Blood Of Systemic Sclerosis Patients Research Articles

Increased oxidative stress response in circulating blood of systemic sclerosis patients – relation to disease characteristics and inflammatory blood biomarkers

BackgroundSystemic sclerosis (SSc) is a rare connective tissue disorder of unknown etiology characterized by organ fibrosis and microcirculation dysfunction. Emerging evidence suggests that SSc is related to increased oxidative stress, which contributes to further tissue and vascular damage. MethodsOxidative stress response in the peripheral blood was assessed in patients with SSc (n = 55) and well-matched controls (n = 44) using real-time monitoring of protein hydroperoxide (HP) formation by the coumarin boronic acid (CBA) assay. We also analyzed the relationship between HP generation and SSc clinics, systemic inflammation, and cellular fibronectin, an emerging biomarker of endothelial damage. ResultsSSc was characterized by a significantly faster (2-fold) fluorescent product generation in the CBA assay and higher cumulative HP formation (3-fold) compared to controls (p<0.001, both). The dynamics of HP generation were not associated with the form of the disease (diffuse vs. limited SSc), current immunosuppressive therapy use, presence of abnormal nailfold capillaries, and autoantibody profile. Still, it was enhanced in patients with more severe illness and certain clinical manifestations (i.e., pulmonary hypertension, digital ulcers, and cyclophosphamide treatment) and in smokers (current or past). Higher serum CRP, blood eosinophil count, and cellular fibronectin with lower hemoglobin levels were independent determinants of increased HP formation. ConclusionsOur data indicate a pro-oxidant imbalance in SSc, likely related to systemic inflammation and endothelial injury. However, extensive prospective studies are needed to verify whether it is also associated with clinical disease progression.

Peripheral blood IL-6 levels in systemic sclerosis patients: Correlation between IL-6 levels and clinical phenotypes.

Detect the expression of Interleukin-6 (IL-6) in the serum of patients with systemic sclerosis (SSc) and assess its association with clinical and laboratory features of the disease. The clinical data from 50 patients with SSc in the affiliated hospital of Xuzhou Medical University, China were retrospectively analyzed. The level of IL-6 in peripheral blood of systemic sclerosis patients was compared between the different clinical phenotypes groups. Spearman correlation test was used to analyze the correlation of the IL-6 with C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), creatinine, and cystatin C. Interleukin-6 increased in the disease course ≥5 years, systemic sclerosis-Interstitial lung disease (SSc-ILD), pulmonary arterial hypertension (PAH), gastrointestinal involvement, and cardiac involvement group compared with the group with disease course <5 years, no SSc-ILD, PAH, gastrointestinal involvement, and no cardiac involvement group. The differences were statistically significant (p< 0.05). Correlation analysis showed that IL-6 in the group with disease course ≥5 years had a positive correlation with ESR (Rs=0.438, p=0.022) and CRP (Rs=0.825, P < 0.001), whereas it was negatively correlated with creatinine (Rs=-0.481, p=0.011). Nevertheless, when it came to disease course <5 years, the figures had no statistically significant difference. Serum IL-6 in patients with systemic sclerosis is related to different clinical phenotypes. IL-6 helps to diagnose various phases of diseases, monitor severities of diseases, and predict the prognosis of patients.

Platelet Phagocytosis via P-selectin Glycoprotein Ligand 1 and Accumulation of Microparticles in Systemic Sclerosis.

