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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.
B lymphocytes serve as key defenders against infection. Once activated, B cells undergo a series of transformations that culminate in antibody production. Cox-2 protein levels are dramatically elevated shortly after B lymphocyte activation and Cox-2 expression is a prerequisite for optimal antibody production . While Cox-2 expression is increased in activated B lymphocytes, cyclooxygenase-1 (Cox-1) is ubiquitously expressed in most cell types and does not appear to be essential for optimal human antibody synthesis. Both Cox-1 and Cox-2 are involved in the conversion of arachidonic acid to prostaglandins (PGs). PGs are small lipid mediators which regulate a wide array of physiological and immune functions . PGE2, an abundant PG, is produced by many cell types and is a mediator of pain, fever and swelling, which are common features of inflammatory diseases . Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen and indomethacin target the activity of Cox-1/Cox-2 and reduce PGE2 synthesis. Therefore, NSAIDs are recommended in cases of pain and fever and are prescribed for inflammatory-related diseases. About half of NSAID prescriptions are for people over 60 years old .
Aging has a profound impact on the immune system. Immunosenescence is characterized by quantitative (decrease in cell number) and qualitative (decline in cell activity) changes [5, 6] which are accompanied by a pro-inflammatory environment [7, 8]. These changes result in decreased immunity and, in general, poor responses to vaccinations. B cells consist of a heterogeneous population comprised of naïve (newly generated) and memory (antigen experienced) cells. The ratio between naïve and memory B cells varies in human subjects and is influenced by age [9, 10].
Cox-2 inhibition, in the case of Cox-2 deficient mice or caused by Cox-2 selective drugs or by NSAIDs, dramatically hinders antibody production in human peripheral blood mononuclear cells and B cells in vitro and decreases antibody titers to vaccination in mice and humans [11, 12]. Studies to date on Cox-2 expression in B cells were done using only young human subjects (18-35 years old) who show a predominant naïve B cell phenotype. There are no data regarding the expression of Cox-2 in B cells from older subjects. Recent research showed that Cox-2 expression is reduced in the femurs of old mice which results in impaired bone healing in case of femur fracture . In B cells, a decrease in Cox-2 expression/activity would be expected to impair antibody synthesis which could, at least in part, explain why the elderly produce less antibody. Herein, we investigated Cox-2 protein expression and activity (by measuring PGE2 production) and antibody production in naïve and memory B cells from older people (over 60 years old). Since immunoscenescence can be characterized by changes in cytokine synthesis, IL-6 and IL-10 levels in activated B cells from older versus younger people were also determined. High levels of IL-6 are indicative of a pro-inflammatory environment whereas IL-10 promotes antibody synthesis and blunts inflammation.
F(ab′)2 goat anti-human IgG, F(ab′)2 (herein called anti-Ig) was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA), CpG oligodeoxynucleotide 2395 (5′-TCGTCGTTTTCGGCGCGCGCCG-3′) from Coley Pharmaceutical Group (Wellesley, MA) and arachidonic acid from (Nu-Check-Prep, Elysian, MN). Human B cells were stained with the following antibodies: CD19 (APC conjugated), CD27 (Pe conjugated), CD69 (APC-Cy7 conjugated) (BD Biosciences, San Jose, CA) and Cox-2 (Fitc conjugated; Cayman Chemical, Ann Arbor, MI). Human IgM and IgG quantitation kit was purchased from Bethyl Laboratories (Montgomery, TX).
