Amélie Servettaz

Selective Oxidation of DNA Topoisomerase 1 Induces Systemic Sclerosis in the Mouse

Systemic sclerosis (SSc) is a connective tissue disorder of great clinical heterogeneity. Its pathophysiology remains unclear. Our aim was to evaluate the relative roles of reactive oxygen species (ROS) and of the immune system using an original model of SSc. BALB/c and immunodeficient BALB/c SCID mice were injected s.c. with prooxidative agents (hydroxyl radicals, hypochlorous acid, peroxynitrites, superoxide anions), bleomycin, or PBS everyday for 6 wk. Skin and lung fibrosis were assessed by histological and biochemical methods. Autoantibodies were detected by ELISA. The effects of mouse sera on H2O2 production by endothelial cells and on fibroblast proliferation, and serum concentrations in advanced oxidation protein products (AOPP) were compared with sera from patients with limited or diffuse SSc. We observed that s.c. peroxynitrites induced skin fibrosis and serum anti-CENP-B Abs that characterize limited SSc, whereas hypochlorite or hydroxyl radicals induced cutaneous and lung fibrosis and anti-DNA topoisomerase 1 autoantibodies that characterize human diffuse SSc. Sera from hypochlorite- or hydroxyl radical-treated mice and of patients with diffuse SSc contained high levels of AOPP that triggered endothelial production of H2O2 and fibroblast hyperproliferation. Oxidized topoisomerase 1 recapitulated the effects of whole serum AOPP. SCID mice developed an attenuated form of SSc, demonstrating the synergistic role of the immune system with AOPP in disease propagation. We demonstrate a direct role for ROS in SSc and show that the nature of the ROS dictates the form of SSc. Moreover, this demonstration is the first that shows the specific oxidation of an autoantigen directly participates in the pathogenesis of an autoimmune disease.

Antibodies to fibroblasts in idiopathic and scleroderma-associated pulmonary hypertension

The aim of the present study was to investigate the presence of anti-fibroblast antibodies in patients with idiopathic or scleroderma-associated pulmonary arterial hypertension (PAH) and healthy controls. PAH was documented by right-heart catheterisation (mean pulmonary artery pressure at rest >25 mmHg). Serum immunoglobulin (Ig)G and IgM reactivities of patients with idiopathic PAH (n = 35), scleroderma-associated PAH (n = 10), diffuse (n = 10) or limited cutaneous (n = 10) scleroderma without PAH and age- and sex-matched healthy individuals (n = 65) were analysed by cell-based ELISA and immunoblotting on normal human fibroblasts. As assessed by ELISA, 14 out of 35 (40%) patients with idiopathic PAH and three out of 10 (30%) patients with scleroderma-associated PAH expressed anti-fibroblast IgG antibodies. IgG from all individuals bound to one major 40-kDa protein band. IgG from patients with idiopathic PAH bound to two 25- and 60-kDa bands with a higher intensity than IgG from other individuals. In conclusion, immunoglobulin G anti-fibroblast antibodies are present in the serum of patients with pulmonary arterial hypertension. Immunoglobulin G from patients with idiopathic pulmonary arterial hypertension or scleroderma-associated pulmonary arterial hypertension express distinct reactivity profiles with fibroblasts antigens, suggesting distinct target antigens.

Targeting ADAM-17/notch signaling abrogates the development of systemic sclerosis in a murine model

OBJECTIVE Systemic sclerosis (SSc) is characterized by the fibrosis of various organs, vascular hyperreactivity, and immunologic dysregulation. Since Notch signaling is known to affect fibroblast homeostasis, angiogenesis, and lymphocyte development, we undertook this study to investigate the role of the Notch pathway in human and murine SSc. METHODS SSc was induced in BALB/c mice by subcutaneous injections of HOCl every day for 6 weeks. Notch activation was analyzed in tissues from mice with SSc and from patients with scleroderma. Mice with SSc were either treated or not treated with the γ-secretase inhibitor DAPT, a specific inhibitor of the Notch pathway, and the severity of the disease was evaluated. RESULTS As previously described, mice exposed to HOCl developed a diffuse cutaneous SSc with pulmonary fibrosis and anti-DNA topoisomerase I antibodies. The Notch pathway was hyperactivated in the skin, lung, fibroblasts, and splenocytes of diseased mice and in skin biopsy samples from patients with scleroderma. ADAM-17, a proteinase involved in Notch activation, was overexpressed in the skin of mice and patients in response to the local production of reactive oxygen species. In HOCl-injected mice, DAPT significantly reduced the development of skin and lung fibrosis, decreased skin fibroblast proliferation and ex vivo serum-induced endothelial H(2)O(2) production, and abrogated the production of anti-DNA topoisomerase I antibodies. CONCLUSION Our results show the pivotal role of the ADAM-17/Notch pathway in SSc following activation by reactive oxygen species. The inhibition of this pathway may represent a new treatment of this life-threatening disease.

