Aging and developmental transitions in the B cell lineage

Kara M. Johnson1,4, Kevin Owen2 and Pamela L. Witte3,4

Departments of 1 Microbiology and Immunology, 2 Anesthesiology, and 3 Cell Biology, Neurobiology and Anatomy, and 4 Program for Immunology and Aging, Loyola University Medical Center, Maywood, IL 60153, USA

Correspondence to: P. Witte, Department of Cell Biology, Neurobiology and Anatomy, Program for Immunology and Aging, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153, USA. E-mail: pwitte{at}lumc.edu
Transmitting editor: C. J. Paige


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
One explanation for the deterioration of the humoral immune response in elderly individuals is that B lymphopoiesis declines with increasing age. Recent studies report a dramatic decline in pre-B cell numbers in old mice. Surprisingly, the number of mature B cells does not decline with age. To determine if new B cells are made in aged animals despite the drop in pre-B cells, we used 5'-bromo-2-deoxyuridine labeling to determine the production rate of B cells in the bone marrow and spleen of young and old mice. Because of the great variability in the number of early B lineage cells in old mice, we acquired data on >60 young and 50 old mice throughout these experiments. The transitional and mature B cell compartments in the spleen have slower labeling kinetics in old mice as compared to young. By the end of 4 weeks of labeling, an average of only 15% of the mature B cell compartment consists of newly made cells compared to 30% in young mice. However, in contrast to an earlier report, our results indicate that there is no statistical difference in the rate of production of new immature B cells in the marrow of young and old animals. In total, our results confirm previous work showing that mature B cells in old mice have a slower turnover, but more importantly suggest that the defect in mature B cell turnover is not due to a decline in B lymphopoiesis, but rather an inability of the newly made cells to replenish the peripheral compartments.

Keywords: 5'-bromo-2-deoxyuridine, aging, B lymphopoiesis, cellular differentiation, flow cytometry


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Aging impacts the homeostatic function of many complex systems in the body, including the immune system. Elderly individuals experience a higher incidence of disease than younger individuals, including autoimmune disease. In addition, immunizing yields a diminished protective immune response in old as compared to young individuals (13). Deficiencies in both the humoral and cell-mediated immune response have been documented. Specifically, Th cell function declines with age (46), and B cell alterations include production of a greater proportion of low-affinity antibodies (7,8), different VH gene usage (9,10) and an increased incidence of autoreactive antibodies (11,12).

B cells develop in the bone marrow where they progress through a series of stages defined by Ig gene rearrangement status and phenotypic marker expression (13,14). The pro-B cell compartment contains cells undergoing µ heavy chain gene rearrangement. Cytoplasmic µ expression and assembly of the pre-B cell receptor mark the progression of pro-B cells into the pre-B cell compartment. After a proliferative burst, these cells become small resting pre-B cells and undergo light chain gene rearrangement. Development into subsequent stages proceeds without proliferation. Immature B cells express surface IgM and are exported to the periphery. The immature B cells in the periphery are referred to as new migrants or transitional B cells and are defined by high levels of heat-stable antigen (HSA) and low levels of B220. These cells mature in the periphery into functional B cells (1517).

Unlike the thymus, which atrophies with age (18), the bone marrow remains intact and shows no signs of physical atrophy over the lifetime of a rodent. However, it has been well documented that the numbers of B cell precursors in the bone marrow change with age. While the numbers of pro-B cells remain the same in young and old animals, there is a profound decrease in the number of pre-B cells in aged animals (1923). Paradoxically, this decrease is not accompanied by a similar decline in the immature B cell compartment of the bone marrow or the transitional and mature cells of the periphery (21). Data showing changes with age in the B lineage based on phenotype alone provide only a snapshot picture of the current status of the B cell precursors, yet provide evidence for altered population dynamics during B cell development. Possible interpretations of these previous results include: (i) aged bone marrow can compensate for the reduction in the number of pre-B cells by allowing a greater proportion of pre-B cells to mature into newly formed B cells or (ii) new B cells are not being made in old animals, but rather a long-lived subset of B cells fills these later developmental compartments.

