Karina de Leeuw

Reduced number and impaired function of circulating progenitor cells in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is associated with premature and accelerated atherosclerosis. Circulating progenitor cells (CPCs) are circulating bone-marrow derived cells that play an important role in the repair of vascular damage that underlies the development of atherosclerosis. The objective of this study was to determine the number and functionality of CPCs in patients with SLE. The study included 44 female SLE patients in an inactive stage of disease and 35 age-matched female controls. CPC numbers in the circulation were determined by FACS with monoclonals against CD14, CD34 and CD133. Peripheral blood-derived mononuclear cell (PBMNC) fractions were cultured in angiogenic medium. The endothelial-like phenotype was confirmed and the colony forming unit (CFU) capacity, migratory capacity and the potential to form clusters on Matrigel were determined. Expression of apoptosis inhibiting caspase 8L was analyzed in PBMNCs and CPCs by gene transcript and protein expression assays. The number of CD34–CD133 double-positive cells (P < 0.001) as well as the CFU capacity (P = 0.048) was reduced in SLE patients. Migratory activity on tumor necrosis factor-α tended to be reduced in patient CPCs (P = 0.08). Migration on vascular endothelial growth factor showed no significant differences, nor were differences observed in the potential to form clusters on Matrigel. The expression of caspase 8L was reduced at the transcriptional level (P = 0.049) and strongly increased at the protein level after culture (P = 0.003). We conclude that CPC numbers are reduced in SLE patients and functionality is partly impaired. We suggest these findings reflect increased susceptibility to apoptosis of CPCs from SLE patients.

Early atherosclerosis in systemic sclerosis and its relation to disease or traditional risk factors

IntroductionSeveral systemic autoimmune diseases are associated with an increased prevalence of atherosclerosis which could not be explained by traditional risk factors alone. In systemic sclerosis (SSc), microvascular abnormalities are well recognized. Previous studies have suggested an increased prevalence of macrovascular disease as well. We compared patients with SSc to healthy controls for signs of early atherosclerosis by measuring intima-media thickness (IMT) of the common carotid artery in relation to traditional risk factors and markers of endothelial activation.MethodsForty-nine patients with SSc, of whom 92% had limited cutaneous SSc, and 32 healthy controls were studied. Common carotid IMT was measured by using B-mode ultrasound. Traditional risk factors for cardiovascular disease were assessed and serum markers for endothelial activation were measured.ResultsIn patients with SSc, the mean IMT (median 0.69 mm, interquartile range [IQR] 0.62 to 0.79 mm) was not significantly increased compared with healthy controls (0.68 mm, IQR 0.56 to 0.75 mm; P = 0.067). Also, after correction for the confounders age, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein cholesterol (P = 0.328) or using a different model taking into account the confounders age, HDL cholesterol, and history of macrovascular disease (P = 0.474), no difference in IMT was present between SSc patients and healthy controls. Plaques were found in three patients and not in healthy controls (P = 0.274). In patients, no correlations were found between maximum IMT, disease-related variables, and markers of endothelial activation. Endothelial activation markers were not increased in SSc patients compared with controls.ConclusionSSc is not associated with an increased prevalence of early signs of atherosclerosis.

Longitudinal study on premature atherosclerosis in patients with systemic lupus erythematosus

OBJECTIVES To determine risk factors of accelerated atherosclerosis and progression of intima-media thickness (IMT) in patients with systemic lupus erythematosus (SLE). METHODS 74 SLE patients, age ranging from 13 to 69 years, and 74 age- and sex-matched controls were included. IMT of the common carotid artery was determined by B-mode ultrasound imaging. Traditional risk factors for atherosclerosis and disease-related factors were recorded. Cardiovascular risk was estimated using systematic coronary risk evaluation (SCORE). Markers of inflammation (C-reactive protein, CRP) and endothelial activation (thrombomodulin, vascular cell adhesion molecule-1, and von Willebrand factor) were determined. Measurements were repeated in 52 patients after a follow-up of 32+/-7 months. RESULTS IMT was increased in SLE patients compared to controls. Prevalence of smoking and hypertension, use of lipid-lowering drugs and SCORE were higher in patients, as well as levels of CRP and markers of endothelial activation. The age-related increase in IMT was significantly higher in patients than in controls. In multivariate analysis, age and disease duration was independently related to IMT. Increase in IMT during follow-up was related to age only. CONCLUSION The age-related increase in IMT is higher in SLE, indicating that atherosclerosis is accelerated in SLE patients. This is mainly due to disease-related risk factors, as disease duration was independently associated with IMT.

