Scleroderma Peripheral B Lymphocytes Secrete Interleukin-6 and Transforming Growth Factor ß and Activate Fibroblasts.

OBJECTIVE: To study the role of B lymphocytes in systemic sclerosis (SSc). METHODS: Peripheral B cell subpopulations and the production of interleukin-6 (IL-6) and transforming growth factor ß (TGFß) were analyzed using flow cytometry and multiplex assay. The fibroblast proliferation rate upon incubation with supernatants from B cells isolated from SSc patients or healthy controls was assessed using XTT, bromodeoxyuridine, and Ki-67. Collagen production was assessed using a collagen assay. RESULTS: Ninety untreated patients (12 males) fulfilling the American College of Rheumatology/European League Against Rheumatism criteria for SSc (23 with diffuse cutaneous SSc [dcSSc] and 67 with limited cutaneous SSc [lcSSc]) and 30 healthy controls were recruited. Increased proportions of B cells expressing CD69 and CD95 were identified among the patients with SSc. B lymphocytes from dcSSc patients versus lcSSc patients and healthy controls expressed increased proportion of cells positive for CD5 (mean ± SD 24.12 ± 7.93% versus 14.09 ± 6.58% [P = 0.03] and 14.21 ± 5.34% [P = 0.01]), CD86 (39.89 ± 22.11% versus 17.72 ± 13.98% [P = 0.0007] and 11.68 ± 11.09% [P < 0.001]), IL-6 receptor (IL-6R; 33.64 ± 23.12% versus 17.91 ± 13.62% [P < 0.0001] and 12.08 ± 8.68% [P = 0.0009]), or IL-21R (32.55 ± 20.19% versus 5.76 ± 4.40% [P < 0.0001] and 5.93 ± 3.29% [P < 0.0001]). In addition, the levels of IL-6 (mean ± SD 314.3 ± 317.8 pg/ml versus 6.10 ± 2.58 pg/ml; P = 0.0007) and TGFß (mean ± SD 1,020 ± 569 pg/ml versus 163.8 ± 98.69 pg/ml; P = 0.001) secreted by B lymphocytes from patients with SSc were increased compared to healthy controls. Fibroblast proliferation and collagen production were also significantly increased in the presence of B cell supernatant from SSc patients as compared to healthy controls. CONCLUSION: The numbers of activated B cells were increased in SSc patients, and the up-regulation of CD5, CD86, IL-6R, and IL-21R discriminated between patients with dcSSc and those with lcSSc. Peripheral B lymphocytes from SSc patients secreted both IL-6 and TGFß, and they activated fibroblasts in vitro.

Proteomic analysis of vascular smooth muscle cells in physiological condition and in pulmonary arterial hypertension: Toward contractile versus synthetic phenotypes.

Vascular smooth muscle cells (VSMCs) are highly specialized cells that regulate vascular tone and participate in vessel remodeling in physiological and pathological conditions. It is unclear why certain vascular pathologies involve one type of vessel and spare others. Our objective was to compare the proteomes of normal human VSMC from aorta (human aortic smooth muscle cells, HAoSMC), umbilical artery (human umbilical artery smooth muscle cells, HUASMC), pulmonary artery (HPASMC), or pulmonary artery VSMC from patients with pulmonary arterial hypertension (PAH-SMC). Proteomes of VSMC were compared by 2D DIGE and MS. Only 19 proteins were differentially expressed between HAoSMC and HPASMC while 132 and 124 were differentially expressed between HUASMC and HAoSMC or HPASMC, respectively (fold change 1.5≤ or -1.5≥, p < 0.05). As much as 336 proteins were differentially expressed between HPASMC and PAH-SMC (fold change 1.5≤ or -1.5≥, p < 0.05). HUASMC expressed increased amount of α-smooth muscle actin compared to either HPASMC or HAoSMC (although not statistically significant). In addition, PAH-SMC expressed decreased amount of smooth muscle myosin heavy chain and proliferation rate was increased compared to HPASMC thus supporting that PAH-SMC have a more synthetic phenotype. Analysis with Ingenuity identified paxillin and (embryonic lethal, abnormal vision, drosophila) like 1 (ELAVL1) as molecules linked with a lot of proteins differentially expressed between HPASMC and PAH-SMC. There was a trend toward reduced proliferation of PAH-SMC with paxillin-si-RNA and increased proliferation with ELAVL1-siRNA. Thus, VSMCs have very diverse protein content depending on their origin and this is in link with phenotypic differentiation. Paxillin targeting may be a promising treatment of PAH. ELAVL1 also participate in the regulation of PAH-SMC proliferation.