To form a more accurate account of the population dynamics in the B lineage that occur with age, we used 5'-bromo-2-deoxyuridine (BrdU) labeling to assess the kinetics of B cell development in young and old animals. BrdU is a thymidine analog that is incorporated into the DNA of dividing cells. During B cell development, BrdU incorporates into early progenitor cells and remains with the cell through each developmental stage, effectively marking the cells as ‘newly made’. Kinetic BrdU-labeling experiments have been used in the past to examine B cell longevity (24,25) and to follow B cell precursors through development (16) in young mice. These studies demonstrated a rapid renewal of the bone marrow B cells in which 95% of B220lo cells were labeled with BrdU within 3 days. The new migrant B cells were found to have a slightly longer half-life of 4–7 days, while the mature compartment consisted of mostly long-lived cells with an average half-life of 4–6 weeks. Here, we extend this experimental approach to include both young and old mice. We present a BrdU-labeling time-course experiment ranging from 4 h to 4 weeks, which allowed us to assess the early kinetics of the rapidly renewing B cell populations in the bone marrow and determine the proportion of B cells that join the mature B cell pool in the spleen. Kline et al. used a similar approach to measure the lifespan of B cells in mice of different ages (26). Unlike the previous study, we looked at the early kinetics (12 h to 11 days) of the bone marrow B cell populations, allowing us to calculate production rates. Additionally, in contrast to the Kline study, we examined a large number of mice (up to 12) at each time point, providing us with a statistical basis for our conclusions and allowing us to comment on the implications of the extreme mouse-to-mouse variability in conducting aging studies. In light of these differences, our data suggest a different conclusion than the previous study. While Kline et al. suggest that new B cell generation in the bone marrow declines with age, we provide evidence that new B cells are generated in old animals at the same rate as in young mice, but are altered in their ability to replenish the peripheral transitional and mature B cell pools. The distinction between these two conclusions is critical for determining the direction of future research.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
Female BALB/c mice of 2, 12 and 22 months of age were purchased from the National Institute of Aging (Bethesda, MD), whose colony is maintained by Harlan (Indianapolis, IN). Upon receipt, the animals were housed at the Animal Research Facility at Loyola University Medical Center under specific pathogen-free conditions.

BrdU administration
For early time points, mice received i.p. injections of 0.8mg of BrdU (Sigma, St Louis, MO) resuspended in 0.2 ml of PBS. Mice loaded with BrdU for a duration of up to 7 days were injected every 12 h. Mice loaded with BrdU for 11 days were injected every 24 h. Mice loaded with BrdU for 2–4 weeks were given drinking water containing 0.25 mg/ml of BrdU. The water was shielded from light and changed every 2–3 days for the duration of the experiment. This concentration of BrdU was established by conducting a pilot study using BrdU at 0.8 and 0.25 mg/ml. When we fed BrdU at 0.8 mg/ml over the course of 4 weeks, we observed a decline in the number of total bone marrow cells, specifically B cell precursors. However, when we fed BrdU at 0.25 mg/ml, no decline in precursor B cell number was observed during a course of 4 weeks of labeling and the intensity of BrdU labeling detection was not compromised (data not shown).

Isolation of bone marrow and spleen cells
Spleens were removed and single-cell suspensions were made by rubbing spleens between the frosted ends of two glass slides. Old mice with gross splenic tumors were not used in these experiments. Spleen cells were kept in RPMI 1640 supplemented with 5% FBS (lot 10M44; Summit Bio technology, Fort Collins, CO), 1% L-glutamine, 1% penicillin/streptomycin and 5 x 10–5 M ß2-mercaptoethanol. Cells were pelleted and resuspended in 10 ml of the above media for counting. Bone marrow was isolated by flushing tibias and femurs with the above media using a 5-ml syringe and a 26-gauge needle. Aggregated cells were dissociated by pipetting up and down with a 5-ml pipette. Cells were then centrifuged and the cell pellets were resuspended in 5 ml of media for counting. The cells were counted by Trypan blue exclusion.