One unit of blood was obtained from healthy donors as approved by the University of Rochester Institutional Review Board and Office for Human Subjects Protection. We considered young donor individuals to be between 18-35 years old (n=10; 6 males and 4 females) and older individuals to be those over 60 years old (60-72 years old; n= 9; 3 males and 6 females). Given the sample size (n= 4-10) Mann-Whitney test and Student's t test were chosen for statistical analysis. Donors who were taking NSAIDs (including low dose aspirin) or had an iron level lower then 12.3 were excluded from the study. Volunteers were healthy, were not obese or diabetic, were not taking steroids or diabetic medication and women volunteers were not pregnant. The racial category of the participating subjects was white or other (choose not to answer). All blood donations were collected in the morning (8-9 AM). An aliquot of blood (1 ml) was used to prepare plasma which was used for ELISA. Peripheral blood mononuclear cells were obtained by density-gradient centrifugation of buffy coat using Ficoll-Paque Plus. CD19 positive cells were captured with a magnet, washed and detached using CD19 Detachabead (Dynal Inc, Brown Deer, WI). B cells were stained with CD19 and CD27 antibodies and then sorted on a FACS Aria Cell Sorter (BD Biosciences) into naïve (CD19+CD27−) and memory (CD19+CD27+) B cells (> 98% purity).
Naïve and memory B cells (1 × 106 cells/ml, unless otherwise specified) were cultured in RPMI1640 media (Invitrogen Life Technologies) supplemented with 10% FBS, 50 μM β-mercaptoethanol (Eastman Kodak, Rochester, NY), 10 mM Hepes (U.S. Biochemical, Cleveland, OH), 2 mM L-glutamine (Invitrogen Life Technologies, Carlsbad, CA), 50 μg/ml gentamicin (Invitrogen Life Technologies, Carlsbad, CA) and 10 μM arachidonic acid. B cells were stimulated with anti-Ig (2 μg/ml) plus ODN CpG 2395 (1 μg/ml) (concentrations determined by a titration experiment) for variable time points as described in the figure legends.
B lymphocytes were cultured for 2 days in the presence of mitogens (anti-Ig plus CpG 2395). Cells were surface stained for CD69 (BD Biosciences) for 20 minutes at 4°C, washed in staining buffer (PBS with 0.3% BSA) and then pelleted by centrifugation. For Cox-2 intracellular staining, cells were fixed and permeabilized (Caltag Laboratories) followed by staining with Cox-2 antibody (1 μg/ 106 cells; Cayman Chemicals). An equal number of live cells (based on forward and side scatter) were acquired and were analyzed on a LSRII (BD Bioscience, San Jose, CA) flow cytometer using FlowJo software (Tree Star).
B cells were stimulated with anti-IgG plus CpG 2395 for 2 days after which supernatants were harvested. PGE2 synthesis was determined using an enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI), as recommended by the manufacturer.
Activated B cells (1× 105 cells/ ml) were cultured in triplicate in 96-well round bottom plates. Cell cultures were pulsed with [3H]-thymidine (1 μCi/well) for 24h and the incorporation was determined by scintillation spectroscopy.
Activated B cells were cultured in triplicate in 96-well plates for 7 days. Supernatants were collected and used for IgM and IgG detection using the human-specific ELISA kit (Bethyl Laboratories) as recommended by the manufacturer.
Stimulated (anti-Ig plus CpG 2395) naïve and memory B cells were cultured for two days in vitro after which supernatants were harvested. IL-6 and IL-10 production was determined by ELISA as recommended by the manufacturer (BD Pharmingen, San Jose, CA).
Mann-Whitney test was used for statistical analysis and p values of *, p<0.05; **, p<0.001; ***, p<0.0001 are considered significant. A second test, Student's t test was also used and the results of the t test are shown in the figure legends.