It is unclear why activated platelets and platelet-derived microparticles (MPs) accumulate in the blood of patients with systemic sclerosis (SSc). This study was undertaken to investigate whether defective phagocytosis might contribute to MP accumulation in the blood of patients with SSc. Blood samples were obtained from a total of 81 subjects, including 25 patients with SSc and 26 patients with stable coronary artery disease (CAD). Thirty sex- and age-matched healthy volunteers served as controls. Studies were also conducted in NSG mice, in which the tail vein of the mice was injected with MPs, and samples of the lung parenchyma were obtained for analysis of the pulmonary microvasculature. Tissue samples from human subjects and from mice were assessed by flow cytometry and immunochemical analyses for determination of platelet-neutrophil interactions, phagocytosis, levels and distribution of P-selectin, P-selectin glycoprotein ligand 1 (PSGL-1), and HMGB1 on platelets and MPs, and concentration of byproducts of neutrophil extracellular trap (NET) generation/catabolism. Activated P-selectin+ platelets and platelet-derived HMGB1+ MPs accumulated in the blood of SSc patients but not in the blood of healthy controls. Patients with CAD, a vasculopathy independent of systemic inflammation, had fewer P-selectin+ platelets and a negligible number of MPs. The expression of the receptor for P-selectin, PSGL-1, in neutrophils from SSc patients was significantly decreased, raising the possibility that phagocytes in SSc do not recognize activated platelets, leading to a failure of phagocytosis and continued neutrophil release of MPs. As evidence of this process, activated platelets were not detected in the neutrophils from SSc patients, whereas they were consistently present in the neutrophils from patients with CAD. HMGB1+ MPs elicited generation of NETs, which were only detected in the plasma of SSc patients. In mice, P-selectin-PSGL-1 interaction resulted in platelet phagocytosis in vitro and influenced the ability of MPs to elicit NETs, endothelial activation, and migration of leukocytes through the pulmonary microvasculature. The clearance of activated platelets via PSGL-1 limits the undesirable effects of MP-elicited neutrophil activation. This balance is disrupted in patients with SSc. Its reconstitution might curb vascular inflammation and prevent fibrosis.

Blood Monocyte Subsets are Dysregulated and Respond with High IL-6 Secretion in Patients with Systemic Sclerosis

Abstract Background Most of the blood monocytes are classical monocytes (CM), minor subpopulations are intermediate monocytes (IM) and non-classical monocytes (NCM). CD56+ monocytes are a subpopulation of CM. Aim of the study was to analyze monocyte subpopulations in Systemic Sclerosis (SSc) patients and to evaluate their cytokine production potential. Methods 25 SSc patients and 16 age-matched healthy controls were included in the study. Monocyte subpopulations were analyzed by flow cytometry (antibody staining of CD14, CD16, CD56). Magnetic bead separation was used to isolate monocytes. The cells were stimulated with TLR4 ligand LPS and TLR8 ligand ssRNA40 and cytokines were measured by ELISA. Results The absolute number of circulating monocytes was significantly increased in SSc patients compared to healthy controls (684/μl ± 61 vs. 512/μl ± 38, p=0.033). Calculated absolute number of CM and CD56+ monocytes was not different between SSc patients and controls. In contrast, the calculated absolute numbers of IM (72/μl ± 13 vs. 32/μl ± 5, p=0.0003) and NCM was increased in SSc patients compared to controls (97/μl ± 12 vs. 45/μl ± 8, p=0.0011). The cytokine response of SSc monocytes to TLR4 ligand LPS was equal to control monocytes but SSc monocytes produced significantly more IL-6 in response to TLR8 ligand ssRNA40 than control monocytes. In addition, SSc monocytes already secrete IL-6 without further stimulation. IL-6 was also increased in the serum of SSc patients compared to healthy controls. Conclusion Total monocytes are expanded in the blood of SSc patients, and this increase is caused by an expansion of IM and NCM. The monocytes might contribute to the presence of IL-6 in SSc patients due to their increased potential to secrete this cytokine.

Treg/Th17 cell balance and phytohaemagglutinin activation of T lymphocytes in peripheral blood of systemic sclerosis patients.