Staining cells for flow cytometry analysis
Two different protocols were used for staining cells for BrdU incorporation. In either case, bone marrow cells were stained with anti-B220–PerCP (RA3-6B2; PharMingen, San Diego, CA), anti-CD43–biotin (S7; PharMingen), anti-IgM–phycoerythrin (PE) (Jackson ImmunoResearch, West Grove, PA) and anti-BrdU–FITC (B44; Becton Dickinson, San Jose, CA) to distinguish between BrdU-labeled pro-, pre- and immature-B cells. Spleen cells were stained with anti-B220–PE (RA3-6B2; PharMingen), anti-HSA–biotin (M1/69; PharMingen) and anti-BrdU–FITC to distinguish between BrdU-labeled transitional and mature B cells. All antibodies were tittered to determine appropriate dilution. Cells (1 x 106) were first stained with anti-IgM–PE (bone marrow) and anti-B220–PE (spleen). After a 20-min incubation on ice, cells were washed in 500 µl of staining buffer (HBSS with calcium/magnesium, 5% heat-inactivated FBS and 0.1% sodium azide). Next, bone marrow cells were stained with anti-CD43–biotin and spleen cells with anti-HSA–biotin for 20 min on ice. At this point one of two protocols was used to permeabilize cells and detect incorporated BrdU. Either the PharMingen BrdU Flow Kit was used according the manufacturer’s instructions or the following protocol was used [kindly supplied by Dr Michael Cancro, based on previously published protocols (16,27)]. After the above antibody stain, cells were washed with 500 µl of cold PBS. The cells were resuspended in another 500 µl of cold PBS and permeabilized with 1.2 ml of 95% ice-cold ethanol added at 1 drop/s while slowly vortexing. After a 20-min incubation on ice, cells were washed with 500 µl of cold PBS. Then 100 Kunitz U DNase (Sigma) was added to each sample and incubated at 25°C for 20 min to denature the DNA and expose incorporated BrdU. Next, cells were fixed in 1 ml of 1% paraformaldehyde plus 0.05% Tween and incubated at room temperature for 20 min followed by a 20-min incubation on ice. Independent of the protocol used, all cells were stained with anti-BrdU–FITC and incubated on ice for 20 min. For the final stain, the bone marrow cells were stained with anti-B220–PerCP plus streptavidin–allophycocyanin to develop the anti-CD43–biotin. Spleen cells received streptavidin–allophycocyanin to develop the HSA–biotin. For each experiment, a set of samples was stained for surface phenotype markers without the permeabilization and DNase steps to control for effects of these treatments on cell surface profiles. These samples were fixed in 1% paraformaldehyde. Flow cytometry analysis was done using a FACSCalibur and data was analyzed using CellQuest software (Becton Dickinson).

Calculation of production rate
The production rate was calculated using least-squares regression analysis of the linear portion of the plot ‘number of labeled cells’ versus ‘time’. The regression line was fitted through at least three points and was not forced through the origin. The regression coefficient represents the number of cells produced per day.

Statistical analysis
To compare numbers of B lineage cells, and total numbers of bone marrow and spleen cells between age groups, Student’s t-test was used. Production rates were compared using analysis of covariance (comparison of slopes). P < 0.05 was considered significant. InStat by GraphPad and SPSS software was used for analysis.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Numbers of B-lineage subsets in the bone marrow of young and old BALB/c mice
One hallmark change within the B cell lineage of an old mouse is a decline in the number of pre-B cells. Published studies on absolute numbers of B cells in the bone marrow of old mice report no change in the number of pro-B cells, but a 2- to 4-fold decrease in the number of pre-B cells in old mice as compared to young (1923). The number of immature B cells, however, has been shown to decrease <2-fold in old mice (21). The experiments performed here on 2- and 22-month-old BALB/c mice are generally in agreement with previous reports. Our study, which took place over a 30-month period, included >60 young and 50 old mice purchased from the National Institute of Aging’s aging colony at Harlan. Similar to previous studies, we found an overall increase in the number of bone marrow cells in old animals as compared to young (Table 1) and the number of pre-B cells was significantly decreased by ~30% in old mice (Table 2). Despite a significant decrease in the percent of pro- and immature B cells in old mice, there was no significant decrease in the actual numbers of pro- and immature B cells as compared to young mice (Table 2) due to the increase in the overall cellularity. The number of B220hi mature recirculating bone marrow B cells was increased in old animals, accounting for ~10% of the increase in total bone marrow cellularity (Table 2).


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Table 1. Numbers of cells in the bone marrow and spleen of 2- and 22-month-old micea
 

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Table 2. Percent and number of cells at each stage of development in the bone marrow and spleen of 2- and 22-month-old micea
 
Labeling of precursor B cells in the bone marrow of young and old mice
In order to determine if new B cells are made from the reduced pool of precursors in old mice, we compared the kinetics of B cell development in 2- and 22-month-old animals by labeling young and old mice with BrdU over a course of 4 weeks. For the early time points (4 h to 11 days), BrdU was administered by i.p. injection, but for the later time points (2 and 4 weeks) BrdU was fed in the drinking water to avoid toxicity. Bone marrow cellularity and total numbers of B220+ cells were maintained throughout BrdU treatment, suggesting negligible toxic effects. We analyzed >100 mice combined in these experiments and at the pivotal early time points (12 h to day 3) we analyzed six to eight mice of each age group.

