin. Furthermore, this study suggests a portion of PBMCs in SSc patients abnormally differentiates into CD14^brightCD163⁺CD204⁺ subset. The subset specific to SSc may play an important role in the pathogenesis of this disease, as the source of CD163⁺ or CD204⁺ macrophages in the skin.     

Characterization of monocyte/macrophage subsets in the skin and peripheral blood derived from patients with systemic sclerosis

Introduction

Recent accumulating evidence indicates a crucial involvement of macrophage lineage in the pathogenesis of systemic sclerosis (SSc). To analyze the assembly of the monocyte/macrophage population, we evaluated the expression of CD163 and CD204 and various activated macrophage markers, in the inflammatory cells of the skin and in the peripheral blood mononuclear cells (PBMCs) derived from patients with SSc.

Methods

Skin biopsy specimens from 6 healthy controls and 10 SSc patients (7 limited cutaneous SSc and 3 diffuse cutaneous SSc) were analyzed by immunohistochemistry using monoclonal antibody against CD68 (pan-macrophage marker), CD163 and CD204. Surface and/or intracellular protein expression of CD14 (marker for monocyte lineage), CD163 and CD204 was analysed by flow cytometry in PBMCs from 16 healthy controls and 41 SSc patients (26 limited cutaneous SSc and 15 diffuse cutaneous SSc). Statistical analysis was carried out using Mann-Whitney U test for comparison of means.

Results

In the skin from SSc patients, the number of CD163⁺ cells or CD204⁺ cells between the collagen fibers was significantly larger than that in healthy controls. Flow cytometry showed that the population of CD14⁺ cells was significantly greater in PBMCs from SSc patients than that in healthy controls. Further analysis of CD14⁺ cells in SSc patients revealed higher expression of CD163 and the presence of two unique peaks in the CD204 histogram. Additionally, we found that the CD163⁺ cells belong to CD14^brightCD204⁺ population.

Conclusions

This is the first report indicating CD163⁺ or CD204⁺ activated macrophages may be one of the potential fibrogenic regulators in the SSc skin. Furthermore, this study suggests a portion of PBMCs in SSc patients abnormally differentiates into CD14^brightCD163⁺CD204⁺ subset. The subset specific to SSc may play an important role in the pathogenesis of this disease, as the source of CD163⁺ or CD204⁺ macrophages in the skin.     

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

Introduction  

Systemic sclerosis (SSc) is characterized by fibrosis and microvascular abnormalities including dysregulated angiogenesis. Chemokines, in addition to their chemoattractant properties, have the ability to modulate angiogenesis. Chemokines lacking the enzyme-linked receptor (ELR) motif, such as monokine induced by interferon-γ (IFN-γ) (MIG/CXCL9) and IFN-inducible protein 10 (IP-10/CXCL10), inhibit angiogenesis by binding CXCR3. In addition, CXCL16 promotes angiogenesis by binding its unique receptor CXCR6. In this study, we determined the expression of these chemokines and receptors in SSc skin and serum.     

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

Introduction  

Systemic sclerosis (SSc) (scleroderma) is a complex autoimmune disease that clinically manifests as progressive fibrosis of the skin and internal organs. Anti-centromere antibodies (ACAs), anti-topoisomerase antibodies (ATAs), and anti-RNA polymerase III antibodies (ARAs) are three mutually exclusive SSc-associated autoantibodies that correlate with distinct clinical subsets characterized by extent of cutaneous involvement and pattern of organ involvement. The current report sought to determine whether plasma cytokine profiles differ in SSc patients grouped according to these SSc-associated autoantibody subsets.     

Enhanced angiogenic potency of monocytic endothelial progenitor cells in patients with systemic sclerosis

Introduction  

Microvasculopathy is one of the characteristic features in patients with systemic sclerosis (SSc), but underlying mechanisms still remain uncertain. In this study, we evaluated the potential involvement of monocytic endothelial progenitor cells (EPCs) in pathogenic processes of SSc vasculopathy, by determining their number and contribution to blood vessel formation through angiogenesis and vasculogenesis.     