AIMTo investigate T-cell activation, the percentage of peripheral T regulatory cells (Tregs), Th17 cells and the circulating cytokine profile in systemic sclerosis (SSc).METHODSWe enrolled a total of 24 SSc patients and 16 healthy controls in the study and divided the patients as having diffuse cutaneous SSc (dcSSc, n = 13) or limited cutaneous SSc (lcSSc, n = 11). We performed a further subdivision of the patients regarding the stage of the disease - early, intermediate or late. Peripheral venous blood samples were collected from all subjects. We performed flow cytometric analysis of the activation capacity of T-lymphocytes upon stimulation with PHA-M and of the percentage of peripheral Tregs and Th17 cells in both patients and healthy controls. We used ELISA to quantitate serum levels of human interleukin (IL)-6, IL-10, tissue growth factor-β1 (TGF-β1), and IL-17A.RESULTSWe identified a decreased percentage of CD3+CD69+ cells in PHA-stimulated samples from SSc patients in comparison with healthy controls (13.35% ± 2.90% vs 37.03% ± 2.33%, P < 0.001). However, we did not establish a correlation between the down-regulated CD3+CD69+ cells and the clinical subset, nor regarding the stage of the disease. The activated CD4+CD25+ peripheral lymphocytes were represented in decreased percentage in patients when compared to controls (6.30% ± 0.68% vs 9.36% ± 1.08%, P = 0.016). Regarding the forms of the disease, dcSSc patients demonstrated lower frequency of CD4+CD25+ T cells against healthy subjects (5.95% ± 0.89% vs 9.36% ± 1.08%, P = 0.025). With regard to Th17 cells, our patients demonstrated increased percentage in comparison with controls (18.13% ± 1.55% vs 13.73% ± 1.21%, P = 0.031). We detected up-regulated Th17 cells within the lcSSc subset against controls (20.46% ± 2.41% vs 13.73% ± 1.21%, P = 0.025), nevertheless no difference was found between dcSSc and lcSSc patients. Flow cytometric analysis revealed an increased percentage of CD4+CD25-Foxp3+ in dcSSc patients compared to controls (10.94% ± 1.65% vs 6.88% ± 0.91, P = 0.032). Regarding the peripheral cytokine profile, we detected raised levels of IL-6 [2.10 (1.05-4.60) pg/mL vs 0.00 pg/mL, P < 0.001], TGF-β1 (19.94 ± 3.35 ng/mL vs 10.03 ± 2.25 ng/mL, P = 0.02), IL-10 (2.83 ± 0.44 pg/mL vs 0.68 ± 0.51 pg/mL, P = 0.008), and IL-17A [6.30 (2.50-15.60) pg/mL vs 0 (0.00-0.05) pg/mL, P < 0.001] in patients when compared to healthy controls. Furthermore, we found increased circulating IL-10, TGF-β, IL-6 and IL-17A in the lcSSc subset vs control subjects, as it follows: IL-10 (3.32 ± 0.59 pg/mL vs 0.68 ± 0.51 pg/mL, P = 0.003), TGF-β1 (22.82 ± 4.99 ng/mL vs 10.03 ± 2.25 ng/mL, P = 0.031), IL-6 [2.08 (1.51-4.69) pg/mL vs 0.00 pg/mL, P < 0.001], and IL-17A [14.50 (8.55-41.65) pg/mL vs 0.00 (0.00-0.05) pg/mL, P < 0.001]. Furthermore, circulating IL-17A was higher in lcSSc as opposed to dcSSc subset (31.99 ± 13.29 pg/mL vs 7.14 ± 3.01 pg/mL, P = 0.008). Within the dcSSc subset, raised levels of IL-17A and IL-6 were detected vs healthy controls: IL-17A [2.60 (0.45-9.80) pg/mL vs 0.00 (0.00-0.05) pg/mL, P < 0.001], IL-6 [2.80 (1.03-7.23) pg/mL vs 0.00 pg/mL, P < 0.001]. Regarding the stages of the disease, TGF-β1 serum levels were increased in early stage against late stage, independently from the SSc phenotype (30.03 ± 4.59 ng/mL vs 13.08 ± 4.50 ng/mL, P = 0.017).CONCLUSIONIt is likely that the altered percentage of Th17 and CD4+CD25-FoxP3+ cells along with the peripheral cytokine profile in patients with SSc may play a key role in the pathogenesis of the disease.

