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Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Much of the alteration in T cell responsiveness has been attributed to the long term consequences of decreased T cell generation following thymic involution in young adults 2, 3, 4, 5, 18 . Although bone marrow B cell generation decreases from the first to the fourth month of age 19, 20, 21, 22, 23, 24, 25, 26, 27 , a further dramatic decrease in B cell generation as mice age has not been documented. Thus, while several investigators have reported a progressive decrease in the population of bone marrow pre-B cells after 12 mo of age 22, 23, 24, 25, 26, 27 , it has been reported that the populations of both mature and immature splenic B cells remain relatively constant as mice age 27 .
Recently, Fulcher and Basten have analyzed the life span of cells within various B cell subsets by assessing the proportion of labeled cells of mice fed 5'-bromo-2-deoxyuridine (BrdU)3 for 1–7 wk 21 . Their findings confirmed earlier demonstrations of an increase in the half-life of splenic B cells from less than 2 wk in 6-wk-old mice to 4–6 wk in 3- to 10-mo-old mice 20 .
To determine whether aging is accompanied by a further increase in B cell longevity, we have analyzed the distribution of cells within various bone marrow and spleen cell subsets of aged mice as well as BrdU incorporation into cells of these subsets. Consistent with earlier studies, the population of mature peripheral B cells is maintained in aged mice despite a four- to sixfold decrease in the bone marrow pre-B cell population. However, in contrast to earlier conclusions 27 , our findings indicate that although newly generated B cells accumulate with time in the subset of least mature bone marrow B cells (sIgMvery low), there is a four- to sevenfold depletion of cells representative of both the later stages of bone marrow development (sIgMint-high) and the least mature subset of B cells in the spleen 28, 29 . In addition to a decrease in the proportion of B cells that complete their bone marrow maturation and subsequently enter the spleen, the half-life of mature B cells within the spleen of aged mice is increased dramatically compared with that of mature splenic B cells in 4- to 8-mo-old mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals (1.5–26 mo old) were obtained through the National Institute of Aging; 1.5-mo-old animals were also acquired from the rodent breeding facility at The Scripps Research Institute (La Jolla, CA).
BrdU treatment
Mice were administered drinking water containing 0.8 mg/ml of BrdU (Sigma, St. Louis, MO). This water was changed every 2–3 days throughout the time course of the experiment.
Isolation of spleen and bone marrow cells
Spleens were removed and placed in HBSS without phenol red, homogenized individually into a single cell suspension, passed through a Nytex mesh (Tetko, Lancaster, NY), and then pelleted by centrifugation. The pelleted spleen cells were resuspended in 2 ml of 0.14 M NH4Cl and 0.017 M Tris-Cl, pH 7.2, and incubated for 2 min at 37°C to remove RBCs. After this, the cells were again pelleted by centrifugation and immediately washed in 6 ml of HBSS. The spleen cells, now free of RBCs, were resuspended to a concentration of 10 x 106 cells/ml in HBSS supplemented with 0.1% BSA (fraction 5; Sigma) and 0.2% sodium azide (HBSS/BSA/Az). Bone marrow cells were isolated by flushing the femurs and tibias with HBSS/BSA/Az using a 3-cc syringe with an attached 26-gauge needle. Clumps were dispersed by repeatedly drawing up and expelling the cell suspension with the syringe and needle. Following this, the cells were filtered through a Nytex mesh and subjected to centrifugation. The pelleted cells were then resuspended to a concentration of 10 x 106 cells/ml in HBSS/BSA/Az for further manipulation.
