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Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33136.
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Abstract |
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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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E47 was first identified as a protein that binds Ig and insulin
gene-regulatory elements (2, 5, 6). In B lymphocytes, the
active DNA-binding complex consists of E47 homodimers
(7, 8, 9); whereas in non-B cells, E47 is unable to bind DNA
or it binds DNA as a heterodimer with cell-restricted bHLH proteins,
such as MyoD or NeuroD (10, 11). The formation and
function of the homodimer or heterodimer depend on the balance between
the E2A-encoded proteins, other class I bHLH proteins (class I bHLH
transcription factor and E2-2), and the E protein-inhibitory
proteins, Id 1–3, which lack the DNA-binding domain and function as
dominant negative inhibitors of E proteins (12). E2A-null
mutant mice fail to generate precursors and mature B cells. The arrest
of B cell development occurs at an early stage, given that no Ig DJ
rearrangements or expression of the B lineage-specific marker B220 can
be detected in homozygous mutant mice (13, 14).
Reconstitution of normal numbers of peripheral mature B cells requires
the presence of both E12 and E47 (15). E2A transcription
factors have been shown to regulate the expression of several B lineage
genes such as 
5, early B cell factor, TdT, and
recombination-activating gene-1 (15, 16, 17, 18, 19), to regulate Ig
rearrangements (20, 21), and to promote Ig class switch
recombination (2).
Aging is associated with decreased production of precursor B lineage
cells in the bone marrow (22, 23, 24, 25, 26, 27). However, despite the
decrease in newly emerging cells from the bone marrow, the population
of mature splenic B cells is maintained because of increased life span
(23, 28). Reduced diversification of B cells has also been
reported to occur in the germinal center (29), where
affinity maturation and isotype switching take place
(30, 31, 32). We have previously demonstrated that pre-B cell
numbers, as well as the surrogate L chain, composed of the
variable-like region of the pre-B cell receptor (VpreB) and 5
peptides, which is critical for Ig variable heavy chain selection,
cellular proliferation, and survival at the pre-B cell stage, are
significantly reduced in aged mice (22, 26, 33). We
further showed that low surrogate L chain mRNA and protein levels, as
measured in IL-7-expanded cultures of B cell precursors from aged mice,
are associated with decreased protein levels of the E2A-encoded
transcription factors E47 and E12 (26).
Here, we have investigated the expression and function of the E2A-encoded transcription factors E12 and E47 in IL-7-expanded pro-B/pre-B cell precursors and in splenic B cells, unactivated or LPS activated, from aging mice. We examined whether the decrease seen in E2A protein levels in aged bone marrow could also be reflected in functional DNA binding and if these measurements of E2A (E12/E47) would also be affected in splenic B cells from aged mice. Moreover, we investigated the nature of the complex, i.e., whether E12/E47 or E47/E47 dimers were involved in DNA binding in B cell precursors in the bone marrow and in mature B cells in the spleen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male and female young (2–4 mo of age) and old (24–27 mo of age) BALB/c mice were obtained from the National Institute on Aging (Bethesda, MD).
Splenic B cell purification
B cells were isolated from the spleens of young and old mice. Briefly, cells were washed twice with medium (RPMI 1640; Life Technologies, Gaithersburg, MD) and incubated (108 cells/ml) for 20 min at 4°C with 100 µl of anti-B220 Microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany), according to the MiniMacs protocol (Miltenyi). Cells were then purified using magnetic columns. At the end of the purification procedure, cells were found to be almost exclusively (85–90%) B220 positive by cytofluorimetric analysis. After the isolation procedure was ended, cells were maintained in serum-free medium for 3 h at 4°C to minimize potential effects of anti-B220 Abs on B cell activation.