A1.13 Increased presence of CD206+macrophage subset in peripheral blood of systemic sclerosis patients

Background and objectives In systemic sclerosis (SSc), the immune-inflammatory infiltrate is primarily made of macrophages and T cells. 1 Recently, alternatively activated macrophages (M2) have been shown to enhance fibrosis through the production of pro-fibrotic molecules (i.e. fibronectin, metalloproteinases and TGFβ. 2 M2 are characterised by an increased expression of specific markers, primarily CD206 (macrophage mannose receptor-1) and macrophage scavenger receptors (CD204 and CD163). The study investigated the presence of M2 in the peripheral blood (PB) of SSc patients. In addition, the capability of SSc serum to induce the expression of M2 markers and the production of pro-fibrotic metalloproteinase (MMP)-9 in cultured PB mononuclear cells (PBMCs) of healthy subjects (HS) were investigated. Materials and methods Eight SSc patients (mean age 65 ± 7 years) who fulfilled the new criteria of EULAR/ACR 3 and five age matched HS were enrolled into the study, after signing an Informed Consent. SSc patients were treated with vasodilators and endothelin-1 blockers. PB from SSc patients and HS was analysed by flow cytometry (FC) to detect the presence of cells positive for CD206, CD204 and CD14 (a marker of monocyte/macrophage lineage). PBMC-derived monocytes isolated from the enrolled HS (2 × 10 6 cells/well) were maintained for 12 h in growth medium at 10% of fetal bovine serum and then stimulated for 48 h with the serum from SSc patients and HS themselves. The expression of CD206, CD204 and CD68 (a macrophage marker) was evaluated by immunocytochemistry (ICC). MMP-9 production was evaluated by zymography. Results In the PB of SSc patients, the CD206 + cell percentage was found significantly higher than that of HS (p + cells co-expressed CD204, belonging to the monocyte/macrophage lineage due to their expression of CD14. SSc serum stimulated PBMC-derived monocytes to express CD206, CD204 and CD68 thus indicating their activation into macrophages. Conversely, serum from HS was unable to stimulate the expression of CD206 and it induced a limited expression of CD204 in these cells. Zymography showed that treatment with SSc serum induced an increased production and secretion of MMP-9 compared to the treatment with HS serum in PBMC-derived macrophages. Conclusion These preliminary results show an increased presence of the CD206 + cell subset in the PB of SSc patients. The ability of SSc serum to induce the expression of this M2 macrophage marker and to increase the production of pro-fibrotic MMP-9 might support a possible involvement of this cell subset in the pathogenesis of SSc. References Wynn TA, Ramalingam TR et al . Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med 2012; 18 :1028–40. Fairweather D, Cihakova D et al . Alternatively activated macrophages in infection and autoimmunity. J Autoimmun 2009; 33 :222–30. van den Hoogen F, Khanna D, Fransen J et al . 2013 classification criteria for systemic sclerosis: an American College of Rheumatology/European League against Rheumatism collaborative initiative. Arthrit Rheum 2013; 65 :2737–47.

SP0164 Inflammatory-Driven Myocardial Fibrosis – the Role of Inflammatory Progenitors

BackgroundHeart-specific inflammation-myocarditis is a common cause of pathological tissue remodelling and heart failure with the phenotype of inflammatory dilated cardiomyopathy (iDCM). During the last years there was a shift in...

AB0144 Increased frequency of interleukin-2 production by th17 and tc17 lymphocytes in peripheral blood of systemic sclerosis patients

BackgroundThe pathogenesis of systemic sclerosis (SSc) is largely unknown, although proinflammatory cytokines are considered to play a central role. We hypothesized that Th17 and/or Tc17 cell populations and cytokine expression...