Staining for FACS analysis
For cell surface staining of spleen cells, in which expressions of heat-stable antigen (HSA), CD23, and B220 were used to subdivide various B cell populations, the following staining regimen was used. Spleen cells (1 x 106) were first incubated with culture supernatant containing the rat anti-HSA hybridoma Ab J11D 30 for 15 min on ice. Three milliliters of HBSS/BSA/Az was then added to the cells, which were centrifuged and resuspended in 100 µl of HBSS/BSA/Az before addition of biotinylated mouse anti-rat IgM (clone G53-238, PharMingen, San Diego, CA). The cells were then incubated for 15 min on ice, again washed, and resuspended in 100 µl of HBSS/BSA/Az for staining with streptavidin-Cy5 (Zymed, South San Francisco, CA) to reveal bound anti-HSA. After a 15-min incubation on ice, excess streptavidin-Cy5 was removed by washing before the simultaneous addition of anti-B220 CyChrome (clone RA3-6B2, PharMingen) and anti-CD23 PE (clone B3B4, PharMingen). The cells were then incubated on ice for 15 min, washed, and resuspended in 100 µl HBSS/BSA/Az before beginning the anti-BrdU staining (described below). All the other combinations of Ab staining followed the same methodology as that outlined above. For analysis of IgM, IgD, and B220 expression on spleen cells, cells were first stained with anti-IgDa biotin (clone AMS 9.1, PharMingen). After a 15-min incubation on ice, the cells were washed and then incubated with streptavidin-Cy5 to reveal the anti-IgDa staining. After this, the cells were stained with anti-B220 Cychrome, incubated on ice for 15 min, and washed. The cells were then stained with anti-IgMa PE (clone D5-1, PharMingen), incubated for 15 min on ice, washed in HBSS/BSA/Az, and resuspended in 100 µl of HBSS/BSA/Az. For analysis of HSA, IgM, IgD, and B220 expression on spleen cells, cells were first incubated with culture supernatant containing the rat anti-HSA Ab clone J11D for 15 min on ice. The cells were washed and then stained with biotinylated mouse anti-rat IgM (clone 653-238, PharMingen) for 15 min on ice. After washing, cells were stained with both streptavidin-613 (Life Technologies, Grand Island, NY) to reveal bound anti-HSA and goat anti-mouse IgM-AMCA (Chemicon, Temecula, CA). After a 15-min incubation on ice, the cells were again washed and incubated simultaneously with anti-B220-Cy5 (clone RA3-6B2 purified, PharMingen; conjugated with the Cy5 mAb labeling kit, Amersham, Pittsburgh, PA) and anti-IgDa-PE (clone AM59.1, PharMingen) for 15 min on ice. The cells were washed and resuspended in 100 µl of HBSS/BSA/AZ.
For the staining of total bone marrow cells, 1 x 106 cells from each animal were incubated with anti-IgMa PE or anti-CD43 PE (clone S7, PharMingen, San Diego, CA) for 15 min on ice. The cells were washed with 3 ml of HBSS/BSA/Az, centrifuged, and resuspended in 100 µl of HBSS/BSA/Az. The cells were then stained with anti-IgDa in the case of the cells stained with anti-IgMa or with goat anti-mouse Ig (multiple absorbed, PharMingen) for those cells stained with anti-CD43 PE. After a 15-min incubation on ice, the cells were washed with 3 ml of HBSS/BSA/Az. Finally, both sets of cells were simultaneously stained with anti-B220 Cychrome and with streptavidin-Cy5 to reveal the biotinylated Abs. Whole bone marrow cells from each animal were also stained with TER-119 biotin (PharMingen) for 15 min on ice and washed with 3 ml of HBSS/BSA/Az, followed by staining with streptavidin PE (Biomeda, South San Francisco, CA). After a 15-min incubation on ice, excess streptavidin PE was removed by washing with 3 ml of HBSS/BSA/Az, following which the cells were resuspended in 100 µl of HBSS/BSA/Az before beginning the anti-BrdU staining.
