Skewed peripheral B- and T-cell compartments in patients with ANCA-associated vasculitis.

OBJECTIVES: To characterize lymphocytes dysregulation in patients with granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). METHODS: Using flow cytometry, we analysed B- and T-cell subsets in peripheral blood from 37 untreated patients with active disease (29 GPA and 8 MPA) and 22 healthy controls (HCs). RESULTS: GPA patients had increased Th2 (1.8 vs 1.0%, P = 0.02), Th9 (1.1 vs 0.2%, P = 0.0007) and Th17 (1.4 vs 0.9%, P = 0.03) cells compared with HC. Patients with MPO-ANCAs had significantly more CD21- B cells than HC or PR3-ANCA patients (6.9 vs 3.3% and 4.4%, P = 0.01). CD69 expressing B cells were significantly higher in GPA and MPA (3.0 and 5.9 vs 1.4%, P = 0.02 and P = 0.03, respectively) compared with HC, whereas B-cell activating factor-receptor expression was decreased in GPA and MPA (median fluorescence intensity ratio 11.8 and 13.7 vs 45.1 in HC, P < 0.0001 and P = 0.003, respectively). Finally, IL-6-producing B cells were increased in GPA vs HC (25.8 vs 14.9%, P < 0.0001) and decreased in MPA vs HC (4.6 vs 14.9%, P = 0.005), whereas TNF-α-producing B cells were lower in both GPA and MPA patients compared with controls (15 and 8.4 vs 30%, P = 0.01 and P = 0.006, respectively). CONCLUSION: Skewed T-cell polarization towards Th2, Th9 and Th17 responses characterizes GPA, whereas B-cell populations are dysregulated in both GPA and MPA with an activated phenotype and a decreased B-cell activating factor-receptor expression. Finally, inflammatory B cells producing IL-6 are dramatically increased in GPA, providing an additional mechanism by which rituximab could be effective.

Proteomic Analysis of Human Scleroderma Fibroblasts Response to Transforming Growth Factor-ß.

PURPOSE: Systemic sclerosis (SSc) is characterized by autoimmunity, vasculopathy and fibrosis. Fibrosis is due to an activation of fibroblasts by the transforming growth factor-ß (TGF-ß). This study investigates the proteomic response of SSc fibroblasts to TGF-ß. EXPERIMENTAL DESIGN: Skin fibroblasts from diffuse SSc patients and healthy controls (HC) are cultured with or without TGF-ß. Two-dimensional differential in-gel electrophoresis and mass spectrometry (MS) combined with Ingenuity Pathway analysis (IPA) and Panther/David software analyze proteins differentially expressed between groups. Real-time cell analyzer (RTCA) assesses fibroblast proliferation and viability. RESULTS: Two-hundred-and-seventy-nine proteins are differentially expressed between groups. Principal component analysis shows significant differences between groups. IPA shows specific process networks such as actin cytoskeleton and integrin signaling. Panther and David software show predominant biological processes such as cellular and metabolic processes. TGF-ß enhances protein synthesis and protein pathways. IPA and RTCA suggest the involvement of epidermal growth factor receptor (EGFR) and phosphatidylinositol 3 kinase (Pi3K). CONCLUSIONS AND CLINICAL RELEVANCE: That the proteome of fibroblasts differs between SSc patients and HC is confirmed, and it is demonstrated that fibroblasts exacerbate their proteomic phenotype upon stimulation with TGF-ß. EGFR and Pi3K are highlighted as proteins of interest in SSc fibroblasts.

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.

Implication of B lymphocytes in the pathogenesis of ANCA-associated vasculitides.

ANCA-associated vasculitis (AAV) is a subgroup of vasculitides characterized by the detection of anti-neutrophil cytoplasm antibodies (ANCA). Until recently, the pathogenesis of AAV mainly involved neutrophils, T cells, and ANCA. Importantly, data were recently published supporting B-cell implication in this setting. Thus, the identification of activated B lymphocytes in granulomatous lesions and the efficacy of B-cell depletion using rituximab in the treatment of patients with AAV changed our mind. However, the impact of B lymphocytes on disease activity and its specific role in the pathogenesis of AAV remains unclear, at least in part as the consequence of the limited number of patients investigated and the restricted number of studies investigating B-cell subsets. Perturbations of B-cell homeostasis have been identified in AAV with increased expression of CD38 and decreased expression of CD5 in active phase, contrasting with increased expression of CD25 and CD86 in remission state. Although decreased secretion of interleukin (IL)-10 has also been reported during disease flares, these data remain controversial and the cytokines secretion profile of B-cells needs to be further investigated. Herein, we summarize recent advances in the understanding of the implications of B-cells in the field of AAV and propose new fields of investigation for a better understanding of B lymphocytes in the pathogenesis of AAV.

Pathophysiology of systemic sclerosis: state of the art in 2014.

Major work has been done in order to improve the understanding of systemic sclerosis (SSc) pathogenesis. A number of new experimental models have been set up, that should help to understand the disease pathogenesis and test new therapeutic targets. Reactive oxygen species represent a hallmark of the pathogenesis of SSc, both at the fibroblast and at the endothelial cell levels. Although a large number of genetic studies have been conducted, it is still difficult to identify a genetic background specific to SSc, and the major progress in this setting is probably the identification of an interferon signature. Besides endothelial cells and fibroblasts, major development has been made in the understanding of the role of B cells and autoantibodies in the pathogenesis of SSc. Plasmacytoid dendritic cells seem to play a major role in the pathogenesis of SSc through the secretion of CXCL4, although these data will need to be confirmed in the near future.