After BrdU treatment, bone marrow was harvested and cell surface phenotype and BrdU incorporation was determined by flow cytometry analysis. A sample profile, including the gates we used to distinguish the different B cell subsets, is shown in Fig. 1. In both young and old mice, the pro-B cell compartment (B220lo/CD43+/IgM, Hardy fractions A-C'), which contains the rapidly proliferating compartment of developing B cells, fully labeled within 1–2 days of BrdU administration (Fig. 2A). The pre-B cell compartment (B220lo/CD43/IgM, Hardy fraction D) in both age groups was fully labeled by day 3 (Fig. 2B). The immature B cells (B220lo/CD43/IgM+, Hardy fraction E) of young mice reached maximum BrdU labeling at ~95% by day 7 (Fig. 2C), while in old mice maximum labeling plateaued at 75% of the total immature B cell compartment after 7 days (Fig. 2C). The labeling curve of immature B cells in 22-month-old mice is indicative of two populations of cells—one that rapidly renews with BrdU-labeled cells (linear curve) and one that never acquires new labeled cells (unlabeled 20%) (28).



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Fig. 1. Representative FACS profiles of bone marrow from young and old mice. Pro- (B220lo/CD43+/IgM), pre- (B220lo/CD43/IgM) and immature (B220lo/CD43/IgM+) B cells were first gated on B220lo, followed by gating based on differential expression of IgM and CD43. The histograms represent BrdU incorporation of each B cell subset after 2 days of BrdU labeling. The light gray peak in the pro-B cell histograms represents the isotype control.

 


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Fig. 2. Kinetics of BrdU labeling of the pro-, pre- and immature B cell compartments of young and old mice. Two- and 22-month-old BALB/c mice were administered BrdU (see Methods) for 4 weeks. Bone marrow was harvested at 12 h, 1, 2, 3, 7 and 11 days, and 2 and 4 weeks post-labeling, and cells were stained with antibodies to phenotypic markers to distinguish (A) pro-, (B) pre- and (C) immature B cell subsets, and stained with anti-BrdU–FITC to detect BrdU incorporation as described in Methods. The percent labeled cells was determined by flow cytometry using CellQuest software. n = 6–12 mice at 12 h, 1, 2, 3 and 7 days, and 2 weeks; n = 3 at 11 days and 4 weeks. In (C), the BrdU labeling profile of 12-month-old mice (to day 7) is included (n = 3–12 mice at each time point). Data represent means ± SE.

 
An earlier publication suggested a more severe defect in immature B cell production in old animals than what we report here, specifically in an IgMhi subset (26). For comparison, we divided the immature B cells into IgMlo and IgMhi populations. In our experiments, we saw similar numbers of IgMlo B cells in young and old mice (Table 2), and little difference in the labeling kinetics—95% of the IgMlo cells labeled by day 11 in both age groups (Fig. 3A). There were, however, ~30% fewer IgMhi immature B cells in old mice when compared to young IgMhi immature cells (Table 2). The IgMhi cells from old mice reached maximum labeling at only 70% after 7 days of BrdU injection compared to maximum labeling at 95% in young mice (Fig. 3B).



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Fig. 3. Kinetics of BrdU labeling of the IgMhi and IgMlo immature B cell subsets of young and old mice. The profiles generated as described in Fig. 2 were reanalyzed, and the immature B cell population was divided into (A) IgMhi and (B) IgMlo cells. Because the IgM+ cells did not fall into two distinct populations, IgMlo cells were arbitrarily defined as the lower one-fourth of the total IgM+ gated population (see Fig. 1). n = 6–12 mice at 12 h, 1, 2, 3 and 7 days, and 2 weeks; n = 3 at day 11 and 4 weeks. Data represent means ± SE.

 
The early time points in the labeling experiments presented here allowed us to predict the production rate (number of cells produced per day in two tibias and two femurs) from the total number of labeled cells in each bone marrow compartment over time (Table 3). Similar numbers of new pro-B cells were made per day in young and old animals (1.46 x 106 and 1.28 x 106 respectively). Pre-B cell production was significantly decreased from 3.57 x 106 cells/day in young mice to 2.56 x 106 cells/day in old mice. In old animals, 0.82 x 106 immature B cells were produced per day, which was a slight increase over the 0.73 x 106 produced per day in young mice. This increase in production paralleled the IgMlo subpopulation—0.25 x 106 cells were produced per day in young mice versus the 0.37 x 106 produced per day in old mice. Approximately 0.4 x 106 IgMhi cells were produced per day in both young and old animals. This rate reflects only the rapid labeling cells within the IgMhi compartment (linear portion of curve), and suggests that a similar number of cells enter this compartment per day in young and old mice despite the presence of a separate, kinetically stagnant pool of immature B cells in old mice.


