Marek Bielecki

CD163 and its role in inflammation

Mononuclear phagocytes represent a heterogeneous population of cells with individual subpopulations exerting different pro- or anti-inflammatory functions. CD163 is a monocyte/macrophage specific marker expressed predominantly on cells which possess strong anti-inflammatory potential. The expression of CD163 is strongly induced by anti-inflammatory mediators such as glucocorticoids and interleukin-10, while being inhibited by pro-inflammatory mediators such as interferon-gamma. CD163-expressing mononuclear phagocytes, as well as soluble CD163, may both take part in downregulating an inflammatory response. It seems, therefore, that CD163 may be an interesting target for therapeutic modulation of the inflammatory response.

Angiopoietins-1 and -2 are differentially expressed in the sera of patients with systemic sclerosis: high angiopoietin-2 levels are associated with greater severity and higher activity of the disease

OBJECTIVES The aim of the study was to investigate serum concentration of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in SSc patients and to correlate Ang-1 and -2 levels with clinical manifestations. METHODS Serum levels of Ang-1 and -2 were determined by ELISA in 47 SSc patients and 27 healthy controls matched for age and sex. RESULTS In SSc patients, Ang-1 was significantly lower [mean (s.d.): 21,666.09 (11,516.06) pg/ml], while Ang-2 was significantly increased [2739.60 (1009.25) pg/ml] when compared with the control group [Ang-1: 28607.13 (10,506.93) pg/ml; Ang-2: 1706.28 (556.28) pg/ml, P < 0.01, for both comparisons]. No correlation was observed between Ang-1 and -2 levels. Serum concentrations of Ang-2 correlated with the modified Rodnan skin score (P < 0.01, r =0.422), the European Scleroderma Study Group (EScSG) disease activity index score (P < 0.001, r =0.403), ESR (P < 0.05, r = 0.366) and CRP concentration (P < 0.05, r =0.292), and showed inverse correlation with diffusing capacity for carbon monoxide (DL(CO)) (P < 0.01, r = -0.281). Ang-2 was significantly increased in SSc patients with more advanced capillary damage, as indicated by the presence of late capillaroscopic pattern, than in those with less severe microangiopathy (active pattern), and in SSc patients with intermediate/late stage of disease than in SSc subjects with early disease. In multivariate regression analysis, Ang-2 was independently associated with the EScSG activity index [β-coefficient (β = 0.305, P= 0.038], ESR (β= 0.390, P =0.006) and, inversely, with the presence of digital ulcers (β =-0.426, P = 0.003). CONCLUSIONS Differential expression of Ang-1/Ang-2 might contribute to the pathogenesis of SSc. Ang-2 might be a new biomarker of disease activity in SSc.

Peripheral blood mononuclear cells from patients with systemic sclerosis spontaneously secrete increased amounts of vascular endothelial growth factor (VEGF) already in the early stage of the disease.

PURPOSE To investigate the capacity of the peripheral blood mononuclear cells (PBMC) from patients with systemic sclerosis (SSc) to produce vascular endothelial growth factor (VEGF), and to identify clinical associations of altered production of VEGF by PBMC in SSc. In addition, correlation with another pro-angiogenic cytokine, TNF-related weak inducer of apoptosis (TWEAK), was evaluated. METHODS PBMC were isolated from 25 patients with SSc and 17 healthy controls (HC). VEGF and TWEAK were measured in the supernatants of cultured PBMC using commercially available ELISA kits. RESULTS PBMC from SSc patients spontaneously released significantly greater amounts of VEGF as compared with HC. Production of VEGF was comparable between patients with early SSc and those with longer disease duration, and in both SSc groups higher than in HC. Patients without active digital ulcers produced significantly greater amounts of VEGF as compared with HC, while there was no significant difference in the production of VEGF between SSc patients with active digital ulcers and HC. VEGF/TWEAK ratio was significantly higher in PBMC from SSc patients than in HC indicating that high production of VEGF is not paralleled by increased release of TWEAK in SSc. CONCLUSIONS PBMC form SSc patients produce increased amounts of VEGF already in the early stage of disease. There is an imbalance in the profile of pro-angiogenic mediators produced by PBMC in SSc which might contribute to the pathogenesis of SSc. Further studies should address clinical significance of our findings.