Recent published data indicate that Cox-2 levels decrease in mouse bone tissue with age . We were therefore interested in whether or not B cells from elderly humans would also have a deficit in Cox-2 expression. In order to investigate Cox-2 expression and activity in B cells from older people, we enrolled older volunteers (over 60 years old). Younger subjects (18-35 years old) were used as controls. The percentage of naïve and memory B cells in the peripheral blood is reported to change with age; there is an increase in the percentage of memory B cells in the older individuals [9, 10]. Therefore, we first analyzed the percentage of naïve and memory B cells in the peripheral blood of young and older subjects enrolled in the study. To indentify memory B cells we used the CD27 marker in combination with CD19. CD27 is absent on naïve B cells but is highly expressed on memory B cells [10, 14]. PBMC were isolated from young and older subjects and were stained with anti-CD19 and anti-CD27 antibodies. Figure 1 (A and B) shows that younger volunteers have ~ 75% naïve B cells (CD19+CD27−) and ~ 25% memory B cells (CD19+CD27+) in the peripheral blood. Older subjects showed a scattered range in terms of the percentage of naïve and memory B cells; older volunteers had between 2-70% naive B cells and between 30-98% memory B cells. We did not detect a significant change in the number of B cells isolated from older compared to young volunteers. There was no correlation between the B cell number and racial category, gender and actual age of young and older subjects.
Because of the significant difference in the percentage of naïve and memory B cells in young and older volunteers it was essential to isolate B cells based on their naïve and memory phenotype. Highly purified B cells were stained with CD19 and CD27 and were sorted into naïve (CD19+CD27−) and memory (CD19+CD27+) B cells. In order to determine Cox-2 expression, naïve and memory B cells were activated with broad polyclonal B cell activators, namely anti-Ig plus oligodeoxynucleotide (ODN) CpG 2395 for 48h after which cells were harvested and stained for Cox-2. Figure 2A shows that anti-Ig plus CpG 2395 activated naïve B cells from young and older people upregulated Cox-2 expression. Similarly, memory B cells from both young and old increased Cox-2 expression upon activation (Figure 2B). Naïve and memory B cell population upregulated Cox-2 to similar levels. Overall though there was no difference in Cox-2 expression levels between naïve or memory B cells from young or old.
In order to see if the level of Cox-2 protein expression paralleled Cox-2 activity, PGE2 production was determined in both activated naïve and memory B cells from young and older subjects. Figure 3 (A and B) shows that PGE2 levels were increased in activated naïve and memory B cells from young and older people. There was no difference in PGE2 production (and hence Cox-2 activity) between naïve and memory B cells or between young and older people. There was no difference in Cox-2 expression and activity in naïve and memory B cells from young and older volunteers exposed to lower CpG 2395 concentrations (0.25 and 0.5 μg/ml) with or without anti-Ig (data not shown).
It was reported that immunosenescence causes a decrease in cell proliferation [15, 16]. Therefore, we analyzed the expression of activation markers and the proliferation of naïve and memory B cells from young and older subjects during the culture period. The activation marker used was CD69 which is elevated on activated B cells. Figure 4 (A-D) shows that in both naïve and memory B cells CD69 expression is increased upon activation and there is no difference between naïve and memory B cells from young and older people.
Cell proliferation was assayed using the [3H]-thymidine assay (Figure 4E and 4F). On day 2 naïve and memory B cells from young and older people had ~ 2,000 cpm after which it increased dramatically reaching a peak on day 3 (~ 15,000 cpm for young and ~12,000 cpm for older people). Memory B cells from young and older people showed a comparable proliferation profile. There was no significant difference in proliferation of B cells from older people compared to young subjects.
Several reports have shown that the elderly produce less antibody compared to younger subjects [17, 18]. Taken into consideration that Cox-2 expression is linked to optimal antibody synthesis  and Cox-2 expression and activity showed no differences in naïve and memory B cells in older subjects as compared to young volunteers in this study (Figure 2 and and3),3), we hypothesized that older people would produce similar levels of antibody in vitro. In order to test our hypothesis, naïve and memory B cells from young and older volunteers were activated with anti-Ig plus CpG 2395 for 7 days after which supernatants were harvested and used for antibody ELISA. Activated naïve B cells (5A and 5B) from older people produced ~12,000 ng/ml IgM and ~200 ng/ml IgG. These antibody levels were comparable to those synthesized by naïve B cells isolated from young subjects (~7,300 ng/ml IgM and ~240 ng/ml IgG). Memory B cells from older people synthesized more IgM (~70,000 ng/ml) compared to young subjects (~31,000 ng/ml) (Figure 5C). However, because of the scattered profile, the difference is not statistically significant. In contrast, IgG production in memory B cells from young and older people show very similar levels (~ 4,000 ng/ml in young and ~5,400 ng/ml in older people) (Figure 5D).
