Figure 6: Selective sensitivity of mature and MZ B cells to β−adrenergic stimulation.

Homogenate from equal weights of spleen tissue was prepared to measure catecholamine levels. (a) Splenic noradrenaline concentration is significantly increased 1–5 days after MCAO in comparison to sham-operated controls. (b) No difference in splenic adrenaline concentration was detected (sham n=4, 1d n=4, 2d n=9, 5d n=5, 7d n=7). (c) Splenocyte suspensions were prepared from naïve C57BL/6 mice and labelled to differentiate B-cell subsets (Supplementary Fig. 2b) and expression of β2-AR by flow cytometry to determine the percentage of β₂-AR⁺ MZ and follicular B cells (n=12, 4 biological replicates with 3 technical replicates each). (d) Mixed splenocytes were cultured with increasing concentrations of noradrenaline for 4 h and viability of B cells was determined by flow cytometry using B-cell markers described previously (Supplementary Fig. 2b) and Alexa Fluor-488-Annexin V dead cell apoptosis kit. Noradrenaline significantly reduced viability of MZ and follicular B cells in a dose-dependent manner (n=3 biological replicates). (e) Bone marrow cells were prepared from naïve C57BL/6 mice and labelled to determine B-cell subset expression of β2-AR (Supplementary Fig. 2c). Levels of β₂-AR were higher on mature recirculating naïve B220⁺CD23⁺CD21⁺ and CD138⁺B220⁺CD21^−/lo plasma B cells. Negligible expression of β2-AR was detected on B220⁺CD23⁻CD21⁺ immature and CD138⁺B220⁻CD21^−/lo long-lived memory B cells (n=12, 4 biological replicates with 3 technical replicates each). (f) Bone marrow cells were gated on B220 and plotted against CD23 and CD21/35 to distinguish naïve and immature B cells. Cells were also gated to distinguish plasma from memory B cells (Supplementary Fig. 2c). (g) MCAO-induced significant reductions in B220⁺CD23⁺CD21⁺ naïve and CD138⁺B220⁺CD21^-/lo plasma B cells (sham n=4, MCAO n=6). B220⁺CD23⁻CD21⁺ immature and CD138⁺B220⁻CD21^−/lo memory B cells were not significantly altered. (h) Healthy human donor blood was labelled for flow cytometry analysis of β₂-AR expression on CD19⁺ B cells. (i) 80% of CD19⁺ B cells in human blood express β₂-AR (n=12, 4 biological replicates with 3 technical replicates). Data show mean+s.d. (b,c,e) One-way ANOVA with Bonferroni correction, (f) One-way ANOVA with TUKEY correction (d,h) unpaired t-test *P<0.05, **P<0.01, ***P<0.001.