High serum sCD163/sTWEAK ratio is associated with lower risk of digital ulcers but more severe skin disease in patients with systemic sclerosis

IntroductionSystemic sclerosis (SSc) is an autoimmune disease characterized by chronic inflammation, vascular injury and excessive fibrosis. CD163 is a scavenger receptor which affects inflammatory response and may contribute to connective tissue remodelling. It has recently been demonstrated that CD163 can bind and neutralize the TNF-like weak inducer of apoptosis (TWEAK), a multifunctional cytokine which regulates inflammation, angiogenesis and tissue remodelling. We aimed to investigate the relationships between serum levels of soluble CD163 (sCD163) and soluble TWEAK (sTWEAK) in relation to disease manifestations in SSc patients.MethodsThis study included 89 patients with SSc who had not received immunosuppressive drugs or steroids for at least 6 months and 48 age- and sex-matched healthy controls (HC) from four European centres. Serum concentrations of sTWEAK and sCD163 were measured using commercially available ELISA kits.ResultsThe mean serum concentrations of sTWEAK were comparable between SSc patients (mean +/- SD: 270 +/- 171 pg/mL) and HC (294 +/- 147pg/mL, P >0.05). Concentration of sCD163 and sCD163/sTWEAK ratio were significantly greater in SSc patients (984 +/- 420 ng/mL and 4837 +/- 3103, respectively) as compared to HC (823 +/- 331 ng/mL and 3115 +/- 1346 respectively, P <0.05 for both). High sCD163 levels and a high sCD163/sTWEAK ratio (defined as > mean +2SD of HC) were both associated with a lower risk of digital ulcers in SSc patients (OR, 95%CI: 0.09; 0.01, 0.71, and 0.17; 0.06, 0.51, respectively). Accordingly, patients without digital ulcers had a significantly higher sCD163 concentration and sCD163/sTWEAK ratio as compared to SSc patients with digital ulcers (P <0.01 for both) and HC (P <0.05 for both). A high sCD163/sTWEAK ratio, but not high sCD163 levels, was associated with greater skin involvement.ConclusionsThe results of our study indicate that CD163-TWEAK interactions might play a role in the pathogenesis of SSc and that CD163 may protect against the development of digital ulcers in SSc. Further studies are required to reveal whether targeting of the CD163-TWEAK pathway might be a potential strategy for treating vascular disease and/or skin fibrosis in SSc.

Increased Production of a Proliferation-inducing Ligand (APRIL) by Peripheral Blood Mononuclear Cells Is Associated with Antitopoisomerase I Antibody and More Severe Disease in Systemic Sclerosis

Objective. A proliferation-inducing ligand (APRIL), a member of the tumor necrosis factor (TNF) family, plays a crucial role in the survival of peripheral B cells, and may contribute to the pathogenesis of systemic sclerosis (SSc) through upregulation of autoantibody production and maintenance of autoimmune phenomena. We evaluated the capacity of peripheral blood mononuclear cells from patients with SSc (SSc-PBMC) to produce APRIL; and investigated correlations between production of APRIL by SSc-PBMC and clinical and laboratory features of the disease. Methods. PBMC from 20 patients with SSc and 14 healthy subjects were incubated in fetal calf serum-supplemented RPMI medium. APRIL levels were determined in cell culture supernatants by ELISA. Results. PBMC from patients with SSc produced significantly more APRIL (961 ± 151 pg/ml/105 cells) than control PBMC (798 ± 219 pg/ml/105 cells; p < 0.01). In patients with SSc, increased production of APRIL was associated with the presence of diffuse skin involvement, scleroderma lung disease, peripheral vasculopathy, greater capillary damage on capillaroscopy, and presence of anti-topoisomerase I (anti-topo I) antibodies. Multivariate regression analysis revealed anti-topo I antibodies as the only independent predictor of high production of APRIL by PBMC. Conclusion. Production of APRIL is increased in SSc-PBMC and is associated with the presence of anti-topo I antibodies and more severe disease. Targeting the APRIL pathway might represent a therapeutic possibility for treatment of patients with SSc, in particular those with anti-topo I antibodies.