Levels of adiponectin,a marker for PPAR-gamma activity,correlate with skin fibrosis in systemic sclerosis: potential utility as biomarker?

Introduction

Progressive fibrosis in systemic sclerosis (SSc) is linked to aberrant transforming growth factor beta (TGF-beta) signaling. Peroxisome proliferator-activated receptor gamma (PPAR-gamma) blocks fibrogenic TGF-beta responses in vitro and in vivo. Reduced expression and function of PPAR-gamma in patients with SSc may contribute to progression of fibrosis. Here we evaluated the levels of adiponectin, a sensitive and specific index of PPAR-gamma activity, as a potential fibrogenic biomarker in SSc.

Methods

Adiponectin levels were determined in the sera of 129 patients with SSc and 86 healthy controls, and serial determinations were performed in 27 patients. Levels of adiponectin mRNA in skin biopsies from SSc patients were assessed in an expression profiling microarray dataset. Regulation of adiponectin gene expression in explanted human subcutaneous preadipocytes and fibroblasts was examined by real-time quantitative PCR.

Results

Patients with diffuse cutaneous SSc had reduced serum adiponectin levels. A significant inverse correlation between adiponectin levels and the modified Rodnan skin score was observed. In longitudinal studies changes in serum adiponectin levels were inversely correlated with changes in skin fibrosis. Skin biopsies from a subset of SSc patients showed reduced adiponectin mRNA expression which was inversely correlated with the skin score. An agonist ligand of PPAR-gamma potently induced adiponectin expression in explanted mesenchymal cells in vitro.

Conclusions

Levels of adiponectin, reflecting PPAR-gamma activity, are correlated with skin fibrosis and might have potential utility as a biomarker in SSc.     

CD109, a TGF-�� co-receptor, attenuates extracellular matrix production in scleroderma skin fibroblasts

Introduction

Scleroderma or systemic sclerosis (SSc) is a complex connective tissue disease characterized by fibrosis of skin and internal organs. Transforming growth factor beta (TGF-β) plays a key role in the pathogenesis of SSc fibrosis. We have previously identified CD109 as a novel TGF-β co-receptor that inhibits TGF-β signaling. The aim of the present study was to determine the role of CD109 in regulating extracellular matrix (ECM) production in human SSc skin fibroblasts.

Methods

CD109 expression was determined in skin tissue and cultured skin fibroblasts of SSc patients and normal healthy subjects, using immunofluorescence, western blot and RT-PCR. The effect of CD109 on ECM synthesis was determined by blocking CD109 expression using CD109-specific siRNA or addition of recombinant CD109 protein, and analyzing the expression of ECM components by western blot.

Results

The expression of CD109 proteinis markedly increased in SSc skin tissue in vivo and in SSc skin fibroblasts in vitro as compared to their normal counterparts. Importantly, both SSc and normal skin fibroblasts transfected with CD109-specific siRNA display increased fibronectin, collagen type I and CCN2 protein levels and enhanced Smad2/3 phosphorylation compared with control siRNA transfectants. Furthermore, addition of recombinant CD109 protein decreases TGF-β₁-induced fibronectin, collagen type I and CCN2 levels in SSc and normal fibroblasts.

Conclusion

The upregulation of CD109 protein in SSc may represent an adaptation or consequence of aberrant TGF-β signaling in SSc. Our finding that CD109 is able to decrease excessive ECM production in SSc fibroblasts suggest that this molecule has potential therapeutic value for the treatment of SSc.     

Increased frequency of circulating Th22 in addition to Th17 and Th2 lymphocytes in systemic sclerosis: association with interstitial lung disease

Introduction  

T cell abnormalities have been associated with the pathogenesis of systemic sclerosis (SSc). Recently, besides T helper (Th)17 cells, the Th22 subset has been identified in humans. Our purpose was to investigate the pattern of cytokines produced and chemokine-receptors expressed by peripheral blood (PB) Th cells in SSc and healthy donors (HD) focusing on cells producing interleukin (IL)-17 and IL-22 and to identify specific clinical associations.     