Anti-BrdU staining
This protocol is the same as that used by Sprent and Tough 31 , which is a modification of that used by Carayon and Boyd 32 . After the cell surface staining was completed, the cells were resuspended in 0.5 ml of ice-cold 0.15 M NaCl followed by the dropwise addition of 1.2 ml of ice-cold 95% ethanol while gently vortexing. The cells were then incubated for 30 min on ice before being pelleted by centrifugation and were washed with PBS. The cells were resuspended in 1 ml of PBS supplemented with 1% paraformaldehyde and 0.01% Tween-20 and incubated for 30 min at room temperature. The cells were pelleted, and the PBS solution was removed by decanting and suspended in a solution containing 0.15 M NaCl, 4.2 mM MgCl2, and 50 U/ml of DNase (RNase free; Boehringer Mannheim, Indianapolis, IN). The cells were incubated for 10 min at room temperature before being spun and then washed in PBS. The pelleted cells were resuspended in 100 µl of PBS, and 10 µl of anti-BrdU-FITC (Becton Dickinson, San Jose, CA) was added. The cells were incubated for 30 min at room temperature, washed with PBS, and resuspended in 400 µl of HBSS/BSA/Az for FACS analysis. All FACS analyses were conducted using a dual laser FACSCaliber or FACSVantage in a triple laser configuration (Becton Dickinson), and data points were plotted using CricketGraph III (Cricket Software, Malvern, PA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous analyses of the turnover of splenic B cells in mice fed
BrdU for 1–7 wk demonstrated that in 6- to 8-wk-old mice, 50% of
B220+ cells were BrdU+ between 1 and 2 wk,
whereas by 3 mo and up to 10 mo of age, 50% of B220+
splenic B cells were BrdU+ only after 4–6 wk of feeding
BrdU 21 . To determine whether the turnover of splenic B cells is
altered in aged mice, 15 23- to 26-mo-old mice were fed BrdU for
periods of 8 days to 8 wk. For comparative purposes, 1.5- to 3.5-mo-old
and/or 4- to 8-mo-old mice were also analyzed at each time point.
Although, on the average, the total number of spleen cells was similar
for 1.5- to 3.5-mo-old mice (91 x 106) and 4- to
8-mo-old mice (104 x 106), the total number of spleen
cells was over twofold lower in 23- to 26-mo-old mice (38 x
106). Representative FACS profiles of B220+
splenic B cells of aged and younger mice fed BrdU for 8 days to 8 wk
are shown in Fig. 1
. The percentage of
BrdU-labeled cells in each of the individual mice of various ages used
in this study is plotted in Fig. 2.
Although the rate of accumulation of BrdU-labeled cells in BALB/c mice
appears to be somewhat lower than the rate reported by Fulcher and
Basten for C57BL/6 and B6 x SJL mice 21 , 50% of total splenic
B cells of 1.5- to 3.5-mo-old mice were BrdU labeled by 6–7 wk, and
40% of splenic B cells of 4- to 8-mo-old mice were BrdU labeled by 8
wk (Fig. 2A). However, the rate of accumulation of
BrdU-labeled splenic B cells was significantly reduced in aged mice, in
which only 12% of splenic B cells were labeled by 8 wk.
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), were analyzed for BrdU
incorporation, the pattern closely followed that of total splenic B
cells, in that 50% of HSAlow-int cells of 1.5- to
3.5-mo-old mice were labeled within 7 wk (Fig. 2B). Although
precise estimates of half-life are not possible when 50% BrdU labeling
was not achieved over the time course of the experiment, extrapolation
of the data in Fig. 2B suggests that whereas 50% of mature
B cells of 4- to 8-mo-old mice would be BrdU labeled after 11–12 wk,
it would require >24 mo before 50% of mature splenic B cells of aged
mice would be BrdU labeled. This finding suggests that the increased
longevity of total splenic B cells in aged mice, while reflecting in
part a shift in the population from immature to mature cells, is
primarily the consequence of a marked increase in the longevity of
mature B cells per se in aged mice. Analysis of the population of immature splenic B cells in aged mice
The relatively slow rate at which BrdU-labeled cells accumulate in the spleens of aged mice suggests that with age, fewer and fewer cells enter the mature B cell pool. This could either be the result of a decreased frequency of newly generated B cells entering the spleen from the bone marrow of aged individuals or, alternatively, the result of a decrease in the frequency of B cells that further mature once they enter the spleen. The least mature population of splenic B cells has been identified as cells that express the highest levels of surface HSA, first as CD23- and then as CD23+ cells 28, 29, 33 . Typically, these cells can also be identified by the absence or low expression of sIgD. This population generally represents 10–20% of splenic B cells in young mice 28 . Recent studies have been interpreted as indicating that this population remains intact in aged mice 27 , which, if true, would suggest that the frequency of B cells newly emigrating from the bone marrow to the spleen remains unchanged. However, these previous studies arbitrarily used high expression of HSA as the sole marker for immaturity and assumed that the 10–20% of cells with highest levels of HSA in spleens of aged mice were equivalent to the least mature splenic B cells of young mice.