B cell culture
B cells were cultured in complete medium (RPMI 1640, supplemented with 10% FCS, 10 µg/ml penicillin-streptomycin and 2 mM L-glutamine). For the determination of the optimal culture conditions, cells (2 x 104 or 2 x 105 in 200 µl of complete medium) were stimulated for 24, 48, or 72 h in 96-well culture plates with LPS (0.1, 1 or 10 µg/ml; Sigma-Aldrich, St. Louis, MO). In the last 4 h of incubation, cultures were pulsed with 1 µCi of [3H]thymidine to determine cell proliferation. Our previous results (34) indicate that optimal B cell proliferation is induced when 1 x 106 cells/ml in a final volume of 200 µl are activated by 10 µg/ml LPS for 48 h. Doses of 0.5 and 2 x 106 cells/ml in a final volume of 200 µl were also evaluated but yielded lower B cell proliferation than did the dose of 1 x 106 cells/ml. For the preparation of nuclear and cytoplasmic extracts, B cells (1 x 106/ml) were stimulated for 48 h in six-well culture plates with 10 µg/ml LPS. These conditions were optimal for cell proliferation. After this time, cells were lysed, and extracts were prepared.
Bone marrow cell culture and Ab staining
Bone marrow cells (3 x 105) from 28 pairs of young and old mice were analyzed for surface CD43, B220, and IgM. Briefly, cells were incubated with PE-labeled anti-CD43 Abs (1/40 diluted; BD PharMingen, San Diego, CA), CY-conjugated anti-B220-Abs (1/40 diluted; BD PharMingen), and FITC-labeled anti-µ Abs (1/100 diluted; Jackson ImmunoResearch Laboratories, West Grove, PA) in FACS buffer (HBSS, buffered in 0.02% sodium azide and 0.1% BSA) for 30 min on ice, using the protocol described elsewhere (26). After staining, cells were fixed with 0.25% paraformaldehyde. Samples of 104 cells were analyzed on a LSR flow cytometer (BD PharMingen) using a logarithmic amplification. The percentage of pre-B cells (IgM-CD43-B220low) from total nucleated bone marrow cells was significantly decreased by aging in 23 of 28 mice, similar to previous results (26, 33). Thus, old mice were classified as severely depleted mice (>80% loss in pre-B cells) or moderately depleted mice (20–80% loss in pre-B cells). Of 28 mice, 5 were nondepleted. Table I summarizes the results obtained with these mice. Expanded populations of pro-B/pre-B cells from young and old mice were obtained as previously described (26, 27). Briefly, bone marrow cells were cultured in six-well culture plates at the concentration of 106/ml in complete medium in the presence of 5 ng/ml murine IL-7 (BioSource International, Camarillo, CA). Seven days later, nonadherent cells were harvested, analyzed by flow cytometry, and lysed; nuclear and cytoplasmic extracts were prepared. The expansion of pro-B/early pre-B cells with IL-7 was decreased significantly in the bone marrow from old as compared with young mice beginning at day 3 after culture initiation and continuing through day 7. At day 7, we observed the maximum difference in expansion between young and old mice (26). After day 7, a great proportion of dead cells were found in both young and old mice.
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Nuclear and cytoplasmic extracts were prepared from cultured bone marrow or spleen cells essentially as previously published (35); briefly, cells were harvested and centrifuged in an Eppendorf 5415C microfuge (Brinkmann Instruments, Westbury, NY; New York; 2000 rpm, 5 min). The pellet was resuspended in 30 µl of solution A containing 10 mM HEPES (pH 7.9), 10 mM KCl, 1.0 mM EDTA, 1 mM DTT, 1.5 mM MgCl2, 1 mM PMSF, 1 tablet of protease inhibitor mixture (Boehringer Mannheim, Mannheim, Germany), and Nonidet P-40 (0.1%); briefly vortexed; and centrifuged (8000 rpm, 5 min, 4°C). The supernatant containing the cytoplasmic extract was removed, and the pellet containing the nuclei was resuspended in solution B containing 20 mM HEPES (pH 7.9), 0.1 mM EDTA, 1 mM DTT, 1.5 mM MgCl2, 2 mM PMSF, 1 tablet of protease inhibitor mixture, and glycerol (10%). The lysate was incubated on ice for 20 min, protein sonicated for a few seconds, and centrifuged (14,000 rpm, 15 min, 4°C). Aliquots of the nuclear extract were stored at -80°C. For the preparation of total cell lysates, cultured cells were harvested and centrifuged in an Eppendorf 5415C microfuge (2000 rpm, 5 min). The pellet was resuspended in 30 µl of a solution containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Nonidet P-40, 0.5% sodium deoxycholate, 1 tablet of protease inhibitor mixture (Boehringer Mannheim), and 1 mM PMSF; briefly vortexed; and centrifuged (14,000 rpm, 15 min, 4°C). Protein content was determined by the Bradford assay.