Differentiation potential of CD14+ monocytes into myofibroblasts in patients with systemic sclerosis

Background

Circulating monocytes are a highly plastic and functionally heterogeneic cell type with an activated phenotype in patients with systemic sclerosis (SSc). CD14⁺ monocytes have the potential to differentiate into extra-cellular matrix (ECM) producing cells, possibly participating in fibrogenesis.

Aim

To study the effect of GM-CSF, IL-4 and endothelin -1 (ET-1) alone or in combination on monocyte differentiation into myofibroblasts.

Methods

CD14⁺ cells were isolated from peripheral blood from 14 SSc patients and healthy controls by positive selection and incubated with different combinations of GM-CSF, IL-4 and ET-1 for 14 days. Type-1 collagen and α-SMA were detected by Western blot, qPCR and confocal microscopy. HLA-DR, CD11c and CD14 expression was analysed by flow cytometry. A collagen gel contraction assay was performed for functional myofibroblast assessment.

Results

GM-CSF both induced collagen and α-SMA expression after 14 days. ET-1 further increased GM-CSF-induced collagen expression in a dose dependent manner up to 30-fold. IL-4/GM-CSF combination leads to a more DC-like phenotype of monocytes associated with reduced collagen and α-SMA expression compared to GM-CSF alone. Collagen and α-SMA expression was higher in monocytes from SSc patients and monocytes were more prone to obtain a spindle form. In contrast to controls, ET-1 and IL-4 alone were sufficient to induce α-SMA expression in monocytes from SSc patients. Despite the induction of α-SMA expression, monocyte-derived myofibroblasts only had a moderate capability of contraction in functional analyses.

Conclusion

SSc monocytes display increased maturation towards myofibroblasts demonstrated by their phenotype and α-SMA expression when compared to monocytes from healthy controls, however only with minor functional contraction properties.     

Tumour necrosis factor-α production in fibrosing alveolitis is macrophage subset specific

Background  

Previous studies have revealed that tumour necrosis factor (TNF)-α is upregulated in fibrosing alveolitis (FA) in humans. The aim of this study was to compare the TNF-α secretory profile of alveolar macrophages (AMs) and peripheral blood monocytes (Mos) of patients with cryptogenic FA and systemic sclerosis (SSc), a rheumatological disorder in which lung fibrosis can occur. In particular, we wished to assess whether TNF-α levels differ between SSc patients with FA (FASSc) and a nonfibrotic group.     

Early systemic sclerosis: short-term disease evolution and factors predicting the development of new manifestations of organ involvement

Introduction

We investigated early systemic sclerosis (SSc) (that is, Raynaud''s phenomenon with SSc marker autoantibodies and/or typical capillaroscopic findings and no manifestations other than puffy fingers or arthritis) versus undifferentiated connective tissue disease (UCTD) to identify predictors of short-term disease evolution.

Methods

Thirty-nine early SSc and 37 UCTD patients were investigated. At baseline, all patients underwent clinical evaluation, B-mode echocardiography, lung function tests and esophageal manometry to detect preclinical alterations of internal organs, and were re-assessed every year. Twenty-one early SSc and 24 UCTD patients, and 25 controls were also investigated for serum endothelial, T-cell and fibroblast activation markers.

Results

At baseline, 48.7% of early SSc and 37.8% of UCTD patients had at least one preclinical functional alteration (P > 0.05). Ninety-two percent of early SSc patients developed manifestations consistent with definite SSc (that is, skin sclerosis, digital ulcers/scars, two or more teleangectasias, clinically visible nailfold capillaries, cutaneous calcinosis, X-ray bibasilar lung fibrosis, X-ray esophageal dysmotility, ECG signs of myocardial fibrosis and laboratory signs of renal crisis) within five years versus 17.1% of UCTD patients (X² = 12.26; P = 0.0005). Avascular areas (HR = 4.39 95% CI 1.18 to 16.3; P = 0.02), increased levels of soluble IL-2 receptor alpha (HR = 4.39; 95% CI 1.03 to 18.6; P = 0.03), and of procollagen III aminopropeptide predicted disease evolution (HR = 4.55; 95% CI 1.18 to 17; P = 0.04).

