Aged mice exhibit distinct peripheral B-cell phenotypes differing in apoptotic susceptibility: An ex vivo analysis

Abstract

Background: Age-related changes in the antibody response have been classically associated with alterations in T-cell help, but increasing evidence shows that intrinsic B-cell defects exist. This article analyzes the apoptotic susceptibility of peripheral B-cells in aged and young control mice. Materials and Methods: Freshly isolated lymphocytes from spleen and Peyer's patches (PPs) were labeled for B-cell lineage (B220+ cells) and germinal center B subset (GCs, 8220+/PNA+ cells). Alternatively, splenic B-cells purified by MACS were used. Apoptosis was monitored by the Annexin V binding, incorporation of 3,3′-dihexyloxacarbocyanine iodide (DiOC 6(3)), propidium iodide (PI) staining, and morphological changes. Moreover, intracellular Bcl-2 expression and Bad phosphorylation status were also analyzed in B-cells. Results: We showed in aging mice an enhanced Annexin V+/PI- cell percentage in splenic B-lymphocytes, which was correlated with a lower ΔΨm. By contrast, no change in apoptosis was observed in compartments known to be enriched in activated B-cells (GCs and PPs). Analysis of Bcl-2 levels revealed no modification. When using B-cells purified by MACS, we strongly confirm data obtained on staining cells. Moreover, enhanced spontaneous apoptosis of splenic B-cells in aged mice was found to be correlated with a reduced phosphorylated Bad expression. Conclusion: Increased apoptosis of resting B-cells in old mice may be determined by an altered Bad phosphorylation, which in turn contributes to cell death by lowering the mitochondrial threshold for apoptosis. © 2006 International Society for Analytical Cytology.