Figure 5. OMV containing MID activate tonsillar B cells.

(A) The mitogenic effect of M. catarrhalis OMV isolated from different strains on B cells was analyzed by measuring thymidine uptake at 96 h. Error bars indicating SEM from 6 different donors, * p≤0.05, OMV versus control. (B) The viability of B lymphocytes after OMV stimulation was assessed by trypan blue exclusion staining. (C) Purified B cells were stimulated with different concentrations of OMV with or without MID (OMV Δmid), whole M. catarrhalis wild type or its isogenic mutant or recombinant MID^962-1200 as indicated. The OMV concentrations used were in the range of 0.1–25 µg/ml. Error bars indicate SEM from 5 different donors. (D) OMV activated B cells mainly produce IL-6. Purified B cells were incubated with 10 µg/ml OMV wild type (OMV wt) or MID-deficient OMV (OMV Δmid) for 96 h. Thereafter, the cytokine contents in the B cell culture supernatants were determined using a human cytokine protein array. The protein microarray cut-off controls are indicated as positive controls. Complete medium and a culture supernatant from unstimulated B cells were included as a negative control. A minor up-regulation of IL-10 and GRO was also detected. (E–H) OMV binding to B cells induces IL-6 and IgM secretion. The stimulatory effect of M. catarrhalis OMV on B cells was analyzed by measuring IL-6 secretion at 48 h (E) and IgM (F), IgG (G) or IgA (H) production at 96 h. B cells were activated with 10 µg/ml OMV isolated from MID-expressing bacteria (wt) or a MID-deficient mutant (Δmid). In addition, whole M. catarrhalis wild type (wt), its isogenic mutant (Δmid), or recombinant MID^962-1200 were included in the analysis. Error bars indicate SEM from 5 (E) and 3 (F–H) different donors.