Identification of target antigens of anti-endothelial cell antibodies in patients with anti-neutrophil cytoplasmic antibody-associated vasculitides: a proteomic approach.

Anti-endothelial cell antibodies (AECAs) have been reported to cause endothelial cell dysfunction, but their specific targets have never been identified in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs). Proteins from human umbilical vein endothelial cells (HUVECs) were separated by 2-dimensional electrophoresis (2-DE). 2-D immunoblots were used to compare serum IgG reactivities from 30 patients with AAV and 12 healthy controls (HCs). Proteins identified as target antigens by MALDI- TOF-TOF mass spectrometry included lamin A/C, vimentin, α-enolase, far upstream binding protein 2 (FUBP2) and protein disulfide-isomerase A3 precursor (PDIA3). Antibodies targeting lamin A, vimentin, α-enolase, FUBP2 and PDIA3 were identified in 57.1%, 64.3%, 35.7%, 50% and 0% of patients with microscopic polyangiitis and 8%, 3.3%, 7.2%, 0% and 6.7% of HCs respectively. IgG from patients with microscopic polyangiitis had stronger reactivity against HUVEC than other groups and HCs and induced stronger Erk phosphorylation in HUVECs than IgG from HCs.

Proteomes of umbilical vein and microvascular endothelial cells reflect distinct biological properties and influence immune recognition.

Human umbilical vein endothelial cells (HUVEC) are widely used as a source of endothelial cells (EC). However, HUVEC characteristics cannot be extrapolated to other types of EC, particularly microvascular ECs. Our objective was to compare the proteomes of microvascular ECs and HUVEC. Proteomes of HUVEC and human microvascular pulmonary EC (HMVEC-P) and dermal EC (HMVEC-D) from healthy Caucasian donors were compared by 2D DIGE and MS. Fatty acid binding proteins 4 and 5 were among the 159 and 30 proteins spots found to have at least twofold change in expression between HUVEC and HMVEC-D and between HUVEC and HMVEC-P samples, respectively. Eight protein spots showed twofold changed expression between HMVEC-D and HMVEC-P samples. Ingenuity® analysis revealed that proteins differentially expressed between HUVEC and HMVEC-D samples interact with retinoic acid. In vitro tubulogenesis assays showed a differential effect of retinoic acid between HUVEC and HMVEC. Moreover, serum IgG from patients with a rare vascular disease, systemic sclerosis, showed distinct reactivity profiles in HUVEC and HMVEC-D protein extracts. The proteome profiles of HUVEC and microvascular EC differ noticeably, which reflects distinct biological properties and influence immune recognition.

Identification of target antigens of anti-endothelial cell and anti-vascular smooth muscle cell antibodies in patients with giant cell arteritis: a proteomic approach.