Increased release of soluble CD163 by the peripheral blood mononuclear cells is associated with worse prognosis in patients with systemic sclerosis

PURPOSE CD163 is a scavenger receptor which is exclusively expressed on monocytes/macrophages and participates in modulation of inflammatory response. We aimed to evaluate ex vivo production of soluble CD163 (sCD163) by peripheral blood mononuclear cells (PBMC) from patients with systemic sclerosis (scleroderma, SSc). MATERIAL/METHODS Concentration of sCD163 was measured by commercially available ELISA kit in the PBMC suparnates from 23 SSc patients and 16 age- and sex-matched healthy controls (HC). Eighteen SSc patients were subsequently followed for at least three years or until death whichever happened earlier. Disease progression was defined as death due to SSc-related organ complication, development of a new or progression of pre-existing SSc-related organ involvement. RESULTS PBMC from SSc patients released significantly greater amounts of sCD163 as compared with HC (p<0.05). No significant associations between release of sCD163 by PBMC and baseline clinical or laboratory parameters of the disease could be found. However, concentration of sCD163 in cell culture supernates was significantly higher in 6 SSc patients who experienced subsequent progression of the disease as compared with 12 SSc patients with stable disease course over a 3-year follow-up period (p<0.05). CONCLUSIONS We show, for the first time, that PBMC from SSc release significantly greater amounts of sCD163 than do PBMC from healthy subjects. Evaluation of sCD163 production by PBMC ex vivo may serve as a new biomarker of disease progression. Further studies are required to evaluate the role of sCD163 in the development of SSc.

The role of leukotrienes in the pathogenesis of systemic sclerosis

Systemic sclerosis (SSc, scleroderma) is an autoimmune disease characterized by widespread vascular injury and progressive fibrosis of the skin and internal organs. SSc-related involvement of the lungs, heart, kidneys and/or the gastrointestinal system accounts for the increased mortality of scleroderma patients. Despite the progress which has recently been made in this field, the treatment of SSc is still unsatisfactory due to the low efficacy and/or high toxicity of available therapies. Leukotrienes are a family of lipid mediators synthesized from arachidonic acid in a process mediated by 5-lipoxygenase; they include leukotriene B4 and a group of cysteinyl leukotrienes: C4, D4, and E4. Leukotrienes play an important role in the regulation of all the processes vital to the pathogenesis of SSc, namely inflammation, vascular function and connective tissue remodeling. The available data suggests that an excessive synthesis of leukotrienes may contribute to the development and progression of SSc. Accordingly, blockade of leukotriene pathways appears to be a new, promising target for the treatment of SSc.

The arachidonate 5-lipoxygenase activating protein gene polymorphism is associated with the risk of scleroderma-related interstitial lung disease: a multicentre European Scleroderma Trials and Research group (EUSTAR) study

