Decreased Lyn expression and translocation to lipid raft signaling domains in B lymphocytes from patients with systemic lupus erythematosus

OBJECTIVE: B lymphocytes from patients with systemic lupus erythematosus (SLE) are hyperactive and produce anti-double-stranded DNA (anti-dsDNA) autoantibodies. The cause or causes of B cell defects in SLE are unknown. In this study, we determined the level and subcellular distribution of Lyn protein, a key negative regulator of B cell receptor signaling, and assessed whether altered Lyn expression is characteristic of B cells in the setting of SLE. METHODS: Negative selection was used to isolate B lymphocytes from blood. Lipid raft signaling domains were purified from B cells obtained from 62 patients with SLE, 15 patients with rheumatoid arthritis, and 31 healthy controls, by gradient ultracentrifugation. The total Lyn protein level was determined by Western blotting, confocal microscopy, and fluorescein-activated cell sorting (FACS). The distribution of Lyn into lipid raft and nonlipid raft domains was determined by Western blotting and confocal microscopy. Lyn content in B cell subpopulations was determined by FACS. In order to assess B lymphocyte activity, we used (3)H-thymidine incorporation and enzyme-linked immunosorbent assay to measure spontaneous proliferation and IgG and cytokine production by B cells. RESULTS: This study revealed that B lymphocytes from a majority of patients with SLE have a reduced level of Lyn and manifest altered translocation to lipid rafts. An investigation into the mechanisms of Lyn reduction suggested that increased ubiquitination is involved. This was evident from increased ubiquitination of Lyn and translocation of c-Cbl into lipid rafts. Studies of B cell responses showed that altered Lyn expression was associated with heightened spontaneous proliferation, anti-dsDNA autoantibodies, and increased interleukin-10 production. CONCLUSION: This study provides evidence for altered Lyn expression in B cells from a majority of patients with SLE. Altered Lyn expression in SLE may influence the B cell receptor signaling and B cell hyperactivity that are characteristic of the disease.