Memory B cells from older people express normal levels of cyclooxygenase-2 and produce higher levels of IL-6 and IL-10 upon in vitro activation

Abstract

Worldwide the elderly population is increasing. The elderly show deficiencies in immune function. B lymphocytes are essential elements of the immune system responsible for antibody production. This laboratory previously showed that activated human B cells isolated from young adults express cyclooxygenase-2 (Cox-2) and that Cox-2 is essential for optimal antibody responses. Recent data suggests that Cox-2 expression decreases with age in mouse bone tissue. There is no information regarding Cox-2 expression in B cells from older human subjects. We investigated the expression and activity of Cox-2 in naïve and memory B cells from older people. We show that B cells from older subjects show similar Cox-2 protein expression and activity, antibody production and proliferation compared to younger people. However, we found that activated memory B cells from older people produce higher levels of IL-6 and IL-10 compared to young adults. Therefore, the dysregulated cytokine production could contribute to immune senescence in the elderly.

Figure 1. Older people show a lower percentage of naïve B cells but a higher percentage of memory B cells in their peripheral blood

Peripheral blood was collected from young (18-35 years old; n= 8) and older (over 60 years old; n= 9) people and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation, stained with CD19 and CD27 antibodies and analyzed on a Becton Dickinson LSRII. A. Young volunteers have ~75% naïve B cells (CD19⁺CD27⁻) whereas older volunteers have a significantly lower percentage of naïve B cells (~40%). B. Older volunteers have an increased percentage (~60%) of memory B cells (CD19⁺CD27⁺) whereas young volunteers have ~ 25% memory B cells in the peripheral blood. Graphs show the mean and SEM. *, p< 0.05 was calculated using the Mann-Whitney test. Student's t test gave similar results (**, p= 0.0087 for Figure 1A and **, p=0.0059 for Figure 1B).

Figure 2. Naïve and memory B cells from older people increase Cox-2 expression upon activation

Sorted naïve and memory B cells from young (n= 8) and older (n= 8) people were stimulated with anti-Ig plus CpG 2395 for 2 days. Cells were stained with an anti-Cox-2 antibody and analyzed on a Becton Dickinson LSRII. Graphs show the mean fluorescence intensity (MFI) of Cox-2 staining. A and B. Naïve and memory B cells from both young and older people increased Cox-2 expression upon activation. Untreated cells show a lower MFI (~500) compared to activated cells (~ 800 for young and ~1,100 for memory from older people). Graphs show the mean and SEM. *, p< 0.05 and **, p< 0.005 was calculated using the Mann-Whitney test. Analysis using Student's t test resulted in: Figure 2A: ***, p= 0.0002 (when comparing young (no treatment) to young (aIg+ CpG 2395) and *, p= 0.0472 (when comparing older (no treatment) to older (aIg+CpG 2395) samples; Figure 2B: **, p= 0.0037 for young (no treatment) versus young (aIg+CpG) and p= 0.064 (not significant) when comparing older (no treatment) to older (aIg+CpG 2395). There is no significant statistical difference between young (aIg+ CpG 2395) and older (aIg+ CpG 2395) samples.

Figure 3. Naïve and memory B cells from older people increase PGE₂ synthesis upon activation

Sorted naïve and memory B cells from young (n= 9) and older (n= 8) people were stimulated with anti-Ig plus CpG 2395 for 2 days after which supernatants were harvested and used for PGE₂ detection. Unactivated naïve and memory B cells produce ~ 75 pg/ml PGE₂ whereas activated naïve and memory B cells (from both young and older subjects) produce ~150 pg/ml PGE₂. Graphs show the mean and SEM. *, p< 0.05, **, p< 0.005 and ***, p<0.0001 was calculated using the Mann-Whitney test. According to Student's t test the p values are: Figure 3A: *, p=0.013 for young (no treatment) versus young (aIg+ CpG 2395) and **, p= 0.004 for older (no treatment) versus older (aIg+ CpG 2395); Figure 3B: ***, p=0.003 for young (no treatment) versus young (aIg+ CpG 2395) and ***, p< 0.0001 for older (no treatment) versus older (aIg+ CpG 2395). There is no significant statistical difference between young (aIg+ CpG 2395) and older (aIg+ CpG 2395) samples.

Figure 4. Naïve and memory B cells from older people increase expression of CD69 and proliferate after in vitro activation

Sorted naïve and memory B cells from young and older people were activated in vitro with anti-Ig plus CpG 2395. On day 2 cells were harvested and stained for CD69 after which cells were analyzed on a Beckton Dickinson LSRII. A and B show the histograms of CD69 expression in anti-Ig plus CpG 2395 activated (black line) compared to no treatment (gray contour) naïve and memory B cells. The histograms are representative for naïve and memory B cells in young and older people. C and D show the fold induction in CD69 MFI of activated cells versus no treatment cells. There is an increase in CD69 MFI in both naïve and memory B cells from young (n=7) and older (n=7) subjects. [³H]-thymidine was added to the activated naïve and memory B cells from young and older people and proliferation was assayed (E and F). Graphs show the mean and SEM of counts per minute (cpm) from young (n= 4) and older (n= 5) people. Naïve and memory B cells showed similar proliferation patterns and there are no significant differences between the two age groups.

Figure 5. B cells from older people produce similar levels of antibodies compared to younger subjects

Sorted naïve and memory B cells from young and older people were activated with anti-Ig plus CpG 2395 for 7 days after which supernatants were harvested and used to determine IgM and IgG production. A and B. Activated naïve B cells from young and older people synthesize similar levels of IgM (~8,000 ng/ml) and IgG (~200 ng/ml). C and D. Activated memory B cells from older people produce more IgM (~70,000 ng/ml) but similar levels of IgG (~4,000 ng/ml) compared to young subjects (~25,000 ng/ml and ~ 4,000 ng/ml, respectively). Graphs show the mean and SEM.

Figure 6. Activated memory B cells from older people increased IL-6 and IL-10 levels

Sorted naïve and memory B cells from young and older people were stimulated with anti-Ig plus CpG 2395 for 2 days after which supernatants were harvested and used for IL-6 and IL-10 detection. A Naïve B cells from young and older subjects produce similar levels of IL-6 (~2,000 pg/ml). B. Memory B cells from older people produce significantly more IL-6 (~3,000 pg/ml) as compared to young subjects (~1,000 pg/ml). C. Naïve B cells from older people produce slightly more IL-10 (~1,500 pg/ml) compared to naïve B cells from young people (~700 pg/ml). D. Memory B cells from older people produce significantly more IL-10 (~1,600 pg/ml) compared to young subjects (~250 pg/ml). **, p< 0.005 was calculated using the Mann-Whitney test. The p values according to t test were: Figure 6B, ***, p= 0.0009 and Figure 6D, *, p= 0.035.