Objectives The arachidonate 5-lipoxygenase activating protein (ALOX5AP) regulates synthesis of leukotrienes (LTs), which are important mediators of inflammation and connective tissue remodelling. The aim of this study was to evaluate if single nucleotide polymorphisms (SNPs) of ALOX5AP confer risk of SSc and/or SSc-related organ involvement. Methods Seven SNPs of ALOX5AP (rs17222814, rs17216473, rs10507391, rs4769874, rs9551963, rs9315050 and rs7222842) were genotyped in a cohort of 977 patients with SSc and 558 healthy controls from centres collaborating within the European Scleroderma Trials and Research group. In 22 SSc patients, concentrations of cysteinyl LTs and LT B4 (LTB4) were measured in the supernatants of ionophore-stimulated peripheral blood mononuclear cells (PBMCs) by means of commercially available enzyme immunoassay kits. Results Significant association was found between rs10507391 polymorphism (T/A) of ALOX5AP and the risk of SSc [odds ratio (OR) 1.27 (95% CI 1.07, 1.50), P < 0.05 vs controls], the presence of SSc-related interstitial lung disease on high-resolution CT of the lungs [OR 1.45 (95% CI 1.17, 1.79), P < 0.05 vs patients without SSc-related interstitial lung disease] as well as with restrictive ventilatory defect [forced vital capacity <70% of predicted; OR 1.51 (95% CI 1.16, 1.97), P < 0.05 vs SSc patients without pulmonary restriction]. PBMCs from SSc carriers of rs10507391 allele A synthesized greater amounts of cysteinyl LTs as compared with SSc patients with rs10507391 TT genotype ( P < 0.05). Synthesis of LTB4 did not differ significantly between the two groups. Conclusion The results of our study indicate that the genetic variants of ALOX5AP might play a role in the development of SSc-related pulmonary fibrosis.

OP0090 Serum CD163/Tweak Ratio Predicts Clinical Course of Digital Ulcers in Patients with Systemic Sclerosis

Background TNF-like weak inducer of apoptosis (TWEAK) regulates inflammation, angiogenesis and tissue remodeling. CD163 molecule, a scavenger receptor and marker of alternatively activated macrophages, modulates TWEAK activity through binding and internalization of TWEAK molecule. We have previously shown that, in a crossectional study, high serum concentrations of soluble CD163 (sCD163) and high CD163/TWEAK ratio were associated with lack of digital ulcers (DU) in patients with systemic sclerosis (SSc) [1]. Objectives To investigate whether serum levels of soluble CD163 (sCD163), soluble TWEAK (sTWEAK) or sCD163/sTWEAK ratio can predict clinical course of DU in patients with (SSc). Methods 50 patients fulfilling the ACR/EULAR classification criteria of SSc who were followed for at least 1 year were included. Serum levels of sCD163 and sTWEAK were measured using commercially available ELISA kits. Digital ulcers were defined as painful area of loss of tissue on the volar surface of fingertips or around the nail distal to the proximal interphalangeal digital crease, present at the time of assessment. In addition, evaluation of SSc patients included assessment of disease subset (diffuse/limited SSc), modified Rodnan skin score, the presence of interstitial lung disease, pulmonary hypertension, results of pulmonary function tests, echo-Doppler, autoantibody testing (anticentromere antibodies, anti-Scl-70 antibodies) and erythrocyte sedimentation rate. Results At baseline DU were present in 18 patients. During follow-up DU healed in 12 SSc patients and persisted in the remaining 6. In addition, 5 patients without DU at baseline developed new DU during follow-up. Because of relatively low numbers, patients with recurrent/persistent DU and those with new DU were analyzed together (n=11). In the remaining 27 patients DU were not found at any visit. There were no significant differences in baseline sCD163 levels between SSc patients in whom DU healed at follow-up, those with persisted/new DU or SSc patients without DU at any visit. In contrast, baseline serum levels of sTWEAK were significantly higher in SSc patients who subsequently experienced healing of DU compared with SSc patients with DU at follow-up and with SSc patients who never had DU (p<0.05 for both comparisons). Accordingly, baseline sCD163/sTWEAK ratio was significantly lower in SSc patients with healed DU as compared with those with persisted/new DU at follow-up and with those who never had DU (p<0.05 for both comparisons). In univariate analysis baseline DU, anticentromere antibodies (ACA) and sCD163/sTWEAK ratio were significantly associated with outcome of DU. In multivariate analysis including baseline DU, ACA and sCD163/sTWEAK ratio, baseline DUs were the strongest independent predictor of risk of DU at follow-up. Conclusions Our results suggest that CD163-TWEAK interactions might play a role in the development/healing of DU in SSc and indicate that sCD163–TWEAK serum ratio is a potential biomarker of peripheral vascular disease in SSc. References Arthritis Res Ther. 2013; Jun 24;15(3):R69. [Epub ahead of print] Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5343