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Table 3. Production rates of B lineage cells in the bone marrow and spleen of 2- and 22-month-old micea
 
Numbers and kinetics of B-lineage subsets in the spleens of young and old animals
To determine if the newly made immature B cells accumulate in the spleen of old mice, we examined the magnitude and kinetics of the transitional and mature B cell compartments. On average, the old mice had a significant increase in the number of spleen cells as compared to young mice (Table 1). No significant difference was seen in the frequency of B220+ cells in the spleens of 2- and 22-month-old animals (data not shown). Data published previously by our laboratory showed that the number of transitional and mature peripheral B cells does not change significantly with age (21). Here, as before, we defined transitional B cells as HSAhi and B220lo, while the mature subset was defined as HSAlo and B220hi (see Fig. 4 for sample profiles and gating). The population of cells expressing both high levels of HSA and B220 was excluded from these gates because we have observed that these cells have an activated phenotype (high levels of surface IgM and a high forward scatter, indicative of large cell size; unpublished data). When gated in this way there was no significant difference in the frequency or number of transitional and mature B cells between young and old animals (Table 2).



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Fig. 4. Representative FACS profiles of spleens from young and old mice. Transitional (HSAhi/B220lo) and mature B cells (HSAlo/B220hi) are gated based on HSA and B220 expression. The histograms represent BrdU incorporation of each B cell subset after 11 days of BrdU labeling. The light gray peak in the transitional B cell histograms represents the isotype control.

 
At each time point over the course of 4 weeks of BrdU labeling, spleen cells were stained to discriminate between BrdU-labeled transitional B cells and mature B cells. The frequency of labeled transitional B cells (HSAhi/B220lo) continued to increase over time in the young animals, plateauing at 70% by 2 weeks (Fig. 5A). In contrast, transitional B cells in the old animals plateaued at ~40% in this same time period (Fig. 5A). It was considered that marginal zone B cells fall within the transitional B cell gate and that this population increases with age. The majority of CD23/CD21hi marginal zone B cells fell within the mature HSAlo/B220hi gate, indicating that marginal zone B cells are not contributing to the population dynamics of the transitional B cell pool in either age group (data not shown). New, labeled mature B cells (HSAlo/B220hi) continued to accumulate in young and old mice, and maximum labeling was not reached in this 4-week period. Thirty percent of the mature B cell compartment in young mice renewed with BrdU-labeled cells after 4 weeks, but in old mice only 15% of the mature compartment renewed in this time (Fig. 5B).



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Fig. 5. Kinetics of BrdU labeling of the transitional and mature B cell compartments of young and old mice. Spleens were harvested from the mice described in Fig. 1. Spleen cells were stained with antibodies to phenotypic markers to distinguish (A) transitional and (B) mature B cell subsets, and stained with anti-BrdU–FITC to detect BrdU incorporation as described in Methods. The percent of labeled cells was determined by flow cytometry using CellQuest software. n = 6–12 mice at 12 h, 1, 2, 3 and 7 days, and 2 weeks; n = 3 at 11 days and 4 weeks. In (B), the BrdU labeling profile of 12-month-old mice (to day 7) is included (n = 3–12 mice at each time point). Data represent means ± SE.

 
The production rate was also calculated for the splenic B cell subsets in young and old mice. This rate represents the number of new cells (assumed to be coming from the bone marrow) that appear in the spleen over time. As shown in Table 3, 43% fewer transitional B cells accumulated per day in old mice as compared to young and the mature B cell compartment showed an even greater kinetic deficiency. Fifty-eight percent fewer newly made cells appear in the mature B cell compartment of old animals compared to young (Table 3).

Comparison of middle-aged mice to young and old
In order to address when during the lifetime of a mouse the changes in B cell development begin, we studied the labeling kinetics of a set of middle-aged (12-month-old) BALB/c mice. The middle-aged mice show an intermediate decline in the relative size of the pre- and immature compartments and an increase in the B220hi mature recirculating bone marrow compartment (data not shown). The labeling kinetics of the immature and transitional B cell compartments over seven days in 12-month-old mice also demonstrates an intermediate phenotype to young and old mice (Figs 2C and 5A). Together, this data suggest that the alteration in B cell development begins prior to 12 months of age and new B cell production progressively slows with time.

