Representative blots from three impartial experiments are shown. While proximal signaling proteins showed stronger activation in the absence of ezrin, components of the distal BCR signaling components displayed distinct effects. Ezrin deficiency resulted in increased B cell proliferation and differentiation into antibody-secreting cells cellular behavior. These studies underscore the importance of understanding how BCR signaling, B cell activation and humoral immunity proceed in the absence of ezrin. Here, we addressed the role of ezrin in B cell antibody response by generating conditional knockout mice that lack ezrin expression exclusively in the B cell lineage. We report that the size of BCR microclusters, and magnitude of BCR signaling and antigen-specific antibody production are increased in the absence of ezrin. Our data demonstrate the physiological relevance of ezrin-mediated control of BCR microclustering and membrane dynamics in optimizing the Dabigatran ethyl ester B cell response to antigen. MATERIALS AND METHODS Mice Ezfl/fl mice (24) were backcrossed with C57BL/6 mice for seven generations before breeding with MB1cre/+ mice (25) to generate the Ezfl/flMB1cre/+ mice (Ez-def). MB1cre/+ mice were used as controls in all experiments. All animals were used in compliance with the guidelines approved by the Cleveland Clinic Institutional Animal Care and Use Committee. Flow cytometry, B cell subset analysis and immunization Purified B cells were stained with FITC-, PE- or APC-conjugated antibodies to sIgM, CD19, CD21, CD40, CD62L and ICAM2 (BD Pharmingen) for marker analysis. Developmental stages of B cells, and mature B cell subsets were identified based on gating strategies previously described (26). Plasma cells in the bone marrow were identified as Dabigatran ethyl ester B220loCD138+ cells. All flow cytometry data were analyzed using FlowJo (Tree Star). MB1cre/+ and Ez-def mice were immunized with either 50 g of 4-hydroxy-3-nitrophenyl (NP)-Ficoll or 50 g of NP-chicken gamma globulin (CGG) along with 10 g of LPS. Sera were collected every week and NP-specific IgG antibodies quantified by ELISA. B cell stimulation and immunoblotting Splenic B and T cells were MACS purified by unfavorable selection (Miltenyi Biotec). B cells were stimulated with 10 g/ml or 50 g/ml (for JNK activation) of anti-IgM, or primed with 10 g/ml of LPS for 48 h, followed by stimulation with 10 g/ml or 50 g/ml (for JNK activation) of anti-IgM for indicated times. Lysates were prepared and immunoblotting performed as described (18). To assess cell proliferation, purified B cells were labeled with 1 M CFSE and stimulated with 10 g/ml of anti-IgM Dabigatran ethyl ester for 5 days. Cells were analyzed every 24 h by flow cytometry and number of cells at each division quantified using FlowJo. ELISPOT assay Purified B cells were primed with 0.1 g/ml of LPS for 48 h followed by stimulation with 10 g/ml of anti-IgM for 24 h, and transferred to ELISPOT plates pre-coated with unlabeled anti-mouse Ig for 16C18 h at 37 C. The plates were washed, incubated with HRP-conjugated anti-IgM and anti-IgG antibodies for 2 h at room temperature, and developed with AEC Chromogen (BD Biosciences). The plates were imaged and analyzed using an Immunospot plate reader (Cellular Technology Ltd). TIRF imaging Purified B cells were labeled with 2 g/ml of Cy5-conjugated goat anti-mouse IgM ( chain-specific) Fab fragment for 20 min at 4 C. For stimulation, cells were added to glass-bottomed petri dishes (MatTek Corporation) coated with 10 g/ml of goat anti-mouse IgM (H+L Rabbit Polyclonal to MEKKK 4 specific) F(ab)2 fragment. Cells were allowed to settle for 2C3 min and images collected every 5 s for a period of 15 min. Images were acquired in warm imaging buffer (RPMI without phenol red, 10% FBS, 2 mM glutamine, 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) using a Leica-AM TIRF microscope DMI6000 (Leica Microsystems) with an attached Hamamatsu EM-CCD camera, and the Leica acquisition software LAS AF Version 2.2.0. An HCX PL APO 100 oil objective (NA=1.47) was used at an additional 1.6 magnification with appropriate filter cubes. The images were digitally deconvolved using Metamorph and analyzed further for cluster area, intensity and velocity with ImagePro Plus 7.0. BCR cluster stability was measured at 8 min of stimulation by quantifying the subsequent number of frames for which each BCR cluster persisted. To test association of BCRs with tyrosine-phosphorylated proteins (pY).