To assess the expression of immature splenic B cells in aged vs young
mice we have used three independent criteria. First, we have
established a FACS gate for high HSA expression independently for
spleen cells of each animal using
B220highCD23- splenic B cells that are greatly
enriched for immature B cells. Second, we have used the rapid
accumulation of BrdU-labeled cells as a confirmational marker that
cells designated as HSAvery high are actually newly
generated cells. Third, we have assessed the expression of sIgD (a
marker for more mature cells) on cells within the HSAvery
high population. The results of these analyses for 1.5- to
3.5-mo-old, 4- to 8-mo-old, and 23- to 26-mo-old mice are shown in
Figs. 1 and 3.
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, the total
proportion of splenic B cells that were recent bone marrow
émigrés by the criteria of being HSAvery high
and BrdU labeled within 8 wk decreased from 11.7% in 1.5- to
3.5-mo-old mice and 5.4% in 4- to 8-mo-old mice to 1.4% in 23- to
26-mo-old mice. In absolute numbers of cells, this represents a
decrease from an average of 6.1 x 106 cells in the
spleens of 1.5- to 3.5-mo-old mice to 3.6 x 106 cells
in the spleens of 4- to 8-mo-old mice and 0.3 x 106
cells in the spleens of aged mice. This progressive decrease in newly
emigrated cells is confirmed by the paucity of sIgDneg-low
cells and is consistent with the conclusion that the steady state of
splenic B cells in aged mice is maintained by both an increased
half-life of mature B cells and a decrease in the emigration to the
spleen of newly generated B cells from the bone marrow. Bone marrow B cell development in aged mice
The finding that the population of recent émigrés from the bone marrow to the spleen is decreased in aged mice suggests that either bone marrow B cell development is decreased in aged mice, or that a smaller percentage of newly generated cells peripheralizes from the bone marrow or enters the spleen. Several investigators have demonstrated that the population of pre-B cells is decreased in aged mice. However, there is disagreement as to whether the population of newly generated sIg+ cells is also decreased 21, 22, 23, 24, 25, 26, 27 . In part, disparate results concerning B cell development in the bone marrow of aged mice is due to the variability in bone marrow B-lineage cells from individual to individual aged mouse even within an inbred strain. Additionally, most aged mice display an increase in mature sIgDhigh cells, most of which represent mature B cells that have recirculated to the bone marrow. These cells were not discriminated from newly generated sIgM+ cells in some early investigations.
To evaluate whether the generation and maturation of B cells are
altered in aged mice, cells were isolated from the femurs and tibias of
14 aged mice and younger control mice fed BrdU for 8 days to 8 wk.
Consistent with previous reports, the overall cellularity (total number
of nucleated cells) was not decreased in the bone marrow of aged mice,
averaging 22 x 106 cells per femur compared with
19 x 106 cells/femur in 1.5- to 3.5-mo-old mice 9, 10, 23, 24, 27 . The aforementioned increase in the population of
sIgDhigh cells can be seen in the FACS profile of the bone
marrow cells of aged vs young adult mice in Fig. 4. The lack of BrdU-labeled cells in this
population of aged mice even after 8 wk of feeding BrdU (Figs. 4 and 5)
confirms that these are mainly mature recirculating cells. This finding
also confirms the conclusion, obtained from analyses of splenic B
cells, that mature B cells of aged mice have a prolonged half-life.
