Nicolas Tamas

Antifibroblast antibodies from systemic sclerosis patients bind to α-enolase and are associated with interstitial lung disease

Objective: To identify target antigens of antifibroblast antibodies (AFA) in systemic sclerosis (SSc) patients. Patients and Methods: In the first part, sera from 24 SSc patients (12 with pulmonary arterial hypertension (PAH) and 12 without) and 36 idiopathic PAH patients, tested in pooled sera for groups of three, were compared with a sera pool from 14 healthy controls (HC). Serum IgG reactivity was analysed by the use of a two-dimensional electrophoresis and immunoblotting technique with normal human fibroblasts antigens. In the second part, serum IgG reactivity for two groups: 158 SSc, 67 idiopathic PAH and 100 HC; and 35 SSc and 50 HC was tested against α-enolase from Saccharomyces cerevisiae and recombinant human (rHu) α-enolase, respectively, on ELISA. Results: In the first part, α-enolase was identified as a main target antigen of AFA from SSc patients. In the second part, 37/158 (23%) SSc patients, 6/67 (9%) idiopathic PAH patients and 4/100 (4%) HC (p<0.001) had anti-S cerevisiae α-enolase antibodies; 12/35 (34%) SSc patients and 3/50 (6%) HC had anti-rHu α-enolase antibodies (p = 0.001). In SSc, the presence of anti-S cerevisiae α-enolase antibodies was associated with interstitial lung disease (ILD), decreased total lung capacity (73.2% vs 89.7%; p<0.001) and diffusion capacity for carbon monoxide (47.4% vs 62.3%; p<0.001), and antitopoisomerase 1 antibodies (46% vs 21%; p = 0.005) but not anticentromere antibodies (11% vs 34%; p = 0.006). Results were similar with rHu α-enolase testing. Conclusion: In SSc, AFA recognise α-enolase and are associated with ILD and antitopoisomerase antibodies.

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 μg/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 (β‐actin, α‐tubulin, and vimentin); glycolytic enzymes (glucose‐3‐phosphate‐deshydrogenase and α‐enolase); and prolyl‐4‐hydroxylase β 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.

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 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 β‐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 α‐enolase, RNA processing and cytoskeletal proteins such as lamin‐A/C.

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

Abstract:  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 μg/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 Edmans 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.

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.

Contribution of antiferritin antibodies to diagnosis of giant cell arteritis

Establishing the diagnosis of giant cell arteritis (GCA) may be challenging. In the absence of validated biological marker1 and despite imaging technique contribution,2 the diagnosis of GCA currently relies on temporal artery biopsy (TAB). Autoantibodies have been identified in GCA3–6 and recently, Baerlecken et al 7 detected IgG antibodies directed against a peptide of the human ferritin heavy chain (FTH1) in 92% of untreated GCA and polymyalgia rheumatica at first diagnosis versus 1% of healthy controls (HC). In order to evaluate the diagnosis value of these antibodies, we tested sera from 122 consecutive patients suspected of GCA at the time of TAB. Based on the American College of Rheumatology (ACR) criteria,8 40 patients had biopsy-proven GCA (TAB+GCA), 29 patients had biopsy-negative GCA (TAB−GCA), 47 patients received another diagnosis than GCA (GCA controls) and 6 patients had polymyalgia rheumatica (collection dc-2010–1079). Sera from 40 healthy blood donors were used as HCs. All patients and controls gave signed informed consent. The study was approved by the ethics …

Scleroderma Peripheral B Lymphocytes Secrete Interleukin-6 and Transforming Growth Factor β and Activate Fibroblasts: IL-6 AND TNFβ SECRETION BY SSc PERIPHERAL B LYMPHOCYTES

To study the role of B lymphocytes in systemic sclerosis (SSc).

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.

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.