Correlation between the number of pre-B cells and severity of the kinetic defect in old mice
One puzzling inconsistency in the data from this study and that from the study by Kline et al. is the discrepancy between the severity of the production of new B cells in old mice (26). Kline et al. suggested that old mice have a severe defect in the production of new immature B cells based on a group of mice that all had a severe defect in pre-B cell numbers. In contrast, our data suggest that, on average, B cell production in the bone marrow is only modestly affected by age. While some old mice in our study had a severe drop in pre-B cell numbers, others fell within the range of young mice (Fig. 6A). We investigated the possibility that new B cell production correlates with the severity of the pre-B cell deficiency. Sherwood et al. have shown a similar correlation between pre-B cell numbers and defects in old mice (23).



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Fig. 6. Correlation between pre-B cell numbers and number of labeled immature, transitional and mature B cells in young and old mice. This figure represents data collected from the mice described in Figs 2 and 5. (A) The number of pre-B cells is plotted for 56 young and 51 old mice. The number of pre-B cells was calculated by multiplying the percent of pre-B cells (generated by CellQuest) by the total number of bone marrow cells. Each point represents an individual mouse and the horizontal bars represent the average of all mice in that group. In (B) and (C), bars represent old mice with either ‘high’ numbers of pre-B cells (~3 x 106/two femurs and two tibias) or ‘low’ numbers of pre-B cells (<3 x 106/two femurs and two tibias). The average number of labeled (B) immature and (C) transitional B cells in each group is plotted over time. Number of labeled cells was calculated by multiplying the percent labeled immature or transitional B cells (generated by CellQuest) by total numbers of immature or transitional B cell. The average number of labeled cells over time for young mice (line graph overlay) is given for reference. (D) Average number of labeled transitional B cells in young mice with either ‘low-end’ (<5 x 106/two femurs and two tibias) or ‘high-end’ (>5 x 106/two femurs and two tibias) pre-B cell numbers. *P < 0.0001.

 
In order to determine if the number of pre-B cells correlates with the number of new B cells that are made, we grouped all our old mice into those with ‘low’ pre-B cell numbers (range = 1.1–2.85 x 106) and those having ‘high’ pre-B cell numbers (range = 3.38–8.25 x 106), and plotted the number of labeled immature and transitional B cells over time for each group. Figure 6(B and C) illustrates the compared production of immature and subsequent accumulation of transitional B cells in the spleen between these two groups. Fewer BrdU-labeled immature B cells accumulated over time in the ‘low’ pre-B cell number group as did in the ‘high’ pre-B cell number group, but the number of labeled immature B cells accumulating over time in the ‘high’ group was similar to young mice (Fig. 6B). The difference between the ‘low’ and ‘high’ pre-B cell number groups was more dramatic in the transitional B cell compartment of the spleen. Approximately half as many BrdU-labeled cells accumulated in the ‘low’ pre-B cell group at the later time points compared to the ‘high’ pre-B cell group, yet the appearance of new transitional B cells in the periphery was still deficient in the ‘high’ pre-B cell group as compared to young (Fig. 6C). Young mice similarly split based on low-end (range = 1.67–4.85 x 106) and high-end (range = 5.00–10.30 x 106) pre-B cell numbers did not show this correlation (Fig. 6D). Therefore, the previous study (26), which looked at fewer old mice than reported here, may have been limited to an unrepresentative distribution of aged animals, which, by chance, fell within the low range of pre-B cell numbers.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
New B cell production in the bone marrow of aged mice
In order to form a more accurate representation of the population dynamics in the B lineage with age, we assessed the kinetics of B cell development in young and old animals by measuring the accumulation of BrdU-labeled cells at each developmental stage. While the number of B cells in the bone marrow and spleen does not change with age, it cannot be assumed that new B cells are being made or that new mature B cells replenish residing cells in the spleen. If we are to understand how humoral immune system function declines with age, we must first document the changes in B cell development.