Because older people have a higher percentage of memory B cells in their peripheral blood and because memory B cells from older subjects produced slightly higher levels of antibody in vitro, the levels of antibody in the plasma of young and older subjects were also determined and there were no differences between IgM and IgG levels in the plasma of young versus older volunteers (data not shown).
IL-6 participates in a plethora of functions including inflammation and immunity . IL-6 is required for B cell differentiation as well as for antibody synthesis . Due to the fact that IL-6 overproduction is associated with autoimmune diseases  and that older people are more likely to develop rheumatoid arthritis, we investigated the levels of IL-6 in activated B cells from older subjects. Naïve and memory B cells from young and older people were activated in vitro with anti-Ig plus CpG 2395 for 2 days after which supernatants were tested for IL-6 using ELISA. Figure 6A shows that naïve B cells from young and older subjects produce similar levels of IL-6 upon activation (~ 2,000 pg/ml). However, IL-6 production in activated memory B cells was significantly different between young and older subjects. Memory B cells from older people produced higher levels of IL-6 after activation (~ 3,000 pg/ml) compared to young subjects (~ 1,000 pg/ml) (Figure 6B).
IL-10 is considered an “anti-inflammatory” cytokine that promotes antibody production and is synthesized by B cells upon activation . IL-10 is involved in the maintenance of optimal immune responses and is considered to act as a feed-back control cytokine . Since memory B cells from older people showed a heightened production of IL-6, IL-10 levels in naïve and memory B cells from young and older subjects were also assayed. Activated naïve B cells from older people showed an increase in IL-10 synthesis (~1,500 pg/ml) compared to naïve B cells from young subjects (~700 pg/ml) (Figure 6C). However, memory B cells from older people showed an even more pronounced increase (~ 1,600 pg/ml) compared to young subjects (~ 250 pg/ml) (Figure 6D).
Aging is associated with decreased immunity, poor response to vaccination and increased susceptibility to infections [17, 18, 23]. While immunosenescence is well documented in human T cells (partly due to the involution of the thymus) , most of the studies focusing on B cell activation, proliferation and antibody production associated with aging were done using mouse models [5, 15, 16].
We recently showed that activated human B lymphocytes from young human beings express Cox-2 and that Cox-2 expression is required for optimal antibody production and inhibition of Cox-2 by NSAIDs hinders human antibody production [11, 12]. There are no data regarding Cox-2 expression in B cells in the human elderly population. So far, Cox-2 expression was analyzed only in the bone and macrophages of old mice. It was reported that Cox-2 expression was decreased in the femur of old mice which increases the risk of fractures . Conversely, mouse macrophages upon activation showed increased Cox-2 activity, demonstrated by increased PGE2 production . PGE2 is a proinflammatory and protumorigenic mediator and therefore NSAIDs, which inhibit Cox-2 activity and decrease PGE2 levels, are recommended as chemo-preventive agents . NSAIDs are also prescribed for inflammation and pain (e.g. rheumatoid arthritis) and the largest group of NSAIDs users are people over 60 years old [4, 27]. These facts raise an interesting scenario: what if B cells from older people express less Cox-2 (which may lead to decreased antibody synthesis) should NSAIDs be recommended for the elderly? On the other hand, if B cells from the elderly produce elevated levels of PGE2 (similar to mouse macrophages) should the elderly be encouraged to use more NSAIDs?
Our compelling data demonstrate that Cox-2 expression and activity (as shown by PGE2 production) is increased upon activation at similar levels in naïve and memory B cells. Furthermore, Cox-2 expression and activity in B cells from older people is comparable to B cells from young subjects. Ibuprofen, the popular over-the-counter NSAID, equally inhibited Cox-2 activity in naïve and memory B cells from young and older people (data not shown). Consequently, NSAIDs are effective in inhibiting Cox-2 expression and activity in both young and older people, independent of the percentage of naïve and memory B cells in their peripheral blood.