Preparation of the DNA probes
The µE5 probe was prepared as follows: 100 µl of each single
strand (26 bp), at a concentration of 100 ng/µl, were annealed volume
for volume at temperatures of 85°C (2 min), 65°C (15 min), 37°C
(15 min), and 25°C (15 min) and on ice (15 min) and then end labeled
for 40 min at 37°C, using T4 DNA polynucleotide kinase in the
presence of 1 µl of [
-32P]ATP. The probe was then
purified over a Sepharose G-25-50 column (Sigma-Aldrich). The
ku probe was prepared as follows. A double strand
ku DNA fragment (56 bp), 10 pmol of the 56-oligomer, was end
labeled for 30 min at 37°C with T4 DNA polynucleotide kinase in the
presence of 2 µl of [-32P]ATP, then incubated with
20 pmol of the complementary oligonucleotide at 85°C for 5 min, and
subsequently cooled at room temperature. The probe was then purified
over a Sepharose G-50-80 spin column (Sigma-Aldrich). The sequences
of the probes were: 5'-CCC-GGC-GCGGGG-GCG-ATT-TCG-AGT-CA-3' (µE5,
present in the IgH intronic enhancer) (36) and
5'-GAT-CAG-TGA-TGG-AGT-TGG-CCA-CTC-CCTCTC-TGC-GCG-CTC-GCT-CGC-TCA-CTG-AGG-CC-3'
(ku). The 56-oligomer sequence was constructed by modifying
the sequence originally described (37) to obtain optimal
ku binding to DNA, as heterodimer ku 70/80 (38).
EMSA
A gel mobility shift assay to determine DNA binding of E47 and of ku was performed as follows. The radiolabeled DNA probe was incubated with 10 µg of nuclear extract in the presence of 0.5 µg of poly(dI-dC) or of the circular plasmid pUC9 as nonspecific competitors for µE5 and ku, respectively. The reaction was performed at room temperature in 15 µl of binding buffer (10 mM Tris-HCl (pH 7.5), 75 mM NaCl, 1 mM DTT, 1 mM PMSF, 6% glycerol). To supershift E47 and/or E12, the nuclear extracts were preincubated with rabbit anti-mouse polyclonal E47 (2 µl; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse polyclonal E12 (2 µl; Santa Cruz Biotechnology) Abs, before the addition of the radiolabeled probes. These Abs were the best supershifting Abs, compared with other anti-E47/anti-E12 Abs (BD PharMingen), based on the evidence that they completely prevented the binding of the µE5 complexes in IL-7-expanded pro-B/pre-B cells, similar to the anti-E47 and anti-E12 mAbs of BD-PharMingen, but could be used at a concentration of 50-fold less protein concentration (4-fold less supplied concentration). To supershift the ku 70/80 heterodimers, the nuclear extracts were preincubated with the polyclonal anti-ku 80 (2 µl; Serotec, Oxford, U.K.), before the addition of the radiolabeled probes. The samples were electrophoresed in 5% polyacrylamide gel at 175 V for 3 h at room temperature. The gels were dried on Whatman 3M paper and exposed to Kodak x-ray films (Eastman, Rochester, NY) at -80°C overnight.