One explanation for the decline in B cell function with age is that new B cell production ceases in old individuals, as has been recently proposed (26). Our data challenge this conclusion based on the finding that there are only moderate kinetic differences in the immature B cell compartment of young and old mice. Specifically, similar numbers of new BrdU-labeled immature B cells are made per day in old mice as compared to young mice, despite a population of immature B cells in old mice that never accumulates labeled cells. It may appear counter-intuitive that the production rate is the same, when, in the old mice, maximum labeling is never reached. However, the kinetically active (linear portion) and the stagnant (unlabeled 25%) populations are distinct from one another and are therefore considered independently (28). Further analysis revealed that the kinetically stagnant population resides in the IgMhi subgroup of immature B cells. Although the identity of this unlabeled fraction of cells is unclear, we propose two possibilities that could explain their presence. One proposition is that these long-lived cells may be recirculating mature B cells. Because they are gated on B220lo, an established indicator of immature B cells, this possibility would be correct only if a proportion of mature B cells in old mice can change their cell surface phenotype and simultaneously down-regulate B220 and up-regulate IgM—a phenomena not reported in young mice. Another proposition that we offer is that this is a population of immature B cells with increased longevity. While we cannot dismiss the possibility that a fraction of mature recirculating B cells change their phenotype and are captured in our immature B cell gate, there is no supporting evidence in the literature for such a phenotypic change with age. We argue that an increased longevity within the immature compartment is a more likely explanation based on unpublished data from our laboratory. We have seen that early B lineage cells from old mice survive longer in culture than those from young mice.

Our data provide evidence that the production of new cells in the bone marrow of aged mice is sustained despite the decreased size of the pre-B cell pool. In normal young mice, millions of new pre-B cells are made every day (29), but over half of these cells are lost before exiting the bone marrow due to unsuccessful gene rearrangement (30,31) or negative selection upon encounter with self-antigen (32,33). Because fewer pre-B cells are made per day in old animals without affecting new immature B cell production, it is likely that the mechanisms that tightly regulate the production of new B cells are relaxed in old mice. This has important implications for proposing models of immune dysfunction with age. For example, in old mice (i) a greater proportion of pre-B cells could be recruited into the immature B cell pool or (ii) fewer immature B cells could succumb to selection against self-antigen. In either case, important quality control checkpoints may be compromised in exchange for quantity of output, potentially leading to the production of autoreactive and functionally defective B cells.

Renewal of the transitional and mature B cell compartments in aged mice
The number of spleen cells and the proportion of splenic B lineage cells in our study did not decline with age. This is in striking contrast to the number of spleen cells reported by Kline et al. On average, the old mice in our study had increased numbers of total spleen cells as compared to young mice (young = 129 x 106 versus old = 157 x 106 cells). In studies using hundreds of aged mice over several years, we never observed spleens as acellular as those in the report by Kline et al. (old = 38 x 106 cells) (26). Additionally, in contrast to the previous report, but corroborating previous data from this laboratory (21), we detected no significant decline in the number of transitional B cells when we used HSA and B220 as phenotypic markers to distinguish transitional and mature B cells [as established by Allman et al. (15,16)]. The rapid accumulation of BrdU-labeled cells in the HSAhi/B220l° population in young mice supports this designation as transitional B cells.

Despite the maintenance of peripheral B cell numbers and despite the relatively conserved production of immature B cells in the bone marrow of old mice, the ability of new cells to replenish the splenic B cell pools is compromised. The transitional B cell compartment in young mice reaches maximum labeling within 2 weeks, which corroborates previous kinetic analysis of this compartment (16,25). However, the kinetic profile of the transitional B cells in old mice is markedly different from their young counterpart. The production rates indicate that almost half as many new transitional B cells are produced per day in old mice as compared to young. The transitional B cell compartment in old mice reaches maximum labeling within 2 weeks, as is the case with young mice, but plateaus at only 40% of the total gated population. This kinetic pattern suggests, again, the presence of two populations of cells in old mice—a kinetically active population that labels slower than their young counterpart (represented by the more negative slope) and a large (60% of the population) stagnant pool that fails to renew with new cells after 4 weeks of labeling. A transitional B cell compartment that contains long-lived cells has important functional implications. It has been shown that transitional B cells remain functionally immature and undergo apoptosis upon IgM cross-linking (16,17). B cells that reside in this compartment for an extended amount of time may be less sensitive to negative selection mechanisms and thus become recruited into the mature B cell pool as functionally defective or even autoreactive cells.

Based on the finding that fewer new, BrdU-labeled transitional B cells are observed in the spleens of old mice, one would predict that fewer labeled cells would also accumulate at the mature B cell stage in these mice. Confirming this prediction, BrdU-labeled cells account for only 15% of the total mature B cell compartment in aged mice after a 4-week period as compared to 30% in the mature B cell compartment of young mice. The accumulation of new mature B cells in young and old mice continues to increase linearly during the 4-week labeling period, but at different rates. Fifty-eight percent fewer new mature B cells are produced per day in old mice as compared to young mice. Because the size of the mature B cell compartment is the same in both age groups, we can conclude that cells in this compartment of old mice are also longer lived than mature B cells in young mice. Increased survival of mature B cells may have profound effects on effector function. Although the physiological mechanism by which an increased cell life span would affect B cell function is not known, we speculate that the documented decline in B cell function with age is related to the failure of the compartment to replenish with newly made cells every 4–6 weeks.