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; summarized in Fig. 5). For
this analysis, cells expressing high levels of sIgD (see above) were
considered to be mature B cells that had recirculated to the bone
marrow and, therefore, were excluded from the analysis of developing
B-lineage cells. Pro-B cells were identified as sIg- cells
that express the pan B cell marker B220 along with CD43. Cells at later
maturational stages become CD43-, and among
B220+sIg- cells, pre-B cells were
distinguished from pro B cells by their lack of CD43 (data not shown).
Following the pre-B cell stage, B cells express sIgM, and levels of
sIgM increase as newly generated B cells mature.
As summarized in Fig. 5 and consistent with earlier studies 22, 23, 24, 25, 26, 27 ,
the population of B220+CD43+ pro-B cells, while
highly variable among bone marrow cells of aged mice, showed no overall
decrease. However, the population of
B220+sIg-CD43- pre-B cells,
although also highly variable in bone marrow cells of aged mice, was
reduced four- to sixfold compared with that in the same population in
younger mice. Thus, whereas the mean percentages of
B220+sIg-CD43- cells among total
nucleated bone marrow cells of 1.5- to 3.5-mo-old and 4- to 8-mo-old
mice were 16.2 and 13.4%, respectively,
B220+sIg-CD43- pre-B cells
represented only 2.8% of the total nucleated bone marrow cells of aged
mice. In terms of absolute numbers of cells this represents, on the
average, 3.2 x 106 cells/femur for 1.5- to 3.5-mo-old
mice, 2.6 x 106 cells/femur for 4- to 8-mo-old mice,
and 0.7 x 106 cells/femur for aged mice.
Maturation of sIgM+ cells in aged mice
Although the population of pre-B cells was reduced, on the
average, by fourfold in aged mice, previous studies have suggested that
the population of newly generated B cells (sIgM+) is only
marginally reduced 27 , a finding that has been used in support of the
argument that the generation of new B cells may be only minimally
compromised in aged mice. To determine whether there are alterations in
the maturation of sIg+ cells in the bone marrow that would
be consistent with the finding that very few immature B cells enter the
spleen in aged mice, we examined the distribution of
sIgM+sIgD- cells and the kinetics of their
accumulation of BrdU in aged and younger control mice. The total number
of sIgM+sIgD-B220+ bone marrow
cells (sIgMvery low plus
sIgMint-high; Fig. 5) and their proportionate
representation among all newly generated B220+ cells were
decreased by 40–60% in aged (23- to 26-mo-old) vs younger (1.5- to
8-mo-old) mice. Although the decrease observed in sIgM+
sIgD- cells between aged and 4- to 8-mo-old mice was
somewhat greater than previously reported 27 , consistent with
previous reports, it was far less dramatic than the decrease in the
population of sIg-CD43- pre-B cells (see
Fig. 5).
Whereas the overall decrease in total
sIgM+sIgD- B cells was approximately twofold
for aged vs young mice, careful examination of the FACS profiles of
these cells suggested a heretofore unrecognized disparity in the
proportion of relatively mature sIgMint-high cells vs
less mature sIgMvery low cells within this population of
bone marrow cells of aged vs young mice (see Figs. 4 and 5). To
quantify differences in these populations, we arbitrarily defined
sIgMvery low cells as those whose anti-IgM PE
fluorescence was less than 0.3 log units greater than the upper limit
found for anti-IgM PE fluorescence for truly IgM-
cells (B220- cells) in each stained bone marrow cell
preparation. As can be seen in Figs. 4 and 5, the total number and
proportionate representation of cells designated as sIgMvery
low among newly developing B220+ cells was not
reduced among bone marrow cells of aged mice compared with cells of
either 1.5- to 3.5-mo-old or 4- to 8-mo-old mice. However, the
proportionate representation of more mature
sIgMint-high bone marrow cells was reduced sevenfold in
aged mice compared with that in juvenile mice and fourfold compared
with that in 4- to 8-mo-old mice. Thus, consistent with the finding
that few newly generated B cells enter the spleen in aged mice,
similarly low numbers of newly generated B cells appear to enter the
pool of more mature sIgMint-high B cells within the bone
marrow. The findings that the population of sIgMvery low
cells is maintained in aged mice, while the numbers of both pre-B cells
and more mature B cells are greatly reduced, suggests that newly
generated sIgMvery low B cells in aged mice are blocked
in their further maturation or mature more slowly than in younger mice,
and with time, cells within this population "pile up" (see below).