SAT0033 Allogenic mesenchymal stem cells-mediated immunoregulation involves FAS/FASL-induced T cell apoptosis in systemic sclerosis

BackgroundSystemic infusion of allogenic mesenchymal stem cells (MSC) have shown therapeutic effects on a variety of autoimmune diseases, but the underlying mechanisms of MSC-based immunoregulation are not fully understood.ObjectivesTo investigate...

Tumor necrosis factor–costimulated T lymphocytes from patients with systemic sclerosis trigger collagen production in fibroblasts

The role of tumor necrosis factor (TNF) in systemic sclerosis (SSc) remains controversial. The present study was undertaken to investigate the influence of TNF receptor (TNFR)-costimulated lymphocytes on collagen expression in fibroblasts. TNFR expression on mononuclear cells from the dermis and blood of SSc patients was assessed by flow cytometry. Peripheral blood CD3+ lymphocytes were activated with CD3/CD28 beads and costimulated with TNFR-selective variants. Expression of interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R), IL-10, and IL-13 was detected by enzyme-linked immunosorbent assay or quantitative reverse transcription-polymerase chain reaction. Healthy fibroblasts were incubated with conditioned media from TNFR-costimulated T lymphocytes, and type I collagen expression was quantified. TNFRI and TNFRII were up-regulated on dermal T lymphocytes from patients with diffuse cutaneous SSc. TNFRII expression correlated with skin thickening. After CD3/CD28 activation, peripheral blood lymphocytes from SSc patients produced more IL-6, sIL-6R, and IL-13 compared to healthy lymphocytes. Costimulation with TNFRI-selective ligands and soluble TNF further increased IL-6 expression, whereas costimulation with TNFRII led to greater release of sIL-6R. IL-10 expression, which normally occurs after TNFRII costimulation, was impaired in SSc T cells. Supernatants of TNF-costimulated SSc lymphocytes induced higher type I collagen expression in fibroblasts, which was partially reversible by dual inhibition of IL-6 and IL-13. Expression of TNFR and IL-6 in the dermis was reversible in a patient who received lymphoablative therapy prior to autologous hematopoietic stem cell transplantation. TNF-costimulated T lymphocytes from SSc patients have a propensity to secrete profibrotic cytokines, while the ability to produce IL-10 is weakened. These results suggest that T lymphocytes in SSc support fibrosis, but might lack the capacity to resolve inflammation.

Circulating platelets as a source of the damage-associated molecular pattern HMGB1 in patients with systemic sclerosis

The link between platelet activation and vascular injury in Systemic Sclerosis (SSc) is poorly characterized. Here we report that platelet activation results in i) the translocation from the cytoplasm to the surface of HMGB1, a prototypical DAMP signal associated with tissue regeneration and ii) the release of platelet derived microparticles (PDμP) expressing HMGB1. Decreased HMGB1 content (334.6 ± 21.2 vs 587.1 ± 11.1 AUF, P < 0.001) and HMGB1 translocation to the outer leaflet of the plasma membrane (17.8 ± 3.5 vs 4.5 ± 0.5%, P < 0.001) characterize circulating platelets of SSc patients (n = 29) when compared with age-matched healthy controls (HC, n = 20). Conversely, a significantly higher fraction of PDμP in the blood of SSc patients, but not of HC, consistently expose (HMGB1 (MFI 62.8 ± 3.95 vs 4.3 ± 0.7). Platelet HMGB1 depletion is significantly associated in SSc patients with degranulation and with expression of P-selectin and of tissue factor as well as with fibrinogen binding to their plasma membrane. These findings indicate that platelets represent a source of HMGB1, an ancestral signal of necrosis, in the vasculature of SSc patients, possible contributing to persistent microvascular injury and endothelial cell activation.