Variability in pre-B cell numbers and correlation to kinetic deficiencies
One consistent sign of aging repeatedly reported in the literature is the sharp decline in pre-B cell numbers (1923). However, within our study group of 51 old mice, pre-B cell numbers range from 1.11 to 8.25 x 106/two tibias and two femurs, with some mice falling within the average range of pre-B cell numbers reported in young mice. Sherwood et al. addressed the correlation between pre-B cell numbers and B cell defects, and demonstrated that old mice with low pre-B cell numbers have less expression of {lambda}5 protein, while old mice with ‘young-like’ pre-B cell numbers express similar levels of {lambda}5 as young mice (23). In our study, old mice with ‘high’ pre-B cell numbers accumulate a greater number of labeled immature cells over time as compared to old mice with ‘low’ pre-B cell numbers, suggesting that new B cell production may be dependent on the severity of the pre-B cell defect. This correlation is even more dramatic in the splenic transitional B cell compartment of old mice. Half as many new transitional B cells accumulate over time in old mice with ‘low’ numbers of pre-B cells compared to old mice with ‘high’ numbers of pre-B cells. Notably, the old mice with ‘high’ pre-B cell numbers (average 4.98 x 106 cells) are still kinetically impaired compared to young mice (average 5.00 x 106 pre-B cells). Additionally, young mice with low-end pre-B cell numbers are not kinetically different from young mice with high-end pre-B cell numbers. Together, this suggests that pre-B cell number alone does not govern the ultimate ability of these cells to progress through development, but rather intrinsic and/or environmental deficiencies in old mice beyond the pre-B cell stage play a role in this development defect. Moreover, we still maintain that the major defect in B cell development of old mice is the inability of newly made cells to join the peripheral B cell compartments.

Potential mechanisms for altered mature B cell turnover
The main contribution of our studies is the evidence that the decline in mature B cell turnover is not accompanied by a severe decline in new B cell production. This evidence refutes previous work. Kline et al. suggest that there is a severe defect in new B cell production in the bone marrow of old mice. The distinction between these two conclusions is critical for determining the direction of future research. Our current hypotheses are directed toward determining mechanisms for the altered survival of immature B cells and/or the altered migration of newly made B cells to the spleen of aged mice. The alternate conclusion would warrant focusing efforts on determining the mechanism of altered B lymphopoiesis with age.

We propose several explanations for why newly made cells do not replenish the mature B cell compartment. The specific cues for the recruitment of transitional B cells into the long-lived mature B cell pool have recently been shown to include the expression of a functional BCR (34) and the environmental survival signal BAFF (35). Because new immature B cells are being made from a reduced population of precursors in aged animals, the regulation at the pre- to immature B cell transition may be less stringent, allowing cells with defective receptors to enter the periphery, just to fail at a later check point. Alternatively, signals provided by the surrounding environment/accessory cells may be altered with age. Another likely mechanism for the failed replenishment of peripheral B cells is a defective homing mechanism. Newly made B cells may be unable to exit the bone marrow and die in situ after a few days. Alternatively, assuming new B cells successfully leave the bone marrow, they may be unable to respond to homing signals required for the migration out of the blood and into the spleen. An interesting question that remains unexplored is whether B cell longevity increases in aged mice in response to a diminished supply of new emigrants from the bone marrow or whether longevity is pre-programmed and thus new immature B cells are unable to successfully compete for space with the residing cells.


    Acknowledgements
 
The authors wish to thank Matt Ewert, Jeannette Pifer and Zheng Yu for technical assistance; John Galvin and Dr Susan Fisher for statistical advice; and Kirstin Gray Parkin, Heather Minges Wols, Paul Jasper and Dr Phong Le for many helpful discussions and critical reading of the manuscript. We also thank Dr Thomas Ellis and Patricia Simms of the Loyola University FACS Core Facility for the excellent help with flow cytometry analysis, and Dr Michael Cancro for sharing his BrdU-labeling protocol at the beginning of these experiments. This work was supported by National Institutes of Health grants RO1AG13874 and KO7AG00997, and NIH Training grant T32AI07508 (to K. M. J.).


    Abbreviations
 
BrdU—5'-bromo-2-deoxyuridine

HSA—heat stable antigen

PE—phycoerythrin


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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