Western blotting
For the evaluation of the amounts of E47 and ku 80 proteins in
the cytoplasm and in the nucleus of splenic B cells, cytoplasmic and
nuclear extracts at equal protein concentration were denatured by
boiling for 4 min in Laemmli sample buffer and subjected to SDS-PAGE
using 4–12% polyacrylamide gel under reducing conditions. Proteins
were then electrotransferred onto nitrocellulose filters (Bio-Rad,
Hercules, CA). Nonspecific sites were blocked by incubation of the
membranes with PBS-Tween (1x PBS containing 0.05% Tween 20)
containing 5% milk for 1 h at room temperature (blocking
solution). Filters were incubated with mouse monoclonal anti-E47
Abs (1/400 diluted, BD PharMingen), mouse monoclonal anti-E12 Abs
(1/400 diluted, BD PharMingen), rabbit polyclonal anti-ku 80 Abs
(1/500 diluted; Serotec), goat polyclonal anti-Ikaros Abs (1/200
diluted; Santa Cruz Biotechnology), or hamster monoclonal FS1
anti-5 Ab (1/5 diluted; gift from Dr. J. Jongstra). These
anti-E47 and anti-E12 Abs gave the best signal by Western
analyses. Mouse monoclonal anti-
-actin (1/8000 diluted;
Sigma-Aldrich) and mouse monoclonal anti-histone H-1 (1/200
diluted; Santa Cruz Biotechnology) were used to detect -actin and
histone H-1, respectively, as loading controls. After overnight
incubation with the primary Ab, immunoblots were incubated with the
appropriate HRP-labeled secondary Abs (Rockland, Gilbertsville, PA;
1/16,000 diluted, for mouse primary Abs; Santa Cruz Biotechnologies,
1/200 diluted, for rabbit primary Abs; Santa Cruz Biotechnology 1/200
diluted, for goat primary Abs) for 3 h at 4°C, developed by
enzyme chemiluminescence, and exposed to CL-XPosure Film (Pierce,
Rockford, IL).
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Results |
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DNA binding of E12 and/or E47 are decreased in both bone marrow and spleen of aged mice
Nuclear extracts of whole bone marrow cells from young and old
mice, as well as of pro-B/pre-B cells from young and old mice, in vitro
expanded with IL-7 for 7 days, were prepared and analyzed by EMSA,
using the µE5 sequence as probe. Results were obtained at day 7
because it is very difficult to measure the levels of E2A in IL-7
cultures, at least before day 6, because the small number of cells
recovered is not sufficient to allow Western analyses. In addition, the
ratios of pro-B vs pre-B cells change during culture, pro-B and pre-B
cells express different levels of E2A (12), and therefore
cultured cells would have to be sorted before analysis. Results in Fig. 1A show that the sensitivity
of our EMSA analysis can measure DNA binding only in nuclear extracts
from IL-7-stimulated pro-B/pre-B cells, but not from whole bone marrow
cells. Typically, IL-7-stimulated bone marrow results in about a 10x
expansion of precursor cells, which gives pro-B cells (
80%) and
early pre-B cells (20%) (26). We have previously shown
a reduction in pro-B/pre-B cells of old bone marrow cells expanded on
IL-7 culture as compared with young bone marrow (26, 33)
and a decreased protein level of E12/E47 in old IL-7 blasts
(26). Here we show that IL-7-stimulated pro-B/pre-B cells
from old mice exhibit a significantly reduced ability to bind the µE5
sequence compared with young mice (Fig. 1A). Both E12 and
E47 are in the DNA-binding complex in these expanded pro-B/pre-B cells,
as indicated by the use of anti-E12 and anti-E47 Abs in the
supershift in Fig. 1A.
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Results in Fig. 1C, showing 8 of 20 pairs of mice in this study, indicate a comparable age-related reduction in the ability of nuclear extracts from both IL-7 blasts and LPS-stimulated splenic B cells from the same old mice to bind the µE5 sequence. Even if the age of the mice is toward the end of BALB/c life expectancy (up to 30 mo), we still see in that range of ages old mice that are not depleted in pre-B cells (Fig. 1C, pair 4, where the old mouse is 25 mo old). We do not know when the reduction of E2A-encoded factors is first observed. However, we were able to see decrease in E2A-binding activity in 18-mo-old mice (data not included). From the data published elsewhere (26), altered expression of E2A in old mice is completely correlated with reduction of surrogate light chains observed in in vitro expanded pro-B/early pre-B cells. This in turn contributes to the decreased B lymphopoiesis; therefore, we can expect that the decline in E2A expression likely precedes that of pre-B cell depletion. When the same nuclear extracts were analyzed by EMSA with a 56-oligonucleotide probe, previously described to bind the DNA repair enzyme ku 70/80, no effects of aging or even of LPS stimulation were observed (Fig. 1D). This is consistent with previous reports that ku 70/80 is unaffected during aging in human PBMC (38).