Accumulation of BrdU-labeled cells in bone marrow B-cell lineage subsets of aged mice
Consistent with earlier studies, 87% of pre-B cells and >97% of
newly generated sIgM+sIgD- B cells within the
bone marrow of young mice were BrdU labeled within 8 days of feeding
BrdU (see Figs. 4 and 5). When a similar analysis was conducted with
bone marrow cells of aged mice, only 67% of all pre-B and 57% of
immature B cells of aged mice were BrdU labeled after 8 days, whereas
by 2 wk approximately 70% of pre-B cells and 90% of immature B cells
were labeled (see Fig. 5). That cells of aged mice may accumulate BrdU
less efficiently than cells of younger mice was ruled out by the
demonstration that within 3 days Ter 119+ erythroid
precursors labeled as well as those of younger mice (data not shown).
The relatively slow accumulation of BrdU-labeled pre-B and immature B
cells is further evidence that in aged mice cells accumulate in and
leave these populations slowly.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The severalfold increase in longevity found for mature B cells in aged
mice would ultimately lead to a substantial increase in the total
population of peripheral B cells unless the rate of entry of B cells
into the mature B cell population were concomitantly decreased. Since
the pool of mature peripheral B cells remains relatively constant as
mice age, considering that in aged mice the decline in the number of
mature B cells in the spleen may be offset by the increase in the
number of mature, presumably recirculating sIgDhigh cells
in the bone marrow (Fig. 4), it must be assumed that fewer cells enter
the mature B cell pool. In contrast to a previous report that suggested
that the population of recent bone marrow émigrés in the
spleen was not markedly reduced in aged mice 27 , we found a
significant reduction in this population. As in the previous report we
assessed the proportion of cells expressing very high levels of HSA.
Although recent bone marrow émigrés express relatively high
levels of HSA 28, 29 , since staining with anti-HSA Abs may vary
from experiment to experiment, designating cells HSAvery
high and, therefore, newly generated, can be arbitrary and thus
merely include the 10–20% of cells within any population expressing
the highest level of HSA. As cells leave the bone marrow and enter the
spleen they are initially CD23- as well as
HSAvery high 33 . Because of this, the
population of CD23- cells is enriched for HSAvery
high cells, which enables a more precise delineation of
HSAvery high cells. When CD23- cells are used
to standardize HSA levels, the proportion of splenic B cells in both
CD23- and CD23+ subsets that were HSAvery
high decreased from 13% in juvenile mice to 7% in adult mice
and 5.4% in aged mice. Consistent with the conclusion that within the
HSAvery high population of aged mice, very few cells were
newly generated, only 14% of these cells designated HSAvery
high in aged mice were sIgDneg-low. Most convincing,
when the accumulation of BrdU-labeled splenic B cells in aged vs young
mice was evaluated, the majority of HSAvery high cells were
rapidly BrdU labeled in juvenile and young adult mice, whereas in aged
mice only 24% of the cells considered HSAvery high were
BrdU labeled even after 2 mo of feeding BrdU. Thus, only a minority of
cells judged to be HSAvery high in the spleens of
aged mice were newly generated, and overall, the percentage of splenic
B cells that were recent bone marrow émigrés by the
criterion of being HSAvery high as well as BrdU labeled
within 8 wk decreased from 11.7% in juvenile mice to 5.4% in 4- to
8-mo-old mice to 1.4% in 23- to 26-mo-old mice. Additionally,
consistent with the conclusion that transit through the various stages
of B cell maturation may be retarded in aged mice, the accumulation of
BrdU-labeled cells into the HSAvery high population was
slower in aged than in younger mice.