Targeting the cannabinoid pathway limits the development of fibrosis and autoimmunity in a mouse model of systemic sclerosis.

Our aim was to evaluate the roles of the cannabinoid pathway in the induction and propagation of systemic sclerosis (SSc) in a mouse model of diffuse SSc induced by hypochlorite injections. BALB/c mice injected subcutaneously every day for 6 weeks with PBS or hypochlorite were treated intraperitoneally with either WIN-55,212, an agonist of the cannabinoid receptors 1 (CB1) and receptors 2 (CB2), with JWH-133, a selective agonist of CB2, or with PBS. Skin and lung fibrosis were then assessed by histological and biochemical methods, and the proliferation of fibroblasts purified from diseased skin was assessed by thymidine incorporation. Autoantibodies were detected by ELISA, and spleen cell populations were analyzed by flow cytometry. Experiments were also performed in mice deficient for CB2 receptors (Cnr2(-/-)). Injections of hypochlorite induced cutaneous and lung fibrosis as well as increased the proliferation rate of fibroblasts isolated from fibrotic skin, splenic B cell counts, and levels of anti-DNA topoisomerase-1 autoantibodies. Treatment with WIN-55,212 or with the selective CB2 agonist JWH-133 prevented the development of skin and lung fibrosis as well as reduced fibroblast proliferation and the development of autoantibodies. Experiments performed in CB2-deficient mice confirmed the influence of CB2 in the development of systemic fibrosis and autoimmunity. Therefore, we demonstrate that the CB2 receptor is a potential target for the treatment of SSc because it controls both skin fibroblast proliferation and the autoimmune reaction.

Physiopathologie de la sclérodermie systémique: état des lieux sur une affection aux multiples facettes

Points essentiels La sclerodermie systemique est une affection rare qui se traduit par des lesions de fibrose et une hyperreactivite vasculaire. Des dysfonctionnements des cellules endotheliales, des fibroblastes et des lymphocytes ont ete identifies. Le dysfonctionnement des fibroblastes se caracterise par une activation incontrolee de la voie du Transforming Growth Factor-s (TGF-s), une synthese inadaptee de Connective Tissue Growth Factor (CTGF) et de radicaux libres, favorisant la synthese de matrice extracellulaire en exces. Les cellules endotheliales synthetisent en exces de l’endotheline 1, vasoconstricteur puissant, produisent de grandes quantites de NO-synthase inductible et subissent une apoptose precoce. Le stress oxydatif semble jouer un role majeur dans la progression de la sclerodermie systemique. Des taux eleves d’interleukine 4, cytokine profibrosante, sont trouves dans le plasma et le derme de malades atteints de sclerodermie systemique. Des autoanticorps sont detectables dans le serum de la majorite des patients sclerodermiques. Certains sont diriges contre des proteines nucleaires ubiquitaires bien identifiees mais n’ont pas de role pathogenique demontre. D’autres autoanticorps reconnaissent les cellules endotheliales et/ou les fibroblastes et pourraient avoir un role pathogene.

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.

Pathogénie des vascularites systémiques primitives (I) : vascularites ANCA-positives