The DNA-binding complex is different in B cell precursors and in mature splenic B cells
We then examined the presence of E47 and/or E12 in the DNA-binding complexes in splenic B cells from young and old mice. Results in Fig. 2 show that in both young and old mice, anti-E47 Abs effectively prevent DNA binding from splenic nuclear extracts, whereas anti-E12 Abs do not modify the DNA binding in the complex, confirming previous reports that only E47/E47 complexes bind DNA in mature B cells (2). Interestingly, and in contrast to splenic B cells, either anti-E47 or anti-E12 Abs were able to completely supershift the band due to DNA binding of E12/E47 proteins in IL-7-blasts, suggesting that the composition of E complexes able to bind DNA differs in B cell precursors (Fig. 1A) as compared with mature B cells. Specificity was shown by the ku probe not being shifted/removed by anti-E47 Abs and by anti-class I bHLH transcription factor Abs not preventing the binding to the µE5 probe (data not shown).
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The expression of the E2A-encoded proteins by Western blot in
total cell lysates from IL-7-expanded pro-B/pre-B cells shows that the
levels of E47 and E12 proteins are decreased in old pro-B/pre-B
extracts (Fig. 3A). Western
analysis was also performed for Ikaros and mouse 5 proteins. Results
show that Ikaros is present at comparable levels in IL-7-expanded
pro-B/pre-B cells from young and old mice. Mouse 5, as already
demonstrated previously (26), is decreased by aging,
although in this sample to a lesser degree than normally seen in aged
pro-B/pre-B cells.
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E12 is expressed at lower levels, as compared to E47, in LPS-stimulated splenic B cells from both young and old mice and is down-regulated by aging
To examine whether E12 is modulated by aging in splenic B cells, nuclear extracts from splenic B cells, LPS activated during a period of 48 h, were analyzed for E47 and E12 protein expression by Western analysis (Fig. 4). Results show that, unlike E47, E12 appears to be present at very low levels in LPS-activated splenic B cells from young mice. Aging significantly impairs the expression of both proteins in nuclear extracts of LPS-activated splenic B cells.
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We next addressed whether the different DNA binding of E47 seen in
young and old mice may occur through different cellular
compartmentalization of this E2A-encoded protein. To this purpose,
nuclear and cytoplasmic extracts of LPS-activated splenic B cells as
well as IL-7 blasts from both young and old mice were analyzed in EMSA
and Western experiments. Results in Fig. 5A indicate that only the
nuclear extracts of unstimulated and LPS-activated splenic B cells from
young mice exhibit DNA binding; this result correlates with Western
data showing that nuclear extracts of LPS-activated splenic B cells
exhibit higher expression of E47 than do cytoplasmic extracts (Fig. 5B). In this experiment, cytoplasmic and nuclear extracts of
B cells from young and old mice show comparable levels of -actin.
Although -actin must be present at higher concentrations in the
cytoplasm than in the nucleus, -actin is found also in the nucleus,
and the possible contamination of nuclear extracts is irrelevant to our
conclusions, because cytoplasmic extracts of splenic B cells do not
display DNA binding. B cells from old mice exhibited neither
DNA-binding nor E47 expression in these experiments, but in other
studies (see Fig. 1C), some (four of eight) nuclear extracts
from old mice were also able to show DNA binding. Conversely, in
pro-B/pre-B cells, at least from young mice, both nuclear and
cytoplasmic extracts display DNA binding and comparable levels of E47
(Fig. 6). Because in almost all IL-7
nuclear and cytoplasmic extracts neither DNA binding nor expression by
Western analysis could be seen for E47/E12, we were not able to
determine whether both nuclear and cytoplasmic extracts had
E47/E12.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of aging on murine B cell precursors in the bone marrow
have been well characterized. The transition from the early pre-B
(CD43+) cell stage to the late pre-B (CD43-)
cell stage, which depends on the expression of the pre-BCR
(µ/5/VpreB) (41, 42, 43), is significantly less efficient
in aging (33). In vivo, CD43+B220+
pro-B/pre-B cells from old mice have reduced surface and cytoplasmic
expression of 5, as well as reduced 5 mRNA (33).
