Representative blots from two impartial experiments are shown in the left panels

Representative blots from two impartial experiments are shown in the left panels. an aggressive autoimmune disorder that afflicts 1.5 million Americans. B cell hyperactivity and anti-dsDNA autoantibody production are hallmarks of SLE (1C3), signifying that altered B cell Acalisib (GS-9820) function is usually central to the pathogenesis of SLE. Human GWAS studies and genetic knockouts have revealed a powerful biochemical pathway that maintains B cell antigen receptor (BCR) signaling under check (4C6); Lyn tyrosine kinase is usually a key component of this pathway. Lyn phosphorylates CD22 on inhibitory tyrosines (7), which then recruits the phosphatase Shp-1 resulting in downregulation of BCR signaling (8). Genetic variations in Lyn (9) and reduced expression of Lyn (10, 11) are associated with strong susceptibility to human SLE, and mice deficient in Lyn, CD22 or Shp-1 exhibit SLE-like disease, including hyperactive BCR signaling, autoantibody production and glomerulonephritis (12C18). As Lyn?/? mice represent a clinically relevant model of SLE, insights from characterization of proteins and processes that regulate B cell hyperactivation and autoantibody production in this model may lead to better molecular understanding of SLE. Ezrin is a member of the ERM family of membrane-cytoskeleton cross-linking proteins. It contains an N-terminal FERM domain that binds transmembrane proteins such as CD44 (19), and Cbp/PAG (20). The C-terminal domain contains a conserved threonine residue (T567) whose phosphorylation is critical for its conformational activation and binding to filamentous actin (F-actin) (19). We have previously reported that ezrin modulates B cell function due to its ability to regulate BCR organization, signaling (21), B cell chemotaxis (22), and IL-10 production (23). Moreover, we recently showed that ezrin supports pathogenic BCR signaling in germinal Acalisib (GS-9820) center-derived diffuse large B cell lymphoma (DLBCL) (24). However, it is not known whether ezrin regulates abnormal BCR signaling and B cell-associated pathogenesis in Lyn?/? mice. In this study we employed Lyn?/? mice in which ezrin was conditionally deleted in B cells to investigate the Acalisib (GS-9820) impact on BCR signaling, autoantibody levels and kidney pathology. Our data show that loss of ezrin in Lyn?/? B cells resolves these major hallmarks of SLE-associated autoimmune pathology. MATERIALS AND METHODS Mice Ezfl/flMB1cre/+ (Ez-def), MB1cre/+, and Lyn?/? mice have been previously described (12, 13, 21). Two to eight month old animals were used, and all experiments performed in compliance with the guidelines approved by the Cleveland Clinic Institutional Animal Care and Use Committee. Flow cytometry Single-cell suspensions were prepared from spleens, blocked with anti-CD16/32 (clone 2.4G2), and stained with FITC- or PE-conjugated antibodies to surface IgM (BD Pharmingen). Developmental and mature B cell subsets were identified by flow cytometry based on previously described gating strategies (21). Flow cytometry data were collected on BD LSR Fortessa and analyzed using FlowJo software (Tree Star). BCR stimulation, immunoblotting, and calcium flux Splenic B and T cells were purified by negative selection using CD43 beads or mouse pan T cell isolation kit II (Miltenyi Biotecc), respectively. Purified B cells were stimulated with 10 g/ml F(ab)2 fragment of anti-mouse IgM (Jackson ImmunoResearch Laboratories) for the indicated times. B and T cell lysates were prepared and immunoblotting performed Acalisib (GS-9820) as described (21). All immunoblotting antibodies were from Cell Signaling Technology, except for actin (Santa Cruz Biotechnology), Ig (Abcam), phosphotyrosine (pY) and ezrin (EMD Millipore). To measure intracellular-free calcium levels, purified HGFR splenic B cells were loaded with Fluo-3 AM (Molecular Probes) at 37C for 20 min. Cells were washed, resuspended in DMEM supplemented with 1% BSA (Sigma) and 20 mM HEPES (Sigma), warmed to 37C for 5 min, and analyzed by flow cytometry. After the baseline was established for 30C40 sec, cells were stimulated with 10 g/ml F(ab)2 fragment of anti-mouse IgM for the indicated duration. Autoantigen array and autoantibody ELISA The profiling of IgM and IgG autoantibodies in sera was done using 98-plex autoantigen arrays. Arrays were hybridized with mouse sera, detected by Cy5-labeled anti-mouse IgM and Cy3-labeled anti-mouse IgG antibodies, and scanned with GenePix? 4400A Microarray Scanner to generate Tiff images. The images were analyzed using Genepix Pro 6.0 software to generate GPR files. The averaged net fluorescent intensity (NFI) of each autoantigen was normalized to internal controls (IgM or IgG). ELISA plates coated with purified dsDNA (Invitrogen) were used to quantify serum anti-dsDNA Acalisib (GS-9820) antibodies, and those coated with antibodies to IgM or IgG, respectively, to quantify total IgM and IgG levels. Histopathology and immunofluorescent staining Kidneys were fixed in 10% formalin (Sigma) for.