Accelerated atherosclerosis in patients with systemic autoimmune diseases

Abstract: Systemic autoimmune diseases such as systemic lupus erythematosus and Wegeners granulomatosis are associated with a significantly increased prevalence of cardiovascular disease (CVD) compared with age‐ and sex‐matched controls. Many risk factors are involved in the pathogenesis of atherosclerosis, the major underlying cause of CVD. In patients with systemic autoimmune diseases, it has been shown that traditional risk factors for CVD cannot completely explain the prevalence of atherosclerosis. Therefore, in addition to these traditional factors, nontraditional risk factors are suggested to contribute to atherogenesis. All risk factors, traditional and nontraditional, contribute to endothelial activation that, followed by endothelial dysfunction, is seen as one of the first steps in this process. This review updates information on the factors that contribute to accelerated atherosclerosis in patients with systemic autoimmune diseases, such as disease‐related factors, inflammatory mediators, and advanced glycation end products.

Fc-gamma receptor polymorphisms differentially influence susceptibility to systemic lupus erythematosus and lupus nephritis

OBJECTIVE To determine relevant Fc-gamma receptor (FcγR) polymorphisms in relation to susceptibility to SLE and LN, and to determine the functional consequences of genetic associations found. METHODS Using multiplex ligation-dependent probe amplification, copy number regions (CNRs) and relevant known functional single nucleotide polymorphisms of FcγRII and FcγRIII were determined in a LN-enriched cohort of 266 Dutch Caucasian SLE patients and 919 healthy Caucasian controls. Expression of FcγRs on leukocytes was assessed using flow cytometry. RESULTS In multivariable analysis, low copy number of CNR1 (including FCGR3B; odds ratio (OR) 2.04; 95% CI: 1.29, 3.23), FCGR2A-131RR (OR 2.00; 95% CI: 1.33, 2.99), and the 2B.4 haplotype of FCGR2B (OR 1.59; 95% CI: 1.13, 2.24), but not FCGR2C open reading frame, were significantly (all P < 0.01) and independently associated with susceptibility to SLE. The 2B.4 haplotype was negatively associated with LN and led to surface expression of FcγRIIb on neutrophils and monocytes. CONCLUSION This study is the first to investigate the most relevant and functional single nucleotide polymorphisms and copy number variations of FcγRII and FcγRIII polymorphisms in one study population, enabling the determination of the individual contribution of each polymorphism in multivariable analysis. Three polymorphisms were shown to be independently associated with susceptibility to SLE. The novel findings of a negative association of the 2B.4 haplotype with LN, and increased expression of FcγRIIb on neutrophils and monocytes as a result of this 2B.4 haplotype warrant future research in the role of these cells and FcγRs in the pathogenesis of SLE and LN.

Standardised assessment of membrane proteinase 3 expression. Analysis in ANCA-associated vasculitis and controls

Objectives: Increased numbers of neutrophils expressing proteinase 3 on their membrane (mPR3) have been reported in anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) and are suggested to be involved in AAV immunopathogenesis. In most studies, neutrophils were analysed for mPR3 expression without priming with TNFα, suggesting that mPR3 expression on neutrophils is dependent on other priming events, such as isolation procedures . These priming events can be variable. Therefore, we analysed mPR3 expression on neutrophils before and after priming with TNFα to assess whether standardised assessment of mPR3 expression requires priming. Using neutrophils before and after priming with TNFα, we assessed percentages of mPR3+ neutrophils in patients with AAV and in disease and healthy controls. Methods: Neutrophils from patients with PR3-AAV and MPO-AAV, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), and from healthy controls were analysed before and after priming with TNFα for mPR3 expression. Results: 42% of all individuals analysed showed minimal expression for mPR3 on all neutrophils before priming with TNFα, whereas after priming a clear mPR3+ subset was observed next to mPR3– neutrophils, corresponding to bimodal mPR3 expression. In patients with PR3-AAV or MPO-AAV, the percentage of mPR3+ neutrophils after priming with TNFα was significantly increased (p<0.01 and p<0.05, respectively) compared with healthy controls. Percentages of mPR3+ PMN were also increased in patients with SLE (p<0.01) but not in RA. Conclusion: Standardised assessment of proteinase 3 on the membrane of neutrophils requires priming with TNFα. Percentages of mPR3+ PMN are increased in AAV and SLE, but not in RA.

Impact of Serum High Mobility Group Box 1 and Soluble Receptor for Advanced Glycation End-Products on Subclinical Atherosclerosis in Patients with Granulomatosis with Polyangiitis

The objective of this study was to evaluate whether levels of high mobility group box 1 (HMGB1) in granulomatosis with polyangiitis (GPA) patients are associated with carotid atherosclerosis, related to levels of soluble receptor for advanced glycation end-products (sRAGE) and influenced by immunosuppressive or lipid-lowering therapy. Twenty-three GPA patients and 20 controls were evaluated for HMGB1- and sRAGE levels and for carotid atherosclerosis using ultrasound to determine intima-media thickness (IMT). In vitro the effect of atorvastatin on the production of HMGB1 by lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVEC) was assessed. Serum HMGB1 and sRAGE levels did not differ between patients and controls. A negative correlation was found between sRAGE and maximum IMT but HMGB1 and carotid IMT were not related. HMGB1 levels were reduced in GPA patients on statins and prednisolone. In vitro, atorvastatin reduced HMGB1 levels in supernatants of activated HUVEC. In conclusion, carotid IMT is inversely correlated with sRAGE levels but not with HMGB1 levels. Statins and prednisolone are associated with reduced serum HMGB1 levels and atorvastatin decreases HMGB1 release by activated HUVEC in vitro, indicating an additional anti-inflammatory effect of statins.