A decrease in the number of splenic B cells that had recently emigrated to the spleen implies that either bone marrow B cell generation is decreased in aged mice or newly generated B cells are impaired in their ability to either leave the bone marrow or enter the spleen once they have left the bone marrow. We have observed, as have others previously 20, 21, 22, 23, 24, 25, 26, 27 , that the population of pre-B cells is reduced severalfold in aged mice and that there is a less dramatic decline in newly generated (sIgM+, IgD-) B cells in the bone marrow of aged vs juvenile or adult mice. However, upon careful analysis, both the phenotypic characteristics of the sIg+ cells present in the bone marrow of aged mice and the kinetics of their BrdU labeling have led us to conclude that B cell generation is indeed markedly impaired and that the population of sIgM+ cells is maintained by the accumulation of immature sIgMvery low cells that fail to undergo the later stages of bone marrow B cell maturation or progress through these stages very slowly.
The maturation of B cells within the bone marrow has been studied
extensively in both sIg transgenic and conventional mice by numerous
laboratories 34, 35, 36, 37, 38 . Following L chain gene rearrangement and L chain
expression, cells express very low levels of sIgM. Maturation of newly
generated B cells is denoted by an increased expression of sIgM and,
ultimately, sIgD expression as well. The physiological significance of
the sIgMvery low stage of development has been suggested by
findings, primarily in sIg transgenic mice, that in the presence of
tolerogenic self antigens, B cell development can proceed through the
sIgMvery low developmental stage, since in some instances
these cells persist or accumulate, while sIgMint-high cells
are essentially eliminated 37, 38 . Inspection of the FACS profiles of
B220+ bone marrow cells of 23- to 26-mo-old mice vs 1.5- to
8-mo-old mice suggests that whereas the population of sIgMvery
low cells is maintained in aged mice, the population of
sIgMint-high cells is diminished (Figs. 4 and 5). To
quantify this apparent alteration in B cell maturation, we arbitrarily
chose to enumerate cells as sIgMvery low if they fell
within 0.3 units on a log fluorescence scale of truly sIg-
cells (B220- cells) on a FACS profile (Fig. 4). When
sIg+ bone marrow cells from aged vs younger mice were
assessed using these criteria, the disparities were readily apparent.
Among bone marrow cells of 1.5- to 3.5-mo-old or 4- to 8-mo-old mice,
approximately 60% of all sIg+ sIgD- bone
marrow cells fall into the sIgMint-high category by this
criterion, while IgMvery low cells represent only 40% of
all newly generated sIg+ cells. In the bone marrow of aged
mice, on the other hand, while the population of sIgMvery
low bone marrow cells is at least equivalent to that found in
younger mice, the population of sIgMint-high cells is
decreased fourfold compared with that in young adults and sevenfold
compared with that in juvenile mice.
Although the phenotypic characteristics of the predominant sIgMvery low bone marrow cells found in aged mice mimic those of the population of cells that persist in tolerant sIg transgenic mice 38 , it is not yet possible to determine the basis for the failure of newly generated B cells in aged mice to further mature. It is possible that newly generated B cells of aged mice are impacted to a greater extent than those of younger mice by environmental self Ags or the anti-idiotypic recognition that is abundant in aged mice 6, 7, 8 . However, any of numerous other mechanisms could account for the failure of sIgMvery low B cells of aged mice to further mature, including failure to receive appropriate maturational signals from the bone marrow stroma or other cells within their developmental milieu.
