Mathieu C. Tamby

New insights into the pathogenesis of systemic sclerosis.

Systemic sclerosis (SSc) is a connective tissue disorder characterized by vascular abnormalities and excessive collagen synthesis. Extracellular matrix overproduction by fibroblasts results from abnormal interactions among endothelial cells, mononuclear cells (lymphocytes and monocytes) and fibroblasts, in a setting of vascular hyperreactivity and tissue hypoxia. Many autoantibodies have been identified in the sera of SSc patients; some of them are specific to the disease, such as anti-centromere antibodies in limited SSc, anti-topoisomerase 1 and anti-RNA polymerase I/III antibodies in diffuse SSc. Their pathogenetic role(s) remains uncertain. However, genetic, environmental and possibly alloreactive factors might also contribute to disease susceptibility.

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.

Identification of Target Antigens of Antifibroblast Antibodies in Pulmonary Arterial Hypertension

RATIONALE Pulmonary arterial hypertension (PAH) may be classified as idiopathic (IPAH) or familial (FPAH) or associated with various conditions and exposures such as dexfenfluramine intake (Dex-PAH) or systemic sclerosis (SSc-PAH). Because fibroblast dysfunction has been identified in SSc and IPAH and antifibroblast antibodies (AFAs) with a pathogenic role have been detected in the serum of SSc patients, we used a proteomic approach combining two-dimensional electrophoresis and immunoblotting to identify the target antigens of AFAs in such patients. OBJECTIVES To identify target antigens of antifibroblast antibodies in pulmonary arterial hypertension. METHODS Sera from 24 patients with IPAH, 6 with FPAH, 6 with Dex-PAH, and 12 with SSc-PAH were collected. We pooled sera from sets of three patients with PAH classification and SSc-PAH based on autoantibody profile. Sera from 14 healthy blood donors were also pooled and used as a control. MEASUREMENTS AND MAIN RESULTS Serum IgG antibodies in the pools of patients with IPAH (n = 8), FPAH (n = 2), Dex-PAH (n = 2), and SSc-PAH (n = 4) recognized 103 +/- 31, 63 +/- 20, 78 +/- 11, and 81 +/- 12 protein spots, respectively, whereas serum IgG antibodies from healthy control subjects recognized 43 +/- 22 protein spots. Twenty-one protein spots were specifically recognized by the serum IgG antibodies from patients with PAH. We identified 16 of the protein spots as vimentin, calumenin, tropomyosin 1, heat shock proteins 27 and 70, glucose-6-phosphate-dehydrogenase, phosphatidylinositol 3-kinase, DAP kinase, and others. These proteins are involved in regulation of cytoskeletal function, cell contraction, oxidative stress, cell energy metabolism, and other key cellular pathways. CONCLUSIONS AFAs detected in patients with PAH recognize cellular targets playing key roles in cell biology and maintenance of homeostasis.

Pathogenesis of giant cell arteritis: More than just an inflammatory condition?

Giant cell arteritis (GCA) is characterized by intimal hyperplasia and luminal obstruction leading to ischemic manifestations involving extra-cranial branches of carotid arteries and aorta. Histopathological lesions involve all layers of the arterial wall and are associated with multinucleated giant cells, fragmented internal elastic lamina and polymorphic cellular infiltrates, including T lymphocytes and macrophages. The pathophysiology of GCA is still poorly understood. After dendritic cell activation, CD4(+) T lymphocytes, T helper 1 (Th1) cells, produce interferon gamma and modulate macrophage activation and functions, and Th17 cells produce interleukin 17 (IL-17), which can induce cytokine production by macrophages and fibroblasts. Macrophages in the adventitia produce pro-inflammatory cytokines such as IL-1, IL-6 and tumor necrosis factor alpha. These cytokines promote arterial wall and systemic inflammation. Questions remain regarding the nature of the antigen(s) triggering dendritic cell activation and the mechanisms underlying vascular remodeling. Here we review recent advances in the pathogenesis of GCA, with emphasis on the interactions between cells of the immune system and components of the vessel wall, including vascular smooth muscle cells and endothelial cells, leading to vascular remodeling. Finally, we propose new areas of investigation that could help understand the triggering factors and key pathogenic events in GCA.

The Role of Inflammation and Autoimmunity in the Pathophysiology of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension is characterized by a remodeling of pulmonary arteries with endothelial cell, fibroblast, and vascular smooth muscle cell activation and proliferation. Since pulmonary arterial hypertension occurs frequently in autoimmune conditions such as systemic sclerosis, inflammation and autoimmunity have been suspected to play a critical role in both idiopathic pulmonary arterial hypertension and systemic sclerosis-associated pulmonary arterial hypertension. High levels of pro-inflammatory cytokines such as interleukin-1 and interleukin-6, platelet-derived growth factor, or macrophage inflammatory protein 1 have been found in lung samples of patients with pulmonary arterial hypertension, along with inflammatory cell infiltrates mainly composed of macrophages and dendritic cells, T and B lymphocytes. In addition, circulating autoantibodies are found in the peripheral blood of patients. Thus, autoimmunity and inflammation probably play a role in the development of pulmonary arterial hypertension. In this setting, it would be important to set-up new experimental models of pulmonary arterial hypertension, in order to define novel therapeutics that specifically target immune disturbances in this devastating condition.

Targets of anti-endothelial cell antibodies in pulmonary hypertension and scleroderma

Anti-endothelial cell antibodies (AECAs) have been identified in patients with systemic sclerosis (SSc) with and without pulmonary arterial hypertension (PAH) and in patients with idiopathic pulmonary arterial hypertension (iPAH). However, their target antigens remain poorly identified. Sera from 24 patients with SSc without PAH, 20 patients with SSc with PAH, 30 with iPAH and 12 healthy controls were collected. Target antigens were identified by two-dimensional electrophoresis and immunoblotting in protein extracts of human umbilical vein endothelial cells. Targeted antigens were identified by mass spectrometry. Serum immunoglobulin G from patients with SSc with or without PAH and patients with iPAH specifically recognised 110, 82 and 37 protein spots, respectively. Among others, target antigens of AECAs included lamin A/C, tubulin &bgr;-chain and vinculin. One-dimension immunoblotting experiments confirmed the identification of lamin A/C and tubulin &bgr;-chain. In conclusion, our results confirm the presence of AECA in patients with systemic sclerosis with and without pulmonary arterial hypertension and in those with idiopathic pulmonary arterial hypertension, and provide evidence for the identification of target antigens of these autoantibodies including lamin A/C and tubulin &bgr;-chain.

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.

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.

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.