Conclusion

Most early SSc but only a few UCTD patients progress to definite SSc within a short-term follow-up. Measurement of circulating markers of T-cell and fibroblast activation might serve to identify early SSc patients who are more likely to develop features of definite SSc.     

Cartilage oligomeric matrix protein-induced complement activation in systemic sclerosis

Introduction

Complexes between cartilage oligomeric matrix protein (COMP) and the complement activation product C3b have been found in the circulation of patients with rheumatoid arthritis and systemic lupus erythematosus. In systemic sclerosis (SSc) COMP expression in the skin is upregulated both in lesional and non-lesional skin, which is also reflected in an increased amount of circulating COMP. We investigated the presence of COMP-C3b complexes in serum and skin biopsies of patients with SSc.

Methods

The presence of COMP and COMP-C3b complexes in the serum of 80 patients with limited cutaneous SSc (lcSSc, n = 40) and diffuse cutaneous SSc (dcSSc, n = 40) and 97 healthy controls was measured by ELISA and correlated to different clinical parameters. Samples were collected both at baseline and after three to five years to assess longitudinal changes in COMP-C3b complex levels. Furthermore, skin biopsies from seven patients with dcSSc and three healthy controls were analyzed for expression of COMP and deposition of C3b and IgG.

Results

Serum levels of COMP-C3b were found to be elevated in both dcSSc and lcSSc compared to healthy controls and decreased at the second measurement in patients on immunosuppressive therapy. No co-localization of COMP and C3b was found in the skin biopsies, indicating that the COMP-C3b complexes are formed upon release of COMP into the circulation.

Conclusion

COMP-C3b complexes are found in the serum of patients with SSc. The lack of co-localization between COMP and C3b in the skin suggests that COMP does not drive complement activation in the skin in SSc.     

Elevated Levels of CD4+CD25+FoxP3+ T Cells in Systemic Sclerosis Patients Contribute to the Secretion of IL-17 and Immunosuppression Dysfunction

Objective

Immune imbalance between regulatory T (Treg) and Th17 cells is a characteristic of systemic sclerosis (SSc). The functional heterogeneity among Treg can be elucidated by separating Treg into different subsets based on the expression of FoxP3 and CD45RA. The aim of this study was to investigate the role of Treg subsets in the immune imbalance in naïve SSc.

Methods

Peripheral blood mononuclear cells (PBMCs) of 31 SSc patients and 33 healthy controls were analyzed for the expression of CD4, CD25, CD45RA, CTLA-4, FoxP3, and IL-17 using flow cytometry. Treg immunesuppression capacity was measured in co-culture experiments. The expression of FoxP3, CTLA-4, IL-17A, and RORC mRNA was measured by real-time PCR.

Results

The frequency of CD4⁺CD25⁺FoxP3⁺ Treg cells was significantly elevated in patients with SSc (3.62±1.14 vs 1.97±0.75, p<0.001) with diminished immunosuppression capacity. In SSc, the proportion of FoxP3^highCD45RA⁻ activated Treg cells (aTreg) was decreased, the proportion of FoxP3^lowCD45RA⁻ T cells was increased, and the proportion of FoxP3^lowCD45RA⁺ resting Treg cells (rTreg) was decreased. The immune suppression capacity of aTreg and rTreg was diminished, while FoxP3^lowCD45RA⁻ T cells exhibited a lack of suppression capacity. The immune dysfunction of aTreg was accompanied by the abnormal expression of CTLA-4. Th17 cell numbers were elevated in SSc, FoxP3^lowCD45RA⁻ T cells produced IL-17, confirming their Th17 potential, which was consistent with the elevated levels of FoxP3⁺IL-17⁺ cells in SSc.