INTRODUCTION: Immunological studies of giant cell arteritis (GCA) suggest that a triggering antigen of unknown nature could generate a specific immune response. We thus decided to detect autoantibodies directed against endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in the serum of GCA patients and to identify their target antigens. METHODS: Sera from 15 GCA patients were tested in 5 pools of 3 patients' sera and compared to a sera pool from 12 healthy controls (HCs). Serum immunoglobulin G (IgG) reactivity was analysed by 2-D electrophoresis and immunoblotting with antigens from human umbilical vein ECs (HUVECs) and mammary artery VSMCs. Target antigens were identified by mass spectrometry. RESULTS: Serum IgG from GCA patients recognised 162 ± 3 (mean ± SD) and 100 ± 17 (mean ± SD) protein spots from HUVECs and VSMCs, respectively, and that from HCs recognised 79 and 94 protein spots, respectively. In total, 30 spots from HUVECs and 19 from VSMCs were recognised by at least two-thirds and three-fifths, respectively, of the pools of sera from GCA patients and not by sera from HCs. Among identified proteins, we found vinculin, lamin A/C, voltage-dependent anion-selective channel protein 2, annexin V and other proteins involved in cell energy metabolism and key cellular pathways. Ingenuity pathway analysis revealed that most identified target antigens interacted with growth factor receptor-bound protein 2. CONCLUSIONS: IgG antibodies to proteins in the proteome of ECs and VSMCs are present in the sera of GCA patients and recognise cellular targets that play key roles in cell biology and maintenance of homeostasis. Their potential pathogenic role remains to be determined.

Identification of target antigens of self-reactive IgG in intravenous immunoglobulin preparations.

Intravenous immunoglobulin (IVIg) contains a wide range of self-reactive immunoglobulins (Ig) G. Acidic pH is known to increase the reactivity of purified IgG with self-antigens. We describe here the target antigens of IgG autoantibodies in IVIg and analyze the influence of acidic pH on IgG reactivities. We used 2-DE and immunoblotting with protein extracts of human umbilical vein endothelial cells (HUVEC) and HEp-2 cells. Two IVIg preparations obtained by ethanol fractionation [one with an acidic pH step (acidic-IVIg) and one with beta-propiolactone (propiolactone-IVIg)] and a pool of sera from 12 healthy individuals were tested. Serum IgG of 3 healthy individuals and IgG purified from the same sera with elution at pH 2.8 were also tested individually. Finally, propiolactone-IVIg was acidified at pH 2.8. IgG obtained with a step at low pH recognized many more target spots than IgG obtained without acidic pH. Our findings demonstrate that an acidic pH step artificially enlarges the repertoire of self-reactive IgG. Thus, protein spots recognized by IgG in propiolactone-IVIg represent the core set of self-antigens targeted by IVIg. Overall, 96 proteins were identified by MS. Fourteen were recognized in both extracts including glycolysis proteins such as alpha-enolase, RNA processing and cytoskeletal proteins such as lamin-A/C.

Natural anti-endothelial cell antibodies.

Anti-endothelial cell antibodies (AECA) are detectable in a heterogenous group of autoimmune and inflammatory conditions. These antibodies have also been detected in healthy individuals. Nevertheless, most of the literature focuses on AECA in pathological conditions and their targets and functions in healthy individuals remain to be clarified. Recently, proteome-based technologies have been successfully used for the identification of antigens targeted by natural AECA. Thus, it has been shown that IgG AECA can be detected in sera from healthy individuals that recognize a restricted set of proteins among a whole protein extract of umbilical vein endothelial cells. These proteins correspond to ubiquitous proteins belonging to highly conserved protein families and exerting pivotal roles in cell physiology, including cytoskeletal proteins (beta actin, vimentin, alpha tubulin) and glycolytic enzymes (glyceraldehyde-3-phosphate-deshydrogenase and alpha-enolase). As reported for other types of natural autoantibodies, natural AECA could exert anti-inflammatory and anti-thrombotic functions. In addition, they could play a role in the control of EC activation.

Identification of target antigens of antiendothelial cell antibodies in healthy individuals: A proteomic approach.

In order to identify target antigens of anti-endothelial cell (anti-EC) antibodies (AECA) in healthy individuals, we have used a proteomic approach combining 2-DE and immunoblotting. Whole cell protein extracts obtained from human umbilical vein EC (HUVEC) cultures were used as a source of antigens. Serum IgG from 12 healthy blood donors were tested at a concentration of 200 microg/mL. Targeted spots were identified by MS. The HUVEC proteome was composed of 884 protein spots. Among these, 61 +/- 25.8 (mean +/- SD) spots were recognized by serum IgG from healthy individuals, with marked differences from one individual to another. Among these spots, 11 were recognized by serum IgG from all healthy individuals tested. These spots corresponded to six different proteins with several spots corresponding to different isoforms of the same protein. Target antigens were: cytoskeletal proteins (beta-actin, alpha-tubulin, and vimentin); glycolytic enzymes (glucose-3-phosphate-deshydrogenase and alpha-enolase); and prolyl-4-hydroxylase beta subunit, a member of the disulfide isomerase family. This study shows that the repertoire of IgG AECA is heterogeneous among healthy individuals. IgG from all of the healthy individuals tested recognized a restricted set of highly conserved ubiquitous proteins playing key roles in cell biology and maintenance of homeostasis.