Also in vitro IL-7-expanded pro-B/pre-B cells from old mice show
decreased expression of both 5 and VpreB at the mRNA level,
suggesting that the age-related reduction in surrogate light chains
results, in part, from dysfunctional transcriptional regulation
(26). A decrease in the expression of the E2A-encoded
transcription factors E47 and/or E12 during aging correlates with a
reduced expression of the 5 component of the surrogate light chain
and, in turn, with reduced pre-B cell numbers (26). Here
we show that nuclear extracts of IL-7-stimulated pro-B/pre-B cells from
old mice, exhibiting reduced amounts of E47 and E12 expression, also
display reduced ability to bind DNA. The decrease in E47/E12 protein
itself can explain the decrease in EMSA activity; we do not yet know
about posttranslational modifications of these transcription factors.
In these cells, Ikaros, a zinc finger transcription factor exerting a
critical role during the early stages of B cell development, is
unaffected by aging, demonstrating that not all proteins or
transcription factors are down-regulated during aging.
Little is known concerning the effects of aging on E2A activity in mature B cells. This is the first study describing that the DNA binding of the E2A-encoded protein E47 is reduced in splenic B cells from old as compared with young mice. This reduction reflects decreased levels of E47 expression in the nuclear extracts of splenic B cells from old mice and likely contributes to the reduced B cell proliferation and/or class switch observed in aging mice (43, 44). It might also contribute to the age-related changes in the quality of the Ab response, because the expression of the Ab repertoire changes with age and shifts with respect to Ab specificity, affinity, and heterogeneity. Old mice produce reduced serum levels of Abs against exogenous Ags, these Abs being also of reduced affinity, and higher serum levels of autoantibodies (45, 46, 47). Moreover, the Abs produced in old mice are often oligoclonal (48). Several factors contribute to these changes including thymic involution and reduced T cell functions, which are critical for B cell development, somatic mutation, and isotype switching. It has not yet been well clarified whether B cells from old mice may function normally if adequate T cell help is provided. Nonetheless, several recent reports have shown intrinsic changes in the B cell pool affecting B cell responses. These include clonal expansion of CD5+ B cells (48, 49), impaired signal transduction events through the BCR (50), reduced inducibility of costimulatory molecules (30), and also reduced expression and activity of E2A-encoded transcription factors (herein). Moreover, it has been demonstrated that peripheral B cells from old mice are phenotypically distinct from those found in young mice and that these unique B cell populations arise and are maintained as a consequence of chronic antigenic stimulation (51). Even though we have shown that both bone marrow B cell precursors and mature splenic B cells have a decrease in E2A DNA binding, we do not think that the E2A-deficient splenic B cells are derived only from E2A-deficient recent immigrants from the bone marrow given that these would be only a small population of the total mature B cells we are analyzing in the B220-purified splenic cells (23, 52).