Conclusion

A decrease in aTreg levels, along with functional deficiency, and an increase in the proportion of FoxP3^lowCD45RA⁻ T cells, was the reason for the increase in dysfunctional Treg in SSc patients, potentially causing the immune imbalance between Treg and Th17 cells.     

Prevalence, correlates and clinical usefulness of antibodies to RNA polymerase III in systemic sclerosis: a cross-sectional analysis of data from an Australian cohort

Introduction

The prevalence of antibodies to RNA polymerase III (anti-RNAP) differs among systemic sclerosis (SSc) cohorts worldwide. Previously reported associations of anti-RNAP include diffuse cutaneous disease, tendon friction rubs and renal crisis, with recent reports suggesting a close temporal association between malignancy and SSc disease onset among patients with anti-RNAP.

Methods

Patients with SSc were tested for the presence of anti-RNAP at recruitment into the Australian Scleroderma Cohort Study. We used univariate and multivariable methods to identify and quantify clinical and laboratory correlates of anti-RNAP in SSc. Diagnostic testing procedures were used to determine the usefulness of these antibodies in estimating the likelihood of clinically important outcomes.

Results

There were 451 patients with mean ± standard deviation age and disease duration at recruitment of 58.1 ± 12.4 and 11.6 ± 10.0 years, respectively; 151 (33.5%) patients were recruited within 5 years of diagnosis of SSc. Overall, 69 (15.3%) patients had anti-RNAP. Univariate associations of anti-RNAP were diffuse disease (75.4% vs. 20.9%, P < 0.0001), joint contractures (73.9% vs. 30.1%, P < 0.0001), greater highest-recorded modified Rodnan skin score (20.6 ± 12.4 vs. 10.1 ± 7.9, P < 0.0001), synovitis (31.9% vs. 19.9%, P = 0.03), myositis (2.9% vs. 0.5%, P = 0.05), systemic hypertension (59.4% vs. 39.7%, P = 0.002), renal crisis (24.6% vs. 1.8%, P < 0.0001) and malignancy diagnosed within 5 years of onset of SSc skin disease (13.3% vs. 3.9%, P = 0.01). In multiple regression analysis, after adjustment for other covariates, anti-RNAP were independently associated with renal crisis (odds ratio (OR) 3.8, 95% confidence interval (CI) 1.2 to 11.5, P = 0.02; positive predictive value (PPV) 24.6%, negative predictive value (NPV) 98.2%), diffuse disease (OR 6.4, 95% CI 2.9 to 13.8, P < 0.0001; PPV 75.4%, NPV 20.9%), joint contractures (OR 2.5, 95% CI 1.2 to 5.3, P = 0.02; PPV 73.9%, NPV 69.9%) and malignancy diagnosed within 5 years of onset of SSc skin disease (OR 4.2, 95% CI 1.3 to 13.4, P = 0.01; PPV 13.3%, NPV 96.1%).

Conclusions

Anti-RNAP status is a clinically useful prognostic marker in SSc and enables clinicians to identify patients at high risk of developing renal crisis, synovitis, myositis and joint contractures. Patients with anti-RNAP also have an increased risk of malignancy within a 5-year timeframe before or after onset of SSc skin changes.     

Decreased expression of the endothelial cell-derived factor EGFL7 in systemic sclerosis: potential contribution to impaired angiogenesis and vasculogenesis

Introduction

Microvascular damage and defective angiogenesis and vasculogenesis have a major role in the pathogenesis of systemic sclerosis (SSc). Epidermal growth factor-like domain 7 (EGFL7) is a proangiogenic molecule which is predominantly expressed and secreted by endothelial cells and their progenitors and controls vascular development and integrity. In this study, we investigated the possible involvement of EGFL7 in SSc.

Methods

Serum EGFL7 levels from 60 patients with SSc and 35 age- and sex-matched healthy controls were examined by colorimetric sandwich enzyme-linked immunosorbent assay. The expression of EGFL7 in forearm skin biopsies (n = 16 SSc, n = 10 controls), cultured dermal microvascular endothelial cells (MVECs) (n = 3 SSc, n = 3 controls) and late-outgrowth peripheral blood endothelial progenitor cell (EPC)-derived endothelial cells (n = 15 SSc, n = 8 controls) was investigated by immunofluorescence and Western blotting.