SAT0024 Increased production of leukotrienes by peripheral blood mononuclear cells is associated with more severe disease and worse prognosis in patients with systemic sclerosis

Background Leukotrienes (LTs) are a group of pro-inflammatory and pro-fibrotic, arachidonic acid-derived lipid mediators. We have shown that LTs are increased in the lungs of patients with interstitial lung disease (ILD) associated with systemic sclerosis (SSc). Objectives Since leukocytes are considered a major source of LTs in humans and since inflammatory infiltrates consisting of mononuclear cells are found in the skin and internal organs in early SSc, we undertook this study to investigate the relationship between the synthesis of LTs by peripheral blood mononuclear cells (PBMC) and clinical features of SSc. Methods Leukotriene B4 (LTB4) and cysteinyl leukotrienes (CysLTs) were measured by ELISA in the supernatants from ionophore-stimulated PBMC of 39 patients with SSc and 24 age- and sex-matched healthy controls (HC). Only patients, who had not received immunosuppressive drugs, aspirin or other NSAIDs for at least 6 months, 1 month and 7 days respectively before the study, were included. Follow-up data were available in 25 SSc patients (mean ± SD follow-up time: 34±18 months). Disease progression was defined as death due to SSc-related organ complication, development of a new or progression of pre-existing SSc-related organ involvement (>10% decrease in FVC, increase by at least one WHO class, increase by ≥120% of upper normal limit in serum creatinine and/or loss of >10% of weight after excluding other causes). Results Concentration of LTB4 was significantly higher in PBMC cultures from patients with SSc (640±518 pg/mL/105cells) as compared with HC (353±216 pg/mL/105cells, p<0.05). Higher LTB4 levels were associated with the presence of diffuse SSc, restrictive ILD (FVC<80% predictive and FEV1/FVC >0.8), pulmonary hypertension (PASP>45 mmHg by echo), and more severe microangiopathy in capillaroscpy. Mean concentration of LTB4 was higher in 7/25 SSc patients who experienced subsequent progression of the disease (918±665 pg/mL/105cells) compared with the remaining 18/25 with stable disease (569±549 pg/mL/105cells), but the difference was not significant (p=0.1). We did not find significant differences in the synthesis of CysLTs between SSc patients (313±162 pg/mL/105cells) and HC (266±138 pg/mL/105cells, p>0.05), nor significant associations of CysTLs concentration and SSc-related organ involvement. However, concentration of CysLTs was significantly higher in PBMC cultures from SSc patients with subsequent progression of the disease (452.0±197.3 pg/mL/105cells) as compared with those with stable SSc (275.6±138.9 pg/mL/105cells, p<0.05). Conclusions The results of our study indicate that increased synthesis of LTs might be involved in the pathogenesis and progression of SSc. Consequently, inhibition of LTs synthesis or action might represent a new, promising target in the treatment of SSc. Disclosure of Interest A. Lapinska: None Declared, M. Bielecki: None Declared, O. Distler Grant/Research support from: PD Dr. O. Distler has consultancy relationship and/or has received research funding from Actelion, Pfizer, Ergonex, BMS, Sanofi-Aventis, United BioSource Corporation, medac, 4D Science, Boehringer-Ingelheim, Active Biotech and Roche in the area of potential treatments of scleroderma and its complications. Speakers Bureau: PD Dr O. Distler has received lecture honoraria from Actelion, Pfizer, Encysive and Ergonex., I. Domyslawska: None Declared, L. Chyczewski: None Declared, S. Sierakowski: None Declared, S. Gay: None Declared, K. Kowal: None Declared, O. Kowal-Bielecka Grant/Research support from: Dr O Kowal-Bielecka work was supported by research grant (2-PO5B-050-27) from the Polish State Committee for Scientific Research.