Points essentiels ● La pathogenie des vascularites systemiques associees aux anticorps anti-cytoplasme de polynucleaires neutrophiles (ANCA) est incompletement elucidee. ● Parmi les vascularites ANCA-positives qui interessent les vaisseaux de petit calibre, on distingue la granulomatose de Wegener (GW), la polyangeite microscopique (MPA) et le syndrome de Churg et Strauss (SCS). Au cours de ces pathologies, les ANCA constituent un outil precieux d’aide au diagnostic. ● Des ANCA diriges contre la proteinase 3 sont detectes chez plus de 90 % des malades atteints de forme systemique de GW, tandis que des ANCA anti-myeloperoxydase (MPO) sont presents chez 60 a 75 % des malades atteints de MPA et 40 a 60 % des malades atteints de SCS. ● Le role pathogene des ANCA est bien documente in vivo, et le transfert passif des ANCA anti-MPO est suffisant pour induire des lesions de glomerulonephrite extra-capillaire dans un modele de souris invalidees pour le gene de la MPO et immunisees avec cet antigene. In vitro, chez la souris et chez l’homme, les ANCA anti-proteinase 3 sont capables d’activer les polynucleaires neutrophiles en presence de TNF-α et contribuent a la survenue des lesions. ● Des lymphocytes T (LT) pourraient jouer un role dans la pathogenie de la GW, des LT helper de type 1 ayant ete detectes dans les tissus de patients ayant une GW localisee, tandis que des LT helper de type 2 ont ete identifies au sein de lesions de vascularites de malades ayant une forme systemique de GW. ● Les polynucleaires eosinophiles pourraient jouer un role dans la pathogenie du SCS.The pathogenesis of different types of systemic vasculitis negative for antineutrophil cytoplasm antibodies (ANCA) and involving small or medium-sized vessels is not very well documented. During polyarteritis nodosa (PAN), which is related to hepatitis B virus (HBV) infection, as well as during cryoglobulinemic vasculitides, associated with hepatitis C virus (HCV), and probably during Henoch Schönlein purpura, histological lesions may result from the deposition of immune complexes formed from viral antigens and from antibodies responsible for the activation of the classic complement pathway and for recruitment of polymorphonuclear neutrophils. Two other mechanisms are discussed for other types of ANCA-negative systemic vasculitis: immune complex deposition and sheer stress at arterial bifurcation points. A bacterial superantigen is suspected in Kawasaki disease but remains unproved.

New insights on chemically induced animal models of systemic sclerosis.

Purpose of reviewTo discuss the most recent published studies on chemical-induced animal models of systemic sclerosis (SSc) and to precise the important signalling pathways that lead to the initiation and progression of the disease in these models. Recent findingsEnvironmental factors undoubtedly contribute to the initiation and the development of SSc. Among those factors, bleomycin has been identified as a possible SSc-inducing substance in mice. The bleomycin model mimics the inflammatory changes observed in the early phase of the disease. This model has been extensively studied and has allowed identification of several key pathways activated in the human disease. More recently, a new chemical-induced model of scleroderma has been developed in mice using daily intradermal injections of a solution generating hypochlorous acid (HOCl)-model. This HOCl-model recapitulates the whole spectrum of the human disease, as fibrosis, inflammation, autoimmunity and vasculopathy can be observed in mice and brought new arguments for a major role of reactive oxygen species in the induction of local and systemic fibrosis. SummaryChemically induced models truly develop a SSc-like disease and argue for a crucial role of ROS in SSc.

Serum eosinophil cationic protein: a marker of disease activity in Churg-Strauss syndrome.

Abstract:  The cytotoxic proteins released by activated eosinophils should play a role in the development of Churg‐Strauss syndrome (CSS). Eighteen patients (15 males and 3 females, age 41 ± 13.3 years) with CSS according to the American College of Rheumatology criteria were included in the study. Thirteen serum samples from 11 patients were obtained at the time of disease flare, and the sera from 6 of them were also obtained at the time of clinical remission. Sera from seven other patients were obtained in clinical remission. Anti‐neutrophil cytoplasm antibodies were detected in four (22.2%) patients. Fifteen healthy individuals were used as controls. Mean eosinophil count differed significantly between CSS patients with active disease and patients in clinical remission (3,407/mm3 vs. 258/mm3; P < 0.01), between CSS patients with active disease and healthy individuals (3,407/mm3 vs. 211/mm3; P < 0.01). Mean serum ECP levels differed significantly between patients with active or inactive disease (219 μg/L vs. 56.8 μg/L; P < 0.0001), between patients with active disease and healthy individuals (219 μg/L vs. 26.2 μg/L; P < 0.0001), but not between patients with inactive disease and healthy individuals (ns). Peripheral blood eosinophils count correlated with serum ECP during CSS flares disease (R= 0.6264; P < 0.05) and during periods of remission (R= 0.4798; P < 0.05). Our results support that ECP might be used as a disease activity marker in CSS.