The role of B cells in systemic sclerosis

Systemic sclerosis (SSc) is a connective disease characterized by features of autoimmunity, vasculopathy, inflammation, and fibrosis. The disease typically starts with Raynaud's phenomenon, followed by skin thickening in the extremities due to inflammation and fibrosis. Fibrosis results from excessive collagen production by fibroblasts, which constitutes the final common pathway of complex cellular interactions including B cells. Several studies have indicated that B cells may play a role in SSc. Lesional skin infiltrates from SSc patients consist of a variety of cells, including eosinophils, neutrophils, lymphocytes, plasma cells, and macrophages. Autoantibodies of several specificities are present in the serum of SSc patients of which antitopoisomerase 1 is the most common, and evidence has been gathered for a potential pathogenic role of some autoantibodies, eg, anti-PDGF antibodies. The blood of SSc patients contains an increased proportion of naïve B cells but a decreased proportion of memory B cells. Furthermore, serum levels of interleukin-6, an important pro-inflammatory cytokine, have been shown to correlate with skin fibrosis. Animal models of SSc have provided more in-depth information on the role of B lymphocytes, eg, through disruption of B cell function. In this review we will discuss the evidence that B cells are involved in the pathogenesis of SSc.

Peripheral blood T lymphocytes from systemic sclerosis patients show both Th1 and Th2 activation.

Our objective was to investigate the phenotype of helper T cells in the peripheral blood of patients with systemic sclerosis (SSc). PBMC from 15 patients with SSc and 15 sex- and age-matched controls were investigated for lymphocyte subpopulations (CD3, CD4, CD8, CD19, CD16/CD56, CD3-DR); IL-2, IL-4, and IFN-gamma mRNAs; and the relative cytokines in their cytoplasm. The last assay was carried out both in unstimulated and in PMA-activated PBMC. SSc patients presented a higher percentage of activated T cells, CD3+ DR+ (19.7 +/- 9.9 vs 5.1 +/- 2.5%; P < 0.0001); 12 of them presented IFN-gamma mRNA-positive cells; and none IL-2 or IL-4 mRNAs. Under basal conditions, PBMC from six SSc patients contained IL-2, IL-4, and IFN-gamma (i.e., they showed both Th1 and Th2 activation), and 1 IFN-gamma only. PMA-stimulated PBMC of patients differed from those of controls only in the increased percentage of IFN-gamma positive cells (52 +/- 12 vs 37 +/- 11%; P < 0.01). Our study demonstrates that Thl activation occurs in the peripheral blood of SSc patients. This evidence must be faced with from both a pathogenetic and a therapeutical point of view.

Oligoclonal expansion of V delta 1+ gamma/delta T-cells in systemic sclerosis patients.

Systemic sclerosis (SSc) is a multisystem disease characterized by T-cell infiltration of involved tissues, fibrosis, and small vessel vasculopathy. Using flow cytometric analyses, we found an increased percentage of gamma/delta T-cells expressing the T-cell antigen receptor variable (V) delta 1 gene segment in the peripheral blood and bronchoalveolar lavage fluid of patients with SSc. To estimate clonality of these V delta 1+ T-cells, the diversity of V delta 1 junctional regions (V-Diversity-Joining gene segments) was examined using a reverse transcriptase-polymerase chain reaction to amplify T-cell antigen receptor delta chain transcripts isolated from peripheral blood mononuclear cells, lung, esophagus, stomach, or skin of patients and controls. Limited diversity of V delta 1-J delta junctional regions in SSc patients was demonstrated by the finding of greater restriction in the nucleotide lengths of junctional region cDNAs in individual SSc patients than in controls. Sequence analyses confirmed that V delta 1-J delta junctional regions from the blood of SSc patients had less diversity than those from controls, in that a significantly higher proportion of sequences were repeated in patients (54.4% vs. 19.4% in controls). Evidence for selection of the V delta 1+ T-cells in tissues of individual SSc patients came from the findings that the same V delta 1-J delta junctional sequences could be isolated from the same tissue over time and that identical V delta 1-J delta junctional sequences could be isolated from multiple tissues. These data suggest that expansion of V delta 1+ gamma/delta T cells may be antigen driven in SSc patients.