A main feature of immunosenescence is decreased antibody production caused by quantitative (mainly due to reduced proliferation and isotype switching of B cells) or qualitative changes (reduced antigen-specific antibody titers) [9, 28, 29] leading to a poor response to vaccination and more infections in the elderly [18, 30, 31]. We found that activated B cells from older people produced similar levels of antibody in vitro. Memory B cells from older people also produced more IgM upon activation. However, we found that IgM and IgG levels in the plasma of young and older people were nearly identical. The fact that B cells from older people produced similar antibody levels as young people might be explained by the activators used in our experiments. In order to activate the B cells in vitro, anti-Ig in combination with ODN CpG 2395 was used . Anti-Ig binds to the surface of B cells and ensures activation of both naïve and memory (isotype switched) B cells . ODN CpG serves as a ligand for toll-like receptor 9 (TLR9), increases Cox-2 expression in B cells and is reported to act as an adjuvant in vaccination . Using anti-Ig plus ODN CpG 2395 we show that B cells from older people respond to activation by increasing CD69 expression, proliferation and antibody production similar to young subjects highlighting the role of ODN CpG as a potential adjuvant.
“Inflammaging” refers to increased pro-inflammatory cytokine production in the elderly . IL-6, one of the most studied cytokines, is increased in activated human mononuclear cells  and in human serum in inflammatory diseases . It was reported that an elevated IL-6 environment increases IgG production in a mouse model in vivo . Interestingly, we show that only the memory B cells from older people produced high levels of IL-6. We speculate that activated memory B cells from older people produced high levels of IL-6 in order to promote antibody synthesis. On the other hand, IL-6 is required for experimentally-induced as well as for naturally occurring autoimmune diseases . The fact that memory B cells from older people produce more IL-6 suggest that those elderly which have a higher percentage of memory B cells in their peripheral blood would synthesize more IL-6 upon activation and be more susceptible to developing autoimmune diseases.
IL-10 is considered an anti-inflammatory cytokine dampening the effects of pro-inflammatory mediators  and it was showed that B cell production of IL-10 can prevent arthritis in mice . We found that activated memory B cells from older people, the same cell population which synthesized high levels of IL-6, also produced increased levels of IL-10. Although TNFα, another pro-inflammatory cytokine, is associated with aging, we could not detect TNF-α in activated B cells in vitro (data not shown). PGE2 induces type-2 (IL-10 production) and inhibits type-1 responses (TNF-α) in macrophages . Therefore, it is likely that increased levels of PGE2 produced by activated B cells in vitro would induce IL-10 production and inhibit TNF-α synthesis.
Our study compared the functions of naïve and memory B cells isolated from the peripheral blood of young and older people. In order to discriminate between naïve versus memory B cells, the CD27 marker was used. However, there are several reports showing that the B cell compartment is much more complex, comprising small populations of isotype switched and non-switched naïve and memory B cells [38, 39]. While these studies analyzed the surface markers in total B cell populations, it is challenging to conduct compex in vitro experiments using a low and limited number of cells. Nevertheless, B cells heterogeneity [14, 38] and dysregulated B cell development in bone marrow and spleen of the elderly [5, 24] appear to be contributing factors to the multifaceted process of immune aging. Furthermore, defects in T cell development and function [5; 23] and abnormalities in T cell-B cell interactions  also have to be considered as part of immunosenescence.
Our study highlights the finding that although B cells from older people exhibited a similar pattern of activation, proliferation and antibody production as compared to young subjects, memory B cells from older people showed an increased production of IL-6 and IL-10. Therefore, dysregulation of cytokine production is another participating factor in immunosenescence.
This research was supported by USPHS grant DE011390, ES10247 and The Toxicology Training Grant T32 ES07026
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