Small artery elasticity is decreased in patients with systemic lupus erythematosus without increased intima media thickness

IntroductionThe objectives of this study were to determine small arterial elasticity (SAE) in systemic lupus erythematosus (SLE) and to investigate its relationship with intima media thickness (IMT), accumulation of advanced glycation end products (AGEs), endothelial activation and inflammation.MethodsThirty SLE patients with inactive disease and 30 age- and sex-matched healthy controls were included. Twenty patients with essential hypertension (EH) served as positive control. SAE was assessed by pulse-wave analysis using tonometric recordings of the radial artery. IMT of the carotid arteries was measured by ultrasound. AGE accumulation was assessed with an AGE-reader. Endothelial activation markers and C-reactive protein (CRP) were determined by enzyme-linked immunosorbent assay (ELISA).ResultsSAE was decreased in SLE (P = 0.01) and further decreased in EH (P < 0.01) compared to healthy controls. IMT was increased in EH (P < 0.05), but not in SLE. AGE accumulation was increased in SLE (P < 0.05) and further increased in EH (P < 0.01) compared to healthy controls. Endothelial activation markers and CRP were increased in SLE but not in EH. SAE related to AGE accumulation (r = -0.370, P < 0.05), CRP (r = -0.429, P < 0.05) and creatinine clearance (r = 0.440, P < 0.05), but not to IMT and endothelial activation markers. In multivariate analysis SLE was an independent predictor of SAE.ConclusionsSAE is decreased in SLE patients without increased IMT, independently of traditional cardiovascular risk factors. Longitudinal studies are needed to investigate whether SAE, endothelial activation and AGE accumulation are early markers for cardiovascular disease in SLE.

Auto-antibodies to double-stranded DNA as biomarker in systemic lupus erythematosus: Comparison of different assays during quiescent and active disease

Objective Auto-antibodies directed to dsDNA (anti-dsDNA) are used in diagnosis and follow-up for SLE. However, multiple assays are used. The objective of this study was to determine the best-performing assays, especially in prediction of exacerbations. Methods Seven assays were compared during LN (n = 58). The two assays with the most frequent positive results during active nephritis were selected and tested in 152 SLE patients with quiescent disease, 40 with active disease and 214 disease controls. Furthermore, longitudinal samples of SLE patients with and without exacerbations were examined to determine the positive predictive value of an increase for an exacerbation. Results Of seven assays, results of the Farr (Siemens) and enzyme-labelled anti-isotype assay (EliA) (ThermoFisherScientific) were foremost associated with active nephritis (both 95%). Sensitivity in active SLE was equal using Farr or EliA (95 vs 93%). In quiescent disease, the specificity of EliA was higher (Farr: 53% vs EliA: 91%). In longitudinal analyses, a 25% increase of anti-dsDNA preceded an exacerbation in 75 vs 69% (Farr vs EliA). In SLE patients without exacerbations (n = 42), a rise was seen in 10 vs 12%. Increases in anti-dsDNA occurred more often prior to nephritis (n = 17) compared with non-nephritic flares (n = 17), which was not different between both assays (Farr: 82 and 66%, respectively; EliA: 93 and 43%, respectively). Conclusion Anti-dsDNA is most frequently positive using Farr and EliA during active nephritis, with comparable sensitivity. Both assays performed equally during exacerbations. However, EliA had higher specificity in quiescent disease and had several advantages, including no use of radioactive materials and less time required.

FSAP‐mediated nucleosome release from late apoptotic cells is inhibited by autoantibodies present in SLE

Inefficient clearance of apoptotic cells and the subsequent exposure of the immune system to nuclear contents are crucially involved in the pathogenesis of systemic lupus erythematosus (SLE). Factor VII‐activating protease (FSAP) is activated in serum upon contact with dead cells, and releases nucleosomes from late apoptotic cells into the extracellular environment. We investigated whether FSAP‐mediated nucleosome release from late apoptotic cells is affected in SLE patients. Nucleosome release in sera of 27 SLE patients and 30 healthy controls was investigated by incubating late apoptotic Jurkat cells with serum and analyzing the remaining DNA content by flow cytometry. We found that nucleosome release in sera of SLE patients with high disease activity was significantly decreased when compared with that in SLE sera obtained during low disease activity or from healthy individuals. Upon removal of IgG/IgM antibodies from SLE sera, nucleosome release was restored. Similarly, monoclonal antinuclear antibodies inhibited nucleosome release in healthy donor serum or by plasma‐purified FSAP. This inhibition was lost when Fab fragments were used, suggesting that antigen cross‐linking is involved. In conclusion, FSAP‐mediated nucleosome release from late apoptotic cells is greatly impaired in SLE patient sera, possibly hampering the clearance of these cells and thereby propagating inflammation.