Peter Szodoray1, Stephanie M Stanford2, Øyvind Molberg3, Ludvig A Munthe4, Nunzio Bottini2, Britt Nakken5. 1. Centre for Immune Regulation, Department of Immunology, University of Oslo and Oslo University Hospital-Rikshospitalet, Oslo, Norway. Electronic address: peter.szodoray@medisin.uio.no. 2. Division of Cellular Biology, La Jolla Institute for Allergy and Immunology, La Jolla, Calif. 3. Department of Rheumatology, Oslo University Hospital-Rikshospitalet, Oslo, Norway; Institute of Clinical Medicine, University of Oslo, Oslo, Norway. 4. Centre for Immune Regulation, Department of Immunology, University of Oslo and Oslo University Hospital-Rikshospitalet, Oslo, Norway; Institute of Clinical Medicine, University of Oslo, Oslo, Norway. 5. Centre for Immune Regulation, Department of Immunology, University of Oslo and Oslo University Hospital-Rikshospitalet, Oslo, Norway.
Abstract
BACKGROUND: We recently identified a human B-cell population that is naturally autoreactive and tolerized by functional anergy (BND cells). OBJECTIVE: We sought to identify the molecular mechanism of how anergic autoreactive BND cells escape functional anergy and whether this process is altered in patients with lupus. METHODS: Isolated peripheral blood naive and BND cells were cultured with various stimuli, and their activation status was determined by using an intracellular Ca(2+) mobilization assay. Lyn kinase and Syk activities were assessed by using phospho-flow analysis. CD45 phosphatase activity was determined by using a novel flow-based assay, which takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid, an analog of phosphotyrosine, which can be incorporated into peptides. Real-time quantitative PCR was used to quantitate LYN, SYK, and CD45 mRNA. RESULTS: T-helper signals reversed the state of anergy, allowing BND cells to fully respond to antigenic stimulation by restoring signaling through the B-cell receptor (BCR). The mechanism was dependent on increased activity of the tyrosine phosphatase CD45 and CD45-dependent activation of Lyn and Syk. CD45 phosphatase activity was increased by T-cell help both in BND and naive B cells. Furthermore, we found that BND cells obtained from patients with systemic lupus erythematosus exhibited increased CD45 activity and BCR-signaling capacity, thus being less tolerized than BND cells from healthy control subjects. CONCLUSION: Our findings suggest that CD45 is a key regulator of BCR-signaling thresholds mediated by T-cell help. This raises the possibility that BND cells could represent precursors of autoantibody-secreting plasma cells and suggests a role for these autoreactive B cells in contributing to autoimmunity if not properly controlled.
BACKGROUND: We recently identified a human B-cell population that is naturally autoreactive and tolerized by functional anergy (BND cells). OBJECTIVE: We sought to identify the molecular mechanism of how anergic autoreactive BND cells escape functional anergy and whether this process is altered in patients with lupus. METHODS: Isolated peripheral blood naive and BND cells were cultured with various stimuli, and their activation status was determined by using an intracellular Ca(2+) mobilization assay. Lyn kinase and Syk activities were assessed by using phospho-flow analysis. CD45 phosphatase activity was determined by using a novel flow-based assay, which takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid, an analog of phosphotyrosine, which can be incorporated into peptides. Real-time quantitative PCR was used to quantitate LYN, SYK, and CD45 mRNA. RESULTS: T-helper signals reversed the state of anergy, allowing BND cells to fully respond to antigenic stimulation by restoring signaling through the B-cell receptor (BCR). The mechanism was dependent on increased activity of the tyrosine phosphatase CD45 and CD45-dependent activation of Lyn and Syk. CD45 phosphatase activity was increased by T-cell help both in BND and naive B cells. Furthermore, we found that BND cells obtained from patients with systemic lupus erythematosus exhibited increased CD45 activity and BCR-signaling capacity, thus being less tolerized than BND cells from healthy control subjects. CONCLUSION: Our findings suggest that CD45 is a key regulator of BCR-signaling thresholds mediated by T-cell help. This raises the possibility that BND cells could represent precursors of autoantibody-secreting plasma cells and suggests a role for these autoreactive B cells in contributing to autoimmunity if not properly controlled.
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