IgG from patients with systemic sclerosis bind to DNA antitopoisomerase 1 in normal human fibroblasts extracts.

By using a semi-quantitative immunoblotting technique, we have analyzed serum immunoglobulin G (IgG) reactivities of patients with limited cutaneous systemic sclerosis and anticentromere antibodies, patients with diffuse systemic sclerosis and antitopoisomerase 1 antibodies, patients with diffuse systemic sclerosis without antitopoisomerase 1 or anticentromere antibodies and age- and gender-matched healthy controls with normal human skin fibroblasts and HEp-2 cells antigens. Serum IgG reactivities of patients with diffuse systemic sclerosis and antitopoisomerase 1 antibodies differed significantly from those of healthy controls or systemic sclerosis patients in other groups for reactivity with fibroblast proteins. IgG from patients with antitopoisomerase 1 antibodies bound to a 90 kDa fibroblast band and to a 100 kDa protein band in a HEp-2 cell protein extract. These two bands were further identified as DNA topoisomerase 1. Our results indicate that IgG from patients with diffuse systemic sclerosis bind DNA topoisomerase 1 in normal human fibroblasts extracts.

A combined SDS-PAGE and proteomics approach to identify target autoantigens in healthy individuals and patients with autoimmune diseases.

We here present a method for the identification of antigens recognized by autoantibodies in healthy individuals and patients with autoimmune diseases. We have analyzed IgG reactivities from healthy individuals and patients with autoimmune diseases with endothelial cell antigens by combining a one-dimensional (1D) quantative immunoblotting technique and a 2D immunoblotting technique. Whole-cell protein extracts obtained from human umbilical vein endothelial cells (HUVEC) were used as a source of antigens. Serum IgG from healthy blood donors and from patients with autoimmune diseases were tested at a concentration of 200 microg/mL. One-dimensional immunoblotting was first performed to detect candidate reactivity bands and 2D immunoblotting was secondly performed following 2D electrophoresis to identify protein spots. The gels and 2D blots were scanned and analyzed by imaging software. The matching permitted exact localization of particular relevant protein spots hybridized by antibodies on the 2D blots. The targeted bands from 1D spots and the targeted spots from 2D gels were identified by Edman's N-terminal sequencing and mass spectrometry, respectively. This approach is applicable to both normal and pathological conditions, potentially leading to the identification of new relevant target antigens.

Immunoblotting on HEp-2 cells increases the detection of antitopoisomerase 1 antibodies in patients with systemic sclerosis.

In order to improve the detection of antitopoisomerase 1 antibodies in a cohort of 111 systemic sclerosis patients, we have performed immunoblots on protein extracts of HEp-2 cells. Using indirect immunofluorescence and ELISA, 27 patients (24.3%) had antitopoisomerase 1 antibodies, 32 (28.8%) had anticentromere antibodies, 31 (27.9%) had antinuclear antibodies with none of these two antibodies and 21 (18.9%) had no antinuclear antibody. IgG from 24/27 (88.9%) patients with antitopoisomerase 1 antibodies bound to 2 protein bands of 63 and 100 kDa identified as topoisomerase 1 by N-terminal sequencing. Antitopoisomerase 1 antibodies were detected in 9 (8.1%) patients who had no antitopoisomerase 1 antibody as determined by ELISA. Patients with antitopoisomerase 1 antibodies had an almost similar phenotype without distinction between ELISA or immunoblot approaches. Our results provide evidence for the use of a combination of ELISA and immunoblot approaches for the detection of antitopoisomerase 1 antibodies.