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

Abstract

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 Hthymidine 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 Patients with systemic lupus erythematosus (SLE) manifest immunologic abnormalities that include spontaneous B lymphocyte proliferation, hyperresponsiveness to physiologic stimuli, and altered pattern of production of and responses to cytokines (1-3). One consequence of these abnormalities is the production of pathogenic autoantibodies to nuclear antigens, including double-stranded DNA (dsDNA). Although anti-dsDNA autoantibodies have features indicating T lymphocytedriven responses, several studies suggest that the production of anti-dsDNA autoantibodies could also be attributable to intrinsic B cell defects (4). Furthermore, studies in gene-deficient mice have revealed that defects in negative regulators of B cell receptor (BCR) signaling, CD22, Fc␥ receptor II (Fc␥RII), or CD72, result in production of anti-dsDNA autoantibodies (5-7). In patients with SLE, there is evidence that a reduction in expression of Fc␥RIIA and CD22 relates to anti-dsDNA production (8,9). However, the molecular basis for the relationship between BCR signaling defects and SLE immunopathology remains unclear.

Altered lipid raft–associated proximal signaling and translocation of CD45 tyrosine phosphatase in B lymphocytes from patients with systemic lupus erythematosus

“…B cells were isolated by depleting non-B cells with a cocktail of mouse monoclonal antibodies (mAb) against CD3, CD4, CD8, CD14, CD16, and CD56 (10). The following antibodies were used for immunoprecipitation and Western blotting: rabbit anti-Lyn (sc-15), anti-c-Cbl (C-20), and anti-Csk (C-15), mouse anti-CD45 (clones 35 Isolation and culture of B lymphocytes.…”

“…For some experiments, B cells (1-2 ϫ 10 5 ) were rested for 1 hour at 37°C and then activated with the coated beads (cell: bead ratio 1:3) at 37°C. After stimulation, B cells were either fixed and stained for confocal microscopy or lysed in 1% Triton X-100 buffer for Western blotting (10).…”

“…LRs were purified on a 2.5-ml 5-40% discontinuous sucrose gradient (10). Fractions 2 and 3 (5-30% interphase containing LRs) and fractions 8 and 9 (non-LR fractions) were pooled, and proteins were precipitated with methanol-chloroform.…”

“…However, Lyn also plays an indispensable negative regulatory role in proximal signaling (16). In this respect, it is notable that alterations in the level of Lyn and/or other regulatory molecules in LRs are related to lupus autoimmunity in human patients (10,17,18).…”

Association of antioxidized low‐density lipoprotein antibodies with vascular arterial events and renal manifestations in systemic lupus erythematosus patients: Comment on the article by Frostegård et al

“…We read with great interest the article by Frostegård et al addressing the association between antioxidized lowdensity lipoprotein (anti-OxLDL) antibodies and specific clinical manifestations, including vascular arterial events and renal involvement in patients with systemic lupus erythematosus (SLE) (1). Although the relationship between anti-OxLDL antibodies and vascular arterial events in SLE has previously been recognized in cohorts of European ancestry (2,3), our group (4) as well as Romero et al (5) have failed to corroborate these observations when studying multiethnic lupus cohorts.…”