Data in the present paper confirm the observations (10, 11, 53) that in mature B cells E2A proteins bind DNA as E47/E47 complexes. This likely reflects the fact that E12 binds DNA poorly as a homodimer as a consequence of a negative domain immediately N-terminal to the bHLH motif (54, 55), as well as that E12 is expressed at lower levels than E47 in mature B cells (54). Thus, E2A proteins in B cells are unique in their ability to bind DNA as homodimers, whereas in the muscle, in the brain, or in pancreatic cells they bind DNA as heterodimers (10, 11, 54). As Benezra (56) has demonstrated in cell lines, this homodimer contains two disulfide-linked molecules of E47. It should be pointed out that, despite the large body of results on mature B cells, no evidence exists in the literature on the composition of the complex able to bind DNA in B cell precursors. The EMSA results in this paper demonstrate that in splenic mature B cells only E47/E47 complexes bind DNA, whereas in IL-7-expanded pro-B/pre-B cells E47/E12 complexes are involved in DNA binding, likely because splenic B cells express predominantly E47 whereas pro-B/pre-B cells express comparable levels of E47 and E12. In conclusion, the main findings of this study are: 1) the age-dependent decrease in the DNA binding of E12 and/or E47 in both bone marrow and spleen; 2) the different composition of the DNA-binding complexes in IL-7-expanded pro-B/pre-B cells, where both E47/E12 participate in DNA binding, and in the splenic B cells, where E47/E47 complexes bind DNA; and 3) the observation that both nuclear and cytoplasmic extracts of IL-7-expanded pro-B/pre-B cells, but only, nuclear extracts of LPS-activated splenic B cells, show E2A gene expression and DNA binding. These results provide insight into mechanisms through which aging affects B cell development in the bone marrow and B cell functions in the spleen via a down-regulation of the E12/E47 transcription factors and suggest possible approaches for augmenting the humoral response in aged individuals.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bonnie B. Blomberg, Department of Microbiology and Immunology, RMSB, No. II 3146A, University of Miami School of Medicine, 1600 Northwest 10th Avenue, Miami, FL 33136. E-mail address: bblomber{at}med.miami.edu
3 Abbreviations used in this paper: bHLH, basic helix-loop-helix; BCR, B cell receptor; VpreB, variable-like region of the pre-B cell receptor.
Received for publication August 13, 2002. Accepted for publication November 8, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 motif. Science 247:467.5 surrogate light chains and reduced development within the pre-B cell compartment. J. Immunol. 161:4472.
chain (CD25, TAC) expression defines a crucial stage in pre-B cell development. Int. Immunol. 6:1257. and isoforms of protein kinase C in peripheral blood T and B cells from young and elderly adults. Mech. Ageing Dev. 77:197.[Medline]
cells and lymphocytes. Nucleic Acids Res. 19:3893.This article has been cited by other articles:
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 I. Tsuboi, K. Morimoto, Y. Hirabayashi, G.-X. Li, S. Aizawa, K. J. Mori, J. Kanno, and T. Inoue Senescent B Lymphopoiesis Is Balanced in Suppressive Homeostasis: Decrease in Interleukin-7 and Transforming Growth Factor-{beta} Levels in Stromal Cells of Senescence-Accelerated Mice Experimental Biology and Medicine, June 1, 2004; 229(6): 494 - 502. [Abstract] [Full Text] [PDF]
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D. Frasca, E. Van der Put, R. L. Riley, and B. B. Blomberg Reduced Ig Class Switch in Aged Mice Correlates with Decreased E47 and Activation-Induced Cytidine Deaminase J. Immunol., February 15, 2004; 172(4): 2155 - 2162. [Abstract] [Full Text] [PDF]
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, Young; 
, old.
C, E2A DNA binding in IL-7 blasts and LPS-activated splenic
B cells is equivalently reduced in the same aged individual mice.
Nuclear extracts from equal numbers (107) of IL-7-expanded
pro-B/pre-B cells or splenic B cells stimulated with LPS for 48 h
were run in EMSA (10 µg/lane). Films were scanned and analyzed as
described in B. In IL-7 blasts, the levels of DNA binding of
old vs young were 1–13% in seven of eight mice, with the exception
that in one pair of mice (pair 4), old DNA binding was 83% of young
controls. This mouse was the only one not to be depleted in pre-B cells
(with 11.5% pre-B cells) as evaluated by flow cytometry. In
LPS-activated splenic B cells, the levels of old DNA binding were
2–40% compared with young controls in seven of eight mice; and
similar to IL-7 blasts, in pair 4, DNA binding was 81% of young
controls. D, DNA binding of ku 70/80 is not modified by B
cell activation or by aging. The same nuclear extracts shown in
B were run in EMSA (10 µg/lane) using a 56-oligonucleotide
probe that binds the DNA repair enzyme ku 70/80. The multiple bands are
created by one, two, or more multimers of ku binding to DNA. S,
Supershift performed with the anti-ku 80 Ab (see Materials
and Methods).