The finding that the population of sIgMvery low cells is maintained in aged mice despite the four- to sixfold decrease in the pre-B cell population implies that rather than being eliminated as they enter more mature B cell stages, cells may be prohibited from further maturation and thereby accumulate with time in the sIgMvery low population. Consistent with this interpretation is the finding that only 57% of cells in this population were labeled within 8 days of feeding BrdU. In contrast, over 90% sIgM+ sIgD- cells in 1.5- to 8-mo-old mice were labeled in the current studies within 8 days of feeding BrdU, and a large majority were labeled by 3–5 days in previous studies in which earlier time points were analyzed 21, 28, 29 . The relatively slow accumulation of BrdU-labeled sIgMvery low cells in aged mice suggests that cells leave this population slowly, and a steady state is achieved in which relatively few pre-B cells on a daily basis mature and enter the sIgMvery low pool, and relatively few cells mature sufficiently to leave this population and ultimately enter the spleen.
It is of interest that the population of cells identified as pre-B
cells, by virtue of being B220+sIg- and
CD43-, also accumulates BrdU-labeled cells relatively
slowly (Fig. 5). Since there are so few pre-B cells in the marrow of
aged mice, extensive accumulation of cells in this population is
unlikely. However, recent studies have suggested that sIgMvery
low cells may, in the presence of self Ags, down-regulate their
sIg, up-regulate recombinase-activating gene-1 and -2, and ultimately
re-express their µ-chains with another L chain 39, 40, 41 . Because of
such "receptor editing" it is likely that some cells in the
sIgMvery low population would be in a state of equilibrium
with sIg- cells. If such a phenomenon were a common
occurrence in aged mice, the pace of BrdU labeling of a proportion of
sIg- pre-B cells would be equivalent to the pace of
labeling of sIgMvery low cells.
Although the population of peripheral B cells remains relatively
stable throughout adulthood and even into old age, the possibility that
B cell homeostasis is actively maintained remains controversial 42, 43 . Nonetheless, the demonstration that peripheral B cell turnover is
reduced in aged mice while, concomitantly, the generation and
emigration of maturing B cells from the bone marrow is diminished
suggests a connection between the two phenomenon in the maintenance of
total B cells. However, even if the decrease in B cell generation and
increased B cell longevity are linked, it is not yet known whether 1)
the existence of a stable population of long-lived peripheral B cells
provides signals that lead to a decrease in B cell generation, or 2) a
decrease in B cell generation is the primary defect in aged mice,
ultimately resulting in the increased longevity of the existing
population of mature B cells. While a decrease in pre-B cell generation
in aged mice had been known previously and attributed to either
decreased IL-7 production by bone marrow stromal cells 27 or
decreased 
5 expression in aged pro-B cells 44 , the decreased
maturation and accumulation of sIgMvery low cells had
heretofore not been recognized. Whether this block in B cell maturation
proceeds and leads to decreased pre-B cell generation or increased
peripheral B cell longevity or is caused by either of these phenomena
is also not known. Finally, although stem cells of aged mice have been
shown to be capable of reconstituting irradiated mice 45 , experiments
have not yet been conducted to delineate whether developing B-lineage
cells of aged mice are intrinsically deficient in their ability to
mature or whether deficiencies in the bone marrow environment of
developing B cells in aged mice are responsible for the decrease in B
cell generation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Norman R. Klinman, Department of Immunology, IMM-16, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037.
3 Abbreviations used in this paper: BrdU, 5'-bromo-2-deoxyuridine; sIg, surface Ig; HBSS/BSA/Az, HBSS supplemented with 0.1% BSA and 0.2% sodium azide; HSA, heat-stable Ag; PE, phycoerythrin.
Received for publication July 30, 1998. Accepted for publication December 17, 1998.
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), 4- to 8-mo-old (
),
and 23- to 26-mo-old (
) mice fed BrdU for 8 days to 8 wk are shown.
The splenic B cells in the aged and young mice used in this study are
presented. The data points were plotted and graphed using CricketGraph
III. The formula for the curve of best fit and data extrapolation was
generated by the least squares method, an available function of
CricketGraph III.
