Graduate Student University of Toronto Toronto, Ontario, Canada
Background: We have shown that diphtheria toxin [DT] administration to CD19-Cre/R26-iDTR mice but not control mice leads to B-cell depletion and blunted blood pressure [BP] responses to angiotensin II [Ang-II]-induced hypertension [HTN]. Here we show B-cell subset dynamics in Ang-II-induced HTN using detailed tissue flow cytometry [FC], and in-vitro B-cell culture systems.
METHODS AND RESULTS: To investigate B-cell dynamics during Ang-II-induced HTN, wild-type [WT] C57BL/6J male mice aged 10-12 wk implanted with osmotic minipumps delivering Ang-II 500 ng/kg/min x 28-d vs. saline underwent FC of all B-cell subsets in aorta, spleen, and bone marrow. CD19+ B cells in aorta increased 3.6-fold with Ang-II vs. saline (5.4±2.4E3 vs. 1.5±0.8E3 cells/aorta; N=5/group; p< 0.01). In spleen, CD138+ plasma cells increased 2.3-fold with Ang-II vs. saline (7.6±3.8E5 vs 3.2±1.4E5 cells/spleen; N=5/group; p< 0.001). By contrast, plasma cells were decreased 3-fold in bone marrow after Ang-II vs. saline (0.7±0.7E6 vs. 2.06±3.8E6 cells/bone marrow; N=5/group; p< 0.05). To investigate whether these dynamic shifts in B-cell populations were due to direct effects of Ang-II, splenic B cells were isolated from 9-12 wk old WT mice via negative selection using an antibody-coated magnetic bead cocktail, with purity confirmed by FC (>95%). Purified B cells were cultured in RPMI-1640 medium supplemented with 5% heat-inactivated fetal calf serum, penicillin, and streptomycin (100 U/mL). Cells were stimulated for 24 h with: (a) anti-IgM (0.013 mg/mL), (b) anti-CD40 (0.01 mg/mL), (c) anti-IgM/CD40, or (d) resting media (unstimulated control). A range of Ang-II exposures (10 nM-100 µM) were then applied to each of these culture conditions, and B-cell activation was assessed via surface expression of CD86 and CD69 using FC. Ang-II treatment did not alter CD86 or CD69 expression on B cells under any conditions (N=2-3/group; p>0.05).
Conclusion: In vitro, Ang-II (10 nM–100 µM) does not directly activate B cells via BCR/CD40 pathways, as evidenced by unchanged CD86/CD69 expression on purified splenic B cells. These findings suggest that the in vivo B-cell dynamics observed during Ang-II-induced HTN are indirect- rather than direct-effects of Ang-II. Our data suggest that Ang-II levels regulate peripheral plasma cell homeostasis through migration from/to or expansion within bone marrow and spleen. DT-mediated B-cell depletion and abrogation of Ang-II-induced HTN in CD19-Cre/R26-iDTR mice demonstrate the importance of B-cells in the pathogenesis of HTN, and raise the therapeutic potential of B-cell-targeted treatments of HTN.