Results

Serum EGFL7 levels were detectable in 68.6% of healthy controls and 45% of SSc cases (P < 0.05). Circulating levels of EGFL7 were significantly decreased in SSc patients compared with healthy controls (P = 0.01). Serum levels of EGFL7 were significantly lower in both limited cutaneous SSc and diffuse cutaneous SSc patients than in controls (P = 0.02 and P = 0.04, respectively). In SSc, decreased serum EGFL7 levels were significantly correlated with the severity of nailfold capillary abnormalities. Patients with the most severe capillary changes and digital ulcers had serum EGFL7 levels significantly lower than healthy controls, while the EGFL7 levels did not differ significantly between controls and SSc patients with less capillary damage and lack of digital ulcers. Endothelial EGFL7 expression was strongly downregulated or even almost completely undetectable in SSc-affected dermis compared with controls (P < 0.001). In cultured SSc dermal MVECs and late-outgrowth peripheral blood EPC-derived endothelial cells, EGFL7 was significantly downregulated compared with cells obtained from healthy subjects (P < 0.01 and P < 0.001, respectively).

Conclusions

Our findings suggest that the loss of EGFL7 expression in endothelial cells and their progenitors might play a role in the development and progression of peripheral microvascular damage and the defective vascular repair process characteristic of SSc.     

Functional,radiological and biological markers of alveolitis and infections of the lower respiratory tract in patients with systemic sclerosis

Background

A progressive lung disease and a worse survival have been observed in patients with systemic sclerosis and alveolitis. The objective of this study was to define the functional, radiological and biological markers of alveolitis in SSc patients.

Methods

100 SSc patients (76 with limited and 24 with diffuse disease) underwent a multistep assessment of cardiopulmonary system: pulmonary function tests (PFTs) every 6–12 months, echocardiography, high resolution computed tomography (HRCT) and bronchoalveolar lavage (BAL), if clinically advisable. Alveolar and interstitial scores on HRCT and IL-6 plasma levels were also assessed as lung disease activity indices.

Results

90 SSc patients with abnormal PFTs and 3 with signs and/or symptoms of lung involvement and normal PFTs underwent HRCT and echocardiography. HRCT revealed evidence of fibrosis in 87 (93.5%) patients, with 55 (59.1%) showing both ground glass attenuation and fibrosis. In 42 patients who had exhibited ground glass on HRCT and consented to undergo BAL, 16 (38.1%) revealed alveolitis. 12 (75%) of these patients had restrictive lung disease (p < 0.0001) and presented diffuse skin involvement (p = 0.0009). IL-6 plasma levels were higher in patients with alveolitis than in patients without (p = 0.041). On logistic regression model the best independent predictors of alveolitis were diffuse skin involvement (OR(95%CIs):12.80(2.54–64.37)) and skin score > 14 (OR(95%CIs):7.03(1.40–34.33)). The alveolar score showed a significant correlation with IL-6 plasma levels (r = 0.36, p = 0.001) and with the skin score (r = 0.33, p = 0.001). Cultures of BAL fluid resulted positive in 10 (23.8%) of the 42 patients that underwent BAL and after one year a deterioration in PFTs occurred in 8 (80%) of these patients (p = 0.01). Pulmonary artery systolic pressure ≥ 40 mmHg was found in 6 (37.5%) patients with alveolitis.

Conclusion

We found alveolitis only in 38.1% of the patients who had exhibited ground glass on HRCT and then underwent BAL, probably because the concomitant fibrosis influenced results. A diffuse skin involvement and a restrictive pattern on PFTs together with ground glass on HRCT were judged possible markers of alveolitis, a BAL examination being indicated as the next step. Nevertheless BAL would be necessary to detect any infections of the lower respiratory tract that may cause further deterioration in lung function.