Aged B lymphocytes retain their ability to express surface markers but are dysfunctional in their proliferative capability during early activation events

Abstract

Background: Ageing is associated with dysfunction in the humoral response leading to decreased protection against infectious diseases. Defects in T cell function due to age have been well characterized but it is unclear if dysfunctions in antibody responses are due to deficiencies in a helper environment or intrinsic B cell defects. Previous studies from our laboratory have shown that aged B lymphocytes are able to differentiate into high affinity antibody-secreting cells at a frequency similar to their young counterparts. However, expansion of B cells in vivo was reduced in aged animals when compared to young.

Methods: To further investigate the cause of this reduced expansion, we have now examined early activation events of aged B cells in response to anti-CD40 monoclonal antibody (mAb) and lipopolysaccharide (LPS) stimulation in vitro. To do this spleen cells were harvested from young, middle-aged and aged quasi-monoclonal (QM) mice and cultured in complete RPMI for 24 and 48 hours. Cultures contained either LPS or anti-CD40 mAb and murine IL-4. Cells were collected and analyzed using flow cytometry. To examine the proliferative capacity of aged B cells spleen cells were collected as before and cultured in 96 well microtiter plates with either LPS or anti-CD40 mAb and murine IL-4 for 24 hours. Tritiated thymidine ([3H]-Tdr) was added to each well and incubated for another 24 hours after which cells were collected and analyzed using a scintillation counter.

Results: Resting aged B cells exhibited similar levels of CD40 expression when compared to young cells and efficiently up-regulated CD86 and CD69 and also down-regulated CD38 upon stimulation. However, aged B cells proliferated less than young B cells and showed a consistent, but not statistically significant, reduction in their ability to form blast cells.

Conclusion: Aged B cells exhibited a reduced response in some early activation events but produced at least a partial response in all cases. Thus, therapeutic intervention may be possible, despite intrinsically different responses in aged B cells.

Figure 1

CD40 levels are similar on freshly isolated B cells from normal and Tg animals. Freshly isolated SPC from young and aged C57 (A and B) and QM (C-G) mice, were stained with anti-CD40 mAb along with anti-B220 and NP-PE. Percentages of B220⁺ lymphocytes are shown in (A) and (C), and CD40 expression (B and D) was determined for the B220⁺ population. Percentages of NP-specific QM B cells and non-NP binding cells are shown in E. The levels of CD40 on NP^- and NP⁺/B220⁺ cells are shown in (F) and (G). Thin lines indicate cells from young animals and thick lines indicate cells from aged animals. Representative of 3 separate experiments.

Figure 2

Splenocytes from aged mice are able to upregulate CD86 effectively upon stimulation. SPC from young, middle-aged, and aged QM mice were harvested and cultured for 24 and 48 hours. Splenocytes were stained for expression of cell surface markers. Lymphocytes were gated on FSC/SSC and B cells on B220⁺. Representative of 4 separate experiments.

Figure 3

B cells from aged mice regulate surface marker expression similar to those from young mice. SPC from young and aged QM mice were harvested and cultured with LPS or anti-CD40 with IL-4 for 48 hours. Splenocytes were stained for expression of cell surface markers. Lymphocytes were gated on FSC/SSC and B cells on B220⁺. Data are representative of 4 separate experiments.

Figure 4

Aged B cells are able to produce blast cells when stimulated with LPS. SPC were harvested from young, middle-aged, and aged QM mice and cultured for 24 and 48 hours in stimulating conditions (LPS) and analyzed for side and forward scatter by flow cytometry. Data are representative of 4 experiments.

Figure 5

Aged B cells are able to effectively produce blast cells in response to stimulation. SPC from young and aged QM mice were harvested and cultured for 48 hours. Blast cells were gated using FSC/SSC. Percentage of blast cells in media were divided into percentages of blast cells for each stimulating condition to give fold-increase as shown.

Figure 6

Middle-aged and aged B cells show decreased ability to proliferate under stimulating conditions. SPC from young, middle-aged, and aged QM mice were harvested and cultured in triplicate with anti-CD40 mAb or LPS for 24 hours and incubated with [3H]-Tdr and incubated another 24 hours. CPM was measured as described in material and methods. Error bars are shown for triplicate samples. Middle-aged and aged B cells stimulated with LPS show a significant decrease in proliferative capacity (P = 0.03 and 0.005 respectively). Aged B cells stimulated with LPS show a significant decrease (P = 0.029) while middle-aged cells appear to be decrease are not significantly different (P = 0.09). Data represents 4 experiments. P values determined by student's T-test.