Figure 3. Ca²⁺ mobilization and B cell receptor clustering are induced by OMV.

(A) Mobilization of [Ca²⁺]_i in human tonsillar B cells induced by Moraxella OMV. Purified Fura-2 loaded B cells were stimulated with wild-type OMV (10 µg/ml), MID-deficient OMV (10 µg/ml) or ionomycin (100 nM). Ca²⁺ mobilization was analyzed as described in Materials and Methods. Each experiment was repeated with at least three different donors with comparable results. (B) Aliquots of Triton-insoluble lysates of B cells obtained before (Control) and after stimulation with formaldehyde-fixed M. catarrhalis wild type (Bacteria), OMV or treated with filipin prior to stimulation with OMV (Filipin + OMV) were fractionated on discontinuous sucrose gradients and were immunoblotted with anti-flotillin, anti-caveolin, anti-TLR2, anti-IgD or anti-TLR9 mAbs. (C) Visualization of receptor clustering in lipid rafts. B cells unstimulated (Control), wild type OMV (OMV wt) or MID-deficient OMV (OMV Δmid) stimulated for 30 min were fixed and stained with anti-flotillin (top) or anti-TLR2 (bottom). Alexa Fluor 594 goat anti-mouse IgG pAb were used as a secondary layer. (D) Colocalization of flotillin, TLR2, Rab5 and TLR9 with IgD (BCR) confirmed by confocal microscopy. Purified B cells were incubated with OMV for 30 min, fixed and stained with FITC-conjugated or RPE-conjugated mAbs against IgD (BCR) and FITC-conjugated anti-TLR9 mAb, anti-flotillin mAb or anti-TLR2 mAb using Alexa Fluor 594-conjugated secondary pAb. For the triple staining, stimulated B cells were incubated with rabbit anti-IgD pAb (BCR) and mouse anti-Rab5 mAb (Rab5). After several washes, B cells were incubated with Alexa Fluor 633 goat anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG secondary mAb followed by incubation with FITC-conjugated anti-TLR9 mAb (TLR9). The lymphocytes shown are representatives of more than 90% of the cells imaged in 3 separate experiments with different donors.